Ab185656

Cells were lysed in the RIPA lysis buffer that included a protease inhibitor cocktail on ice. An equivalent amount of total protein was separated from various samples using SDS-PAGE gels at 100 V for 1.5 h and then transferred onto 0.22-μm polyvinylidene difluoride membranes at 280 mA for 1.5 h. The membranes were blocked with 5% non-fat milk for 1 h and then incubated overnight at 4°C with primary antibodies. The primary antibodies were: anti-HOXA9 (ab191178, 1:500), anti-CyclinD1 (ab134175, 1:5000), anti-c-Myc (ab185656, 1:1000), and anti-β-catenin (ab16051, 1:1000; all from Abcam, San Francisco, USA). Membranes were washed and then treated with goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) for 1 h at 37°C. Protein bands were visualized using chemiluminescence HRP substrate, where β-actin served as an internal control. Blot density was then quantified using Image J software.

The cellular protein was isolated with protein lysis buffer (Roche, USA). Subsequently, 50 μg of the total protein was subjected to SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocked with 5% skimmed milk in PBST for an hour, the membranes were washed with TBST three times, and incubated with primary antibodies including Anti-Bax antibody (ab32503, 1 : 1000, Abcam, USA), Anti-Bcl-2 antibody (ab32124, 1 : 1000, Abcam, USA), Anti-Bad antibody (ab32445, 1 : 1000, Abcam, USA), Anti-PDLIM1 antibody (ab129015, 1 : 1000, Abcam, USA), Anti-Amphiregulin antibody (ab89119, 1 : 1000, Abcam, USA), Anti-Myc antibody (ab185656, 1 : 1000, Abcam, USA), Anti-CCND2 antibody (ab267318, 1 : 1000, Abcam, USA), Anti-YAP antibody (ab52771, 1 : 1000, Abcam, USA), Anti-p-YAP antibody (ab254343, 1 : 1000, Abcam, USA), Anti-β-actin antibody (ab115777, 1 : 1000, Abcam, USA), and Anti-Lamin A antibody (ab108595, 1 : 1000, Abcam, USA) at 4°C overnight. The membranes were washed with TBST and then incubated with the horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibody (1 : 3000, Abcam, USA) for an hour at RT. The bands were developed with Pierce™ ECL Western Blotting Substrate (Invitrogen, USA). The gray value of each protein was analyzed using Image J analysis software (National Institutes of Health, USA).