FIG. 1.
Kinase assay of mouse p90rsk-mo-1. Cos-7 cells were mock transfected without DNA (ø) or transiently transfected with pCMV-neo vector control (V) or with pCMV-HA-rskmo-1 (HA-mo-1) as described in Materials and Methods. Cells were stimulated for 5 min with EGF (hatched bars) or with carrier BSA (solid bars) and harvested by sonication in buffer H. Sixty micrograms of the cell extracts obtained by centrifugation at 100,000 × g was immunoprecipitated with either anti-HA (A) or anti-rsk (B) antibody. After two washes, the immunoprecipitates were assayed for S6 kinase activity as described in Materials and Methods. Anti-HA immunoprecipitates (C) or 12 μg of total cell extracts (D) was resolved by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes for analysis. Membrane blots were probed with rabbit anti-rsk antibody to confirm protein expression.