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> Chemicals & Drugs > Inorganic Chemical > Pervanadate

Pervanadate

Pervanadate is a chemical compound that has been used to study signaling pathways and cellular processes.
It is a potent inhibitor of protein tyrosine phosphatases, which play a crucial role in regulating cellular signaling.
Pervanadate can enhance the activity of protein tyrosine kinases, leading to increased phosphorylation of cellular proteins and downstream effects.
Reseachers utilize pervanadate to investigate the role of tyrosine phosphorylation in a variety of biological systems, including cell growth, differentaition, and metabolism.
Understanding the mechanisms of pervanadate action is an active area of research with implications for our knowledge of signal transduction and potential therapeutic applications.

Most cited protocols related to «Pervanadate»

1
Multiparametric Immune Cell Analysis
Cited 26 times
After fixation, cells were permeabilized with methanol for 10 min at 4°C, washed twice in cell staining media (CSM; PBS with 0.5% bovine serum albumin and 0.02% sodium azide), and then incubated for 30 min at room temperature simultaneously with relevant antibodies. KG-1 cells were incubated with antibodies against pH2AX and cleaved poly-ADP ribose polymerase (cPARP) to mark cells that had undergone DNA damage and/or apoptosis. PBMCs were incubated with antibodies against surface markers to delineate immune cell subtypes and pSLP-76, an intracellular signaling molecule and substrate for ZAP-70(24 (link)).
For PBMCs treated with pervanadate the antibodies shown in table 1 were used. For KG-1 cells undergoing DNA damage determination the antibodies shown in table 2 were used. After antibody incubation, cells were washed once in cell staining media (CSM), stained with 1 mL of 1:5000 191/193Iridium (Ir) DNA intercalator (www.dvssciences.com; DVS Sciences, Richmond Hill, Ontario, Canada), diluted in PBS with 1.6% PFA and incubated for 20 min at room temperature or at 4°C overnight. Cells were then washed twice with CSM and finally with water for mass cytometric analysis. In order to accurately assess the durability of cisplatin staining in the absence of antibodies, a mock antibody staining procedure was performed with a 30 minute incubation step in 100 µL CSM followed by all subsequent sample processing steps as described above.
Fienberg H., Simonds E.F., Fantl W.J., Nolan G.P, & Bodenmiller B. (2012). A platinum-based covalent viability reagent for single cell mass cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 81(6), 10.1002/cyto.a.22067.
Publication 2012
Antibodies Apoptosis Cells Cisplatin DNA Damage Immunoglobulins Intercalating Agents Methanol pervanadate Poly(ADP-ribose) Polymerases Protoplasm Serum Albumin, Bovine Sodium Azide ZAP70 protein, human
2
Reverse Phase Protein Microarray Analysis
Cited 11 times
Highly enriched populations of approximately 6000 tumor epithelial cells were obtained from SCCs by Laser Capture Microdissection (LCM) using a PixCell IIa (Molecular Devices, Sunnyvale, CA). An equivalent amount of epithelium from AK and UIA samples was isolated by UV cutting laser microdissection using the Veritas (Molecular Devices, Sunnyvale, CA). Microdissected cells were lysed on the CapSure® Macro LCM Caps (Molecular Devices, Sunnyvale, CA) using with a 1:1 mixture of T-Per™ Tissue Protein Extraction Reagent (Pierce, Rockford, IL) and 2X Tris-Glycine SDS Sample Buffer (Invitrogen, Carlsbad, CA) containing 5% β-mercaptoethanol. Reverse Phase Protein Microarrays (RPMA) were prepared using an Aushon 2470 solid pin microarrayer (Aushon Biosystems, Billerca, MA). For sample set 1, the arrayer was outfitted with 350 μm pins, and for sample set 2, 185 μm pins were used. A series of positive and negative control lysates consisting of cell lines treated or untreated with compounds that cause broad phosphoprotein increases (e.g. pervanadate, calyculin) were also printed and slides were stored dessicated at −20°C prior to staining with antibody. For estimation of total protein amounts, selected arrays were stained with Sypro Ruby Protein Blot Stain (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and visualized on the NovaRay scanner (Alpha Innotech, San Leandro, CA). Printed slides were prepared for staining by treating with 1x Reblot (Chemicon, Temecula, CA) for 15 min, followed by 2×5 min washes with PBS. Slides were treated for at least 5 hours or overnight with blocking solution (1g I-block (Applied Biosystems, Bedford, MA), 0.5% Tween-20 in 500mL PBS) with constant rocking. Blocked arrays were stained with antibodies on an automated slide stainer (Dako North America, Inc., Carpinteria, CA) using the Catalyzed Signal Amplification System kit according to the manufacturer’s recommendation (CSA; Dako). Briefly, endogenous biotin was blocked for 10 minutes with the biotin blocking kit (Dako), followed by application of protein block for 5 minutes; primary antibodies were diluted in antibody diluent and incubated on slides for 30 minutes and biotinylated secondary antibodies were incubated for 15 minutes. Signal amplification involved incubation with a streptavidin-biotin-peroxidase complex provided in the CSA kit for 15 minutes, and amplification reagent (biotinyl-tyramide/hydrogen peroxide, streptavidin-peroxidase) for 15 minutes each. A signal was generated using streptavidin-conjugated IRDye680 (LI-COR Biosciences, Lincoln, NE). Slides were allowed to air dry following development. Slides containing sample set 1 were stained with a set of 51 antibodies against phospho-specific and total proteins, and slides containing sample set 2 were stained with a set of 101 antibodies. The proteins and phosphoproteins measured in the two sample sets are shown in a supplementary Table. All of sample set 1endpoints were repeated in set 2 with the exception of cleaved caspase 3 (D175), HIF-1α, pEGFR (Y992), pFOX01 (S256), pNFkB (S536), and pp90RSK (S380) due to antibody availability. All antibodies were subjected to extensive validation for single band, appropriate MW specificity by Western blot as well as phosphorylation specificity through the use of cell lysate controls (e.g. HeLa +/− pervanadate, Jurkat +/− Calyculin purchased from Cell Signaling as lysates).
Stained slides were scanned individually on the NovaRay scanner (Alpha Innotech) or the Vidar Scanner (Vidar Systems, Herndon, VA). The TIF images for antibody-stained slides and Sypro-stained slide images were analyzed using MicroVigene v2.9.9.9 (VigeneTech, Carlisle, MA, USA). Briefly, Microvigene performed spot finding, local background subtraction, replicate averaging and total protein normalization, producing a single value for each sample at each endpoint. All data was background subtracted (local and slide average), normalized to total protein, and all signal values produced for data analysis were at least 2 standard deviations above background.
Einspahr J.G., Calvert V., Alberts D.S., Curiel-Lewandrowski C., Warneke J., Krouse R., Stratton S.P., Liotta L., Longo C., Pellicani G., Prasad A., Sagerman P., Bermudez Y., Deng J., Bowden G.T, & Petricoin EF I.I.I. (2012). Functional Protein Pathway Activation Mapping of the Progression of Normal Skin to Squamous Cell Carcinoma. Cancer Prevention Research (Philadelphia, Pa.), 5(3), 403-413.
Publication 2012
3
Analyzing Mer Protein Phosphorylation in 697 B-ALL Xenograft Mice
Cited 8 times
NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were transplanted with 2 ×
106 697 B-ALL cells by intravenous injection into the tail
vein, and leukemia was established for 14 days prior to treatment
with a single dose of 3 mg/kg 11 or an equivalent volume
(10 mL/kg) of saline vehicle. Pervanadate solution was prepared fresh,
as described above. Femurs were collected from mice 30 min after treatment,
and bone marrow cells were flushed with 1 mL of room temperature RPMI
medium + 20% FBS + 1 μM MgCl2 + 100 untis/ml DNase
+ 240 μM pervanadate and incubated at room temperature in the
dark for 10 min. Bone marrow cells were collected by centrifugation
at 4 °C, lysates were prepared, Mer protein was immunoprecipitated,
and total and phospho-Mer proteins were detected and quantitated by
Western blot, as described above.
Zhang W., DeRyckere D., Hunter D., Liu J., Stashko M.A., Minson K.A., Cummings C.T., Lee M., Glaros T.G., Newton D.L., Sather S., Zhang D., Kireev D., Janzen W.P., Earp H.S., Graham D.K., Frye S.V, & Wang X. (2014). UNC2025, a Potent and Orally Bioavailable MER/FLT3 Dual Inhibitor. Journal of Medicinal Chemistry, 57(16), 7031-7041.
Publication 2014
Bone Marrow Cells Deoxyribonucleases Femur Leukemia Magnesium Chloride Mus pervanadate Proteins Saline Solution
4
Tyrosine Kinase Receptor Signaling Assay
Cited 7 times
697 B-ALL cells
and Molm-14 AML cells were cultured in the presence of 11 or vehicle-only for 1.0 h. Pervanadate solution was prepared fresh
by combining 20 mM sodium orthovanadate in 0.9× PBS in a 1:1
ratio with 0.3% (w/w) hydrogen peroxide in PBS for 15–20 min
at room temperature. Cultures were treated with 120 μM pervanadate
for 3 min prior to collection, and cell lysates were prepared in 50
mM HEPES (pH 7.5), 150 mM NaCl, 10 mM EDTA, 10% glycerol, and 1% Triton
X-100, supplemented with protease inhibitors (Roche Molecular Biochemicals,
no. 11836153001). Mer and Flt3 proteins were immunoprecipitated with
anti-Mer (R&D Systems, no. MAB8912) or anti-Flt3 (Santa Cruz Biotechnology
no. sc-480) antibody and Protein G agarose beads (InVitrogen). Phospho-proteins
were detected by Western blot using an antiphospho-Mer antibody raised
against a peptide derived from the triphosphorylated activation loop
of Mer8 (link) (Phopshosolutions, Inc.) or an
antibody specific for phosphorylated Flt3 (Cell Signaling Technology,
no. 3461). Nitrocellulose membranes were stripped and total proteins
were detected using a second anti-Mer antibody (Epitomics Inc., no.
1633-1) or anti-Flt3 antibody (Santa Cruz Biotechnology no. sc-480).
Relative phosphorylated and total protein levels were determined by
densitometry using ImageJ, and IC50 values were calculated
by nonlinear regression.
32D Cells expressing a chimeric EGFR-Mer,
EGFR-Axl, or EGFR-Tyro3 receptor were cultured in the presence of 11 or vehicle-only for 1.0 h before stimulation with 100 ng/mL
EGF (BD Biosciences no. 354010) for 15 min. Cells were centrifuged
at 1000g for 5 min and washed with 1× PBS. Cell
lysates were prepared in 20 mM HEPES (pH 7.5), 50 mM NaF, 500 mM NaCl,
5.0 mM EDTA, 10% glycerol, and 1% Triton X-100, supplemented with
protease inhibitors (10 μg/mL leupeptin, 10 μg/mL phenylmethylsulfonyl
fluoride, and 20 μg/mL aprotinin) and phosphatase inhibitors
(50 mM NaF and 1.0 mM sodium orthovanadate). Mer protein was immunoprecipitated
using a custom polyclonal rabbit antisera raised against a peptide
derived from the C-terminal catalytic domain of Mer and Protein A
agarose beads (Santa Cruz Biotechnology). Axl and Tyro3 proteins were
immunoprecipitated using an antibody directed against a FLAG epitope
engineered into the chimeric proteins (Sigma-Aldrich, no. F1804).
Phosphotyrosine-containing proteins were detected by Western blot
with a monoclonal HRP-conjugated antiphosphotyrosine antibody (Santa
Cruz Biotechnology, no. sc-508). Antibodies were stripped from membranes,
and total proteins were detected with the same antibodies used for
immunoprecipitation.
Zhang W., DeRyckere D., Hunter D., Liu J., Stashko M.A., Minson K.A., Cummings C.T., Lee M., Glaros T.G., Newton D.L., Sather S., Zhang D., Kireev D., Janzen W.P., Earp H.S., Graham D.K., Frye S.V, & Wang X. (2014). UNC2025, a Potent and Orally Bioavailable MER/FLT3 Dual Inhibitor. Journal of Medicinal Chemistry, 57(16), 7031-7041.
Publication 2014
Antibodies Antibodies, Anti-Idiotypic Aprotinin Catalytic Domain Cells Chimera Edetic Acid EGFR protein, human FLT3 protein, human G-substrate Glycerin HEPES Immune Sera Immunoglobulins inhibitors Intestinal Atresia, Multiple leupeptin Monoclonal Antibodies Nitrocellulose Orthovanadate Peptides Peroxide, Hydrogen pervanadate Phosphoric Monoester Hydrolases Phosphotyrosine Protease Inhibitors Proteins Rabbits Sepharose Sodium Sodium-20 Sodium Chloride Tissue, Membrane Triton X-100 Western Blotting
5
Signaling pathways in platelet activation
Cited 6 times
Antibodies and Reagents—Anti-human GPVI monoclonal antibody
(mAb) 204-11 has been described previously
(25 (link)). Anti-Syk polyclonal Ab
(pAb) (26 (link)) was kindly provided
by J. B. Bolen (DNAX, CA). Anti-phospho-Syk (Tyr525/526) pAb and
anti-Myc mAb were purchased from Cell Signaling Technology (New England
Biolabs UK Ltd., Herts, UK). T7-Tag mAb was purchased from Novagen
(Nottingham, UK). Anti-phosphotyrosine mAb 4G10, anti-FcR γ-chain pAb,
and normal rabbit IgG were purchased from Upstate Biotechnology (Milton
Keynes, UK). Anti-human CLEC-2 mAb was purchased fromR&D Systems Inc.
(Minneapolis, MN). Anti-PECAM-1 mAb AB468 was from Autogen-Bioclear
(Wiltshire, UK). Horseradish peroxidase-conjugated donkey anti-rabbit
secondary Ab and enhanced chemiluminescence reagents (ECL) were purchased from
Amersham Biosciences. Mouse IgG1 monoclonal was purchased from Abcam
(Cambridge, UK). Fluorescein isothiocyanate-conjugated anti-mouse IgG
secondary antibody was purchased from Sigma. Collagen was obtained from
Nycomed Austria GmbH (Linz, Austria). Rhodocytin was purified from the venom
of Calloselasma rhodostoma (27 (link)). The Src kinase
inhibitors used were, PP1, purchased from BioSource Europe (Nivelles,
Belgium); PP2, purchased from Calbiochem (Nottingham, UK); and PD0173952, a
gift from Pfizer Global Research and Development (Ann Arbor, MI). The Syk
kinase inhibitor, R406, was a kind gift of Dr. D. Simmons (Cellzome UK Ltd.,
Cambridge). FcR γ-chain “knock-out” mice were bred as
heterozygotes as described
(28 (link)). 10 mm Pervanadate was freshly prepared on the day for use by mixing sodium
orthovanadate and hydrogen peroxide in phosphate-buffered saline to final
concentrations 10 mm, then left for 5 min at room temperature and
kept on ice. Other reagents were from previously described sources
(6 (link),
8 (link),
24 ).
Constructs—The human pRc/GPVI, pEF6/FcR γ-chain,
pEF6/CLEC-2, and mutant CLEC-2 (Y7F) expression plasmids have been previously
described (8 (link)). The human
pcDNA3/-G6b-B (kindly given by Prof R. D. Campbell, Oxford, UK) has been
described (9 (link)) and the human
pcDNA3/PECAM-1 (kindly given by C. D. Buckley, Birmingham, UK) has also been
described (29 (link)). The NFAT
luciferase reporter containing three copies of the distal NFAT site from the
interleukin-2 promoter has been described
(30 (link)).
Making Myc-tagged FcR γ-Chain—The c-Myc
epitope tag (EQKLISEEDL) was fused at the amino terminus of FcR γ-chain
by using pEF6/FcR γ-chain as a template and the primers: forward
(5′-GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG CTG GGA GAG CCT CAG
CTC-3′) and reverse (5′-CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC
GGC CGC TGC TTG TTC AAC-3′), and subcloned into pEF6.
Site-directed Mutagenesis of G6b-B—Site-directed mutagenesis
of G6b-B was performed by a QuikChange® Site-directed Mutagenesis Kit
(Stratagene, Cambridge, UK). The primers G6b-B-Y211F-forward (5′-CCG AGC
CTG CTC TTT GCG GAT CTG GAC-3′) and G6b-B-Y211F-reverse (5′-GTC
CAG ATC CGC AAA GAG CAG GCT CGG-3′) were used for tyrosine to
phenylalanine mutation in G6b-B (Y211F) using wild-type human pcDNA/G6b-B as a
template. The primers G6b-B-Y237F-forward (5′-GAT GCC TCC ACC ATC TTT
GCA GTT GTA GTT TG-3′) and G6b-B-Y237F-reverse (5′-CAA ACT ACA ACT
GCA AAG ATG GTG GAG GCA TC-3′) were used for tyrosine to phenylalanine
mutation in G6b-B (Y237F) using wild-type human pcDNA/G6b-B as a template and
they were also used for tyrosine to phenylalanine mutation in G6b-B
(Y211F/Y237F) using mutant pcDNA/G6b-B (Y211F) as a template. All sequences
were verified by sequencing.
Cell Culture—Wild-type (WT), Syk-deficient
(31 (link)), SHP1 and SHP2
double-deficient (32 (link)) (kindly
donated by L. Meyaard, Utrecht, The Netherlands), SHIP-deficient
(33 (link)) (kindly donated by D. K.
Newman, Milwaukee, WI) DT40 chicken B cells were grown in RPMI supplemented
with 10% fetal bovine serum, 1% chicken serum, 100 units/ml penicillin, 100
μg/ml streptomycin, 50 μm β-mercaptoethanol, and 20
mm glutamine.
Luciferase Assay—The NFAT reporter assay was performed as
described (8 (link),
24 ). The indicated amount of
DNA of each construct and 15 μg of NFAT-luciferase reporter construct were
transfected by electroporation at 350 V and 500 microfarads into 2 ×
107 cells of WT, Syk-deficient, and SHP1/SHP2-deficient DT40 cells.
Twenty hours after transfection, live cells were counted by trypan blue
exclusion, and samples divided for luciferase assay (2 × 106 cells/ml), flow cytometry (5 × 105 cells/sample), and Western
blotting (1 × 106 cells/sample). Collagen was used at 10
μg/ml and rhodocytin was used at 50 nm. Luciferase activity was
measured with a Centro LB960 microplate luminometer (Berthold Technologies,
Germany). All results were compared with basal in mock-transfected cells.
Flow Cytometry—Cell surface expression of transfected cells
was analyzed by flow cytometry using 1 μg/ml, GPVI mAb, PECAM-1 mAb, and
Myc mAb to detect CLEC-2 and FcR γ-chain, T7-Tag mAb to detect G6b-B, or
mouse IgG followed by staining with 4 μg/ml fluorescein
isothiocyanate-conjugated anti-mouse IgG secondary antibody, and assessed on a
FACScalibur (Becton Dickinson, San Jose, CA). Data were analyzed using
CellQuest software.
Human and Mouse Platelets—Washed preparations of human and
mouse platelets were prepared as previously described
(5 (link),
8 (link)). Platelets were resuspended
in a modified Tyrodes-HEPES buffer at concentrations of 4 ×
108/ml (human) or 2 × 108/ml (mouse). Platelets
were prewarmed to 37 °C for 5 min and incubated with inhibitors or solvent
controls for up to 10 min.
Immunoprecipitation and Western Blotting—Transfected cells
(1 × 108/ml) were incubated for 30 min in RPMI at 37 °C
before stimulating. After stimulation, transfected cells or platelets were
lysed with ice-cold 2× lysis buffer (2% Triton X-100, 2% dodecyl
maltoside, 4 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 20
μg/ml aprotinin, 20 μg/ml leupeptin, 2 μg/ml pepstatin, 10
mm sodium orthovanadate, pH 7.5) and insoluble material was removed
by centrifugation. For immunoprecipitation, lysates were precleared with
protein A (G)-Sepharose beads for 30 min at 4 °C and mixed with 2 μg of
the indicated antibodies and protein A-Sepharose beads (protein G-Sepharose).
The mixture was rotated for 2 h at 4 °C. Whole cell lysates or
immunoprecipitated lysates were added to 2× Laemmli sample buffer.
Samples were separated by SDS-PAGE on 10 or 4–12% BisTris gels
(Invitrogen) and transferred to polyvinylidene difluoride membrane. Western
blotting was carried out as described previously
(24 ).
Statistical Analysis—Experiments were performed on at least
three occasions and results are shown as mean ± S.E. with the exception
of the representative Western blots and flow cytometry histograms. Statistical
significance was determined using Student's t test.
Mori J., Pearce A.C., Spalton J.C., Grygielska B., Eble J.A., Tomlinson M.G., Senis Y.A, & Watson S.P. (2008). G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2. The Journal of Biological Chemistry, 283(51), 35419-35427.
Publication 2008

Most recents protocols related to «Pervanadate»

1
Pervanadate Boost for Tyrosine Phosphorylation
We used the pervanadate boost method [11 (link)] to increase identification of tyrosine phosphorylated peptides with the isobaric TMT labelling experiments. The pervanadate solution was prepared by adding 10 μl of 0.1 M sodium orthovanadate to 10 μl of 0.2 M hydrogen peroxide (diluted 50 × from a 30% stock). The solution was then incubated at room temperature for 15 min and was added to the PBMCs in PBS. A part of PBMCs from healthy donors were treated with 0.1 mM pervanadate solution in phosphate-buffered saline (pH 7.4) at 37 ℃ for 10 min.
Kaneko T., Ezra S., Abdo R., Voss C., Zhong S., Liu X., Hovey O., Slessarev M., Van Nynatten L.R., Ye M., Fraser D.D, & Li S.S. (2024). Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients. Clinical Proteomics, 21, 13.
Full text: Click here
Publication 2024
2
Culturing Mouse and Human Prostate Cancer Cells
Mouse MyC-CaP (53 (link)) and luciferase-expressing MyC-CaP (MyC-CaP/GFP-Luc) (54 (link)) PCa cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml) and streptomycin (100 μg/ml; 1X Pen/Strep). Both cell lines were confirmed to be negative for mycoplasma, lymphocytic choriomeningitis virus, lactate dehydrogenase virus, mouse hepatitis virus, and mouse parvovirus by IDEXX Bioanalytics. Human C4-2B PCa cells were purchased from American Type Culture Collection and were grown on 0.01% poly-lysine–coated tissue culture plates in RPMI 1640 supplemented with 10% FBS and 1X Pen/Strep. All cells were maintained in a sterile incubator at 37°C and 5% CO2. For serum starvation, cells were cultured in growth media with 0.1% FBS. For pervanadate stimulation, cells were treated with 200 μM pervanadate for 15 min at 37°C before lysis.
Stanford S.M., Nguyen T.P., Chang J., Zhao Z., Hackman G.L., Santelli E., Sanders C.M., Katiki M., Dondossola E., Brauer B.L., Diaz M.A., Zhan Y., Ramsey S.H., Watson P.A., Sankaran B., Paindelli C., Parietti V., Mikos A.G., Lodi A., Bagrodia A., Elliott A., McKay R.R., Murali R., Tiziani S., Kettenbach A.N, & Bottini N. (2024). Targeting prostate tumor low–molecular weight tyrosine phosphatase for oxidation-sensitizing therapy. Science Advances, 10(5), eadg7887.
Publication 2024
3
Phospho-flow Cytometry of pSTAT3
Phospho-flow cytometry was used to assess the levels of phosphorylated STAT3 (pSTAT3). Frozen murine tumors were thawed, washed and cultured for 10 min with pervanadate at 37 °C in complete RPMI. Subsequently, the cells were stained for multiparameter flow cytometry as described above.
Wieboldt R., Sandholzer M., Carlini E., Lin C.W., Börsch A., Zingg A., Lardinois D., Herzig P., Don L., Zippelius A., Läubli H, & Mantuano N.R. (2024). Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2. Cellular and Molecular Immunology, 21(5), 495-509.
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Publication 2024
4
Immunoprecipitation of WEHI3 Proteins
Cell lysates of WEHI3 cells were prepared with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, 150 mM NaCl, 1% Triton-X100, and protease/phosphatase inhibitors. In some experiments, the cells were subjected to pervanadate treatment prior to preparing the lysates. The obtained cell extracts were incubated with antibody-conjugated agarose beads for 1 h at 4°C, and the precipitated proteins were analyzed by SDS−PAGE and immunoblotting.
Karyu H., Niki T., Sorimachi Y., Hata S., Shimabukuro-Demoto S., Hirabayashi T., Mukai K., Kasahara K., Takubo K., Goda N., Honke K., Taguchi T., Sorimachi H, & Toyama-Sorimachi N. (2024). Collaboration between a cis-interacting natural killer cell receptor and membrane sphingolipid is critical for the phagocyte function. Frontiers in Immunology, 15, 1401294.
Full text: Click here
Publication 2024
5
Cultivation and Genetic Manipulation of Mouse Mast Cells
A stable cell line of mouse bone marrow-derived mast cells (BMMCL) was generously provided by Dr. M. Hibbs (39 (link)). The cells were cultivated in freshly prepared RPMI-1640 culture medium, which was supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, MEM nonessential amino acids, 1 mM sodium pyruvate, 10% fetal calf serum (FCS), and 10% WEHI-3 cell supernatant as a source of interleukin-3 (IL-3). Cells were grown at 37°C in 5% CO2 and passaged every 2-3 days. BMMCL were transfected with DNA constructs through nucleofection using a Mouse Macrophage Kit and the Amaxa Nucleofector II (program Y-001; Lonza Cologne AG, Cologne, Germany), following the manufacturer’s instructions. Subsequently, the cells were transferred to culture media supplemented with IL-3 and cultured for 24-48 hours before analysis. In some cases, cells were treated for 30 min with 100 nM Calyculin A to inhibit Ser/Thr phosphatases. Alternatively, cells were pretreated for 30 min with 10 μM LY-333531 to inhibit cPKC before Calyculin A treatment. Cells were also treated for 15 min with freshly prepared pervanadate as described previously (10 (link)) to inhibit Tyr phosphatases. To suppress the activity of Src family kinases, cells were incubated for 60 min with 20 μM PP2 before pervanadate treatment. To activate PKCs, cells were incubated for 15 min with 1 μM PMA.
The HEK 293FT packaging cells, derived from human embryonic kidney tissue, were sourced from Promega Biotec. Cells were grown at 37°C in 5% CO2 in DMEM supplemented with 10% FCS and antibiotics. For lentivirus production, HEK 293FT cells were used at passages 4-15. Cells were transfected with DNA constructs using polyethylenimine as described previously (30 (link)).
Sulimenko V., Sládková V., Sulimenko T., Dráberová E., Vosecká V., Dráberová L., Skalli O, & Dráber P. (2024). Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2. Frontiers in Immunology, 15, 1321321.
Full text: Click here
Publication 2024

Top products related to «Pervanadate»

1
Protease inhibitor cocktail by Roche
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2
Pervanadate by Merck Group
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Pervanadate is a laboratory chemical compound used as a tyrosine phosphatase inhibitor in cellular and biochemical research. It is a potent inhibitor of protein tyrosine phosphatases, which play a crucial role in various signal transduction pathways. Pervanadate is commonly used in cell culture experiments to study the effects of tyrosine phosphorylation on cellular processes.
3
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4
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
7
Protease inhibitor cocktail by Merck Group
Sourced in United States, Germany, China, United Kingdom, Italy, Japan, Sao Tome and Principe, France, Canada, Macao, Switzerland, Spain, Australia, Israel, Hungary, Ireland, Denmark, Brazil, Poland, India, Mexico, Senegal, Netherlands, Singapore
The Protease Inhibitor Cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that can degrade proteins. It is a combination of various chemical compounds that work to prevent the breakdown of proteins in biological samples, allowing for more accurate analysis and preservation of protein integrity.
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Cytofix cytoperm by BD
Sourced in United States, Germany, United Kingdom, France, Canada, Belgium, Australia
Cytofix/Cytoperm is a fixation and permeabilization solution developed by BD for use in flow cytometry and immunohistochemistry applications. It is designed to facilitate the intracellular staining of proteins and other cellular components while preserving cellular structure and antigenicity.
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Ionomycin by Merck Group
Sourced in United States, Germany, United Kingdom, Macao, France, Italy, China, Canada, Switzerland, Sao Tome and Principe, Australia, Japan, Belgium, Denmark, Netherlands, Israel, Chile, Spain
Ionomycin is a laboratory reagent used in cell biology research. It functions as a calcium ionophore, facilitating the transport of calcium ions across cell membranes. Ionomycin is commonly used to study calcium-dependent signaling pathways and cellular processes.
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Dynabeads protein g by Thermo Fisher Scientific
Sourced in United States, Norway, Germany, United Kingdom, China, Canada, Japan, Denmark, Australia, France
Dynabeads Protein G is a magnetic bead-based product used for the purification and isolation of immunoglobulins (Ig) and other proteins that bind to Protein G. It provides a reliable and efficient method for the capture and separation of target proteins from complex samples.

More about "Pervanadate"

Pervanadate, a powerful tool for studying cellular signaling pathways, is a chemical compound that has garnered significant attention in the scientific community.
As a potent inhibitor of protein tyrosine phosphatases (PTPs), pervanadate plays a crucial role in regulating cellular signaling processes, with implications for our understanding of cell growth, differentiation, and metabolism.
Researchers often utilize pervanadate in conjunction with other reagents, such as protease inhibitor cocktails, Lipofectamine 2000, DMSO, FBS, DMEM, Cytofix/Cytoperm, Ionomycin, and Dynabeads Protein G, to investigate the complex mechanisms underlying signal transduction.
By inhibiting PTPs, pervanadate enhances the activity of protein tyrosine kinases, leading to increased phosphorylation of cellular proteins and downstream effects.
The study of pervanadate's mechanisms of action is an active area of research, with the potential to uncover new insights into signal transduction pathways and pave the way for innovative therapeutic applications.
Researchers can leverage the power of AI-driven platforms like PubCompare.ai to identify the most accurate and reproducible protocols from the literature, preprints, and patents, optimizing their pervanadate-related experiments and taking their research to new heights.
Whether you're interested in exploring the role of tyrosine phosphorylation in biological systems or seeking to expand your understanding of cellular signaling, pervanadate and its associated techniques offer a wealth of opportunities for scientific exploration and discovery.