FIGURE 4. ERK2 and MITF co-immunoprecipitate from HEK293 cells, and the ERK-binding domain of MITF is necessary and sufficient for this interaction.
A, V5-epitope-tagged MITF derivatives were expressed in HEK293 cells and immunoprecipitated; co-precipitation of ERK2 was assessed. B, HEK293 cells were co-transfected with plasmid DNA encoding either V5-tagged full-length MITF, MITF-ΔEBD (a derivative lacking residues 98 –198), or empty vector, and plasmid encoding ERK2. Cells were lysed 48 h post-transfection, and MITF was immunoprecipitated using an anti-V5 antibody. Immunoprecipitated complexes (IP) along with 10% of the input lysate (10%) were analyzed by Western blotting using antibodies against the V5 tag and ERK2. Both MITF and MITF-ΔEBD run as doublets. The asterisk indicates a cross-reacting band present in the lysate. C, HeLa cells were transfected with plasmid DNA encoding either V5-tagged full-length MITF, MITF-ΔEBD, or empty vector. Cells were analyzed by immunofluorescence microscopy using an anti-V5 antibody. Cells were also counterstained with 4′,6-diamidino-2-phenylindole to visualize nuclei. Scale bar = 20 μm. D, HEK293 cells were co-transfected with plasmid DNA encoding V5-tagged MITF98 –198 or empty vector, and plasmid DNA encoding ERK2. MITF98 –198 was immunoprecipitated and complexes were analyzed as above.