N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (suc-LLVY-MCA) was from Sigma-Aldrich (St Louis, MO). The antibodies used in this study were anti-CerS2 (Sigma Aldrich, St Louis, MO), anti-cathepsin D, anti-cathepsin L and anti-cathepsin S (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HNE-Michael adducts (Calbiochem, San Diego, CA), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Millipore, Temecula, CA) and anti-F4/80 (Biolegend, San Diego, CA).
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a laboratory reagent used to detect and quantify the GAPDH protein in biological samples. GAPDH is an enzyme involved in the glycolysis pathway and is commonly used as a reference or control protein in various assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Anti flag m2, by Merck Group (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Image studio software version 5, by LI COR (2 mentions)
Anti akr1c3 clone np6 g6 a6, by Merck Group (2 mentions)
Anti γh2ax, by Merck Group (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti erk, by Cell Signaling Technology (2 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
Anti tgf β1, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (2 mentions)
44 protocols using anti glyceraldehyde 3 phosphate dehydrogenase gapdh
1
Cathepsin and Lipid Peroxidation Assay
2
Cell Line Authentication and Characterization
3
Compound Library Screening Protocol
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Thapsigargin (Tg), puromycin dihydrochloride, p38 inhibitor (SB203580), and JNK inhibitor (SP600125) were purchased from Sigma (St. Louis, MO, USA). AZD8055 and refametinib were procured from Cayman Chemical (Ann Arbor, MI, USA). The anti-puromycin and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Millipore (Burlington, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. The anti-phospho 4E-BP1 (Ser65), anti-4E-BP1, anti-phospho ERK 1/2, anti-ERK 1/2, anti-phospho p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho eIF2α, anti-eIF2α, anti-phospho c-Jun, anti-c-Jun, and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG (Fcγ fragment specific) was supplied by Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The Selleck Anti-Cancer Compound Library consisting of 414 drugs was purchased from the Department of Convergence Medicine, ASAN Medical Center, University of Ulsan College of Medicine (Seoul, Korea).
4
Western Blot Analysis of Cellular Proteins
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Cells and spheroids were harvested as previously described [11 (link)]. Whole cell lysates were generated using Tris Glycine SDS sample buffer (Gradipore) by shaking at room temperature for 1 h and further processed via SDS-PAGE as described previously [16 (link)]. The following primary antibodies were used: anti-AKR1C3 (clone NP6.G6.A6, 1:500, Sigma), anti-HMGCS2 (mitochondrial) (ab137043, 1:300, Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:50000, Millipore). Visualization and quantification of protein bands were performed with Image Studio software Version 5.2 (LI-COR Biosciences).
5
Antibody-based Characterization of SR-BI
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The
following antibodies were used: anti-SR-BI
antibody directed against either the C-terminal cytoplasmic domain
or the EC domain (Novus Biologicals, Inc., Littleton, CO), anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (Millipore, Billerica, MA), peroxidase-conjugated
goat anti-rabbit secondary IgG, and peroxidase-conjugated sheep anti-mouse
secondary IgG (GE Healthcare), fluorescein isothiocyanate-conjugated
goat anti-rabbit secondary IgG (BD Biosciences, San Jose, CA), and
Alexa633-conjugated
goat anti-rabbit secondary IgG (Invitrogen). Human HDL (1.063–1.21
g/mL) was purchased from Biomedical Technologies, Inc. [125I]Sodium iodide and [3H]cholesterol were purchased from
PerkinElmer,
and [3H]cholesteryl oleyl ether (COE) was purchased
from American Radiolabeled Chemicals, Inc. (St. Louis, MO). EZ-Link
sulfo-NHS-LC-biotin and streptavidin agarose resin were obtained from
Thermo Fisher Scientific (Rockford, IL). Perfluorooctanoic acid (PFO),
cholesterol oxidase (Streptomyces), acyl:CoA cholesterol
acyltransferase (ACAT) inhibitor (i.e., Sandoz 58-035), and cholesterol,
4-cholesten-3-one, and cholesteryl oleate standards
were obtained from Sigma-Aldrich (St. Louis, MO). All other reagents
were of analytical grade.
following antibodies were used: anti-SR-BI
antibody directed against either the C-terminal cytoplasmic domain
or the EC domain (Novus Biologicals, Inc., Littleton, CO), anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (Millipore, Billerica, MA), peroxidase-conjugated
goat anti-rabbit secondary IgG, and peroxidase-conjugated sheep anti-mouse
secondary IgG (GE Healthcare), fluorescein isothiocyanate-conjugated
goat anti-rabbit secondary IgG (BD Biosciences, San Jose, CA), and
Alexa633-conjugated
goat anti-rabbit secondary IgG (Invitrogen). Human HDL (1.063–1.21
g/mL) was purchased from Biomedical Technologies, Inc. [125I]Sodium iodide and [3H]cholesterol were purchased from
PerkinElmer,
and [3H]cholesteryl oleyl ether (COE) was purchased
from American Radiolabeled Chemicals, Inc. (St. Louis, MO). EZ-Link
sulfo-NHS-LC-biotin and streptavidin agarose resin were obtained from
Thermo Fisher Scientific (Rockford, IL). Perfluorooctanoic acid (PFO),
cholesterol oxidase (Streptomyces), acyl:CoA cholesterol
acyltransferase (ACAT) inhibitor (i.e., Sandoz 58-035), and cholesterol,
4-cholesten-3-one, and cholesteryl oleate standards
were obtained from Sigma-Aldrich (St. Louis, MO). All other reagents
were of analytical grade.
6
Antibodies and Reagents for Cancer Research
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Antibodies recognizing Hexokinase I (C35C4), Hexokinase II (C64G5), phospho-pyruvate kinase isozymes M2 (PKM2) (Tyr105), PKM2 (D78A4), α-tubulin, phospho-EGFR (Tyr1068) (1H12), phospho-AKT (Ser473), and phospho-p44/42 mitogen-activated protein kinase (MAPK) (Thr202/Tyr204) (20G11) were purchased from Cell signaling Technology (Danvers, MA, USA). Anti-Pyruvate Dehydrogenase E1-alpha subunit (S293) and total OXPHOS human antibody cocktail were obtained from Abcam (Cambridge, United Kingdom). Antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was supplied from Millipore (Burlington, MA, USA) and anti-Flag was purchased from Sigma Aldrich (St. Louis, MO, USA). Epidermal growth factor (EGF), sodium 2-oxobutyrate (AKB), and L-aspartic acid were supplied from Sigma Aldrich. Gefitinib and osimertinib were obtained from MedChemExpress (Monmouth Junction, NJ, USA). Erlotinib, phenformin, and rotenone were purchased from Cayman (Ann Arbor, MI, USA).
7
Subcellular Fractionation and Immunoblotting
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MES23.5 cells were treated with P7C3 for 2 h. After washing, the cells were treated with MPP+ for 24 h. The cells were then collected, and cytosolic and mitochondrial fractions were isolated using the Mitochondria Isolation Kit for cultured cells (Beyotime). For immunoblot analysis, TOM20 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-Tubulin (Abcam, Cambridge, UK) were selected as the markers for mitochondria and cytosol, respectively. For obtaining the nuclear and cytoplasmic fractions, cells were treated as described above and then lysed in the fractionation buffer containing 3 mM CaCl2, 2 mM MgAc, 320 mM sucrose, 0.1 mM EDTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5% NP-40 for 20 min on ice. After centrifugation for 15 min at 600 × g at 4 °C, the supernatant was collected as the cytoplasmic fraction. The pellets were washed twice with the fractionation buffer without NP-40 and lysed in the nuclear lysis buffer containing 280 mM KCl, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20 mM Hepes (pH 7.9), 25% glycerol, 1.5 mM MgCl2, and 0.3% NP-40 as the nuclear fraction. Histone 2B (Abcam) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, Billerica, MA, USA) were selected, respectively, as the markers for nucleus and cytoplasm in immunoblot analyses.
8
Glioblastoma Cell Culture and Chemotherapeutic Treatments
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Culture petri dishes were obtained from BD Biosciences. Dulbecco's modified Eagle's medium (DMEM) with high glucose, antibiotics (penicillin/streptomycin), and puromycin were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from HyClone Laboratories (Logan, UT, USA). 8.0-μm pore size transwell inserts were purchased from SPL Life Sciences (Korea). Poly- D-lysine (PDL), Temozolomide (TMZ), BCNU (1,3-Bis (2-chloroethyl)-1-nitrosourea,Carmustine), chromogen, 3,3′diaminobenzidine tetrahydrochloride (DAB), 4′,6-diamidino-2-phenylindole (DAPI), DMSO, MTT, proteinase K, and proteinase inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vectastain ABC kit, FITC-avidin and Cy3-avidin were purchased from Vector Laboratories (Burlingame, CA, USA). Antibodies used for the study are listed as follow: anti-BRCC3 (Abcam, Cambridge, MA, USA; ProSci Incorporated, Poway, CA, USA) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, Billerica, MA, USA), anti-γH2AX (Millipore), horseradish peroxidase (HRP)-conjugated or biotinylated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).
9
Protein Extraction and Western Blot Analysis
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Cells were lysed using RIPA buffer supplemented with 0.5 mM PMSF, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 2.5 mM NaF and 1% Triton X-100 by shaking at 4 °C for 1 h. Fifty micrograms of protein were separated using 4–12% NuPAGE Bis-Tris protein gels (Thermo Fisher ScientificTM, Vienna, Austria) and transferred onto a 0.2 µm nitrocellulose membrane (GE Healthcare, Vienna, Austria). Blocking of membranes and antibody incubation was performed in StartingBlockTM (PBS) blocking buffer (Thermo Fisher ScientificTM, Vienna, Austria) using the following antibodies: anti-AKR1C3 (clone NP6.G6.A6, 1:500, Sigma), anti-androgen receptor (1:500, Cell Signaling), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:50000, Millipore). Visualization and quantification of protein bands were performed with Image Studio software Version 5.2 (LI-COR Biosciences).
10
Recombinant PEPD Protein Expression and Characterization
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hPEPD, hPEPD-G278D, hPEPD-R265X and hPEPD-X265R were produced in E.coli as previously described [13 (link), 17 (link)]. EP and human EGF (236-EG-200) were purchased from Fresenius Kabi and R&D Systems, respectively. The following antibodies were used in the study: anti-PEPD (Abcam, ab86507) which detects hPEPD, hPEPD-G278D and mPEPD, anti-ErbB1 (Cell Signaling, 2232), anti-p-ErbB1 (Y1173) (Cell Signaling, 4407), anti-ErbB2 (Cell Signaling, 2165), anti-p-ErbB2 (Y1221/1222) (Cell Signaling, 2243), anti-AKT (Cell Signaling, 4691), anti-p-AKT (Cell Signaling, 4060), anti-ERK (Cell Signaling, 9102), anti-p-ERK (Cell Signaling, 9101), anti-STAT3 (Cell Signaling, 4904), anti-p-STAT3 (Cell Signaling, 9145), anti-cleaved caspase-3 (Cell Signaling, 9661), anti-BCL-2 (Cell Signaling, 2870), anti-BAX (Cell Signaling, 2772), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Millipore, MAB374), and biotin-conjugated anti-6XHistidines (His)-tag (Bethyl, A190-113B). Horseradish peroxidase (HRP)-conjugated streptavidin (N100) was purchased from Thermo Scientific. A goat anti-rabbit IgG-HRP was purchased from Jackson ImmunoResearch (111-035-003).
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