Radioimmunoprecipitation assay (ripa)

Hepatic tissue samples were removed from BALB/c mice and then were homogenized manually with glass homogenizers. The tissues were processed in Radioimmunoprecipitation Assay (Beyotime Biotechnology Company, Jiangsu, China) and centrifuged at 12000 g for 15 min at 4°C. Supernatant protein concentrations were assessed using BCA Protein Assay Kit (Beyotime Biotechnology Company, Jiangsu, China). Cytoplasmic and nuclear fractionations were performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology) according to the producer's instruction. Proteins were separated in 10% polyacrylamide sodium dodecyl sulfate gels and then were transferred to polyvinylidene fluoride membrane. The membranes were blocked using 5% nonfat milk for 2 hours and then were probed with antibodies against ERK1/2 (1 : 500), p38 (1 : 500), JNK (1 : 500), phospho-Erk1/2 (1 : 500), phospho-p38 (1 : 500), phospho-JNK (1 : 500), NF-κb (1 : 500), IκB-α (1 : 500), β-actin (1 : 1000), and lamin B1 (1 : 500), for an overnight incubation. After that, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 hour. After washing, the proteins were detected with BeyoECL Plus (Beyotime Biotechnology Company, Jiangsu, China) and images were captured with the UVP imaging system.

Radioimmunoprecipitation assay (Beyotime, Shanghai, China) was used for lysing cells or tissues. The mixture was centrifuged at 4 °C, 12,000 × g for 5 min. Protein samples were quantified using the BCA assay. Ten micrograms protein sample was loaded in 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After nonspecific antigen blockage, the membrane was immunoprecipitated with primary antibodies (ProteinTech, Wuhan, China) at 4 °C overnight and secondary antibodies for 2 h. Band exposure was achieved by electrochemiluminescence (Pierce, Rockford, IL, USA) and analyzed by Image-Pro Plus (Media Cybernetics, Silver Springs, MD, USA).

Cells were collected and lysed using Radio Immunoprecipitation Assay (Beyotime Biotechnology Company, China) with proteinase inhibitor according to manufacturer's protocol. Briefly, the lysate was centrifuged at 12000 g for 15min at 4°C. Then supernatant was collected and protein concentration was measured using BCA Protein Assay Kit (Beyotime Biotechnology Company, China). 20 ug protein of each sample was separated in 10% polyacrylamide sodium dodecyl sulfate gels. Then protein was transferred to to polyvinylidene fluoride membrane and blocked with 5% fat-free milk for 3 hours with Tris-buffered saline Tween-20 (TBST) (Beyotime, Beijing, China) at room temperature. The membranes were incubated with antibodies against B7-H1 (1:1000, Cell Signaling Technology, USA), GAPDH (1:3000, 6C5, Abcam, USA) overnight at 4°C and washed 3 times (10 minutes per time) after incubation. After that, The membranes were processed in secondary antibodies conjugated with horseradish peroxidase (HRP) for 1–2 hours at dilution of 1:4000. After washing as previously, the protein was detected using an ECL Kit (Beyotime Biotechnology Company, China) and images were captured by the UVP imaging system. B7-H1 expression was normalized to endogenous reference GAPDH.