Figure 2.

Chitin elicitor activates OsMKK4 and constitutive active OsMKK4 activates OsMPK3 and OsMPK6 in vivo.(a) Cells constitutively expressing hemagglutinin (HA) epitope-tagged mitogen-activated protein kinase kinase (MAPKK) in the wild-type (WT) form were treated with chitin elicitor (Chitin). The MAPKKs were immunoprecipitated and kinase activities were determined by immunoprecipitation (IP) kinase assays using kinase-inactive OsMPK6 (OsMPK6^KR) as a substrate. Phosphorylation of OsMPK6^KR was detected by autoradiography after SDS-PAGE (top panel). Production of the MAPKKs was detected by immunoblot analysis using the anti-HA antibody (third and fifth panels). Equal loading was confirmed by CBB staining (second, fourth and bottom panels).(b) Myelin basic protein (MBP) kinase activities of OsMPK3, OsMPK4 and OsMPK6 in lines expressing OsMKK4^DD after induction by dexamethasone (DEX). Production of OsMKK4^DD was confirmed by immunoblot analysis using the anti-HA antibody. Activation of OsMPK3, OsMPK4 and OsMPK6 was determined using an IP kinase assay. The kinases were immunoprecipitated from 10 μg (OsMPK6), 50 μg (OsMPK3) or 100 μg (OsMPK4) of total proteins using specific antibodies. Phosphorylation of MBP was detected by autoradiography after SDS-PAGE (each top panel). Mitogen-activated protein kinases (MAPKs) were detected by immunoblot analysis (each middle panel). Equal loading was confirmed by CBB staining (each bottom panel).