Horseradish peroxidase conjugated goat anti rabbit secondary antibody

For western blot analysis, cells at 90% confluency were washed in PBS before incubation with RIPA lysis buffer consisting of 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) on ice for 10 min. The cell lysates were clarified by centrifugation at 9000 × g for 10 min, and the supernatants were collected. Protein concentration was measured by bicinchoninic assay (Aidlab, Beijing, China). Equal amounts of total protein were separated on 10% SDS-polyacrylamide gel, and then transferred electrophoretically to nitrocellulose membranes blocked with TBST buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% Tween-20) containing 5% fat-free dry milk for 2 h and incubated for 3 h with rabbit polyclonal anti-human hPer2 antibody (dilution, 1:500) in TBST. Following incubation with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution, 1:2000; catalog no., SA00001-2; ProteinTech Group, Inc.), immunoreactive proteins were visualized with an enhanced chemiluminescence detection system. The western blot experiments were repeated three times. The relative expression of the target protein was calculated as the gray value ratio of target protein content to β-actin content (target protein/β-actin) using Quantity One version 4.62 image analysis software (Bio-Rad Laboratories, Inc.).

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).