Enhanced chemiluminescence blotting analysis system

Cell protein extracts were prepared using M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) according to the manufacturer’s instructions. Total protein was fractionated by SDS-PAGE and transferred onto PVDF membranes. The following primary antibodies were used: PD-L1 (17952-1-AP, ProteinTech), SOX6 (NBP1-85811, Novus), WNK2 (07-2261, Millipore), BTG3 (ab112938, Abcam), RBSP3 (ab106973, Abcam), active β-catenin (05-665, Millipore), OCT4 (ab19857, Abcam). p-AKT (sc-293125), p-ERK1/2 (sc-16982), total β-catenin (sc-7963), ERK1/2 (sc-514302), PTEN (sc-7974), cyclin D1 (sc-8396), p21 (sc-6246), and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology. GAPDH was analyzed to show equal protein loading. Blots were developed with the enhanced chemiluminescence blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Immunoblot images were digitized and quantified using the ImageJ software. Results were expressed as a relative ratio of PD-L1 to GAPDH and the PD-L1/GAPDH ratio in normal cells was set as 1.

Whole-cell or nuclear protein extracts were prepared using the M-Per Mammalian Protein Extraction Reagent (Pierce Biotechnology, Woburn, MA) or the Nuclear Extraction Kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. Total protein (30 μg) and nuclear protein (10 μg) were loaded onto 10–20% SDS-PAGE gels, electrophoresed, and then transferred to nitrocellulose membranes. Antigen-antibody complexes were detected using the enhanced chemiluminescence blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK). The following antibodies were used: EZH2 (Cell Signaling; 5246), H3K27me3 (Cell Signaling; 9733), total histone 3 (Cell Signaling; 9715), E-cadherin (GenScript; A01589), Vimentin (GenScript; A01189), Twist (Abcam; ab50887), p-AKT (Santa Cruz; sc-293125), AKT (Santa Cruz; sc-1618), GAPDH (Santa Cruz; sc-47724), YY1 (Santa Cruz; sc-7341) and lamin B1 (Santa Cruz; sc-20682). GAPDH (whole cell lysate) and lamin B (nuclear protein) were applied as loading controls. Primary antibodies were used at a dilution of 1:1000.