Figure 1.

Effect of the R472H mutation on the autophosphorylation activity of Arabidopsis phot1 expressed in insect cells. A, autoradiograph (left panel) showing light-dependent autophosphorylation activity of phot1 and the R472H mutant in protein extracts from insect cells. Protein extracts were prepared under dim red light, followed by kinase assays given a mock irradiation (D) or irradiated with white light (L). Immunoblot analysis of phot1 protein levels is shown below. B, impact of photochemically inactivating the LOV domain on phot1 R472H kinase activity. The autoradiograph (left panel) shows light-dependent autophosphorylation activity of phot1 R472H harboring the LOV1 mutation C234A or the LOV2 mutation C512A. Immunoblot analysis of phot1 protein levels is shown below. In each case, kinase activity was quantified from the autoradiographs with ImageJ and expressed as a percentage of maximal autophosphorylation (error bars indicate ± S.E., n = 3). Significant differences between autophosphorylation in the irradiated and mock irradiation control are indicated (*, p < 0.05, Student's t test).