Anti hla g

Cells were lysed in lysis buffer (50 mM NaCl, 50 mM EDTA, and 1% Triton X-100) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were then blocked with 5% nonfat milk diluted in PBS for 2 h at room temperature and incubated with the following primary antibodies: anti-IDO-1 (Cat.No.86630S, Cell Signaling Technology, Boston, USA); anti-HLA-G (Cat.No.66447-1-Ig, Proteintech, Wuhan, China), and anti-GAPDH (Cat.No.60004-1-Ig, Proteintech, Wuhan, China). Then, membranes were washed with PBS containing 0.05% Tween and incubated with secondary antibodies conjugated with HRP for 1 h at room temperature. The bands were developed using a chemiluminescence reagent (Thermo Fisher, Waltham, USA).

Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors (P0013B, Beyotime Biotechnology), and the concentrations of total cellular protein were measured using a BCA assay kit (P0010, Beyotime Biotechnology). Total protein samples (40 μg/gel) were separated via 12% SDS/PAGE gel and transferred to PVDF membranes (IPVH00010, Merck Millipore, Germany) by electroblotting. After blocking in TBST with 5% skimmed milk powder for 1 hr at room temperature (RT), the membranes were incubated with primary antibodies, including anti-Fyn (ab125016, 1 : 1000, Abcam, Cambridge, MA, USA), anti-HLA-G (79769, 1 : 1000, Cell Signaling Technology, Danvers, CO, USA), anti-ERK1/2 (ab54230, 1 : 1000, Abcam), anti-p-ERK1/2 (ab201015, 1 : 1000, Abcam), anti-STAT3 (ab68153, 1 : 1500, Abcam), anti-p-STAT3 (9145, 1 : 2000, Cell Signaling Technology), and anti-GAPDH (AB-P-R 001, 1 : 1000, Goodhere Biotechnology, Hangzhou, China), overnight at 4°C. Then, the membranes were washed and incubated with HRP-labelled goat anti-rabbit secondary antibody (BOSTER Biotechnology, Wuhan, China) for 1 hr at RT. The blot signals were detected by the Odyssey infrared imaging system (LI-COR Biosciences) and analysed by Odyssey software (LI-COR Biosciences).