Fig. 3. In vitro phosphorylation by MEK of immunoprecipitated low-activity cellular ERK1 dimer (* – 0) results in the appearance of the shifted fully activated dimerized ERK1 (* – *). Total ERK1 was immunoprecipitated from 30 minute extracts from sea urchin embryos using an anti-ERK antibody and then treated in vitro with either active MEK (IP + MEK) or an inactive (kinase dead) mutant of MEK (IP + k.d.MEK); a control IP in the absence of the anti-ERK antibody is also shown (Control IP-AB). A and B show a comparison between western blots of the same samples with (A) anti-ERK antibody or (B) anti-dualphosphorylated ERK antibody. In both cases, the immunoprecipitates are compared with an untreated aliquot of the 30 minute extract used for immunoprecipitation. Note that immunoprecipitates using anti-dualphosphorylated ERK antibody are highly enriched in the 91 kDa dimeric form. (C) Relative activities of samples treated as in B, washed in MAP kinase buffer and used in protein kinase assays. The sample activated by MEK shows sixfold higher activity than those either untreated or