FIG 4.

Jak-Stat pathway in N167 cells. (A) Immunoblotting of total Stat2 and phosphorylated Stat2 (p-Stat2) in N167 cells treated with IFN-α. Cells were treated for the indicated times with 50 U/ml IFN-α. (B) Immunoblotting of Stat proteins and their activated phosphorylated proteins (p-Stat1 and p-Stat2) in N167 cells treated with IFN-α. Cells in the presence of vehicle (DMSO), 5 μg/ml Gly-9, or 5 μg/ml Gly-14 were treated with IFN-α as described for panel A, for the indicated times. It is noteworthy that Stat proteins in N167 cells were activated by IFN-α irrespective of the presence of glycoside compounds. (C) Immunofluorescence of Stat2 in N167 cells treated with IFN-α. Cells were treated with IFN-α at the designated doses for 20 min before analysis. Nuclei were stained with Hoechst 33258 (H33258). (D) Immunoblotting of phosphorylated Stat proteins (p-Stat1 and p-Stat2) in N167 cells treated with IFN-α and a Jak inhibitor. Cells were treated with 10 U/ml IFN-α for the indicated times in the presence or absence of 5 μg/ml Gly-9 or 50 nM Jak inhibitor I (Jak inh). It is noteworthy that Jak inhibitor I blocked IFN-α-induced activation of Stat proteins irrespective of the presence of Gly-9. (E) Immunofluorescence of Stat2 in N167 cells treated with IFN-α and Jak inhibitor. Cells were treated with 5 μg/ml Gly-9 and 50 U/ml IFN-α for 20 min before analysis, in the presence or absence of 50 nM Jak inhibitor I (Jak inh). Nuclei were stained with Hoechst 33258 (H33258). It is noteworthy that Jak inhibitor I blocked IFN-α-induced translocation of Stat2 into the nucleus.