Figure 8. MK5 interacts with ERK3 in heart.

(A) GST pull-down assays were performed on murine ventricular myocardium lysates (2 mg) using either GST-p38α, ERK3-GST, or GST alone. Bound MK5 was detected by immunoblotting using an anti-MK5 monoclonal antibody. As a control, 50 μg lysate (input) was loaded in lane 1. (B) To validate the GST-pull-down assay, lysates (2 mg) were ‘spiked’ with 1 μg of thrombin-cleaved MK5.1 prior to performing the pull-down assay using either GST-p38α, ERK3-GST, or GST alone. Bound MK5 was detected by immunoblotting using an anti-MK5 monoclonal antibody. As a positive control, 10 ng of MK5.1 was loaded onto the gel. (C) Murine ventricular myocardium lysates were prepared and MK5 immunoprecipitated. Purified rabbit IgG was employed in control immunoprecipitations. Co-immunoprecipitated ERK3, ERK4, or p38α was detected by immunoblotting. (D) GST pull-down assays were performed on murine ventricular myocardium lysates (2 mg) using either GST-MK5.1 or GST alone. Bound ERK3, ERK4, or p38α was detected by immunoblotting. As a control, 50 μg of lysate (input) was loaded in lane 1. (E) Pressure-overload hypertrophy was induced in mice by transverse aortic constriction (TAC). Sham animals underwent the identical surgical procedure, however the aortae were not constricted. After 3-d of TAC, mice were sacrificed, the ventricular myocardium isolated, lysates prepared, and MK5 immunoprecipitated. Purified rabbit IgG was employed in control immunoprecipitations. Co-immunoprecipitated ERK3, ERK4, p38α, phospho p38, and GAPDH were detected by immunoblotting. Aliquots (50 μg) of lysate from TAC and sham hearts were included as controls (Input). Results shown are representative of 3 animals in each group. (F) Lysates (50 μg) from 3-d TAC and sham hearts were analyzed by SDS-PAGE and immunoblotting using an anti-ERK3 or anti-ERK4 antibodies. Shown are the mean ± S.E. (n=3).