Cells were plated at 2×106 per P10 plate. Fresh media (6 ml) was added to the cells and collected after 24 hours. Collected media was spun at 4°C, 5 min, 1200 rpm to remove any cells and cell debris. After debris removal, the protein content in the media was concentrated 6 to 8-fold by using the 10K protein concentrator PES (Thermo Scientific) at 4°C. The protein concentrate was reconstituted in Cell Lysis Buffer (Cell Signaling Technology) and analyzed by Western blot.
Antibodies, Reagents, and Drugs. HIF1A, Lamin A/C, hPDL1, anti-biotin HRP-linked, and anti-rabbit IgG1 HRP-linked antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA); Actin, HuR 3A2, PDL1, donkey anti-goat IgG-HRP, goat anti-mouse IgG1-HRP antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA); the mPDL1/B7-H1 antibody was from B&D systems (Minneapolis, MN, USA); the alpha Tubulin antibody was from Sigma (Sigma-Aldrich). Mouse PDL1 ELISA Kit was purchased from Boster (Pleasanton, CA, USA). MLN4924 was purchased from Active Biochem (Kowloon Bay, Hong Kong); the PD1/PDL1-inhibitor-1 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).
Antibodies, Reagents, and Drugs. HIF1A, Lamin A/C, hPDL1, anti-biotin HRP-linked, and anti-rabbit IgG1 HRP-linked antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA); Actin, HuR 3A2, PDL1, donkey anti-goat IgG-HRP, goat anti-mouse IgG1-HRP antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA); the mPDL1/B7-H1 antibody was from B&D systems (Minneapolis, MN, USA); the alpha Tubulin antibody was from Sigma (Sigma-Aldrich). Mouse PDL1 ELISA Kit was purchased from Boster (Pleasanton, CA, USA). MLN4924 was purchased from Active Biochem (Kowloon Bay, Hong Kong); the PD1/PDL1-inhibitor-1 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).