Fig. 5

IAV infection induces the expression of SOCS1, resulting in JAK1 degradation. (a) 293 T cells were left uninfected or infected with IAV at an MOI of 1 for the indicated times. The protein levels of SOCS1, SOCS3, viral NP and β-actin were analyzed using Western blotting. (b) 293 T cells were left uninfected or infected with IAV at an MOI of 1 for the indicated times. The mRNA levels of SOCS1, SOCS3 were analyzed using real-time qPCR. The error bars represent the means plus standard deviations for three independent experiments. ***, P < .0001. (c) 293 T cells were transfected with siRNAs 1 and 2 targeting SOCS1 or NC (negative control) siRNA. Twenty-four hours later, cells were left uninfected or infected with IAV at an MOI of 1. The protein levels of SOCS1 and β-actin were analyzed by Western blotting at 24 hpi. 293 T cells were transfected with siRNAs 1 targeting SOCS1 or NC siRNA. Twenty-four hours later, cells were left uninfected or infected with IAV at an MOI of 1, and the protein level of SOCS1, JAK1, viral NP and β-actin were detected at 24 hpi. (d) 293 T cells were transfected with siRNA 1 targeting SOCS1 or NC siRNA as indicated. Six hours later, cells were transfected with Flag-tagged JAK1 and HA-tagged ubiquitin. Twenty-four hours post-transfection, cells were left uninfected or infected with IAV at an MOI of 1. At 18 h post infection, the cells were harvested and subjected to immunoprecipitation. The ubiquitination of immunoprecipitated Flag-JAK1 was analyzed using Western blotting using anti-HA tag antibody. The protein level of Flag-JAK1, SOCS1 and β-actin in the whole-cell lysates were also analyzed using Western blotting