Ab205781

Proteins were extracted from the primary cardiomyocytes in RIPA buffer (1% Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH 7.0; Solarbio, China) supplemented with a protease inhibitor cocktail (Cat: I3786-1ML, Sigma). The cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to a PVDF membrane (Millipore Corporation, USA). After blocking with 8% milk in PBS, pH 7.5, the membranes were incubated with the following specific primary antibodies of Bax (ab32503), Bcl-2 (ab59348), cytochrome C (ab13575), Smac/Diablo (ab32023), cleaved-capase-3 (ab13847), cleaved-capase-9 (ab2324), p-p38 (ab47363), p38 (ab31828), p-ERK (ab214362), and ERK1/2 (ab196883; all at a dilution of 1:1000, Abcam, UK). After overnight incubation, the appropriate HRP-conjugated anti-rabbit IgG secondary antibody (ab205781, Abcam, all at a dilution of 1:5000) was subsequently applied and immunodetection was achieved using the ECL Plus detection system (Millipore Corporation) according to the manufacturer's instructions. Band intensity was quantified using Image Lab™ Software (Bio-Rad, China). GAPDH (ab8245, Abcam) was used as an internal control.

Cells were lysed by RIPA lysis buffer (Beyotime) to isolate proteins, and then protein concentrations were analyzed using the bicinchoninic acid (BCA) protein assay kit (Takara). Extracted proteins were separated by an SDS-PAGE minigel, and then transferred onto PVDF membranes. After blocking by 5% fat-free milk powder in TBST buffer, membranes were interacted with primary antibodies against SOX4 (ab80261, Abcam, Cambridge, MA, USA), PCNA (ab29, Abcam), Bax (ab32503, Abcam), Cleaved caspase 3 (ab2302, Abcam), Bcl-2 (ab692, Abcam), N-cadherin (ab18203, Abcam), Vimentin (ab92547, Abcam), E-cadherin (ab15148, Abcam), as well as GAPDH (ab181602, Abcam) at 4 °C overnight, followed by incubated with the HRP-conjugated secondary antibody (ab205781, Abcam). Finally, signal visualization was performed by ECL Substrates (Millipore, Billerica, MA, USA).