Figure 6.

JNK-1 phosphorylates in vitro c-Jun at T91/93 in a T95-dependent manner. (a) NSC-34 cells were transfected with plasmids expressing either c-Jun-wt-HA or c-JunA95-HA-tagged proteins (as indicated) and levels of c-Jun site-specific phosphorylation were analyzed by western blot of total extracts by using the indicated antibodies. (b) Either c-Jun-wt-HA or c-JunA95-HA-tagged proteins were immunoprecipitated by using HA antibodies, then c-Jun-HA immunocomplexes were used as substrates for in vitro phosphorylation assays by using recombinant JNK-1, either alone or in combination with recombinant GSK3β as indicated. Products of in vitro phosphorylation reactions were analyzed by western blot using c-Jun-phospho-specific antibodies for either p-T91/T93, p-S63 or p-239. Total amounts of c-Jun proteins were determined by using the c-Jun total antibody. (c) In vitro phosphorylation assay of recombinant FL-c-Jun protein (Ezio Life Sciences) incubated with recombinant JNK-1 as indicated. Reactions were analyzed by western blot, using either c-Jun-p91/93 or c-Jun-p-S63 phospho-specific antibodies as indicated. Total amounts of c-Jun proteins were determined by using the c-Jun total antibody