Anti socs1

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).

The antibodies anti-FABP4 (number ab92501), anti-JAK1 (number ab47435), anti-JAK3 (number ab45141), anti-STAT1 (number ab30645), anti-STAT3 (number ab76315), anti-SOCS1 (number ab9870), anti-SOCS2 (number ab3692), and anti-Rap1a (number ab197673) were purchased from Abcam (Cambridge, MA). Anti-Src (number 2108S), anti-p-Src family (Tyr416 number 6943S), anti-STAT2 (number 72604S), anti-JAK2 (number 3230S), anti-p-JAK2 (Y1007/1008, number 3771S), and anti-p-STAT2 (number 88410S) antibodies were obtained from Cell Signaling Technology (Danvers, MA), while anti-β-actin (number sc-8432) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

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).

Total protein was extracted from whole cells and 20 μg of isolated protein was separated by SDS-PAGE and electroblotted onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were then probed with antibodies: anti-SOCS1, anti-p21, anti-Cyclin D1 (Abcam, Cambridge, MA, USA). The membranes were stripped and reblotted with an anti-α-tubulin monoclonal antibody (Sigma) as a loading control.