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. 2009 Jul 15;284(37):24972–24980. doi: 10.1074/jbc.M109.042911

FIGURE 1.

FIGURE 1.

Retroviral gene transfer of constitutively active and constitutively inactive mutants of MEK5 into HUVEC alters ERK5 activity. A, the efficiency of retroviral gene transfer indicated by EGFP reporter expression (gray traces) was measured by cytometric analysis 96 h after transduction in comparison with control cells (black traces). B, expression of MEK5 mutants was studied by Western blot analysis 96 h after transduction. Recombinant proteins were detected by a hemagglutinin (HA) tag-specific antibody. Immunoblotting of ERK1/2 served as a loading control. C, endogenous ERK5 expression in cells with MEK5 mutants was analyzed. Note the retarded migration of ERK5 after expression of the constitutively active MEK5 mutant. Cells were starved for 5 h and either kept in suspension (s) or attached to fibronectin for 40 min (a). D, the enzymatic activity of ERK5 was measured in an in vitro immunocomplex kinase assay with recombinant glutathione S-transferase (GST)-MEF2C as a specific substrate. The efficiency of MEF2C phosphorylation was determined by PhosphorImager scanning, and the efficiency of ERK5 immunoprecipitation was measured by Western blotting with a specific antibody.