FIG. 1.

In vivo binding of endogenous GCK and MEKK1: activation of MEKK1 by coexpressed GCK in vivo. (A) Binding of endogenous GCK and MEKK1. RAMOS cells were lysed and subjected to immunoprecipitation (IP) with anti-GCK and immunoblotting (IB) with anti-MEKK1 or immunoprecipitation with anti-MEKK1 and immunoblotting with anti-GCK. Crude lysates were immunoblotted with the GCK and MEKK1 antibodies as well. (B) GCK activates MEKK1 in vivo. 293 cells were transfected with a low dose of HA-MEKK1 plasmid and with either vector or FLAG-GCK as described in Materials and Methods. MEKK1 was immunoprecipitated with anti-HA and assayed for SEK1(K129R) phosphorylation and autophosphorylation (left panel). The right panel documents expression of the transfected plasmids and the level of GST-SEK1(K129R) used in the MEKK1 assays.