Mouse anti pd1 be0146

Cells are lysed in 1X RIPA buffer and supplemented 1:100 Protease/ Phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA). Protein concentrations are determined using the BioRad protein assay kit (Hercules, CA) and 30μg of protein resolved by electrophoresis on a 10% SDS-PAGE. The proteins are transferred onto a nitrocellulose membrane and nonspecific binding is blocked by incubating with 5% nonfat milk in TBS-T buffer (0.01 M Tris–Cl, 0.15 M NaCl, 0.5% Tween-20, pH 8.0) at room temperature for 1h. The membrane is subjected to the indicated antibodies and fluorescence detected using a LI-COR Odyssey Infrared Imaging System. Mouse Anti-PD-L1 (BE0101), mouse anti-PD-1(BE0146) and control rat IgG2A (BE0090) antibodies were purchased from BioXCell (West Lebanon, NH). Anti-Ki-67 (sc-15402) was obtained from Santa Cruz Biotechnology (Dallas, TX). Human Anti-PD-L1 (CST #13684), human anti-PD-1 (CST #86163), Anti-aurora A (CST #14475), Anti-phospho-aurora A (Thr288) (CST #3079), anti-Akt (CST #4691), anti-phospho-Akt (Ser473) (CST #4060), anti-ERK1/2 (CST #4695), anti-phospho-ERK1/2 (CST #4370), anti-PD-L1 (human) (CST #13684), anti-NFκβ (CST #8242) and anti-GAPDH (14C10) (CST #2118), anti-β-Actin (CST #3700) antibodies were purchased from Cell Signaling Technology (Danvers, MA).