Western Blot Analysis of PD-L1 and Cas9 in Osteosarcoma

Osteosarcoma and osteoblast cells were lysed with RIPA Lysis Buffer (Upstate Biotechnology, Charlottesville, VA) plus complete protease inhibitor cocktail tablets (Roche Applied Science, IN). Protein concentrations were calculated by the Bio-Rad Protein Assay (Bio-Rad, CA). Antibodies against PD-L1 (1:1000 dilution), Cas9 (1:1000 dilution) were purchased from Abcam, and β-actin (1:2000 dilution) were purchased from Sigma-Aldrich, respectively. Secondary antibodies IRDye^® 800CW and IRDye^® 680LT were obtained from LI-COR (Biosciences, NE). Western blot analysis was performed as previously reported [35 (link)]. Western blot detection and quantitation was performed by the Odyssey infrared imaging system (LI-COR Biosciences, NE).