Fig. 5. MEF2 proteins physically interact with GATA-4. (A) MEF2A interacts in vivo with GATA-4. Nuclear extracts from 293T cells transfected with empty vectors (Ctl), Flag-GATA-4 and/or HA-MEF2A were immunoprecipitated using an anti-HA antibody, separated on 10% SDS–PAGE, transferred to PVDF membranes, and subjected to immunoblotting using an anti-Flag antibody (top panel). The lower two panels are Western blots carried out on the same nuclear extracts using either HA (to reveal tagged MEF2A proteins) or Flag (to reveal tagged GATA-4 proteins) antibodies. (B) MEF2A proteins interact in vitro with GATA-4. Pull-down assays were performed using immobilized, bacterially produced MBP fusions (MBP-GATA-4 and MBP-LacZ as control) and in vitro translated 35S-labeled MEF2A, MEF2C, MEF2D or luciferase (luc) protein. The protein complexes were resolved on 10% SDS–PAGE. (C) The physical interaction between GATA-4 and MEF2 requires the C-terminal zinc finger DNA-binding domain of GATA-4. Full-length GATA-4 and various GATA-4 mutants (depicted in Figure 6A) were in vitro cotranslated with MEF2A and co-immunoprecipitated using an antibody directed against the extreme C-terminus of GATA-4. The protein complexes were resolved on 15% SDS–PAGE. The asterisks highlight GATA protein bands. (D) The DNA-binding domain of MEF2 is sufficient for interaction with GATA-4. MEF2A DIVE (aa 1–86) retains the MADS and MEF2 domains. Co-immunoprecipitations were performed as described in (C). The asterisks highlight the MEF2A DIVE band. The protein complexes were resolved on 20% SDS–PAGE. (E) MEF2 DNA-binding-defective mutants interact with GATA-4. MEF2C R3T and MEF2C R24L do not bind DNA but are still able to dimerize. A deleted GATA-4 construct [G4 (201–443)] was used to differentiate between GATA-4 and MEF2C, which have similar electrophoretic mobility. Co-immunoprecipitations were performed as described in (C). The protein complexes were resolved on 10% SDS–PAGE.