Figure 3.
Active MAPKKK Is Required for PRKK Activation.
Approximately 106 parsley protoplasts per treatment were transformed to transiently express the constitutively active MAPKKK ΔNPK1 (Kovtun et al., 2000) (lane 2), the HA-tagged version of PRKK (lane 3), or both (lane 4). Protoplasts transformed with the empty vector pRT101 were used as controls (lane 1). After protein extraction, samples were immunoprecipitated with HA antibody. In vitro kinase assays of immunoprecipitated PRKK-HA were performed on 4 μg of kinase-negative GST-SIMK as substrate. Reaction products were analyzed by SDS-PAGE followed by autoradiography (A) and Coomassie blue staining (B). (C) shows PRKK-HA expression. The specificity of immunoprecipitation was controlled by immunoblotting extracts with HA antibody.