Anti pias3

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).

Protein concentrations (n = 6) were determined in the supernatant of colonic tissues by classic BCA protein assay (Beyotime). Equal protein of each sample was fractionated onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane by a Bio-Rad Western blot apparatus. The membranes were blocked with 5% fat-free milk or 5% bovine serum albumin, and then probed with the following primary antibodies for 24 h at 4°C: GAPDH (1:2000), Anti-SOCS1(1:1000), Anti-SOCS3 (1:1000), Anti-JAK2 (1:1000), Anti-JAK2 (phospho Y1007 + Y1008) (1:500), Anti-STAT3 (1:1000), Anti-STAT3 (phospho Y705) (1:800), Anti-STAT6 (1:1000), Anti-STAT6 (phospho Y641) (1:800), Anti-PIAS3(1:1000) (Abcam, Cambridge, UK). The membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000∼1:3000, Abcam, Cambridge, UK), and visualized with an enhanced chemiluminescence (ECL) detection kit (Millipore). Bands were quantified using Image-Pro Plus 5.0 software (Media Cybernetic, Bethesda, MD, USA).