Anti tp73

Cells were harvested from 100 mm dishes by rinsing twice with PBS and then lysed on the plate with 70 μl RIPA buffer (Thermo, USA), and 1× complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) on ice for 30 min. Cell lysates were centrifuged at 4°C at 12000 r/min for 20 min. The supernatant (protein) was retained; protein content was quantified. An aliquot of the total protein (30 μg) was loaded into each lane of a 12% sodium dodecyl sulfate polyacrylamide gel and then electrophoretically transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% nonfat milk in 0.01 mol/L TBS and 0.05% Tween-20 (TBST) at room temperature for 2 h before incubation with anti-BAG1, anti-BCL-2, anti-TP73 (dilution 1:1000; Santa Cruz, California, USA), anti-CASP1, anti-LTBR, or anti-CASP4 (dilution 1:1000; Abcam, Cambridge, MA, USA) antibodies at 4°C overnight, as appropriate. After three quick washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Proteintech, Wuhan, China) for 2 h at room temperature. Bands were visualized using ECL Western Blotting Substrate (Pierce, Rockford, USA).