Journal: Science signaling

Article Title: A spatiotemporal Notch interaction map from plasma membrane to nucleus

doi: 10.1126/scisignal.adg6474

Figure Lengend Snippet: (A) Hierarchical clustering of the proteins characterized by a peak of relative abundance between 2 and 5 min after GSI removal, focusing on neighbors closest to Ezrin (EZR). (B and C) Volcano plots comparing NOTCH2 proximity-labeled proteins enriched at the 5 (B) and 15 (C) min timepoints after GSI removal when compared to GSI (t=0). Proteins related to actin, myosin, or cytoskeletal transport are labeled in blue, and proteins related to endocytosis or vesicular-mediated transport are labeled in orange. P-values are Benjamini-Hochberg corrected (p-value ≤ 0.05, FC ≥ 1.5). (D) Heatmap showing the enrichment pattern of proteins related to nuclear import identified by NOTCH2 proximity labeling. Data in all panels are from simultaneous MS analysis of 14 samples from one labeling timecourse (n=1), with two technical replicates analyzed for each timepoint.

Article Snippet: Silencer © Select siRNAs for Ezrin (s14795), Radixin (s11899), Moesin (s8984), and the siCtrl AM4611 were all purchased from ThermoFisher Scientific.

Techniques: Labeling

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: CLIC5A interacts directly with ezrin, radixin, and moesin. A . yeast two-hybrid (Y2H) assay. Plates contain yeast colonies expressing untagged CLIC5A from the “bait” vector and ezrin 432-586 , radixin 432-583 , or moesin 432-577 from the “prey” vector. Colony growth on double-deficient medium (DDO, lacking leucine and tryptophan) indicates both vectors are present. Blue colonies indicate induced α-galactosidase activity in the presence of X-α-gal (DDO/X) due to direct interactions between bait and prey proteins. Growth on plates containing the antibiotic aureobasidin A (DDO/X/A) indicate a direct interaction between bait and prey proteins resulting in Aureobasidin A resistance. The inset for DDO/X/A plates containing CLIC5A/radixin 432-583 or CLIC5A/moesin 432-577 represents a 2× digital magnification (representative of 3 biologically separate experiments). B , WB with anti(α)-ezrin, α-radixin and α-moesin antibodies of total cell lysates (input) and GST or GST-CLIC5A pulldowns (PD) from untransfected COS-7 cells. Each of the three lanes for input, GST pulldown, and GST-CLIC5A pulldown are from three biologically distinct experiments. C , semiquantitative Y2H mapping of interactions between distinct ERM and CLIC5A constructs. Input and GST-CLIC5A pulldown (PD) is shown (representative of three independent experiments). D , microscale thermophoresis (MST) left panel: total protein stain of recombinant, purified GST-His6x, GST-His6x-CLIC5A, and untagged ezrin 432-586 proteins. Right panel: normalized thermophoresis-induced change in fluorescence for fluorescently labeled GST-His6x or GST-His6x-CLIC5A in the presence of increasing concentrations of purified ezrin 482-586 (H549N/T567E). The calculated affinity (Kd) between GST-His6x-CLIC5A and ezrin 432-586 (H549N/T567E) was 29 ± 12 μM (mean ± SD, n = 3 biological replicates). E , WB of HA-ezrin 297-586 , HA-ezrin 432-586 , and HA-ezrin 432-570 produced by in vitro transcription/translation before (input) and after pulldown (PD) by immobilized GST-CLIC5A. F , α-GFP WB of transiently expressed full-length GFP-ezrin 1-586 , GFP-ezrin 432-586 , and GFP-ezrin 432-570 in COS-7 cells and pulled from the cell lysates by immobilized GST or GST-CLIC5A (PD) (representative of three biologically independent experiments). G , coimmunoprecipitation of HA-ezrin 1-586 , HA-ezrin 432-586 , and HA-ezrin 432-570 coexpressed with GFP-CLIC5A in COS-7 cells (representative of three biological replicates). CLIC, chloride intracellular channel; DDO, double dropout; ERM, ezrin, radixin, and moesin; GST, glutathione S-transferase; WB, Western blot.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Y2H Assay, Expressing, Plasmid Preparation, Activity Assay, Construct, Microscale Thermophoresis, Staining, Recombinant, Purification, Fluorescence, Labeling, Produced, In Vitro, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: ERM knockdown partially displaces CLIC5A from its peripheral location. A , localization of transiently expressed GFP-CLIC5A in living HeLa cells transfected with nonspecific siRNA (Scr siRNA) or triple ezrin, moesin, and radixin siRNA (ERM siRNA) (representative of three biologically distinct experiments). B , quantification of the peripheral: cytoplasmic GFP-CLIC5A ratio in living HeLa cells transfected with GFP-CLIC5A and with Scr siRNA or triple ERM siRNA from a single experiment (Student’s t test). C , mean peripheral: cytoplasmic GFP-CLIC5A in living HeLa cells transfected with GFP-CLIC5A and Scr siRNA or triple ERM siRNA (mean ± SD, n = 3 biologically independent experiments, Student’s t test). D : WB with anti-ezrin, anti-radixin, and anti-moesin antibodies showing individual and triple ezrin, radixin, and moesin siRNA-mediated knockdown of endogenous ezrin, radixin and moesin in HeLa cells. E : Quantification of ERM knockdown from three independent experiments. (One-way ANOVA for ezrin abundance: F = 21.17; p < 0.001, for moesin abundance: F = 11.50; p < 0.0009, for radixin abundance: F = 17.68; p < 0.0002, and for CLIC5A abundance: F = 1.564; p = 0.265. p values for post hoc Dunnett’s multiple comparisons are shown). CLIC, chloride intracellular channel.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Knockdown, Transfection

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: Phosphorylation enhances the CLIC5A/ezrin interaction. A , left panel: WB for endogenous ezrin and endogenous phosphorylated ERM proteins (pERM) in COS-7 cell lysates and GST-CLIC5A pulldowns (PD) from cells treated with vehicle or calyculin A (50 nM) for 30 min prior to lysis. Right panel: densitometric quantification (mean ± SD, n = 3 independent experiments, Student’s t test). B , right panel: α-GFP WB of total cell lysate (input) and GST or GST-CLIC5A pulldown (PD) from lysates of COS-7 transiently expressing GFP-ezrin 432-586 (WT), phosphorylation deficient GFP-ezrin 432-586 (T567A; “A”) or phosphomimetic GFP-ezrin 432-586 (T567D; “D”). Right panel: densitometric quantification of GST-CLIC5A pulldown of transiently expressed GFP-ezrin 432-586 (WT, A or D mutants; one-way ANOVA F = 5.42; p = 0.04, p values for post hoc Dunnett’s multiple comparisons are shown). C , confocal fluorescence microscopy of living HeLa cells transfected with RFP-CLC5A ( magenta ) and/or full-length GFP-ezrin 1-586 ( green ) without (WT) or with a phosphomimetic T567D mutation (the scale bar represents 12 μm). Cells were either transfected separately ( top row ) with RFP-CLIC5A or GFP-ezrin and then mixed 1:1 (control conditions) or they were cotransfected ( bottom row ) with RFP-CLIC5A and GFP-ezrin. The white arrows represent line plots analyzed for pixel intensity (representative of three biologically distinct experiments). D , line plot pixel intensity for RFP ( magenta ) and GFP ( green ) for lines in ( C ). E , quantification of the transiently expressed GFP-ezrin 1-586 (WT or T567D mutant) peripheral: cytoplasmic pixel intensity in the presence and absence of transiently expressed RFP-CLIC5A. Top panel: each data point represents a single cell from a single experiment (two-way ANOVA: interaction F = 19.3, p < 0.0001; CLIC5A effect F = 23.6, p < 0.0001; ezrin mutant versus WT effect F = 156.9; p < 0.0001, p values for pos t hoc Tukey’s multiple comparisons are shown). Bottom panel: each data point represents the mean for one of three biologically independent experiments. (Two-way ANOVA: interaction F = 28.8, p < 0.0007; CLIC5A effect F = 32.7, p < 0.0004; ezrin mutant versus WT effect F = 97.3; p < 0.0001, p values for post hoc Tukey’s multiple comparisons are shown). F , quantification of the RFP-CLIC5A peripheral: cytoplasmic pixel intensity in the presence or absence of transiently expressed GFP-ezrin1-586 (WT or T567D mutant). Top panel: each datapoint represents a single cell from a single experiment (one-way ANOVA F = 0.297, p = 0.744). Bottom panel: each data point represents the mean for one of three biologically independent experiments (one-way ANOVA F = 0.796, p = 0.488). G , Pearson correlation coefficient for colocalization of transiently expressed RFP-CLIC5A and GFP-ezrin (WT or T567D mutant). Right panel: each data point represents a separate image (each image containing 4–10 cells) from a single experiment (two-way ANOVA: interaction F = 13.51, p < 0.0007; separate versus cotransfection F = 1175, p < 0.0001; ezrin mutant versus WT effect F = 22.54; p < 0.0001, p values for post hoc Tukey’s multiple comparisons are shown). Right panel: each datapoint represents the mean from one of three biologically distinct experiments (mean ± SD, Student’s t test). CLIC, chloride intracellular channel; ERM, ezrin, radixin, and moesin; GST, glutathione S-transferase; WB, Western blot.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Phospho-proteomics, Lysis, Expressing, Fluorescence, Microscopy, Transfection, Mutagenesis, Control, Cotransfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: Indirect association of CLIC5A and Rac1. A , WB showing endogenous Rac1 and ezrin as well as transiently expressed GFP-CLIC5A in total cell lysates (TLC) and the same proteins captured by immobilized PAK-protein binding domain (PAK-PBD) from HeLa cells transiently expressing GFP or GFP-CLIC5A. The GFP-CLIC5A expressing cells were treated, or not, with the Rac1 inhibitor NSC23766 (100 μM × 10 min) (representative of three biologically distinct experiments). B , α-GFP WB of GST- or GST-CLIC5A pulldowns from lysates of COS-7 cells transiently expressing WT, constitutively active (L61) or dominant negative (N17) GFP-Rac1 constructs (representative of three independent experiments). C , live-cell imaging of HeLa cells transiently expressing RFP-CLIC5A (magenta) and WT GFP-Rac1 ( green ). The scale bar represents 10 μm. The white lines represent line plots analyzed in ( D ). Colocalization ( white ) in the merged image is observed at the dorsal cell periphery and in the perinuclear location (representative of three biologically distinct experiments). D , pixel intensity of line plots in ( C ) for RFP-CLIC5A ( magenta ) and GFP-Rac1 ( green ). E , Pearson correlation coefficients of all images from three biologically distinct experiments. 1, 2, and 3 = cotransfection of GFP-Rac1 with RFP-CLIC5A; 2’ and 3’ = separate transfection of GFP-Rac1 or RFP-CLIC5A followed by 1:1 mixing of cells. F , in vitro loading of purified, recombinant His-Rac1 with GTPγS in vitro . His-Rac1 (500 ng) was incubated with GDP or GTPγS followed by affinity capture of Rac1-GTPγS by immobilized PAK-PBD. Top panel: protein blot. Bottom panel: α-Rac1 WB. G , α-Rac1 WB of purified His-Rac1 and GST- or GST-CLIC5A pulldowns from cell-free solutions containing WT Rac1, Rac1-GTPγS, or Rac1-GDP (representative of three independent experiments). CLIC, chloride intracellular channel; GST, glutathione S-transferase; WB, Western blot.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Protein Binding, Expressing, Dominant Negative Mutation, Construct, Live Cell Imaging, Cotransfection, Transfection, In Vitro, Purification, Recombinant, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: ERM and Rac-1 activation are amplified by the CLIC5A/ezrin interaction. A , endogenous pERM abundance in lysates and detergent resistant pellets of COS-7 cells transfected with GFP-CLIC5A cDNA and increasing concentrations of GFP-ezrin 432-586 (T567D) cDNA . GFP-CLIC5A cDNA was kept constant and the GFP-CLIC5A: GFP-ezrin 432-586 (T567D) cDNA transfection ratio was 1:1, 1:2, 1:4, and 1:8. Left panel , representative WB. Right panel, densitometric quantification of the endogenous pERM: endogenous ezrin ratio (mean ± SD, n = 3 independent experiments, one-way ANOVA: F = 7.90; p = 0.0007. p values shown represent post hoc Dunnett’s multiple comparisons). B , pERM abundance in lysates and detergent resistant pellets of COS-7 cells transiently transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of GFP- e zrin 432-570 cDNA. Left panel , representative WB. Right panel, densitometric quantification of the endogenous pERM: endogenous ezrin ratio (mean ± S.D., n = 3 biologically independent experiments, two-way ANOVA: interaction F = 12.25; p = 0.01; CLIC5A effect: F = 39.63, p = 0.0002; ezrin 432-570 effect F = 22.58. p = 0.0014; p values shown represent post hoc Tukey’s multiple comparisons). C , endogenous Rac1 WB for COS-7 cell lysates and endogenous Rac1-GTP captured by PAK-PBD pulldown (PD) from COS-7 cells transiently transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of GFP-ezrin 432-586 (T567D) cDNA. Left panel, representative WB. Right panel: densitometric quantification of endogenous Rac1-GTP/total endogenous Rac1 (mean ± SD, n = 3 independent experiments, two-way ANOVA: interaction F = 6.32, p = 0.036; CLIC5A effect F = 29.25, p = 0.001; ezrin 432–586 effect F = 5.88; p = 0.042, p values for post hoc Tukey’s multiple comparisons are shown). D , PAK-PBD pulldown (PD) of endogenous Rac1-GTP from lysates of COS-7 cells transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of transiently expressed GFP-ezrin 432 - 570 cDNA. Left panel, representative WB. Right panel, densitometric quantification of endogenous Rac1-GTP/total endogenous Rac1 (mean ± S.D., n = 3 independent experiments, two-way ANOVA: interaction F = 0.029, p = 0.87; CLIC5A effect F = 62.17 p < 0.0001; ezrin 432–570 effect F = 0.012; p = 0.91, p values for post hoc Tukey’s multiple comparisons are shown). E , endogenous Rac1-GTP abundance in human glomerular endothelial cells (hGENs) determined by Rac1-GTP G-LISA. The cells were transduced with control adenoviral-vector (ad-Vector) or untagged CLIC5A cDNA in the same vector (ad- CLIC5A ) at an increasing multiplicity of infection (MOI) (mean ± SD, n = 3 independent experiments). F , representative WB of lysates from hGEN cells transduced with ad-Vector or ad- CLIC5A with or without ezrin-specific siRNA. G , change relative to baseline of Rac1-GTP in hGEN cells transduced with 30 MOI ad-Vector or ad- CLIC5A with or without ezrin-specific siRNA (mean ± SD, n = 4 independent experiments, two-way ANOVA: interaction F = 6.35, p = 0.027; CLIC5A effect F = 34.18, p < 0.0001; ezrin siRNA effect F = 6.86; p = 0.023, p values shown represent post hoc Tukey’s multiple comparisons). H , coimmunoprecipitation of endogenous Rho-GDI with endogenous ezrin in the presence and absence of transiently expressed GFP-CLIC5A. Left panel: representative α-Rho GDI, α-ezrin, and α-CLIC5A WB of lysates (input) and α-ezrin immunoprecipitates (IP). Right panel: Quantification of endogenous Rho-GDI and endogenous ezrin immunoprecipitated with α-ezrin antibodies (n = 3 biologically distinct experiments, mean ± SD, Student’s t test). CLIC, chloride intracellular channel; PBD, protein binding domain; pERM, phosphorylated ezrin, radixin, and moesin proteins; Rho-GDI, Rho guanine nucleotide dissociation inhibitor; WB, Western blot.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Activation Assay, Amplification, Transfection, Transduction, Control, Plasmid Preparation, Infection, Immunoprecipitation, Protein Binding, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: CLIC5A-dependent feed-forward ezrin activation loop. 1 , the soluble (inactive) conformation of ezrin, in which ezrin N and C termini self-associate with high affinity encounters plasma membrane PI(4,5)P 2 . 2 , a conformational change brought about by binding of the ezrin N-terminal FERM domain to membrane PI(4,5)P 2 unmasks the ezrin C-terminal domain which binds filamentous actin producing the open/active conformation of ezrin. 3 , this open ezrin conformation is subject to phosphorylation of T567 by PKC or Rho-dependent kinase (ROCK) enhancing actin binding. 4 , the actin-associated phosphorylated ezrin C terminus directly binds CLIC5A. 5 , CLIC5A binding stabilizes the open/active conformation of ezrin. 6 , open/active ezrin sequesters inhibitory Rho-GDI (GDI), removing it from Rac-GDP and resulting in spatially restricted Rac1-GTP generation. Open/active ezrin also binds NHERF1 (EBP50) and NHERF2. EBP50 can recruit the Rac GEF ARHGEF7 also known as β-Pix ( , ), which would also enhance Rac1-GTP formation in the absence of the GDI. Rac1-GTP is known to stimulate the localized generation of PI(4,5)P 2 by PI4P5 kinases. By stabilizing the active ezrin hub, CLIC5A promotes Rac1-GTP–stimulated accumulation of PI(4,5)P 2 which then serves to further activate ezrin. Thus, the interaction of CLIC5A with the open/active conformation of ezrin results in feed-forward amplification loop of ezrin-stimulated Rac1 activity and PI(4,5)P 2 generation, in turn serving to stabilize the local cortical actin cytoskeleton. CLIC, chloride intracellular channel; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate; Rho-GDI, Rho guanine nucleotide dissociation inhibitor.

Article Snippet: The siRNAs targeting ezrin in HeLa cells (#SR305077), radixin (#SR304025), and moesin (#SR305077) were purchased from OriGene Technologies, Inc.

Techniques: Activation Assay, Clinical Proteomics, Membrane, Binding Assay, Phospho-proteomics, Amplification, Activity Assay

Journal: Journal of Cancer

Article Title: Nudifloside, a Secoiridoid Glucoside Derived from Callicarpa nudiflora, Inhibits Endothelial-to-Mesenchymal Transition and Angiogenesis in Endothelial Cells by Suppressing Ezrin Phosphorylation

doi: 10.7150/jca.91566

Figure Lengend Snippet: Effects of NDF on the Ezrin in TGF-β1-treated EA.hy 926 cells. A: the effects of NDF on expression of Ezrin and p-Ezrin; B: Effects of NDF on PODXL-Ezrin interaction by immunoprecipitation while anti-Ezrin antibody was used as sedimental protein; C: Effects of NDF on PODXL-Ezrin axis activities by immunoprecipitation while anti-PODXL antibody was used as sedimental protein. *p<0.05 and **p<0.01 compared with control; #p<0.05 and ##p<0.01 compared with TGF-β1; &p<0.05 and &&p<0.01 compared with NDF(5μM). Lane 1: control; lane 2: TGF-β1; lane 3: NDF (5μM); lane 4: NDF (20μM).

Article Snippet: When 70-80% confluency was reached, Ezrin siRNA (SignalSilence® Ezrin siRNA I, CellSignaling Technology) was added at a final concentration of 100 nM by siRNA Transfection Reagent (sc-29528; Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Control

Journal: Journal of Cancer

Article Title: Nudifloside, a Secoiridoid Glucoside Derived from Callicarpa nudiflora, Inhibits Endothelial-to-Mesenchymal Transition and Angiogenesis in Endothelial Cells by Suppressing Ezrin Phosphorylation

doi: 10.7150/jca.91566

Figure Lengend Snippet: Effects of NDF on Ezrin in VEGF-treated EA.hy 926 cells. A: Effects of NDF on expression of Ezrin and p-Ezrin; B: Effects of NDF on PODXL-Ezrin interaction by immunoprecipitation while anti-Ezrin antibody was used as sedimental protein; C: Effects of NDF on PODXL-Ezrin interaction by immunoprecipitation while anti-PODXL antibody was used as sedimental protein. *p<0.05 and **p<0.01 compared with control; #p<0.05 and ##p<0.01 compared with VEGF. Lane 1: control; lane 2: VEGF; lane 3: NDF (5μM); lane 4: NDF (20μM).

Article Snippet: When 70-80% confluency was reached, Ezrin siRNA (SignalSilence® Ezrin siRNA I, CellSignaling Technology) was added at a final concentration of 100 nM by siRNA Transfection Reagent (sc-29528; Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Control

Journal: Journal of Cancer

Article Title: Nudifloside, a Secoiridoid Glucoside Derived from Callicarpa nudiflora, Inhibits Endothelial-to-Mesenchymal Transition and Angiogenesis in Endothelial Cells by Suppressing Ezrin Phosphorylation

doi: 10.7150/jca.91566

Figure Lengend Snippet: Effects of NDF on TGF-β1-induced EndoMT and VEGF-induced angiogenesis when Ezrin was knocked-down in EA.hy 926 cells. Effects of NDF on invasion (A) and expression of EndoMT markers (B, C and D) in Ezrin-knocking-down EA.hy 926 cells at presence of TGF-β1; Effects of NDF on tube formation (E and F) , expression of angiogenic markers (G, H and I) in Ezrin-knocking-down EA.hy 926 cells at presence of VEGF. ##p<0.01 compared with CTRL; *p<0.05 and **p<0.01 compared with siRNA.

Article Snippet: When 70-80% confluency was reached, Ezrin siRNA (SignalSilence® Ezrin siRNA I, CellSignaling Technology) was added at a final concentration of 100 nM by siRNA Transfection Reagent (sc-29528; Santa Cruz Biotechnology).

Techniques: Expressing