Figure 3.

MAP3K19 directly phosphorylates MAP2Ks. A, MAP3K19 was immunoprecipitated (IP) from HEK293T cells and subjected to a kinase assay with kinase-inactive MEK1. B and C, in vitro kinase assay using recombinant MAP3K19 protein and kinase-inactive MEK1 or ERK2, respectively. D, kinase-inactive MEK1 and purified GST-MAP3K19 or GST-MLK1 kinase domain were subjected to in vitro kinase assay in the presence or absence of inhibitors: 5 μm L779450, 1 μm PLX4032, 5 μm U0126, or 2 μm AZD6244. E and F, in vitro kinase assay using recombinant MAP3K19 protein and kinase-inactive MKK7 or JNK1/2, respectively. The data are shown as mean phospho:total protein density ± S.D. Dunnett's multiple comparisons test was used for statistical analysis, with samples in the first lane as control. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ♢, kinase-inactive.