Figure 5

Tyr20 phosphorylation is diminished in the absence of PRD. The constructs pCMV5-ΔCAD and pCMV5-ΔPC (lacking both the CAD and PRD) were transfected into HEK 293T cells. 15 μg of the nuclear extracts from the transfectants were resolved on a 10% NuPAGE gel. Tyrosine phosphorylation was detected by immunobloting with monoclonal antibody 4G10. The same blot was stripped and reprobed with anti-MIXL1-N antibody (1:400). Compared to the vector control, the antibody 4G10 detected a weak signal for the ΔPC mutant, although it is much weaker than that for ΔCAD mutant with the intact PRD domain. V-Vector.