Enhanced chemi luminescence (ecl)

Total protein was extracted from Jurkat cells using a radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membrane was incubated with the following antibodies at 4°C overnight: IL-10 (1:1,000, ABclonal, Wuhan, China) and β-actin (1:10,000, Abways, Shanghai, China). The membranes were then washed and incubated with anti-rabbit horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature. The PVDF membranes were washed with Tris-buffered saline with Tween 20 (TBST), and the blots were visualized using enhanced chemiluminescence (ECL) (Carestream, Wuxi, China). β-actin was used as the internal control. The experiments were independently repeated in triplicate.