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Fig. 2. ERK activity in vivo is associated with its homodimers. Monomers at 44 kDa are largely inactive. (A) Schematic diagram of ERK1 activity during the first mitotic cell cycle of sea urchin embryos. An inset with the original data (Philipova and Whitaker, 1998) is also shown. (B) Sea urchin embryos. Active ERK1 was immunoprecipitated during the first mitotic cell cycle at time points that corresponded to maximum (6 min, NEB) and minimum activity (Unfertilized, 30 min) using an anti-dualphosphorylated ERK antibody and detected by western blotting with a second, different anti-dualphosphorylated ERK antibody or with an anti-MEK antibody (NEB sample). Arrows, active ERK1 dimers. Cell extract: whole-cell extracts are rich in ERK1 monomers as a western blot of a 30 minute post-fertilization cell extract detected with anti-ERK antibody demonstrates. It was necessary to load 150 µg total cellular protein in order to obtain a detectable band at 91 kDa for comparison with the anti-dualphosphorylated ERK antibody immunoprecipitate. The positions of molecular mass markers are shown. (C) HeLa cells. Active ERK was immunoprecipitated from cell extracts using an anti-dualphosphorylated ERK antibody, and proteins detected on western blots using a second anti-dualphosphorylated ERK antibody, an anti-ERK antibody or an anti-MEK antibody. The immunoprecipitate was also run in the absence of ß-mercaptoethanol (ß-ME) before blotting (protein complex). The positions of molecular mass markers are shown (kDa).
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