STAT1 is phosphorylated in response to IFN-a1 and IFNγ. Cells were either treated with IFN-a1 (10 U/ml) or IFN-γ (200 ng/ml) or left untreated. Cells were harvested at indicated time points and endogenous STAT1 were immunoprecipitated with α-STAT1 from the whole cell extracts. Tyrosine phosphorylated STAT1 was detected by immunoblotting using anti-phosphotyrosine antibody (pY, upper panel). The total amount of immunoprecipitated STAT1 was detected with α-STAT1 antibody (lower panel). (A) Head kidney leukocytes. (B) TO cells. (C) TO cells were transfected with a GFP-ssSTAT1a construct. After 48 h, the cells were treated with IFNγ (200 ng/ml) for 30 min, or left untreated. Cells were lysed and GFP-tagged proteins were immunoprecipitated with α-GFP. Phosphorylated GFP-STAT1 was detected by immunoblotting using anti-phosphotyrosine antibody (upper panel). The total amount of immunoprecipitated GFP-STAT1 was detected with α-GFP antibody (lower panel).