MIXL1 is phosphorylated on Tyr20 A) Cartoon depicting MIXL1 constructs generated for mutational analysis. Three MIXL1 mutant constructs were derived from the ΔCAD mutant construct pCMV2-flag-ΔCAD (renamed as Flag-2Y in this figure). Each mutant in the constructs has a Flag epitope (a solid gray bar) attached in frame to the amino-terminus and contains either tyr20 or tyr110 or none as illustrated. B) Detection of tyrosine phosphorylation. The mutant constructs were transiently transfected into HEK 293T cells. 10 μg of nuclear proteins from the transfectants were resolved on a NuPAGE gel. Tyrosine phosphorylation was examined by immunobloting with monoclonal antibody 4G10 (1:3000). The blot was stripped and re-probed with mouse monoclonal antibody anti-flag (1:300). Tyrosine phosphorylation was detected in both the mutant Flag-2Y and the mutant Flag-Y110F but not in the mutant Flag-ΔN25 as well as the mutant Flag-Δ2Y. The results are consistent with Tyr20 being the target of phosphorylation.