Anti phospho jak2

Human IFN-β recombinant protein (rhIFN-β) and human IFN-λ1 recombinant protein (rhIFN-λ1) were purchased from Abbkine (Wuhan, China) and MedChemExpress (Shanghai, China). The Cell Counting Kit (CCK-8) was obtained from Biosharp (Anhui, China). Pyridone 6 (JAK inhibitor I) (A13457) was purchased from Adooq Biosciences (Irvine, CA, USA). Antibodies used for Western blotting, anti-phospho-STAT1 (S727), anti-STAT1, and anti-IFI35 antibodies (all rabbit polyclonal antibodies), were obtained from Abmart (Shanghai, China). Anti-IFIT2, anti-TLR3, anti-IRF7, anti-IRF3, anti-USP18, and anti-ISG15 antibodies (all rabbit) were purchased from ABclonal (Wuhan, Hubei, China). Anti-JAK1, anti-JAK2, anti-phospho-JAK1, and anti-phospho-JAK2 antibodies (all rabbit) were purchased from Abcam (Cambridge, MA, USA). Anti-MDA5 (IFIH1) and anti phospho-IRF3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-RIG-I (DDX58), anti-IFIT1, anti-IFIT3, anti-IFIT5, anti-IRF1, anti-IFITM1, and anti-β-actin antibodies were purchased from Proteintech (Chicago, IL, USA), and the antibodies used are listed in Additional file 7: Table S7. The monoclonal antibody against the JEV envelope (E) was kindly provided by Shengbo Cao (Huazhong Agricultural University, Wuhan, China). For immunofluorescence, rabbit anti-FLAG antibody was purchased from Proteintech.

Oocytes were fixed in 4% PFA for 30 min and permeabilized in 1% Triton X-100 (Sigma) for 15 min at room temperature. Next, oocytes were blocked with 3% bovine serum albumin (BSA) for 1 h and incubated with anti-α-tubulin-FITC (fluorescein isothiocyanate, 1:500; #F2168; Sigma-Aldrich), anti-CREST (1:200; #CA95617; Antibodies Incorporated, Davis, CA), anti-GHR (1:50; #AF1360-SP; R&D Systems, Minneapolis, MN), anti-phospho-JAK2 (1:200; ab32101; Abcam, Cambridge, UK), and anti-phospho- MAPK3/1 (1:200; #4370 T; Cell Signaling Technology) antibodies at 4 ℃ overnight. After washing three times with PBS, oocytes were incubated with the corresponding secondary antibody at room temperature for 1 h. Oocytes were counterstained with an anti-Hoechst 33342 (1:1,000; #C1022; Beyotime, Shanghai, China) antibody for 15 min. Finally, oocytes were examined using a laser-scanning confocal microscope (TCS SP8; Leica, Wetzlar, Germany). Fluorescence intensity was analyzed using ImageJ software (NIH, Bethesda, MD).
For active mitochondria staining, oocytes were cultured in M2 medium (Sigma-Aldrich) containing anti-JC-1 (1:500; #C2005; Beyotime), anti-MitoTracker Red (1:500; #C1049; Beyotime), for 30 min in darkness at 37 ℃ and 5% CO₂. After washing three times with fresh M2 medium (Sigma-Aldrich) for 10 min each, fluorescence intensity was measured as described previously.

Cells were harvested and lysed in PRO-PREP protein extraction solution (iNtRON, Bio Inc, Sungnam, Korea). Protein concentrations were measured with a Bradford Protein Assay Reagent kit (Bio-Rad, Richmond, CA, USA). Proteins were fractionated by 10% SDS-polyacrylamide gels electrophoresis (PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. These were incubated with anti-PCNA, anti-cyclin D1, anti-Bcl-2, anti-phospho-JAK2, anti-β-actin Ab (1:1000; Abcam), anti-gp130, anti-phospho-STAT3 and anti-iNOS antibodies (1:1000; Cell Signaling Technology, Beverly, MA, USA) as primary antibodies. Goat anti-rabbit horseradish peroxidase-conjugated IgG or goat anti-mouse horseradish peroxidase-conjugated IgG (Abcam, Cambridge, MA, USA) served as secondary antibodies. Protein bands were detected with a chemiluminescence reagent kit (SurModics, MN, USA). The data are expressed as means ± SD of three independent experiments.