vSOCS interacted with Jak1 protein and inhibited the Jak1 tyrosine kinase activity <i>in vitro</i>.

<p>(A) Immunoprecipitation (IP) assay. Cells were transfected with pCMV-myc (empty plasmid) as negative control, or with ISKNV-vSOCS. Myc-tagged protein and tubulin were detected by anti-Myc and anti-β-tubulin antibodies in cell lysates (left plane) after 36 h of transfection. The cell lysates were immunoprecipitated with normal rabbit IgG or anti-Jak1 antibody, and then detected by western blotting using anti-Jak1 or anti-myc antibody (right plane). (B) Direct inhibition of Jak kinase activity by ISKNV-vSOCS <i>in vitro.</i> Cells were transfected with empty vector (control) or ISKNV-vSOCSmyc plasmid, and Jak1 was immunoprecipitated with anti-Jak1 antibody. A part of the immunoprecipitation complex was used to incubate with RTK substrate at room temperature for 1 h (left plane). The rest were detected by Western blotting using anti-Jak1 or anti-Myc antibody. Phosphorylated substrates were detected at an absorbance of 450 nm with a reference wavelength of 655 nm after adding mouse anti-PY20-HRP, TMB solution, and Stop Solution. The gray columns represent the cells without IFN-α stimulation, whereas the black columns represent the cells with IFN-α (5000 U) stimulation for 30 min. One out of six representative experiments is shown. Error bars represent the mean ± S.D. (n = 6). *P<0.05.