Fig. 3.

Akt is required for IFN-dependent ISG15 and CXCL10 protein expression. (A) Akt1^+/+2^+/+, Akt1^−/−2^+/+, and Akt1^−/−2^−/− MEFs were treated with mouse IFN-α as indicated. Equal protein aliquots were processed for immunoblotting with an anti-mouse ISG15 antibody. The same blot was reprobed with an anti-tubulin antibody as indicated. The signals for ISG15 and tubulin from three independent experiments (including the one shown) were quantitated by densitometry, and the intensity of ISG15 relative to tubulin was calculated. Data are expressed as means of ratios of ISG15/tubulin ± SE for each experimental condition. (B) Akt MEFs were treated with mouse IFN-γ as indicated. Equal protein aliquots were processed for immunoblotting with an anti-mouse IP10 antibody. Same blot was reprobed with anti-tubulin antibody as indicated. The signals for IP10 and tubulin from two independent experiments (including the one shown) were quantitated by densitometry, and the intensity of IP10 relative to tubulin was calculated. Data are expressed as means of ratios of IP10/tubulin ± SE for each experimental condition. (C) U20S cells were transfected with either the control empty vector or a dominant-negative Akt mutant and treated with human IFN-α as indicated. Equal protein aliquots were processed for immunoblotting with anti-ISG15 antibody. The blot was stripped and probed with anti-GAPDH antibody. Lysates from the same experiment were resolved separately and immunoblotted with an anti-Akt antibody. Also shown is longer exposure of the same blot to demonstrate the presence of endogenous Akt protein.