Peroxidase conjugated anti rabbit and anti mouse igg secondary antibodies

For immunoblotting experiments, cells were treated in 6-cm culture dishes and lysed in RIPA buffer, to which a cocktail of protease and phosphatase inhibitors was added. Samples containing equivalent protein concentrations were resolved by SDS-PAGE and analyzed by immunoblotting. X-ray films were used for the detection of chemiluminescent bands, except when the signals were strong, in which case a C-DiGiT Scanner (LI-COR^®, Lincoln, NE, USA) or AI680 digital imager (GE Lifesciences, Pittsburg, PA, USA) was used to scan the signals. Primary antibodies, at 1:1000 dilutions, used to target PD-L1 (catalog #13684, rabbit mAb) and p53 (catalog #9282, rabbit), were obtained from Cell Signaling Technologies (Danvers, MA, USA). Rabbit anti-GAPDH antibody (catalog # TA802524, used at 1:2000 dilution) was from OriGene Technologies^® (Rockville, MD, USA). Peroxidase-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were from Millipore (Temecula, CA, USA) and used at 1:3000 dilutions.