Figure 2

MIXL1 is phosphorylated on tyrosine residues. Nuclear proteins were extracted from 293T cells transiently transfected with constructs pCMV5-MIXL1 and pCMV5-ΔCAD. Equal amounts of nuclear extracts (10 μg for CMV5 control and MIXL1 and 5 μg for ΔCAD) were loaded in duplicate into a NuPAGE gel and transferred to PVDF membranes. The blot was divided into two halves and hybridized with either anti-MIXL1-N (1:100) or anti-P-Tyr (mouse monoclonal antibody 4G10 at 1:4000). Anti-MIXL1-N antibody detected two species in transfectants expressing full-length MIXL1 and a strong fuzzy band in transfectants expressing the C-terminal truncation. The second half of the blot probed with 4G10 detected a weak but specific signal (arrow) corresponding to the slower migrating form of full-length MIXL1 and a robust signal (arrow) for the C-terminal truncation.