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.