Fig 2.

Identification of IRF4 tyrosine sites responding to c-Src-mediated activation. 293 cells in 12-well plates were transfected with 0.2 μg Flag-IRF4 or its point/deletion mutants, 0.2 μg c-Src, 40 ng pGL3/IFN-β-Luc, and 10 ng Renilla. A dual-luciferase assay was performed. Results are the averages and standard errors (SE) of duplicates. Representative results from at least three independent experiments are shown. The ability of the vector control to activate the promoter construct was set to 1. (A) Y61 and Y124 are crucial for IRF4 activation by c-Src. The tested Flag-IRF4 Y-F point mutants include Y61F, Y121F, Y124F, Y121/124F, Y61/124F, Y151F, Y157F, Y169F, Y180F, Y220F, and Y324F. (B) Y324 is a site critical for IRF4 function independently of phosphorylation. The tested Flag-IRF4 Y-A point mutants include Y36A, Y121A, Y124A, Y191A, Y324A, Y334A, Y427A, and Y439A. (C) c-Src targets multiple sites on IRF4. The tested Flag-IRF4 deletion mutants include 1-190, 1-255, 1-260, 1-412, 1-420, 1-445, Del(140-187), and Del(140-408).