FIG. 5.

Association of ERK1 and -2 with Mxi2. (A) Detection of phosphoproteins in Mxi2 immunoprecipitates (IP). 293T cells transfected with HA-ERK2 or HA-Mxi2 and MEK1E (1 μg) were labeled with ³²P and stimulated with EGF (100 ng/ml) for 5 min where indicated. After anti-HA immunoprecipitation, phosphoproteins were detected in dried gels. The positions of Mxi2 and ERKs are indicated. (B) Detection of phosphorylated ERKs in association with Mxi2. Cells transfected with HA-Mxi2 and stimulated with cotransfected MEK1E or with EGF for 5 min were probed for phospho-ERK in anti-HA immunoprecipitates and their respective total lysates (TL). (C) Association of MAPKs with Mxi2. Cells transfected with HA-Mxi2 were stimulated with EGF for the indicated times, and anti-HA and preimmune (PI) immunoprecipitates were probed for ERK, JNK, and p38. (Bottom) Expression of immunoprecipitated HA-Mxi2. (D) Association of Mxi2 with ERK1 and -2. Cells transfected with HA-ERK1 and -2, in addition to AU5-Mxi2, were stimulated with cotransfected MEK1E or EGF for 5 min and anti-HA and preimmune immunoprecipitates, and their respective total lysates were probed for Mxi2. (E) Association of Mxi2 with ERKs in rat kidney. Lysates from homogenized kidneys (KL) were immunoprecipitated with anti-ERK or preimmune antibodies and probed for coimmunoprecipitating Mxi2. Total lysate from Mxi2-transfected 293T cells was run alongside as a control. (F) Affinity of ERK2 binding to Mxi2. Increasing concentrations of [³⁵S]methionine-labeled ERK2 were allowed to bind in vitro to GST-Mxi2 (open squares) or to GST-p38 (black circles) bound to glutathione-Sepharose beads. Beads were washed, bound proteins were electrophoresed by SDS-PAGE, and bound ERK2 was counted by phosphorimager. The data shown are the average ± the standard error of the mean of three independent experiments. WB, Western blot; c, control.