Polymerase chain reaction (PCR)
RNA was extracted from mouse splenocytes by Trizol reagent (Vazyme Biotech, Nanjing, China) and reverse transcribed by using Superscript qRT SuperMix kit (Vazyme Biotech) according to the manufacturer’s instructions. All the primers were synthesized by GenScript (Nanjing, China) and are listed in Table S3. Quantitative real-time RT-PCR (qRT-PCR) was performed by using the SYBR Green Premix kit (Vazyme) on a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative expression of each gene was determined and normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculated by using the 2-∆∆Ct method.
Western blotting
Mouse splenocytes were lysed and proteins were extracted and blotted with signal transducer and activator of transcription-3 (STAT3) (1:1000), pSTAT3 (1:500), and β-actin (1:2000) antibodies (Affinity Biosciences, USA). Proteins were detected with Super Signal West Pico chemiluminescence Substrate solution (Thermo Fisher Scientific). Immuno-reactive bands were visualized by using a gel documentation system (Syngene, USA). The intensity blots were quantified by densitometry using Image J software.