Rabbit anti jak2

Proteins from the colon samples were extracted in lysis buffer containing protease and phosphatase inhibitors. Protein concentrations (n = 5) were determined in the supernatant of colonic tissues by a classic BCA assay kit (Beyotime, China). Equal amounts of proteins (30 μg) were fractionated by 10% SDS-PAGE gels and transferred onto a polyvinylidene fluoride (PVDF; Millipore, USA) membrane by a Bio-Rad western blot apparatus. The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with the following primary antibodies for 24 h at 4 °C: rabbit anti-JAK2 (1:5000, Abcam, UK), rabbit anti-phospho-JAK2 (1:500–2000, Bioss, China), rabbit anti-TLR4 (1:500–1:2000, Bioss, China), mouse anti-STAT3 (1:500–1000, Bioss, China), rabbit anti-phospho-STAT3 (1:500–2000, Bioss, China), and mouse anti-β-actin (1:1000, ZSGB-BIO, China). After washing with TBST (Tris-buffered solution, pH 7.6, 0.05% Tween 20) 3 times for 10 min, the blots were incubated with anti-rabbit or anti-mouse IgG horseradish secondary antibodies (1/100,000 dilution) for 1 h at room temperature. Finally, the protein bands were visualized with ECL western blot detection reagents (Bridgen, China). The expression levels of the proteins were compared with the control based on the relative intensities of the bands.

The total protein was extracted by using protein lysis buffer from the spinal cord tissues. The protein concentration was measured using BCA Protein Kit (Thermo Fisher). The protein sample (20 µg) was separated on 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were probed with rabbit anti-SOCS3 (Abcam, Cambridge, U.K.; 1:200 dilution), rabbit anti-pSTAT3 (Abcam, 1:1000 dilution), rabbit anti-STAT3 (Abcam, 1:1000 dilution), rabbit anti-pJAK2 (Abcam, 1: 1000 dilution), rabbit anti-JAK2 (Abcam, 1:5000 dilution) for overnight at 4°C. The blots were incubated with horse radish peroxide-conjugated secondary antibody. The imaging was performed with electron chemiluminescence (ECL) emitting solution. Finally, the blots were visualized with an Immobilon Western Chemiluminescent HRP Substrate system (Millipore Corp., Billerica, MA, U.S.A.).