Western Blot Analysis of Immune Markers

Western blot was performed as described before^{6 (link)}. The whole cell extracts were prepared with lysis buffer. The protein concentrations were determined using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). Protein samples (50 μg/lane) were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% resolving gels and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 ℃: PD-1 (1:1000), p-Stat5 (1:1000), Stat5 (1:1000), cleaved Caspase 3 (1:1000), CD4 (1:1000), CD8 (1:1000), and Tubulin (1:1000). All antibodies were purchased from Cell Signaling Technology, Inc. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G secondary antibodies (1:2000; Cell Signaling Technology, Inc.) were used for 1 h at room temperature. Specific immune complexes were visualized using enhanced chemiluminescence (Beyotime Institute of Biotechnology). The results of western blot were semi-quantified with Quantity One software (Version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA).