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. 2011 Apr 29;6(4):e19251. doi: 10.1371/journal.pone.0019251

Figure 5. Hand1 phosphorylation did not affect heterodimer formation with E12 but abolishes DNA binding.

Figure 5

A. HA-Hand1-WT, -AA and -DD plasmids were co-transfected with Myc-E12. HA-Hand1 was pulled down by HA-IP and blotted with an antibody against Myc. Myc-E12 was found to bind to all three HA-Hand1 proteins. B. Gel shift assay. Hand1-AA showed strong DNA binding while phosphorylation (Hand1-DD) abolished the DNA binding (indicated by arrows). Co represents competition oligo. C. ChIP assay. Hand1 wild-type and mutants was expressed with or without Akt in HEK293 cells. Using nuclear extracts from these cells, ChIP assay was performed and the results showed that Hand1-AA had stronger DNA binding than Hand1-DD. Akt also reduced Hand1 DNA binding ability. Abbreviations: Neg con, negative control; Pos con, positive control.