Fig. 4: KIN10 interacts with SPCH in vitro and in vivo.
From: KIN10 promotes stomatal development through stabilization of the SPEECHLESS transcription factor
a KIN10 interacts with SPCH in yeast. b KIN10 directly interacts with SPCH in vitro. MBP and MBP-KIN10 were incubated with GST-SPCH bound to glutathione agarose beads and then eluted and analyzed by immunoblotting using anti-MBP antibody. Red asterisk indicated KIN10-MBP, while blue asterisk indicated MBP. c rBiFC showed that KIN10 interacts SPCH in tobacco leaves. d Coimmunoprecipitation assay of KIN10 and SPCH with seedlings expressing KIN10-YFP and SPCH-myc under 35S promoters or seedlings expressing only SPCH-myc. CoIP was performed using GFP-trap and immunoblotted using anti-GFP and anti-Myc antibodies. e The subcellular location of pKIN10::KIN10-YFP in Arabidopsis cotyledon epidermal cells. Seedlings of pKIN10::KIN10-YFP were grown in ½ MS liquid medium containing 1% Sucrose for 3 days under long-day condition. Scale bar, 20 μm. f, g Quantification of nuclear localization of KIN10 in different scale epidermal cells of (e). Nuclear and cytoplasmic KIN10-YFP signal from more than 200 epidermal cells in 10 cotyledons were analyzed by ImageJ software. Scatter plot (f) and box plot (g) showed negative relationship of KIN10-YFP nuclear/cytoplasmic ratio and the size of epidermal cells. Error bars indicate standard deviation (S.D.). Different letters above the bars indicated statistically significant differences between the samples (ANOVA analysis followed by Uncorrected Fisher’s LSD multiple comparisons test, p < 0.05). h Co-localization of pKIN10::KIN10-YFP and pSPCH::SPCH-RFP in cotyledon epidermal cells. The seedling was grown in ½ MS liquid culture containing 1% Sucrose. Scale bars in confocal images represent 20 μm. Arrow indicates the colocalized KIN10-YFP and SPCH-RFP proteins.
