Fig. 2. Anti-Rsk antibody 2 neutralizes Rsk activity in vitro and in vivo. (A) The anti-Rsk serum 2 inhibits Rsk activity in vitro. Extracts were prepared from immature oocytes (0) and unfertilized mature eggs (150). Immunoprecipitates with anti-Rsk serum 1 and 2 from these extracts were immunoblotted with anti-Rsk serum 2 (upper), or assayed for phosphorylation of S6 peptide (lower). (B) The purified anti-Rsk antibody 2 inhibits Rsk activity in vivo. Immature oocytes were injected with 3 ng of the anti-Rsk antibody purified from the serum 2 (lane 6) or control IgG from preimmune serum 2 (lane 5), or not injected (lane 1-4); oocytes were then treated with 1-MeAde. Extracts were prepared at the indicated times after 1-MeAde addition, and immunoblotted with anti-MAPK antibody (third panel) and anti-active MAPK (fourth panel). Immunoprecipitates with the anti-Rsk serum 1 were immunoblotted with anti-Rsk serum 2 (second panel), and assayed for phosphorylation of GST-S6 (first panel). In lane 4 (210F), mature eggs were inseminated 150 minutes after 1-MeAde addition. In lane 6, Rsk was present in the U form but its activity was almost undetectable.