Journal: The Journal of Biological Chemistry

Article Title: Blue light–excited LOV1 and LOV2 domains cooperatively regulate the kinase activity of full-length phototropin2 from Arabidopsis

doi: 10.1074/jbc.RA117.000324

Figure Lengend Snippet: A, schematic illustration of the arrangement of LOV1, LOV2, Jα, and STK in the amino acid sequence of Atphot2. B, absorption spectra of D450 in the dark (red line) and under BL irradiation (black line) at a fluence of 200 μmol m−2 s−1. The kink appearing at 380 nm was caused by the exchange of a filter. C, kinase assay of phot2 WT and its D720N, C170A, and C426A mutants. Labels “0,” “D,” and “L” indicate the specimens immediately after mixing, after 15-min incubation in the dark, and after 15-min incubation under BL, respectively. The upper panel shows an SDS-PAGE pattern taken using an imaging plate for the same gel stained by Coomassie Brilliant Blue (lower panel). D, result of a gel-shift assay for phot2 WT without and with λPPase treatment and D720N mutant. E, identification of phosphorylated residues in phot2 by MS. Schematics from the top to bottom are D720N mutant as a reference and WT specimens after λPPase treatment, after 1-h incubation of λPPase-treated in the dark, and after 1-h incubation of λPPase-treated under BL.

Article Snippet: In a gel-shift assay, the WT, just after purification, had an apparent molecular weight larger than those of the WT incubated with λ-protein phosphatase (λPPase; New England Biolabs) and the D720N mutant ( D ).

Techniques: Sequencing, Irradiation, Kinase Assay, Incubation, SDS Page, Imaging, Staining, Electrophoretic Mobility Shift Assay, Mutagenesis