Anti jak1

Spleen proteins were extracted using RIPA lysis buffer (Solarbio, Beijing, China) and centrifuged at 12,000 g for 10 min at 4°C. Protein concentration was assessed using the BCA Protein Assay Kit (Biosharp, Beijing, China). Samples containing equal amounts of protein (80 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to 0.45 μm polyvinylidene difluoride membranes (Biosharp, Beijing, China). The non-specific binding sites on the membrane were then blocked with 5% fresh non-fat dry milk in Tris buffered saline TBS with Tween (10 mM Tris, 150 mM NaCl, pH7.4) with 0.1% Tween 20) for 2 h. Subsequently, they were incubated with primary antibodies (anti-IL-2, anti-STAT1, anti-SHP2, anti-JAK1, and anti-p-JAK1; 1:1000 dilution; Abcam, Cambridge, UK) and primary β-actin antibody (1:1,000 dilution; abmart, Shanghai, China) overnight at 4°C, respectively. After this, the membrane was washed followed by three 10 min washes in TBST, and then incubated with anti−mouse secondary antibody (1:2000 dilution; Cell Signaling Technology, Shanghai, China) for 2 h at room temperature followed by three 10 min washes in TTBS. Finally, the chemiluminescent signals were observed with a multifunctional gel imaging system (Bio-Rad, USA) and densitometry for immunoreactive bands was performed with National Institutes of Health software (Image J).

Colon tissues (n = 6) were quickly shredded and mixed with protease inhibitor cocktails, which were randomly selected from every group. The mixture was homogenized by ultrasound on crushed ice and centrifuged at 13000g at 4°C for 10 min; the supernatant was extracted; and the protein concentration was determined by BCA protein assay. Equal amounts of total protein (60 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane using a semidry transfer slot (Bio-Rad, USA). After blocking with 5% nonfat dried milk for 1 h, the membrane was incubated with proportionally diluted antibodies at 4°C overnight. The antibodies included rabbit anti-GAPDH (1:2,000), anti-BCL-2 (1:2,000), anti-BIM-1 (1:1,000), anti-caspase-3 (1:500), anti-BAX (1:1,000), anti-PP2A (1:1,000), anti-β-casein (1:2,500), anti-caveolin-1 (1:1,000), anti-Pim-1 (1:1,000), anti-JAK1 (1:1,000), anti-JAK3 (1:1,000), anti-PIAS1 (1:2,000), anti-PIAS3 (1:2,000), anti-Socs-1 (1:1,000), anti-P-STAT5 (1:1,000), and anti-STAT5 (1:1,000; Abcam). Then, samples were incubated with a corresponding secondary antibody (Abcam) for 1 h. After washing three times with TBST, the blots were visualized by the Proteogel imaging system (FluorChem M, ProteinSimple, USA) and quantified with the Quantity One System (Bio-Rad).

Cell lysates was separated by SDS–polyacrylamide gel (4–10%) electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). The membranes were then blocked with 5% skimmed milk and incubated overnight at 4 °C with the following primary detection antibodies: anti-CENPK (1:1000, Abcam, Cambridge, England), anti-C-MYC (1:1000, Abcam, England), anti-Cyclin D (1:1000, CST, Boston, USA), anti-Cyclin E (1:1000, Abcam, Cambridge, England), anti-Rb (1:1000, CST, Boston, USA), anti-p-Rb (1:1000, CST, Boston, USA), anti-GAPDH (1:10,000, Abcam, Cambridge, England), anti-JAK1 (1:1000, Abcam, Cambridge, England), anti-STAT3 (1:1000, Abcam, Cambridge, England), or anti-p-STAT3 (1:1000, Abcam, Cambridge, England). The species-matched secondary antibodies were then incubated for 1 h at room temperature and the proteins were detected using BeyoECLPlus (Beyotime, Jiangsu, China).