Splenic lymphocytes were diluted in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 0.1 % sodium dodecyl sulfate (SDS), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM EGTA, 5 mM NaF, 1 mM Na
3VO
4, 5 mM Na
2PO
4, and 1× proteinase inhibitor cocktail (Beyotime Institute of Biotechnology, China)) on ice for 30 min and then centrifuged (12,000 rpm, 20 min) at 4 °C. The supernatants were collected, aliquoted, and stored at −80 °C until used. Western blot analysis of Fas and FasL was conducted and quantified as described [
33,
34]. After separating on SDS-PAGE gel electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, USA) and blocked with 5 % nonfat milk for 2 h at room temperature. The following antibodies were used as primary antibodies: rabbit anti-Fas antibody (ab82419, Abcam Ltd, Hong Kong, 1:200 dilution), rabbit anti-FasL antibody (sc-834, Santa Cruz Biotechnology, USA, 1:200 dilution), and mouse anti-beta actin monoclonal antibody (AA128, Beyotime Institute of Biotechnology, China, 1:1,000 dilution), followed by the goat polyclonal secondary antibody to rabbit IgG-H&L-HRP (ab6721, Abcam Ltd, Hong Kong, 1:1,000 dilution) or to mouse IgG-H + L-HRP (A0216, Beyotime Institute of Biotechnology, China, 1:1,000 dilution). Blots were exposed for 60 s to BeyoECL Plus (P0018, Beyotime Institute of Biotechnology, China). The images of Western blots were scanned by ChemiDoc
TM XRS (Bio-Rad, USA), acquired, and analyzed by using Quantity One software (Bio-Rad, USA). Protein levels were expressed as density and were normalized to beta-actin for statistical comparisons [
33].