Ab32372

Briefly, cells were lysed in RIPA lysis buffer (P0013B, Beyotime Institute of Biotechnology, Nantong, China) at 48 hr following transfection as described below and protein concentration was determined by the BSA method (Beyotime Institute of Biotechnology). Equivalent quantities of protein were separated on a 10% SDS‐polyacrylamide gels and then transferred to Polyvinylidene Fluoride (PVDF) Membrane. Membranes were blocked using 5% non‐fat milk and incubated overnight with the appropriate primary antibody. The next day, they were washed three times with TBST and incubated with a HRP‐conjugated secondary antibody (Beyotime Institute of Biotechnology) at 1:5,000 dilution for 1 hr at room temperature. Protein detection was performed using the enhanced chemiluminescence (ECL) system (Millipore, Bedford, MA). Primary immunoblotting antibodies were: anti‐β‐Actin(dilution 1:1,000, 4,970, Cell Signaling Technology, Danvers, MA), anti‐DPP9(dilution 1:1,000, ab42080, Abcam, Cambridge, MA), anti‐p53 (Epitomics, Burlingame, CA), anti‐BAX(dilution 1:1,000, ab32503, Abcam), anti‐APAF1(dilution 1:1,000, ab32372, Abcam), anti‐MUC1(dilution 1:1,000, ab45167, Abcam), anti‐S100A4(dilution 1:1,000, ab124805. Abcam), anti‐E‐caderin(1:50, ab1416), anti‐vimentin (1:1,000, ab92547, Abcam).

Total proteins were extracted from tissue samples or cultured cells with SDS lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice for 20 min and the protein concentrations were determined using BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of total proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 2 h, transferred to 0.22 µm polyvinylidene difluoride membranes (PVDF) (Millipore, Billerica, MA, USA) and incubated with APAF1 antibodies (1:1,000, ab32372; Abcam). Proteins were detected by enhanced chemiluminescence (ECL) as described by the manufacturer (Beyotime Institute of Biotechnology) and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad Laboratories, Hercules, CA, USA). Results were normalized to a constitutive expression gene, GAPHD (1:2,000, ab9485; Abcam).