Anti jak3

HEK293 cells were transfected using the appropriate expression vectors for JAK1 (RC213878; OriGene, Rockville, MD, USA) or JAK3 [19 (link)] prior to lysis with the Triton lysis buffer described above. JAK proteins were immunoprecipitated using anti-JAK3 or anti-myc antibodies and captured using Protein A-Sepharose, as described above. The beads were washed three times with cold lysis buffer and once with ice cold kinase buffer (25 mM of HEPES (pH 7.3), 1% Triton X-100, 100 mM of NaCl, 10 mM of MgCl₂, 3mM of MnCl₂ and 50 µM of sodium orthovanadate). The kinase reaction was initiated via the addition of 100 µM of ATP, and then the beads were incubated at 37 °C for 20 min. The reactions were quenched by washing the Protein A-Sepharose beads with lysis buffer and eluting the material using 2X SDS sample buffer (described above). The samples were resolved using 7.5% SDS-PAGE, and the tyrosine phosphorylation levels of JAK3 were assessed via Western blotting using the anti-pY841 JAK3 monoclonal antibody, anti-pY antibody (Millipore), or anti-JAK3 (Abcam) and anti-myc (Santa Cruz) for 1 h at room temperature. The assays were developed using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (heavy plus light chains; SeraCare), and visualized by using enhanced chemiluminescence and X-ray film.

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).