Gb11176

Samples were frozen in liquid nitrogen and then pulverized. Subsequently, they were lysed with lysis buffer supplemented with a protease inhibitor cocktail to prepare protein extracts. Total proteins were extracted using a total protein sample kit (Servicebio, China), and the protein concentration was measured by the bicinchoninic acid method according to a kit manufacturer’s protocols. Next, total proteins were separated via sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and then transferred to polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies against JAK1 (AF7765, Affinity, China), STAT3 (GB11176, Servicebio, China), OPG (GB113749, Servicebio, China), and RANKL (23,408–1-AP, Proteintech, China) at a dilution of 1:200 and washed with TBST (5 min per wash, three times). Next, the membranes were incubated with the secondary antibodies at room temperature for one hour. Finally, the target protein bands were analyzed using the Bio–Rad ChemiDoc XRS system (Bio-Rad).

Total IPEC-J2 cell proteins were extracted using Radioimmunoprecipitation assay (Beyotime). The extracted proteins were quantified using a bicinchoninic acid protein analysis kit (Beyotime). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) was used to separate equal amounts of proteins, which were then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies overnight at 4°C.
The primary antibodies comprised those recognizing: MX dynamin like GTPase 1 (MX1) (bsm-51528m, Bioss, Woburn, MA, USA; 1:1,000); Janus kinase 1 (JAK1) (bs-1439R, Bioss; 1:1,000); phosphorylated (p-JAK1) (bs-3238R, Bioss; 1:1,000); signal transducer and activator of transcription 3 (STAT3) (GB11176, Servicebio, Wuhan, China; 1:1,000); p-STAT3 (bs-1658R, Bioss; 1:1,000); and GAPDH (GB15002, Servicebio; 1:2,000). After incubation with horseradish peroxidase-labeled secondary antibodies (GB23303, Servicebio; 1:5,000) or (GB25301, Servicebio; 1:5,000), the immunoreactive protein bands were observed using the enhanced chemiluminescence method (Thermo Fisher Scientific, Waltham, MA, USA).