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gst tagged rhoa q63l  (Addgene inc)


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    Addgene inc gst tagged rhoa q63l
    A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to <t>RhoA</t> and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.
    Gst Tagged Rhoa Q63l, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins"

    Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

    Journal: Communications Biology

    doi: 10.1038/s42003-025-08222-5

    A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to RhoA and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.
    Figure Legend Snippet: A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to RhoA and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.

    Techniques Used: Binding Assay, Construct, Sequencing

    A Superimposed 1 H- 15 N HSQC spectra of 50 µM [ U - 15 N]-DAD in the absence (red) and presence (black) of 60 µM GDID (black). The DAD Q1207 cross peak, which is part of the DID-DAD binding interface, undergoes a large change in chemical shift between the bound and free states. B Superimposed 1 H- 15 N HSQC spectra of 5 µM [ U - 15 N]-DAD with 60 µM of RhoA and 50 µM GDID (black) and 5 µM [ U - 15 N]-DAD with 19 µM of RhoA and 13 µM GDID (red). Free DAD is evident at the lower concentration of the RhoA-GDID complex. C NMR-generated isothermal titration of 5 µM [ U - 15 N]-DAD binding to the RhoA-GDID complex and fit using a ‘total binding, accounting for ligand depletion’ model to estimate K d D/R of 27 ± 5 μM for DAD binding to the RhoA-GDID complex. D ELISA-generated isotherm of RhoA binding to 10 nM GDID fit to a “one site-total and nonspecific binding” model to estimate K d R of 8.9 ± 2.4 nM. E ELISA-generated isotherm of RhoA binding to 10 nM GDID-DAD fit to a ‘one site-total and nonspecific binding’ model to estimate K d R/D of 840 ± 200 nM. All values for ELISA experiments are represented as mean ± SD and were performed as N = 3 independent experiments. Values from individual experiments are shown as gray dots. Note that the decrease in the plateau values observed in the ELISA titrations are due to the Hook or Prozone effect in which high ligand concentrations result in a decrease in signal strength .
    Figure Legend Snippet: A Superimposed 1 H- 15 N HSQC spectra of 50 µM [ U - 15 N]-DAD in the absence (red) and presence (black) of 60 µM GDID (black). The DAD Q1207 cross peak, which is part of the DID-DAD binding interface, undergoes a large change in chemical shift between the bound and free states. B Superimposed 1 H- 15 N HSQC spectra of 5 µM [ U - 15 N]-DAD with 60 µM of RhoA and 50 µM GDID (black) and 5 µM [ U - 15 N]-DAD with 19 µM of RhoA and 13 µM GDID (red). Free DAD is evident at the lower concentration of the RhoA-GDID complex. C NMR-generated isothermal titration of 5 µM [ U - 15 N]-DAD binding to the RhoA-GDID complex and fit using a ‘total binding, accounting for ligand depletion’ model to estimate K d D/R of 27 ± 5 μM for DAD binding to the RhoA-GDID complex. D ELISA-generated isotherm of RhoA binding to 10 nM GDID fit to a “one site-total and nonspecific binding” model to estimate K d R of 8.9 ± 2.4 nM. E ELISA-generated isotherm of RhoA binding to 10 nM GDID-DAD fit to a ‘one site-total and nonspecific binding’ model to estimate K d R/D of 840 ± 200 nM. All values for ELISA experiments are represented as mean ± SD and were performed as N = 3 independent experiments. Values from individual experiments are shown as gray dots. Note that the decrease in the plateau values observed in the ELISA titrations are due to the Hook or Prozone effect in which high ligand concentrations result in a decrease in signal strength .

    Techniques Used: Binding Assay, Concentration Assay, Generated, Titration, Enzyme-linked Immunosorbent Assay

    A Plot of the radius of gyration, R G , versus the number of amino acids in a theoretical polypeptide. The parameter ν characterizes the dimensions of the amino acid chain where 0.60, red, represents an expanded coil and 0.30, maroon, represents the most compact globule state. B Monte-Carlo simulation using ν = 0.60 showing the theoretical persistence length between DID and DAD domains as a function of the number of amino acids: ~109 Å for the 285 amino acid DIAPH1 and ~90 Å for the 140 amino acids of DIAPH1/2. C Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, DIAPH1/2, D1/2 and DIAPH1 ΔDAD , D1 ΔD , in the presence of RhoA, +R, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 11 data points to a linear equation yielding slopes of 62 ± 6 AFU*s −1 for A (red), 66 ± 6 AFU*s −1 for D1 (green), 62 ± 7 AFU*s −1 for D1/2 (purple), 85 ± 5 AFU*s −1 for D1 + R (white), 75 ± 4 AFU*s -1 for D1/2 + R (gray) and 104 ± 7 AFU*s −1 for D1 ΔD (orange). D1 ΔD represents 100% activation and D1 or D1/2 represent 0% activation, respectively. Statistical p -values at 95% confidence are shown on the graph and all values are represented as mean ± SD derived from N = 3 independent experiments.
    Figure Legend Snippet: A Plot of the radius of gyration, R G , versus the number of amino acids in a theoretical polypeptide. The parameter ν characterizes the dimensions of the amino acid chain where 0.60, red, represents an expanded coil and 0.30, maroon, represents the most compact globule state. B Monte-Carlo simulation using ν = 0.60 showing the theoretical persistence length between DID and DAD domains as a function of the number of amino acids: ~109 Å for the 285 amino acid DIAPH1 and ~90 Å for the 140 amino acids of DIAPH1/2. C Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, DIAPH1/2, D1/2 and DIAPH1 ΔDAD , D1 ΔD , in the presence of RhoA, +R, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 11 data points to a linear equation yielding slopes of 62 ± 6 AFU*s −1 for A (red), 66 ± 6 AFU*s −1 for D1 (green), 62 ± 7 AFU*s −1 for D1/2 (purple), 85 ± 5 AFU*s −1 for D1 + R (white), 75 ± 4 AFU*s -1 for D1/2 + R (gray) and 104 ± 7 AFU*s −1 for D1 ΔD (orange). D1 ΔD represents 100% activation and D1 or D1/2 represent 0% activation, respectively. Statistical p -values at 95% confidence are shown on the graph and all values are represented as mean ± SD derived from N = 3 independent experiments.

    Techniques Used: Construct, Activation Assay, Derivative Assay

    A Membrane to cytosol ratio of CFP-DIAPH1 fluorescence. The sample sizes for DIAPH1 (D1, green circles), DIAPH1 + CN03 (D1 + CN03, red circles), DIAPH1 ΔDAD (D1 ΔD , cyan squares) and DIAPH1 ΔDAD + CN03 (D1 ΔD + CN03, purple squares) are 31, 44, 51 and 105, respectively. B Membrane to cytosol ratio of phalloidin fluorescence, which binds to F-actin. The sample size for D1 (green circles), D1 + CN03 (red cricles), D1 ΔD (cyan squares) and D1 ΔD + CN03 (purple squares) are 31, 44, 51 and 105, respectively. The analysis uses the ratio of intensities of membrane and cytosol, which decreases the bias caused by alternative actin filaments staining . C Confocal microscope images of AD293 cells transfected with CFP-labeled D1 and D1 ΔD . RhoA activator CN03 was at 1 mg/mL. Cells were stained with Alexa Fluor568 Phalloidin to image filamentous actin and Hoechst 33258 to image the nuclei. Scale bar is 10 µm. The significant cytosolic fraction of overexpressed DIAPH1 after CN03 activation of RhoA or of constitutively active DIAPH1 ΔDAD suggests that there are limited membrane binding sites for DIAPH1 as observed previously . The height of the bar represents the mean and the error bars are ± SEM and statistical p -values at 95% confidence are shown on the graph.
    Figure Legend Snippet: A Membrane to cytosol ratio of CFP-DIAPH1 fluorescence. The sample sizes for DIAPH1 (D1, green circles), DIAPH1 + CN03 (D1 + CN03, red circles), DIAPH1 ΔDAD (D1 ΔD , cyan squares) and DIAPH1 ΔDAD + CN03 (D1 ΔD + CN03, purple squares) are 31, 44, 51 and 105, respectively. B Membrane to cytosol ratio of phalloidin fluorescence, which binds to F-actin. The sample size for D1 (green circles), D1 + CN03 (red cricles), D1 ΔD (cyan squares) and D1 ΔD + CN03 (purple squares) are 31, 44, 51 and 105, respectively. The analysis uses the ratio of intensities of membrane and cytosol, which decreases the bias caused by alternative actin filaments staining . C Confocal microscope images of AD293 cells transfected with CFP-labeled D1 and D1 ΔD . RhoA activator CN03 was at 1 mg/mL. Cells were stained with Alexa Fluor568 Phalloidin to image filamentous actin and Hoechst 33258 to image the nuclei. Scale bar is 10 µm. The significant cytosolic fraction of overexpressed DIAPH1 after CN03 activation of RhoA or of constitutively active DIAPH1 ΔDAD suggests that there are limited membrane binding sites for DIAPH1 as observed previously . The height of the bar represents the mean and the error bars are ± SEM and statistical p -values at 95% confidence are shown on the graph.

    Techniques Used: Membrane, Fluorescence, Staining, Microscopy, Transfection, Labeling, Activation Assay, Binding Assay

    A Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, in the presence of 3 µM ctRAGE, 3 C, 3 µM RhoA, 3 R, or 1, 2, or 3 µM of both ctRAGE and RhoA, 1CR, 2CR, 3 CR, respectively, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 13 data points to a linear equation yielding slopes of 11.1 ± 0.4 AFU*s -1 for A (red), 11.6 ± 0.3 AFU*s −1 for D1 (beige), 13.1 ± 1.0 AFU*s −1 for D1 + 3C (light blue), 14.6 ± 0.8 AFU*s -1 for D1 + 3 R (dark blue), 17.8 ± 0.7 AFU*s −1 for D1 + 1RC (light green), 20.6 ± 0.8 AFU*s -1 for D1 + 2RC (green), and 22.0 ± 0.9 AFU*s −1 for D1 + 3RC (dark green). Statistical p-values at 95% confidence are shown on the graph. B ELISA-based isotherm of RhoA binding to DIAPH1. The data were fit to “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction. C ELISA-based binding isotherm of RhoA binding to DIAPH1 in the presence of ctRAGE. The data were fit to a “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction in the presence of ctRAGE. All values are represented as the mean ± SD and derived from N = 3 independent experiments. Values from individual experiments are shown as gray dots.
    Figure Legend Snippet: A Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, in the presence of 3 µM ctRAGE, 3 C, 3 µM RhoA, 3 R, or 1, 2, or 3 µM of both ctRAGE and RhoA, 1CR, 2CR, 3 CR, respectively, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 13 data points to a linear equation yielding slopes of 11.1 ± 0.4 AFU*s -1 for A (red), 11.6 ± 0.3 AFU*s −1 for D1 (beige), 13.1 ± 1.0 AFU*s −1 for D1 + 3C (light blue), 14.6 ± 0.8 AFU*s -1 for D1 + 3 R (dark blue), 17.8 ± 0.7 AFU*s −1 for D1 + 1RC (light green), 20.6 ± 0.8 AFU*s -1 for D1 + 2RC (green), and 22.0 ± 0.9 AFU*s −1 for D1 + 3RC (dark green). Statistical p-values at 95% confidence are shown on the graph. B ELISA-based isotherm of RhoA binding to DIAPH1. The data were fit to “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction. C ELISA-based binding isotherm of RhoA binding to DIAPH1 in the presence of ctRAGE. The data were fit to a “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction in the presence of ctRAGE. All values are represented as the mean ± SD and derived from N = 3 independent experiments. Values from individual experiments are shown as gray dots.

    Techniques Used: Construct, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay

    A Mechanism of DIAPH1-RhoA localization and activation (i-viii). Color scheme of the DIAPH1 domains is as in Fig. . RhoA-GDP is pink and RhoA-GTP is purple. To localize at the membrane RhoA is farnasylated and unphosphorylated. Other cytoskeleton remodeling proteins, such as profilin, cofilin1/ADF, or cross-linkers that contribute to the described mechanism are omitted for clarity; their roles in this model are a subject for future investigations. B Three-dimensional plot showing K d D/R as a function of α and [RhoGTPase] for DRFs. α and [RhoGTPase] are on a logarithmic scale. The line represents α = 1 and has zero curvature in the [RhoGTPase] dimension. The plot was constructed using K d D and K d R derived from the RhoA-DIAPH1 interaction. Derivations are in Supplementary Appendix . C Three-dimensional plot of unbound DAD, 1-F DB , as a function of RhoGTPase concentration and [DAD] eff . [DAD] eff and [RhoGTPase] are on a logarithmic scale. The gradient from deep blue to yellow shows increasing DIAPH1 activation. Derivations are in Supplementary Appendix . The plot was constructed using K d R , K d D and α derived from the RhoA-DIAPH1 interaction.
    Figure Legend Snippet: A Mechanism of DIAPH1-RhoA localization and activation (i-viii). Color scheme of the DIAPH1 domains is as in Fig. . RhoA-GDP is pink and RhoA-GTP is purple. To localize at the membrane RhoA is farnasylated and unphosphorylated. Other cytoskeleton remodeling proteins, such as profilin, cofilin1/ADF, or cross-linkers that contribute to the described mechanism are omitted for clarity; their roles in this model are a subject for future investigations. B Three-dimensional plot showing K d D/R as a function of α and [RhoGTPase] for DRFs. α and [RhoGTPase] are on a logarithmic scale. The line represents α = 1 and has zero curvature in the [RhoGTPase] dimension. The plot was constructed using K d D and K d R derived from the RhoA-DIAPH1 interaction. Derivations are in Supplementary Appendix . C Three-dimensional plot of unbound DAD, 1-F DB , as a function of RhoGTPase concentration and [DAD] eff . [DAD] eff and [RhoGTPase] are on a logarithmic scale. The gradient from deep blue to yellow shows increasing DIAPH1 activation. Derivations are in Supplementary Appendix . The plot was constructed using K d R , K d D and α derived from the RhoA-DIAPH1 interaction.

    Techniques Used: Activation Assay, Membrane, Construct, Derivative Assay, Concentration Assay

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    Article Snippet: .. GST-Rac1 (Addgene, #12200) and GST-RhoA (Addgene, #12202) were purified from the BL21 (DE3) E. coli strain, and GST-PAK1 CRIB and GST-rhotekin RBD domains were purchased from Cytoskeleton, Inc. AntiFLAG, anti-Myc, anti-p-ERK, anti-p-JNK, anti-ERK (Cell Signaling Technology, Danvers, MA, USA), anti-Rac1, anti-Cdc42 (BD Transduction Laboratories), anti-RhoA (Santa Cruz Biotechnology, Dallas, TX, USA), and antiGFP (Invitrogen) were purchased from the indicated commercial sources. ..

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    A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to <t>RhoA</t> and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.
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    Addgene inc gst-rhotekin construct active rhoa interacting binding domain fused to gst
    A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to <t>RhoA</t> and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.
    Gst Rhotekin Construct Active Rhoa Interacting Binding Domain Fused To Gst, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rhoa gtp pulldown assay pak gst  (Cytoskeleton Inc)
    95
    Cytoskeleton Inc rhoa gtp pulldown assay pak gst
    A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to <t>RhoA</t> and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.
    Rhoa Gtp Pulldown Assay Pak Gst, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/gst+rhoa/Rac1+Pulldown+Activation+Assay+Kit/10__7554_slash_elife__88828-393-2-9
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    glutathione s transferase gst rhotekin  (Cytoskeleton Inc)
    96
    Cytoskeleton Inc glutathione s transferase gst rhotekin
    A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to <t>RhoA</t> and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.
    Glutathione S Transferase Gst Rhotekin, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    protein probes for active rhoa (gst-rhotekin-rho-binding domain)  (Burlington Industries)
    90
    Burlington Industries protein probes for active rhoa (gst-rhotekin-rho-binding domain)
    RhoA, but not <t>Rac1,</t> is a downstream effector of GEF-H1 in antigen-stimulated RBL-2H3 cells. Detection of activated Rac1 ( A ) and RhoA ( B ) was performed by pulldown assays using Rhotekin or <t>PAK1</t> probes that bind RhoA-GTP and Rac1-GTP, respectively. Active levels of both RhoA and Rac1 increased in control cells after 20 min of antigen stimulation (control, stim). Depletion of GEF-H1 did not affect Rac1-GTP levels, but resulted in reduced RhoA-GTP levels (GEF-H1 KD, stim). ( C ) Depletion of GEF-H1 affected the formation of stress fibers. Alexa Fluor 405-phalloidin was used to label F-actin structures in unstimulated cells (left panels) and after 20 min of antigen stimulation (middle panels). Control cells showed the formation of stress fibers that project to the plasma membrane (panels b and c, red arrow). GEF-H1-depleted cells lacked stress fiber formation and showed reduced cell spreading (panels e and f). Scale bar = 20 μm.
    Protein Probes For Active Rhoa (Gst Rhotekin Rho Binding Domain), supplied by Burlington Industries, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    protein probes for active gef-h1 (gst-rhoa-g17a)  (Millipore)
    90
    Millipore protein probes for active gef-h1 (gst-rhoa-g17a)
    RhoA, but not <t>Rac1,</t> is a downstream effector of GEF-H1 in antigen-stimulated RBL-2H3 cells. Detection of activated Rac1 ( A ) and RhoA ( B ) was performed by pulldown assays using Rhotekin or <t>PAK1</t> probes that bind RhoA-GTP and Rac1-GTP, respectively. Active levels of both RhoA and Rac1 increased in control cells after 20 min of antigen stimulation (control, stim). Depletion of GEF-H1 did not affect Rac1-GTP levels, but resulted in reduced RhoA-GTP levels (GEF-H1 KD, stim). ( C ) Depletion of GEF-H1 affected the formation of stress fibers. Alexa Fluor 405-phalloidin was used to label F-actin structures in unstimulated cells (left panels) and after 20 min of antigen stimulation (middle panels). Control cells showed the formation of stress fibers that project to the plasma membrane (panels b and c, red arrow). GEF-H1-depleted cells lacked stress fiber formation and showed reduced cell spreading (panels e and f). Scale bar = 20 μm.
    Protein Probes For Active Gef H1 (Gst Rhoa G17a), supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to RhoA and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.

    Journal: Communications Biology

    Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

    doi: 10.1038/s42003-025-08222-5

    Figure Lengend Snippet: A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to RhoA and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.

    Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

    Techniques: Binding Assay, Construct, Sequencing

    A Superimposed 1 H- 15 N HSQC spectra of 50 µM [ U - 15 N]-DAD in the absence (red) and presence (black) of 60 µM GDID (black). The DAD Q1207 cross peak, which is part of the DID-DAD binding interface, undergoes a large change in chemical shift between the bound and free states. B Superimposed 1 H- 15 N HSQC spectra of 5 µM [ U - 15 N]-DAD with 60 µM of RhoA and 50 µM GDID (black) and 5 µM [ U - 15 N]-DAD with 19 µM of RhoA and 13 µM GDID (red). Free DAD is evident at the lower concentration of the RhoA-GDID complex. C NMR-generated isothermal titration of 5 µM [ U - 15 N]-DAD binding to the RhoA-GDID complex and fit using a ‘total binding, accounting for ligand depletion’ model to estimate K d D/R of 27 ± 5 μM for DAD binding to the RhoA-GDID complex. D ELISA-generated isotherm of RhoA binding to 10 nM GDID fit to a “one site-total and nonspecific binding” model to estimate K d R of 8.9 ± 2.4 nM. E ELISA-generated isotherm of RhoA binding to 10 nM GDID-DAD fit to a ‘one site-total and nonspecific binding’ model to estimate K d R/D of 840 ± 200 nM. All values for ELISA experiments are represented as mean ± SD and were performed as N = 3 independent experiments. Values from individual experiments are shown as gray dots. Note that the decrease in the plateau values observed in the ELISA titrations are due to the Hook or Prozone effect in which high ligand concentrations result in a decrease in signal strength .

    Journal: Communications Biology

    Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

    doi: 10.1038/s42003-025-08222-5

    Figure Lengend Snippet: A Superimposed 1 H- 15 N HSQC spectra of 50 µM [ U - 15 N]-DAD in the absence (red) and presence (black) of 60 µM GDID (black). The DAD Q1207 cross peak, which is part of the DID-DAD binding interface, undergoes a large change in chemical shift between the bound and free states. B Superimposed 1 H- 15 N HSQC spectra of 5 µM [ U - 15 N]-DAD with 60 µM of RhoA and 50 µM GDID (black) and 5 µM [ U - 15 N]-DAD with 19 µM of RhoA and 13 µM GDID (red). Free DAD is evident at the lower concentration of the RhoA-GDID complex. C NMR-generated isothermal titration of 5 µM [ U - 15 N]-DAD binding to the RhoA-GDID complex and fit using a ‘total binding, accounting for ligand depletion’ model to estimate K d D/R of 27 ± 5 μM for DAD binding to the RhoA-GDID complex. D ELISA-generated isotherm of RhoA binding to 10 nM GDID fit to a “one site-total and nonspecific binding” model to estimate K d R of 8.9 ± 2.4 nM. E ELISA-generated isotherm of RhoA binding to 10 nM GDID-DAD fit to a ‘one site-total and nonspecific binding’ model to estimate K d R/D of 840 ± 200 nM. All values for ELISA experiments are represented as mean ± SD and were performed as N = 3 independent experiments. Values from individual experiments are shown as gray dots. Note that the decrease in the plateau values observed in the ELISA titrations are due to the Hook or Prozone effect in which high ligand concentrations result in a decrease in signal strength .

    Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

    Techniques: Binding Assay, Concentration Assay, Generated, Titration, Enzyme-linked Immunosorbent Assay

    A Plot of the radius of gyration, R G , versus the number of amino acids in a theoretical polypeptide. The parameter ν characterizes the dimensions of the amino acid chain where 0.60, red, represents an expanded coil and 0.30, maroon, represents the most compact globule state. B Monte-Carlo simulation using ν = 0.60 showing the theoretical persistence length between DID and DAD domains as a function of the number of amino acids: ~109 Å for the 285 amino acid DIAPH1 and ~90 Å for the 140 amino acids of DIAPH1/2. C Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, DIAPH1/2, D1/2 and DIAPH1 ΔDAD , D1 ΔD , in the presence of RhoA, +R, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 11 data points to a linear equation yielding slopes of 62 ± 6 AFU*s −1 for A (red), 66 ± 6 AFU*s −1 for D1 (green), 62 ± 7 AFU*s −1 for D1/2 (purple), 85 ± 5 AFU*s −1 for D1 + R (white), 75 ± 4 AFU*s -1 for D1/2 + R (gray) and 104 ± 7 AFU*s −1 for D1 ΔD (orange). D1 ΔD represents 100% activation and D1 or D1/2 represent 0% activation, respectively. Statistical p -values at 95% confidence are shown on the graph and all values are represented as mean ± SD derived from N = 3 independent experiments.

    Journal: Communications Biology

    Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

    doi: 10.1038/s42003-025-08222-5

    Figure Lengend Snippet: A Plot of the radius of gyration, R G , versus the number of amino acids in a theoretical polypeptide. The parameter ν characterizes the dimensions of the amino acid chain where 0.60, red, represents an expanded coil and 0.30, maroon, represents the most compact globule state. B Monte-Carlo simulation using ν = 0.60 showing the theoretical persistence length between DID and DAD domains as a function of the number of amino acids: ~109 Å for the 285 amino acid DIAPH1 and ~90 Å for the 140 amino acids of DIAPH1/2. C Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, DIAPH1/2, D1/2 and DIAPH1 ΔDAD , D1 ΔD , in the presence of RhoA, +R, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 11 data points to a linear equation yielding slopes of 62 ± 6 AFU*s −1 for A (red), 66 ± 6 AFU*s −1 for D1 (green), 62 ± 7 AFU*s −1 for D1/2 (purple), 85 ± 5 AFU*s −1 for D1 + R (white), 75 ± 4 AFU*s -1 for D1/2 + R (gray) and 104 ± 7 AFU*s −1 for D1 ΔD (orange). D1 ΔD represents 100% activation and D1 or D1/2 represent 0% activation, respectively. Statistical p -values at 95% confidence are shown on the graph and all values are represented as mean ± SD derived from N = 3 independent experiments.

    Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

    Techniques: Construct, Activation Assay, Derivative Assay

    A Membrane to cytosol ratio of CFP-DIAPH1 fluorescence. The sample sizes for DIAPH1 (D1, green circles), DIAPH1 + CN03 (D1 + CN03, red circles), DIAPH1 ΔDAD (D1 ΔD , cyan squares) and DIAPH1 ΔDAD + CN03 (D1 ΔD + CN03, purple squares) are 31, 44, 51 and 105, respectively. B Membrane to cytosol ratio of phalloidin fluorescence, which binds to F-actin. The sample size for D1 (green circles), D1 + CN03 (red cricles), D1 ΔD (cyan squares) and D1 ΔD + CN03 (purple squares) are 31, 44, 51 and 105, respectively. The analysis uses the ratio of intensities of membrane and cytosol, which decreases the bias caused by alternative actin filaments staining . C Confocal microscope images of AD293 cells transfected with CFP-labeled D1 and D1 ΔD . RhoA activator CN03 was at 1 mg/mL. Cells were stained with Alexa Fluor568 Phalloidin to image filamentous actin and Hoechst 33258 to image the nuclei. Scale bar is 10 µm. The significant cytosolic fraction of overexpressed DIAPH1 after CN03 activation of RhoA or of constitutively active DIAPH1 ΔDAD suggests that there are limited membrane binding sites for DIAPH1 as observed previously . The height of the bar represents the mean and the error bars are ± SEM and statistical p -values at 95% confidence are shown on the graph.

    Journal: Communications Biology

    Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

    doi: 10.1038/s42003-025-08222-5

    Figure Lengend Snippet: A Membrane to cytosol ratio of CFP-DIAPH1 fluorescence. The sample sizes for DIAPH1 (D1, green circles), DIAPH1 + CN03 (D1 + CN03, red circles), DIAPH1 ΔDAD (D1 ΔD , cyan squares) and DIAPH1 ΔDAD + CN03 (D1 ΔD + CN03, purple squares) are 31, 44, 51 and 105, respectively. B Membrane to cytosol ratio of phalloidin fluorescence, which binds to F-actin. The sample size for D1 (green circles), D1 + CN03 (red cricles), D1 ΔD (cyan squares) and D1 ΔD + CN03 (purple squares) are 31, 44, 51 and 105, respectively. The analysis uses the ratio of intensities of membrane and cytosol, which decreases the bias caused by alternative actin filaments staining . C Confocal microscope images of AD293 cells transfected with CFP-labeled D1 and D1 ΔD . RhoA activator CN03 was at 1 mg/mL. Cells were stained with Alexa Fluor568 Phalloidin to image filamentous actin and Hoechst 33258 to image the nuclei. Scale bar is 10 µm. The significant cytosolic fraction of overexpressed DIAPH1 after CN03 activation of RhoA or of constitutively active DIAPH1 ΔDAD suggests that there are limited membrane binding sites for DIAPH1 as observed previously . The height of the bar represents the mean and the error bars are ± SEM and statistical p -values at 95% confidence are shown on the graph.

    Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

    Techniques: Membrane, Fluorescence, Staining, Microscopy, Transfection, Labeling, Activation Assay, Binding Assay

    A Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, in the presence of 3 µM ctRAGE, 3 C, 3 µM RhoA, 3 R, or 1, 2, or 3 µM of both ctRAGE and RhoA, 1CR, 2CR, 3 CR, respectively, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 13 data points to a linear equation yielding slopes of 11.1 ± 0.4 AFU*s -1 for A (red), 11.6 ± 0.3 AFU*s −1 for D1 (beige), 13.1 ± 1.0 AFU*s −1 for D1 + 3C (light blue), 14.6 ± 0.8 AFU*s -1 for D1 + 3 R (dark blue), 17.8 ± 0.7 AFU*s −1 for D1 + 1RC (light green), 20.6 ± 0.8 AFU*s -1 for D1 + 2RC (green), and 22.0 ± 0.9 AFU*s −1 for D1 + 3RC (dark green). Statistical p-values at 95% confidence are shown on the graph. B ELISA-based isotherm of RhoA binding to DIAPH1. The data were fit to “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction. C ELISA-based binding isotherm of RhoA binding to DIAPH1 in the presence of ctRAGE. The data were fit to a “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction in the presence of ctRAGE. All values are represented as the mean ± SD and derived from N = 3 independent experiments. Values from individual experiments are shown as gray dots.

    Journal: Communications Biology

    Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

    doi: 10.1038/s42003-025-08222-5

    Figure Lengend Snippet: A Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, in the presence of 3 µM ctRAGE, 3 C, 3 µM RhoA, 3 R, or 1, 2, or 3 µM of both ctRAGE and RhoA, 1CR, 2CR, 3 CR, respectively, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 13 data points to a linear equation yielding slopes of 11.1 ± 0.4 AFU*s -1 for A (red), 11.6 ± 0.3 AFU*s −1 for D1 (beige), 13.1 ± 1.0 AFU*s −1 for D1 + 3C (light blue), 14.6 ± 0.8 AFU*s -1 for D1 + 3 R (dark blue), 17.8 ± 0.7 AFU*s −1 for D1 + 1RC (light green), 20.6 ± 0.8 AFU*s -1 for D1 + 2RC (green), and 22.0 ± 0.9 AFU*s −1 for D1 + 3RC (dark green). Statistical p-values at 95% confidence are shown on the graph. B ELISA-based isotherm of RhoA binding to DIAPH1. The data were fit to “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction. C ELISA-based binding isotherm of RhoA binding to DIAPH1 in the presence of ctRAGE. The data were fit to a “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction in the presence of ctRAGE. All values are represented as the mean ± SD and derived from N = 3 independent experiments. Values from individual experiments are shown as gray dots.

    Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

    Techniques: Construct, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay

    A Mechanism of DIAPH1-RhoA localization and activation (i-viii). Color scheme of the DIAPH1 domains is as in Fig. . RhoA-GDP is pink and RhoA-GTP is purple. To localize at the membrane RhoA is farnasylated and unphosphorylated. Other cytoskeleton remodeling proteins, such as profilin, cofilin1/ADF, or cross-linkers that contribute to the described mechanism are omitted for clarity; their roles in this model are a subject for future investigations. B Three-dimensional plot showing K d D/R as a function of α and [RhoGTPase] for DRFs. α and [RhoGTPase] are on a logarithmic scale. The line represents α = 1 and has zero curvature in the [RhoGTPase] dimension. The plot was constructed using K d D and K d R derived from the RhoA-DIAPH1 interaction. Derivations are in Supplementary Appendix . C Three-dimensional plot of unbound DAD, 1-F DB , as a function of RhoGTPase concentration and [DAD] eff . [DAD] eff and [RhoGTPase] are on a logarithmic scale. The gradient from deep blue to yellow shows increasing DIAPH1 activation. Derivations are in Supplementary Appendix . The plot was constructed using K d R , K d D and α derived from the RhoA-DIAPH1 interaction.

    Journal: Communications Biology

    Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

    doi: 10.1038/s42003-025-08222-5

    Figure Lengend Snippet: A Mechanism of DIAPH1-RhoA localization and activation (i-viii). Color scheme of the DIAPH1 domains is as in Fig. . RhoA-GDP is pink and RhoA-GTP is purple. To localize at the membrane RhoA is farnasylated and unphosphorylated. Other cytoskeleton remodeling proteins, such as profilin, cofilin1/ADF, or cross-linkers that contribute to the described mechanism are omitted for clarity; their roles in this model are a subject for future investigations. B Three-dimensional plot showing K d D/R as a function of α and [RhoGTPase] for DRFs. α and [RhoGTPase] are on a logarithmic scale. The line represents α = 1 and has zero curvature in the [RhoGTPase] dimension. The plot was constructed using K d D and K d R derived from the RhoA-DIAPH1 interaction. Derivations are in Supplementary Appendix . C Three-dimensional plot of unbound DAD, 1-F DB , as a function of RhoGTPase concentration and [DAD] eff . [DAD] eff and [RhoGTPase] are on a logarithmic scale. The gradient from deep blue to yellow shows increasing DIAPH1 activation. Derivations are in Supplementary Appendix . The plot was constructed using K d R , K d D and α derived from the RhoA-DIAPH1 interaction.

    Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

    Techniques: Activation Assay, Membrane, Construct, Derivative Assay, Concentration Assay

    RhoA, but not Rac1, is a downstream effector of GEF-H1 in antigen-stimulated RBL-2H3 cells. Detection of activated Rac1 ( A ) and RhoA ( B ) was performed by pulldown assays using Rhotekin or PAK1 probes that bind RhoA-GTP and Rac1-GTP, respectively. Active levels of both RhoA and Rac1 increased in control cells after 20 min of antigen stimulation (control, stim). Depletion of GEF-H1 did not affect Rac1-GTP levels, but resulted in reduced RhoA-GTP levels (GEF-H1 KD, stim). ( C ) Depletion of GEF-H1 affected the formation of stress fibers. Alexa Fluor 405-phalloidin was used to label F-actin structures in unstimulated cells (left panels) and after 20 min of antigen stimulation (middle panels). Control cells showed the formation of stress fibers that project to the plasma membrane (panels b and c, red arrow). GEF-H1-depleted cells lacked stress fiber formation and showed reduced cell spreading (panels e and f). Scale bar = 20 μm.

    Journal: Cells

    Article Title: GEF-H1 Transduces FcεRI Signaling in Mast Cells to Activate RhoA and Focal Adhesion Formation during Exocytosis

    doi: 10.3390/cells12040537

    Figure Lengend Snippet: RhoA, but not Rac1, is a downstream effector of GEF-H1 in antigen-stimulated RBL-2H3 cells. Detection of activated Rac1 ( A ) and RhoA ( B ) was performed by pulldown assays using Rhotekin or PAK1 probes that bind RhoA-GTP and Rac1-GTP, respectively. Active levels of both RhoA and Rac1 increased in control cells after 20 min of antigen stimulation (control, stim). Depletion of GEF-H1 did not affect Rac1-GTP levels, but resulted in reduced RhoA-GTP levels (GEF-H1 KD, stim). ( C ) Depletion of GEF-H1 affected the formation of stress fibers. Alexa Fluor 405-phalloidin was used to label F-actin structures in unstimulated cells (left panels) and after 20 min of antigen stimulation (middle panels). Control cells showed the formation of stress fibers that project to the plasma membrane (panels b and c, red arrow). GEF-H1-depleted cells lacked stress fiber formation and showed reduced cell spreading (panels e and f). Scale bar = 20 μm.

    Article Snippet: The glutathione S-transferase (GST)-tagged protein probes for active GEF-H1 (GST-RhoA-G17A), Rac1 (GST-PAK1-Cdc42/Rac-binding domain), and RhoA (GST-Rhotekin-Rho-binding domain) were prepared by expressing proteins in E. coli strain RosettaTM (DE3) (MilliporeSigma, Burlington, MA, USA) [ , , ].

    Techniques: Control, Clinical Proteomics, Membrane