FIG 4.

IAV infection or HA induces phosphorylation and polyubiquitination of IFNGR1. (A) HEK293 cells were transfected with FLAG-tagged IFNGR1. At 24 h posttransfection, cells were left uninfected or infected with IAV at an MOI of 1, as indicated. The cells were then cultured for an additional 24 h and subjected to a denatured IP experiment. NH₄Cl (20 mM) was added to the cell culture 6 h before harvest. The phosphorylation of immunoprecipitated FLAG-IFNGR1 was analyzed by Western blotting (IB) using anti-phosphoserine (p-Serine) antibody. The levels of immunoprecipitated FLAG-IFNGR1, viral M1, and GAPDH in the whole-cell lysates were also detected. (B) HEK293 cells were transfected with FLAG-tagged IFNGR1. At 24 h posttransfection, the cells were transfected with empty-vector control (−) or plasmids encoding HA, as indicated. The cells were cultured for an additional 24 h and subjected to denatured IP. NH₄Cl was added to the cell culture 6 h before harvest. The phosphorylation of immunoprecipitated FLAG-IFNGR1 was detected. (C) HEK293 cells were transfected with FLAG-IFNGR1 and/or tHA-Ub. At 24 h posttransfection, cells were left uninfected or infected with IAV at an MOI of 1, as indicated, for an additional 24 h. All of the transfected cells were treated with NH₄Cl (20 mM) for 8 h before harvest. The whole-cell lysates were subjected to a denatured IP experiment. The levels of ubiquitination of IFNGR1 were analyzed by Western blotting. (D) HEK293 cells were transfected with FLAG-IFNGR1 and/or HA-tagged ubiquitin, as indicated. At 24 h posttransfection, cells were transfected with a control vector or plasmids encoding HA. The cells were harvested at 18 h posttransfection. The whole-cell lysates were subjected to a denatured IP. The levels of ubiquitination of IFNGR1 were detected by Western blotting. Mass, molecular mass (kilodaltons).