Phospho akt

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

Phospho-Akt is a laboratory reagent used for the detection and quantification of phosphorylated Akt (also known as protein kinase B), a serine/threonine-specific protein kinase that plays a key role in cellular processes such as metabolism, cell growth, and survival. This product can be used in various analytical techniques, including Western blotting and ELISA, to measure the levels of activated, phosphorylated Akt in biological samples.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Anti-phospho-Akt (38 citations) Phospho-Akt (Ser473) (17 citations) Phospho-Akt-S473 (8 citations) Anti-phospho-Akt (Ser473, (7 citations) Phospho-Akt (Thr308— (6 citations) Phospho-Akt 1/2/3 (3 citations) Anti-phospho AKT (sc-7985-R) (3 citations) Phospho-Akt (Ser473) and Phospho-Erk1/2 (Thr202/Tyr204) antibodies (2 citations) Phospho-pT308-Akt (2 citations) Phospho-AKT (SC-7985-R) (2 citations)

46 protocols using phospho akt

Protein Expression Analysis: Cell Lysates and Western Blotting

Check if the same lab product or an alternative is used in the 5 most similar protocols

To determine the expression of protein, whole cell extracts (lysate) were prepared from 1 × 10⁶ cells in lysis buffer (20 mM Tris pH7.4, 250 mM sodium chloride, 0.1% Triton-X-100, 2 mM EDTA, 10μg/ml leupeptin, 10 μg/ml aprotinin, 0.5mM phenylmethylsulfonyl fluoride, 4 mM sodium orthovanadate and 1 mM DTT), and 60 μg of the protein was resolved on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were eletro-transferred to nitrocellulose filters, the membrane (Amersham) was blocked with 5% nonfat dry milk in TBST (20 mM Tris, pH 7.6, 137 mM NaCl, 0.05% Tween-20) for 1 h at room temperature, and the proteins were probed with specific antibodies: CyclinD1, p21, PI3K, PTEN, MMP2, FLAG (Bioworld), PCNA, Akt, phospho-Akt (Thr308), phospho-Akt (Ser 473), Alexa Fluor (488) (Santa Cruz), Caspase3, Bax and Bcl-2 (Neomarker). To assaure equal loading, gels were stripped and reprobed with antibodies againstβ-actin (Kangchen Bio-tech Inc., Shanghai, China). All PVDF membranes were detected by chemiluminescence (ECL, Pierce Technology).

He M., Jiang L., Li B., Wang G., Wang J, & Fu Y. (2017). Oxymatrine suppresses the growth and invasion of MG63 cells by up-regulating PTEN and promoting its nuclear translocation. Oncotarget, 8(39), 65100-65110.

+ Open protocol

+ Expand

Gastrocnemius Tissue Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Gastrocnemius tissue (~50 mg) was homogenized in lysis buffer (135 mM NaCl, 1 mM MgCl₂, 2.7 mM KCl, 20 mM Tris base (pH 8), 1% Triton, 10% glycerol, 10.27 mM Na₃VO₃, 3.5 mM PMSF, 1 µM aprotinin, 10 mM Na₄P₂O₇). Homogenates were centrifuged, the infranatant was collected, and an aliquot was used to measure protein by the Bradford method [36 (link)]. Samples were diluted 1:1 (vol:vol) with 2x Laemmli sample buffer (Bio-Rad cat.1610737), heated to 95 °C for 5 min, subjected to SDS-PAGE (60 µg of protein), and transferred to PVDF membranes. The membranes were subsequently probed with the following primary antibodies: AKT1/2/3 total (62 kDa—Abcam cat # 126811), phospho-AKT (Ser473—60 kDa—Santa Cruz cat # 7985-R), GSK-3β total (46 kDa—Santa Cruz cat # sc-9166), phospho-GSK-3β (Ser9—46 kDa—Invitrogen cat # MA5-14873), AMPKα total (63 kDa—Santa Cruz cat # 74461), phospho-AMPKα (Thr172) (63 kDa—Santa Cruz cat # 33524). Blots were visualized using chemiluminescence and were scanned directly into an image quantification program (Scion Image^®). Values were obtained by dividing the values of the phosphorylated protein of interest by its non-phosphorylated content. Values are expressed in arbitrary units (AUs).

Effting P.S., Thirupathi A., Müller A.P., Pereira B.C., Sepa-Kishi D.M., Marqueze L.F., Vasconcellos F.T., Nesi R.T., Pereira T.C., Kist L.W., Bogo M.R., Ceddia R.B, & Pinho R.A. (2022). Resistance Exercise Training Improves Metabolic and Inflammatory Control in Adipose and Muscle Tissues in Mice Fed a High-Fat Diet. Nutrients, 14(11), 2179.

+ Open protocol

+ Expand

Whole Cell Lysate Preparation and Immunoprecipitation

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

To prepare whole cell lysates, cells or tissues were lysed in Buffer B as described previously [7 (link)]. For immunoprecipitations, 200 μg whole cell lysate was diluted in IP buffer and immunoprecipitated with 4G10 anti-phosphotyrosine agarose conjugate (05-777, Millipore) as described previously [9 (link)]. Whole cell lysates and immunoprecipitates were processed by WES to separate and visualize cellular proteins according to a standard instrument protocol. Primary antibodies used were: Ron β (#sc-374626, 1:25), GAPDH (#sc-25778, 1:300), PCNA (#sc-56, 1:25) from Santa Cruz Biotechnology; pan AKT (#4691, 1:25), phospho-AKT (#9271, 1:25), S6 (#2217, 1:25), phospho-S6 (#4856, 1:25), phospho-eIF4B (#3591, 1:25), phosphor-AKT1(# 9018, 1:25), phosphor-AKT2 (# 8599, 1:25), AKT1(# 2938, 1:25), AKT2 (# 3063, 1:25), phospho-S6K1 (#9234, 1:25), S6K1 (#2708, 1:25), cleaved PARP (#9541, 1:25), phospho-GSK3α/β (#9331, 1:25), GSK3α/β (#5676, 1:25), Met (#8198, 1:25), Ret (#14556, 1:25) and Axl (#8661, 1:25) from Cell Signaling Technology. Secondary antibodies were included in a Wes Master Kit (PS-MK14, ProteinSimple).

Wang L., Wang L., Cybula M., Drumond-Bock A.L., Moxley K.M, & Bieniasz M. (2020). Multi-kinase targeted therapy as a promising treatment strategy for ovarian tumors expressing sfRon receptor. Genes & Cancer, 11(3-4), 106-121.

+ Open protocol

+ Expand

Quercetin Modulation of Cell Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dulbecco’s modified Eagle’s media (DMEM), Dulbecco’s phosphate buffer saline (DPBS), fetal bovine serum (FBS), penicillin–streptomycin, quercetin dihydrate (Qu), 3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT), propidium iodide, rhodamine 123, 2,7-dichlorodihydrofluoresceindiacetate (H₂DCF-DA), Fluo-3 AM, proteinase K, ribonuclease A, 1,2-bis-(O-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), β-nicotinamide adenine dinucleotide reduced disodium salt (β-NADH), ascorbic acid, agarose, dimethyl sulfoxide (DMSO) and anti-β-actin antibody were purchased from Sigma–Aldrich Chemicals (St. Louis, MO). Antibodies against Bcl-2, Bax, Bim, AIF, NF-κB, caspase-3/caspase-8, phospho-MEK, MEK1/2, phospho-ERK, ERK 1/2, phospho-p38, p-38, phospho-JNK, JNK, Nrf-2, catalase, Cu/Zn SOD, PI3K, phospho-Akt, Akt and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc,). FITC Annexin V was purchased from BD Pharmingen. Histone H3 antibody was purchased from Cell Signaling Technology.
Stock solution of quercetin (Qu) was prepared in dimethyl sulfoxide (DMSO) and diluted to the desired final concentration with culture medium just before use. The final DMSO concentration did not exceed 0.1% (v/v).

Rafiq R.A., Quadri A., Nazir L.A., Peerzada K., Ganai B.A, & Tasduq S.A. (2015). A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells. PLoS ONE, 10(7), e0131253.

+ Open protocol

+ Expand

Adenoviral Modulation of HGF Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

MM cells were plated at a density of 5 × 10⁵ cells per well in 6-well tissue culture plates and grown in RPMI 1640/5% FBS. Cells were mock-infected or infected with Ad-LacZ or Ad-NK4 at a MOI of 100. After incubating for 30 h, the cells were treated with HGF (10 ng/ml) for 30 min and solubilized with lysis buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, plus protease and phosphatase inhibitors). The resulting cell lysates were separated by gel electrophoresis, transferred to PVDF membranes, and probed with primary antibodies followed by peroxidase-conjugated secondary antibodies. Protein bands were detected by means of an ECL Western analysis system (Amersham Biosciences, Inc.). An anti-β-gal antibody (Molecular Probes) or anti-HGFα (Santa Cruz) was used to detect adenovirus-mediated expression of LacZ and NK4, respectively. Protein bands were detected by using primary antibodies against Met, phospho-Met, AKT, phospho-AKT, ERK, phosphor-ERK, Nanog, Oct4, Myc (all from Santa Cruz Biotechnology), β-catenin (Cell Signaling Technology), and active β-catenin (anti-ABC, Millipore).

Deng X.B., Xiao L., Wu Y., Jin F., Mossman B., Testa J.R, & Xiao G.H. (2014). Inhibition of mesothelioma cancer stem-like cells with adenovirus-mediated NK4 gene therapy. International journal of cancer. Journal international du cancer, 137(2), 481-490.

+ Open protocol

+ Expand

CXCL1 Signaling Pathway Activation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were seeded in 6 well plates at 250,000/well for 24 hours, serum starved for 24 hours, and stimulated with 60 ng/ml mouse recombinant CXCL1 (RnD Systems) for up to 24 hours. Cells were lysed in RIPA buffer containing protease inhibitors (Sigma cat no. P8340) and 10 μM sodium orthovanadate and sonicated. 50 μg protein were resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked in PBS containing 0.05% Tween-20 (PBS-T) containing 3% dry milk for 1 hour and immunoblotted with primary antibodies at a 1:1000 overnight in PBS-T/3% BSA. With the exception of FAK, antibodies were obtained from Cell Signaling Technology: phospho-Src (Tyr416 cat no. 6943), Src (cat no. 2123), phospho-FAK (Tyr397 cat no.8856), FAK (Santa Cruz Biotechnology, cat no. sc558), phospho-p38MAPK (Thr180/Tyr182, cat no. 4511), p38MAPK (cat no. 9212), phospho-IκB (Ser32/36, cat no. 9246), IκB (cat no. 2678), phospho-p42/44MAPK (Thr202/Tyr204, cat no. 9101), p42/44MAPK (cat no. 9102), phospho-AKT (Ser473, cat no. 4060), AKT (cat no. 2965), phospho-Stat3 (Tyr705, cat no. 9145), Stat3 (cat no. 9132). Immunoblots were washed in PBS-T 3 times for 10 minutes each and incubated with secondary anti-rabbit conjugated to horse radish peroxidase at a 1:1000 dilution in PBS-T/3% milk for 2 hours. Reactions were catalyzed with West Femto ECL substrate.

Bernard S., Myers M., Fang W.B., Zinda B., Smart C., Lambert D., Zou A., Fan F, & Cheng N. (2018). CXCL1 derived from mammary fibroblasts promotes progression of mammary lesions to invasive carcinoma through CXCR2 dependent mechanisms. Journal of mammary gland biology and neoplasia, 23(4), 249-267.

+ Open protocol

+ Expand

Protein Expression Analysis of Heart and ADSC

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein samples were extracted from whole heart homogenates (n = 6/group, chosen randomly) and ADSCs as previously described [21 (link)]. Primary antibody against tumour necrosis factor-α (TNF-α) (R&D, Minneapolis, MN, USA), Akt1/2/3 (Santa Cruze, Santa Cruz, CA, USA), phospho-Akt (Santa Cruze), ERK (Cell Signaling), phosphor-ERK (Cell Signaling), signal transducers and activators of transcription 3 (STAT3; Cell Signaling), phosphor- STAT3 (Cell Signaling), Bcl-2 (Santa Cruze), Bax (Santa Cruze) and horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology) were used according to manufacturers’ instructions. GAPDH and β-actin antibody (Cell Signaling Technology) was used to evaluate the amount of protein loaded in each sample (detailed in the supplementary material).

Liu J., Wang H., Wang Y., Yin Y., Du Z., Liu Z., Yang J., Hu S., Wang C, & Chen Y. (2014). The stem cell adjuvant with Exendin-4 repairs the heart after myocardial infarction via STAT3 activation. Journal of Cellular and Molecular Medicine, 18(7), 1381-1391.

+ Open protocol

+ Expand

Pterostilbene Signaling Pathway Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Pterostilbene, β-actin antibodies, dimethyl sulfoxide (DMSO), and other reagents and chemicals used in our experiments were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). All the monoclonal primary antibodies, including NF-kappa (NF-κ) Bp50, NF-κ Bp65, phospho-IκB, phospho-IKK(Ser176), AKT, phospho-AKT, ERK(p44/42), phosphor-ERK(p44/42), Egr-1, secondary antibody (FITC conjugated goat anti-mouse IgG (1: 100 dilution), α-tubulin, and PCNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primers for control, actin, and TNF-α, and 2X SYBR Green PCR Master Mix were obtained from Life Technologies. Horseradish peroxidase (HRP) conjugated secondary antibodies were obtained from Chemicon International (Temecula, CA, USA).

Pei H.L., Mu D.M, & Zhang B. (2017). Anticancer Activity of Pterostilbene in Human Ovarian Cancer Cell Lines. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 23, 3192-3199.

+ Open protocol

+ Expand

Quantitative Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

NPs and cellular protein total protein concentration evaluated by Pierce^TM BCA Protein Assay Kit (Thermo Fisher). NP and cell lysates were loaded into each well and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%). The protein bands were transferred to nitrocellulose membranes after which were blocked in 5% skim milk or 5% BSA. Primary antibodies (dilution 1:1000) against CD 63 (Abcam, Cambridge, UK), HSP 70, phospho-AKT, AKT, phospho-ERK, ERK (Santa Cruz, Dallas, TX, USA), or β-Actin (Abcam, Cambridge, UK) were incubated with the membrane at 4 °C overnight. After being washed three times with TBST (TBS with 0.1% Tween20^®), membranes were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature. After being washed three times with TBST for 15 min each, the reactivity was examined by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA). The image of the membrane was taken using UV or white light on a Davinci-K Gel Imaging System (Davinch-K, Seoul, Korea). The density of bands was quantified by Image J (Version 1.50, National Institutes of Health, Bethesda, MD, USA).

Kim K.H., Park T.S., Cho B.W, & Kim T.M. (2020). Nanoparticles from Equine Fetal Bone Marrow-Derived Cells Enhance the Survival of Injured Chondrocytes. Animals : an Open Access Journal from MDPI, 10(10), 1723.

+ Open protocol

+ Expand

Examining Myocardial Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

At the end of 30 minutes of reperfusion, the left ventricles were homogenized in a lysis buffer containing 10 mmol/L Tris-HCl, 20 orthophosphate, 1 mmol/L ethylene glycol tetraacetic acid, 1 mmol/L ethylenediaminetetraacetic acid, 2 mmol/L Na₃VO₄, and 1 mmol/L phenylmethylsulfonyl fluoride (pH =7.4). After sonication, the lysates were centrifuged, and the proteins were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride-plus membranes. The membranes were blocked with 5% skim milk followed by incubation overnight at 4°C with the antibodies Bcl-2 (1:1000) and Bax (1:1000; Abcam, Cambridge, UK) and Akt (1:500), β-actin (1:1000), phospho-Akt (at Ser473, 1:500), eNOS (1:500), and phospho-eNOS (at Ser1177, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation, the membranes were washed three times with 0.1% Tween-20 for 15 minutes and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2000; Santa Cruz Biotechnology) at 37°C for 2 hours. Relative densitometry was performed using a computerized software package (ImageJ 1.63; National Institutes of Health).

Wu N., Li W., Shu W, & Jia D. (2014). Protective effect of picroside II on myocardial ischemia reperfusion injury in rats. Drug Design, Development and Therapy, 8, 545-554.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!