Ab255818

Total proteins from tissues and cells were lysed conforming to the user’s guidebook of RIPA lysis buffer (Beyotime, China); this was followed by separation with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer with polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After that, membranes were subjected to a standard blocking with 5% non-fat milk, hybridization with primary antibodies at 4 °C overnight, and incubation with secondary antibodies at room temperature for one hour. The bands were detected according to the instructions of the ECL detection kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibodies are presented as follows: Bcl-2 (Bcl-2, 1:1000; ab32124, Abcam, Cambridge, UK), Bcl-9 (Bcl-9, 1:1000; ab37305, Abcam, Cambridge, UK), caspase-3 (caspase-3, 1:1000; ab13847, Abcam), caspase-7 (caspase-7, 1:1,000; ab255818, Abcam, Cambridge, UK), cleaved caspase-3 (cleaved caspase-3, 1:1000; #9661, Cell Signaling Technology, Boston, MA, USA), cleaved caspase-7 (cleaved caspase-7, 1:800; #8438, Cell Signaling Technology, Boston, MA, USA), bax (bax, 1:1000; ab32503, Abcam, Cambridge, UK), and goat anti-rabbit IgG (H + L) secondary antibody (1:5000; ab6721, Abcam, Cambridge, UK ).

Immunohistochemistry was performed as standard protocols. Tissue sections of tumor and para-tumor tissues underwent dewaxing, hydration, antigen repair, and blocking, the sections were incubated overnight at 4 °C in primary antibody (anti-CDH3, ab242060, Abcam; anti-CASP7, ab255818, Abcam; anti-EIF4G1, 15704-1-AP, Proteintech.). After rinsing in phosphate-buffered saline (PBS) three times, the slides were incubated with a secondary antibody and the streptavidin–horseradish peroxidase in turn. At last, the slides were stained with DAB solution. Images were obtained using a direct optical microscope.

Western blot analysis was performed on renal lysates or cell extracts using the following primary antibodies: anti-DcR2 (ab108421; Abcam), anti-FLIP (ab8421; Abcam), anti-cleaved caspase 3 (ab214430; Abcam), anti-caspase 8 (ab25901; Abcam), caspase 3 (ab184787; Abcam), anti-caspase 7 (ab255818; Abcam), anti-cleaved caspase 7 (ab256469; Abcam), Akt (sc5298, Santa Cruz), pAkt (sc135650, Santa Cruz), and anti-GAPDH (BM3876; Boster). The intensity of each band was analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA).