Anti p jak1

Protein analysis was performed using standard immune blotting. The following antibodies were used at the indicated dilution: anti-Noxa (SC-2697) 1:1000; anti-cytochrome (#4212), 1:1000; anti-caspase 3 (#7190), 1:1000; anti-caspase 9 (#9501), 1:1000; anti-PARP (#9542), 1:500 (each Cell Signaling Technology Inc., Danvers, MA, USA); anti-IDO antibody 1:500 (BioGenes, Berlin, Germany); anti-ASK1 (Sc-7931), 1:500; anti-p-ASK1 (Sc-109911), 1:1000; anti-JNK (Sc-474), 1:1000; anti-p-JNK (SC-6254), 1:1000; anti-p38 (Sc-535), 1:1000; anti-p-p38 (Sc-7973), 1:1000; anti-Actin (Sc-1615), 1:5.000; anti-Tom20 (Sc-11415), 1:100; anti-Bap31 (Sc-18579), 1:1000; anti-HO-1 (sc-10789), 1:1000; anti-p-JAK1 (sc-16773), 1:1000, anti-IRE1α (Sc-20790), 1:500; anti-PERK (SC-9477), 1:1000; (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); anti-IκBα (Sc-7182), 1:1000; anti-p-IκBα (AF4809, R&D system), 1:1000; anti-JAK1 antibody (ab47435), 1:1000; anti-IRF1 antibody (ab55330), 1:1000 (each ABCAM).

The BMK-16/myc and SiHa cells were cultivated and treated with 100 ng/ml of ovine IFN-τ for 15 min. Then the cells were lysed with cold RIPA lysis buffer (Santa Cruz Biotechnology) with protease inhibitor cocktail (Sigma Aldrich) by incubating for 30 min at 4ºC, the total proteins were quantified using BCA protein assay kit (Price Rockford, il., USA) according to the manufacturer´s instructions. Fifty micrograms of total protein were separated by SDS-polyacrylamide gel electrophoresis 10% and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). Membranes were blocked with Tris-buffers saline (TBS) containing 0.5% Tween 20 and Blotto, no-fat dry milk (Santa Cruz Biotechnology) and the membrane were incubated with specific antibodies followed by horseradish peroxidase-conjugated secondary antibody incubation. The protein bands were detected using Price ECL Western Blotting substrate (Thermo Scientific). The antibody dilutions used were anti-p-JAK1 (Tyr 1022/Tyr 1023): sc-16773 (dilution 1:200), anti-JAK1 sc:295 (dilution 1:300) 130 kDa, anti-p-STAT1/Tyr-701) sc:7958 (dilution 1:300), anti-STAT1 sc-417 C-11 sc-417, anti-p-TyK2 (Tyr-1054/1055) sc-11763, anti-TyK2 sc-169 130kDa, anti-β actin C-11 sc-1615 (dilution 1:100) 43 kDa (Santa Cruz, Calif., USA).

Hindlimbs were removed from mice at the time of sacrifice and bones were fixed in 10% neutral-buffered formalin, washed and decalcified in a solution of 10% EDTA for 2 weeks, and embedded in paraffin. Immunohistochemical analysis was performed with heat-induced antigen retrieval in sodium-citrate buffer (Dako). Primary antibodies used was anti-phospho-STAT3 at 1:100 (Santa Cruz), anti-IL-11 at 1:200 (Santa Cruz), anti-c-Myc at 1:100 (Santa Cruz), anti-pJAK1 at 1:100 (Santa Cruz). Biotinylated secondary antibody was used with the EnVision + system HRP kit (Dako) and nuclei were counterstained with hematoxylin.