Western Blot Analysis of HIF1α and PD-L1 Expression

Cells were harvested and lysed as described before [20 (link)]. The protein concentration was determined using the Bradford assay (#39222, Serva, Heidelberg, Germany), and equal amounts were loaded on a gel. Protein levels were analyzed by Western blot as described before [21 (link)]. The following primary antibodies were used: mouse anti-human HIF1α (BD Biosciences, #610959), mouse anti-human PD-L1 (Cell Signaling Technology, #29122), mouse monoclonal β-Actin Antibody (C4) (#sc-47778). The following secondary antibodies were used: Donkey-anti-mouse IRDye 800CW (Li-cor Biosciences, Lincoln, NE.) and Donkey-anti-Rabbit IRDye 680CW (Li-cor Biosciences). All antibodies were diluted in 3% non-fat milk in TBST. Immunoblots were analyzed by Odyssey infrared imaging system (Li-cor Biosciences).