Jak 1

RIPA lysis buffer (with 1% phenylmethanesulfonyl fluoride) (Beyotime, Shanghai, China) was used to lyse the cells. After determination of protein concentration with a BCA protein concentration determination kit (Beyotime), the protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transferation onto polyvinylidene fluoride membranes (ThermoFisher). Following blocking with 5% bovine serum albumin, the membranes were incubated with antibodies against NR1D1 (1:1000; Abclonal, Wuhan, China), cyclinD (1:1000; ABclonal), cyclinE (1:1000; Proteintech, Wuhan, China), SOCS3 (1:1000; ABclonal), JAK-1 (1:1000; Affinity, Changzhou, China), p-JAK1 (Tyr 1034/Tyr 1035; 1:1000; Affinity), JAK2 (1:500; Affinity), p-JAK2 (Tyr 1007/Tyr 1008, 1:1000; Affinity), STAT3 (1:500; Affinity), p-STAT3 (Tyr 705, 1:500; Affinity), β-actin (1:2000; Proteintech) at 4 °C overnight. Thereafter, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:10000; Proteintech) at 37 °C for 40 min. Blots were visualized with an enhanced chemiluminescence substrate kit (7 Sea biotech, Shanghai, China).

The tissue immunofuorescence step was performed as described previously (Cao et al., 2021 (link)). Briefly, paraffin-embedded slices were quenched to inactivate endogenous peroxidase for 30 min using 3%H₂O₂; then, the non-specific binding sites were blocked using 3% BSA (bovine serum albumin) at room temperature for .5 h. The sections were incubated overnight at 4°C with STAT3 (1:200; Bioss, Beijing, China), p-STAT3 (1:200; Bioss, Beijing, China), iNOS (1:500; Bioss, Beijing, China), JAK1 (1:200; Affinity, OH, United States), p-JAK1 (1:200; Affinity, OH, United States). After washing, sections were incubated for 50 min in the appropriate secondary antibody at 37°C. Finally, sections were developed with 3,3′-diaminobenzidine (DAB) (G1211, Servicebio, Wuhan, China), and then the nuclei were counterstained with hematoxylin. A light microscope (XSP-C204; CIC, Chongqing, China) was used to observe the sections, and the intensity of the stained areas for each group was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, MD, United States) (3 pictures per animal).