Activity of V5-MEKcr.

figure

posted on 2011-02-18, 02:12 authored by Chih-Shia Lee, Karl J. Dykema, Danielle M. Hawkins, David M. Cherba, Craig P. Webb, Kyle A. Furge, Nicholas S. Duesbery

(A) Phosphorylation and activation of V5-MEKcr in cells. Parental SK-MEL-28 cells and cells stably expressing V5-MEK or V5-MEKcr were treated with PA alone (PA) or LeTx for 24 h. Whole cell lysates were then prepared and immunoblotted with anti-phospho MEK1/2 antibody to examine the phosphorylation status of V5-MEK1cr and V5-MEK2cr in SK-MEL-28 cells (upper panel), or anti-GAPDH antibody for equal loading control (lower panel). (B) Kinase activity of V5-MEKcr. Anti-V5 immunoprecipitates were prepared from SK-MEL-28 cells and then used for in vitro kinase assays (top panels) in the presence or absence of active B-Raf and/or recombinant ERK2. The proteins were then separated in 10% Tris-Glycine gels, and ERK2 phosphorylation was visualized by using a FLA-5000 PhosphoImager. Equal amount of V5 immunoprecipitates were immunoblotted for V5 (bottom panel) as a V5-MEK input control.