Anti jak2

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).

Formaldehyde-fixed aortic tissue was incubated in PBS overnight at 4°C. Then, it was embedded in an OCT compound (Tissue-Tek) and serially sectioned on a cryostat. Serial 8 μM sections were stained with HE staining and immunofluorescence. For immunofluorescence, paraffin-embedded sections were deparaffinised with xylene and rehydrated through graded ethanol and then incubated in 10 mmol/l citrate buffer for antigen retrieval. Primary antibodies (1 : 200, anti-JAK2 (abcam, USA); 1 : 100, anti-STAT3 (Bioworld, China); 1 : 200) were used and then incubation of samples were treated with anti-rat Alexa Fluor 488 secondary antibody (abcam, USA) for 30 min at 37°C. Afterwards, the samples were incubated with DAPI solution for 5 min in the dark; they were washed 3 times with PBS and examined under a fluorescence microscope. Then, cells were plated on autoclaved glass coverslips placed in sterile 6-well plates, fixed in 4% paraformaldehyde, permeabilised in 0.1% Triton, and prevented with 10% goat serum. Subsequently, cells were treated with primary antibodies (1 : 200, anti-JAK2 (abcam, USA); 1 : 100, anti-STAT3 (Bioworld, China)) for 1 h at 37°C. After rinsing with PBS 3 times, the fluorescent secondary antibodies were added and the cells were stained as detailed above.