Journal: PLOS Genetics

Article Title: The RCAR12-CAP1-OST1 module controls ABA-mediated stomatal closure in Arabidopsis

doi: 10.1371/journal.pgen.1012092

Figure Lengend Snippet: (A) Interaction between OST1 and CAP1 validated by BiFC assays. The images are the representative of three independent experiments ( n = 3). OST1 K50R indicates the kinase-dead form of OST1. NLS-mCherry indicates nuclear localization. We repeated the experiment four times with similar results and at least 8 leaves were observed per time. Scale bars = 50 μm. (B) Co-IP assay was performed to test the interaction of CAP1 with OST1 and OST1 K50R , detected with anti-Flag and anti-MYC antibodies. GFP-2Flag was set as a negative control. The images are the representative of two independent experiments ( n = 2) with the same results. (C) In vitro kinase assay showing the phosphorylation of CAP1 by OST1. The phosphorylation of His-CAP1 was examined using an anti-thiophosphate ester (thioP). The loading amounts of His-CAP1 and GST-OST1 were detected using Coomassie Brilliant Blue (CBB) staining. (D) OST1 phosphorylates CAP1 in vitro . CAP1 from OE- CAP1 and ost1 :OE- CAP1 seedlings was immunoprecipitated and separated by a Pho-tag-PAGE gel, then analyzed with anti-Flag antibody. The IP input was analyzed by regular SDS-PAGE gel with anti-Flag antibody. (E) The phospho-sites of CAP1 identified from mass spectrometry analysis. (F) Phosphorylation levels of mutation of phosphosites (serine to alanine, S to A) by OST1, detected with anti-phosphoserine antibody. The loading amounts of His-CAP1 and GST-OST1 were detected by CBB staining. Values indicate mean ± SD ( n = 3 biological repeats). CAP1 (3 μM), ADF4 (3 μM), RCAR12 (1 μM), ABA (1 μM), F-actin (3 μM).

Article Snippet: GST-RCAR12 or GST (5 μg) were incubated with His-CAP1 (5 μg) and 25 μL glutathione S-Sepharose 4B (GS4B, GE Healthcare, PA, USA) resin in 500 μL binding buffer containing 25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and ABA.

Techniques: Co-Immunoprecipitation Assay, Negative Control, In Vitro, Kinase Assay, Phospho-proteomics, Staining, Immunoprecipitation, SDS Page, Mass Spectrometry, Mutagenesis

Journal: PLOS Genetics

Article Title: The RCAR12-CAP1-OST1 module controls ABA-mediated stomatal closure in Arabidopsis

doi: 10.1371/journal.pgen.1012092

Figure Lengend Snippet: (A(A) GST pull-down assay was performed for the interaction of ADF4 with CAP1. In vitro phosphorylation assay was performed, followed by GST pull-down. OST1 K50R indicates the kinase-dead form of OST1. λ-PPase, Lambda Protein Phosphatase. The loading amounts of His-CAP1, GST-ADF4 and GST were detected by CBB staining.(B) Relative protein levels of His-CAP1 in (A). The intensity of His-band was measured using ImageJ software. Three independent experiments were provided for the data statistics. Values are means ± SD ( n = 3). Different letters indicate statistically significant differences by one-way ANOVA with Tukey’s test ( P < 0.05). (C) Analysis of the interaction of CAP1 using Co-IP assay before and after 50 μΜ ABA treatment for 3 h. CAP1-6Flag was immunoprecipitated using anti-Flag-M2 agrose, and immunoblotting assays were performed using anti-Flag and anti-MYC antibodies. (D) Relative protein levels of ADF4 in (C). The intensity was measured using ImageJ software. Four independent experiments were provided for the data statistics. Values are means ± SD (n = 4). Asterisk indicates statistically significant differences (*P < 0.05, Student’s t- test, two-sided). (E) The interactions of the phosphomimic variant of CAP1 S246/249/250/253-256D and CAP1 with ADF4 were analyzed using in vitro GST pull-down assay. The loading was detected by CBB staining. (F) The interactions of the phosphomimic variant of CAP1 S246/249/250/253-256D and CAP1 with ADF4 were analyzed using LCI (Luciferase Complementation Imaging) assay. The indicated constructs were transiently expressed in N. benthamiana leaves. Leaves were photographed 3-day after infiltration. (G) Representative confocal images of F-actin. F-actin was incubated with indicated proteins for 30 min before staining with Alexa488-Phalloidin. Scale bar = 5 μm. (H) Quantification of the length of F-actin in (G) by ImageJ. More than 100 filaments were measured for each treatment. Values are mean ± SEM. * P < 0.05, as determined by one-way ANOVA with Tukey’s test. (I) Depolymerization of F-actin (10% pyrene labeled). F-actin disassembly was monitored by tracking the decrease in pyrene fluorescence per minute. a.u., arbitrary units. Values indicate mean ± SD ( n = 3 biological repeats). CAP1 (3 μM), ADF4 (3 μM), OST1 (1 μM), F-actin (3 μM).

Article Snippet: GST-RCAR12 or GST (5 μg) were incubated with His-CAP1 (5 μg) and 25 μL glutathione S-Sepharose 4B (GS4B, GE Healthcare, PA, USA) resin in 500 μL binding buffer containing 25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and ABA.

Techniques: Pull Down Assay, In Vitro, Phospho-proteomics, Staining, Software, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Variant Assay, Luciferase, Imaging, Construct, Incubation, Labeling, Fluorescence