FIG. 8.

Interaction of Deltex with MEKK1(C). (A) Association of DTX with MEKK1(C) demonstrated by coprecipitation. 293T cells were transfected with pcDNA3 vector, MEKK1(FL), or MEKK1(C) in the presence or absence of Deltex-Myc and total cellular extracts were prepared 24 h after transfection. Extracts (200 μg) were immunoprecipitated using anti-Myc (9E10) antibody. 80% of the precipitate was separated on SDS-PAGE and the level of MEKK1 was determined by immunoblotting (upper panel). Twenty percent of the precipitate was similarly resolved and the contents of Deltex-Myc were detected by anti-Myc (middle panel). The expression of MEKK1(FL) and MEKK1(C) in transfected 293T cell was determined by immunoblotting with anti-MEKK1 (lower panel). (B) Association between Deltex and MEKK1(C) detected by GST pull-down assay. Five micrograms of GST-Deltex (GST-DTX) fusion proteins were first bound to glutathione agarose beads and incubated with 200-μg lysates of 293T cells transfected with the indicated plasmid or with 100-μg extracts of DO11.10 cells. After washing agarose beads, the amounts of MEKK1(C) pulled down were analyzed by immunoblotting with anti-MEKK1 (upper panel), and the quantity of GST-DTX on agarose was assessed by anti-GST (middle panel). The amounts of the transfected and endogenous MEKK1(FL) and MEKK1(C) present in the input cell lysates are shown in the bottom panel. (C) Direct Deltex-MEKK1(C) but not Deltex-MEKK1(FL) binding. Five micrograms of GST, GST-DTX, or GST-DTXΔRF were loaded onto glutathione agarose beads and incubated with 5 μg of His-MEKK1(FL) or His-MEKK1(C). The amounts of His-MEKK1(C) or His-MEKK1(FL) brought down by agarose were determined by immunoblotting with anti-His antibodies (upper panel). Input of GST, GST-DTX, and GST-DTXΔRF was determined by anti-GST antibodies (middle panel), while input of His-MEKK1(FL) and His-MEKK1(C) was assessed by anti-His antibodies (bottom panel).