Figure 6. MEKK4 interaction and regulation of Fln-A phosphorylation via MKK4/SEK1.
(A) Ten μg of GST-MKK4/SEK1 (GST-SEK1) was incubated in either ~500μg of COS-7 lysate or ~1mg E17.5 mouse forebrain (FB) lysate. As controls, equivalent amounts of COS-7 or FB lysates were incubated in glutathione-agarose beads (Beads) or beads pre-incubated in GST (GST-beads). One percent of lysates (1% input) were run for COS-7 or FB lysates. The upper blot was probed with anti-MEKK4 (CT) antibody and shows GST-MKK4/SEK1 precipitated endogenous MEKK4 in both COS-7 and FB lysate. Twenty μg of E15.5 MEKK4−/− FB lysate was run as a control. A doublet band is precipitated in both COS-7 and FB lysate with the upper band corresponding to MEKK4. The lower blot was probed using an anti-Fln-A antibody and showed that GST-MKK4/SEK1 also precipitated endogenous Fln-A from COS-7 and FB lysates.
(B) NIH3T3 cells were mock or siRNA#555-transfected and cultured for 96h. Lysates (20μg/lane) from different mock and siRNA#555 (555)-transfected wells were western blotted and probed for phospho-Fln-A (Ser2152) (upper blot), Fln-A (middle blot), and MEKK4 (lower blot) antibodies. Increased phosphorylation of Fln-A was observed after transfection with siRNA#555.
(C) The relative phospho- to total Fln-A band intensities were quantified for five separate transfections/group which showed increased Fln-A phosphorylation in siRNA#555-transfected cultures.
(D) NIH3T3 cells were either untransfected (Mock) or transfected with dominant-negative SEK1 (dnSEK1), siRNA#555, or dnSEK1 plus siRNA#555. Lysates were collected at 24h, western blotted and probed as in C. The blot shows increased Fln-A phosphorylation (p-Fln-A) after siRNA#555 that was not observed when co-transfected with dnSEK1.
(E) Quantification (as in C) of the results in D for at least 5–6 separate transfections/group. The enhanced Fln-A phosphorylation after siRNA#555 was blocked by co-transfection of dnSEK1.