Fig. 4. (A) The two ERK1 homodimers (* – 0 and * – *) copurify with the ERK1 monomer. Western blots of a fraction with high MAP kinase activity (fraction 23) eluted from a phenyl-Sepharose column during ERK1 purification from mitotic sea urchin embryo extracts probed with (a) an anti-ERK antibody and (b) an anti-dualphosphorylated ERK antibody. 4 µl of the fraction was loaded for each blot. Blot b was detected using the high-sensitivity ECL Advance Kit: a faint band of active monomer is visible (arrow). (B,C) Phosphorylation by MEK and dephosphorylation using CL100 dual specificity phosphatase cause the predicted gel shifts of the ERK1 dimers in the phenylsepharose column fraction. 0.5 µl of fraction 23 was used for each treatment; samples were blotted and probed with (B) anti-ERK antibody and (C) anti-dualphosphorylated ERK antibody. Samples treated with CL100 in the presence of vanadate to block phosphatase activity (CL100+Na₃VO₄) or with mutant-inactive (kinase dead) MEK (k.d. MEK) serve as controls. Treatment with CL100 led to the loss of the more slowly migrating band, while treatment with MEK enhanced the band. Note that after MEK treatment, dimeric forms accumulate in preference to the monomer.