Gadph antibody

Total cell lysates were collected by lysing H9C2 cardiomyocytes with lysis buffer supplemented with Protease Inhibitor Cocktail (Thermo Fisher Scientific) and Phosphatase Inhibitor Cocktail (Roche). The protein concentration of the cell lysate was measured by Bradford assay (Bio-Rad, CA, USA). The extracted protein samples were separated by 8%–12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes for detection with appropriate antibodies. Primary antibodies against total caspase 3 (1 : 1000), cleaved caspase 3 (1 : 1000), Bax (1 : 1000), Bcl-2 (1 : 1000), GADPH antibody (1 : 1000), and horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1 : 3000) were purchased from Cell Signaling Technology. Anti-Ptpn4 antibody (1 : 1000) was purchased from Novus. Blots were visualized with Clarity ECL Western Blotting Detection Reagent (Bio-Rad) and subsequently exposed to X-ray film (Carestream, NY, USA). ImageJ software (National Institutes of Health, MD, USA) was used to analyze the optical densities of the immunoreactive bands. Protein expression was normalized to that of loading control (GAPDH).

Para-nitrophenyl phosphate (pNPP), β-naphthyl phosphate, phenyl phosphate, 4-methylumbelliferyl phosphate and polyethylene glycol (PEG mol. wt. 8000) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). O-phospho-L-tyrosine was purchased from Sigma. Peptides including ppERK2 (TGFLpT²⁰²EpY²⁰⁴VATR) and pp-P38 (TDDEMpT180GpY182VAT) were obtained from China Peptides Co. (China). The BIOMOL green TM reagent for phosphate detection (BML-AK111) was purchased from Enzo Life Sciences. The Flag tag (DYKDDDDK) antibody, GADPH antibody, pp-P38 MAPK (pT¹⁸⁰/pY¹⁸²) antibody and pp-P44/42 MAPK (ERKpT²⁰²/pY²⁰⁴) antibody were purchased from Cell Signaling Technology. Ni-NTA agrose was purchased from Roche. All other materials are from Sigma company. All other materials are from Sigma company.