human rhoa protein  (Cytoskeleton Inc)

Journal: Cell Reports Medicine

Article Title: A Rac-specific competitive inhibitor of guanine nucleotide binding reduces metastasis in triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102233

Figure Lengend Snippet: A41 selectively impairs RAC protein activation (A) Representative surface plasmon resonance (SPR) sensograms of binding of immobilized RHOA or CDC42 with indicated increasing concentrations of A41 ( N > 3). (B) Effect of A41 on GEF-stimulated RAC2, RHOG, RHOA, and CDC42 nucleotide exchange. Purified small G proteins were pre-loaded with GDP and then nucleotide exchange was monitored by the increase in fluorescence following mant-GTP binding in the absence and presence of A41 (5 μM) ( N = 3). K obs is expressed as mean ± SEM. ∗ p < 0.001 vs. control. (C) Effect of A41 on RAC1 G30S activation. RAC1 G30S activation was monitored by the increase in fluorescence following mant-GTP binding in the absence and presence of A41 (5 μM). The GEF TRIO was used to induce nucleotide exchange ( N = 3). Data are expressed as mean ± SEM. (D) Immunoblot analysis and associated quantification of RAC-GTP level and total RAC expression in NIH-3T3 fibroblasts expressing RAC wild-type (RAC1 WT) or RAC1 oncomutants (RAC1 P29S and RAC1b) in the absence (Ctrl) and presence of A41 (10 μM). RAC activation was measured as the ratio of RAC-GTP to total RAC and expressed relative to Ctrl condition. Data are presented as mean ± SEM. ∗∗∗ p < 0.001 vs. controls.

Article Snippet: RAC1, RHOA and CDC42 purified proteins (respectively RH01, RC01 and CD01, Cytoskeleton) were diluted to 5 μg/mL in Na + acetate buffer (pH 5.0) and injected into sensor chip CM5 (GE Healthcare) in a Biacore T200 (GE Healthcare) that was activated with NHS/EDC buffer.

Techniques: Activation Assay, SPR Assay, Binding Assay, Purification, Fluorescence, Control, Western Blot, Expressing

Journal: iScience

Article Title: Regulation of Rho guanine nucleotide exchange factor 3 by phosphorylation in the PH domain

doi: 10.1016/j.isci.2025.112753

Figure Lengend Snippet: S399 phosphorylation reduces ARHGEF3 activity (A) HEK293 cells were transfected with GFP-ARHGEF3 (or GFP as control) for 24 h and then stimulated with 100 nM PMA for 1 h. Cell lysates were subjected to active RhoA pull-down by GST-Rhotekin beads and analyzed by western blotting. Western signals were quantified by densitometry, and relative RhoA activity was expressed as the ratio of active RhoA (pulldown) versus total RhoA (lysate), normalized to control as 1. Data are presented as mean ± SEM ( n = 6). One-sample or paired t test was performed, and p values for significant differences ( p < 0.05) are indicated on the graph. Representative blots are shown. pS399-GFP-ARHGEF3 western blot is also shown. Molecular weight markers (kDa) are indicated on the right side of blots. (B) Cells were transfected with WT-, S399A-, or S399D-GFP-ARHGEF3 (or GFP as control), followed by active RhoA pulldown and quantification as in (A) ( n ≥ 5). Linear mixed-model analysis followed by a Tukey test was performed for pairwise comparison, and p values for significant differences ( p < 0.05) are indicated on the graph. Representative blots are shown. (C) HeLa cells were transfected with WT-, S399A-, or S399D-GFP-ARHGEF3 (or GFP as control) followed by fixation and phalloidin/DAPI staining. Representative images of three independent experiments are shown (scale bar, 2 μm). (D) Fluorescence intensities of stress fibers were quantified for the experiments in (C). Data are shown as mean ± SEM ( n = 3 experiments, >20 cells per experiment). Paired t test was performed, and p values for significant differences ( p < 0.05) are indicated on the graph. (E) HeLa cells were transfected with WT- or S399A-GFP-ARHGEF3 for 24 h and then stimulated with 100 nM PMA for 1 h, followed by fixation and phalloidin/DAPI staining. Representative images of three independent experiments are shown (scale bar, 2 μm). (F) Fluorescence intensities of stress fibers were quantified for the experiments in (E). Data are shown as mean ± SEM (n = 3 experiments, >25 cells per experiment). Two-way ANOVA was performed followed by Tukey test. Significant effect was found with PMA treatment but not with mutation, and p values for significant differences ( p < 0.05) are indicated on the graph.

Article Snippet: Recombinant RhoA , Cytoskeleton Inc , Cat# RH01.

Techniques: Phospho-proteomics, Activity Assay, Transfection, Control, Western Blot, Molecular Weight, Comparison, Staining, Fluorescence, Mutagenesis

Journal: iScience

Article Title: Regulation of Rho guanine nucleotide exchange factor 3 by phosphorylation in the PH domain

doi: 10.1016/j.isci.2025.112753

Figure Lengend Snippet: H427 distinguishes PH domain interaction with PI(3,5)P 2 and PI(4,5)P 2 (A) MD simulations were performed with the ARHGEF3 PH domain bound to membrane bilayers containing PI(4,5)P 2 or PI(3,5)P 2 . The histograms show normalized ensemble-averaged number of contacts formed between residues in the PH domain and the 3- and 4-phosphate groups (3P and 4P) of PI(3,5)P 2 and PI(4,5)P 2 , respectively. Data were averaged over all simulation replicas for each lipid composition. (B) ARHGEF3 PH domain, and highlighted on the ribbon structure are the residues with the highest frequency of interaction with 3P or 4P. (C) HEK293 cells were transfected with WT- or H427D-GFP-ARHGEF3 for 24 h, and cell lysates were subjected to lipid SiMPull assays as in . Data shown are mean ± SEM for one experiment ( n ≥ 10 images). Three independent experiments were performed with similar outcomes. Previously determined threshold for binding (100 GFP counts) is indicated by dotted lines. (D) HEK293 cells were transfected with WT or H427D-GFP-ARHGEF3 (or GFP as control) for 24 h. Cell lysates were subjected to active RhoA pulldown by GST-Rhotekin beads and analyzed by western blotting. Western signals were quantified by densitometry, and relative RhoA activity was expressed as the ratio of active RhoA (pulldown) versus total RhoA (lysate), normalized to control as 1. Data are presented as mean ± SEM ( n = 9). One-sample or paired t test was performed, and p values for significant differences ( p < 0.05) are indicated on the graph. Representative blots are shown. Molecular weight markers (kDa) are indicated on the right side of blots.

Article Snippet: Recombinant RhoA , Cytoskeleton Inc , Cat# RH01.

Techniques: Membrane, Transfection, Binding Assay, Control, Western Blot, Activity Assay, Molecular Weight

Journal: iScience

Article Title: Regulation of Rho guanine nucleotide exchange factor 3 by phosphorylation in the PH domain

doi: 10.1016/j.isci.2025.112753

Figure Lengend Snippet: S399D reduces the catalytic activity of ARHGEF3 (A) Purified WT- and S399D-GST-ARHGEF3 were subjected to in vitro RhoA guanine nucleotide exchange assays. EDTA: positive control for complete nucleotide dissociation. Data from five independent experiments with similar outcomes were fit as a single exponential decay curve (left panel). RhoA nucleotide exchange activity at t = 45 min is shown on the bar graph (right panel), normalized to EDTA as 100%. Data are shown as mean ± SEM ( n = 5). Student’s t test was performed. (B) A model of ARHGEF3 regulation. Left panel: ARHGEF3 activates RhoA at the plasma membrane, requiring binding to PI(4,5)P 2 . PKC-dependent phosphorylation in the PH domain allosterically inhibits the GEF activity of ARHGEF3, dampening RhoA activation and actin stress fiber formation. Right panel: the same phosphorylation also disrupts ARHGEF3 binding to PI(3,5)P 2 (presumably in the late endosome), the role of which is yet to be determined. The graphics were created using BioRender.

Article Snippet: Recombinant RhoA , Cytoskeleton Inc , Cat# RH01.

Techniques: Activity Assay, Purification, In Vitro, Positive Control, Clinical Proteomics, Membrane, Binding Assay, Phospho-proteomics, Activation Assay