epsin1 gfp  (Addgene inc)

Journal: PLOS One

Article Title: Epsin bioactive coating reduced in-stent intimal hyperplasia by promoting early phase reendothelialization and inhibiting smooth muscle cell proliferation

doi: 10.1371/journal.pone.0318019

Figure Lengend Snippet: (A) Immunohistochemical staining with specific anti‐CD31 Epsin1 and Epsin2 antibodies in mice carotid arteries. The relative quantification of Epsin protein expression. (B) and (C) Image J software was applied to evaluate protein expression according to the grayscale values. (D) H&E‐stained carotid arteries treated with sh(Epsin1 + Epsin2) or shNC at 7 days after injury. (E) and (F) Quantification of neointimal area and neointima/media ratio of carotid arteries treated with sh(Epsin1 + Epsin2) or shNC. t-test, n = 6 in each group. * : P < 0.05 versus the Sham group.

Article Snippet: Epsin1 antibody (purchased from Santa Cruz Biotechnology, USA).

Techniques: Immunohistochemical staining, Staining, Quantitative Proteomics, Expressing, Software

Journal: PLOS One

Article Title: Epsin bioactive coating reduced in-stent intimal hyperplasia by promoting early phase reendothelialization and inhibiting smooth muscle cell proliferation

doi: 10.1371/journal.pone.0318019

Figure Lengend Snippet: (A) and (B) Ki67 incorporation assays of EC. Representative immunofluorescence of Ki67 (green) and DAPI (blue). percentages of Ki67 incorporated EC. (C) and (D) Scratch‐wound assays showed the effect of VEGF on the migration of EC infected with siEpsin1 + siEpsin2. (E) and (F): expression levels of Epsin1, Epsin2, VEGFR2 and Erk were determined by Western blotting using the appropriate antibodies. GAPDH served as a loading control. GAPDH served as a loading control. t-test, n = 3, in each group. * : P ＜ 0.05 vs. sinormal control (NC); #: P ＜ 0.05 vs. siEpsin1 + siEpsin2; &: P ＜ 0.05 vs. siNC+VEGF.

Article Snippet: Epsin1 antibody (purchased from Santa Cruz Biotechnology, USA).

Techniques: Immunofluorescence, Migration, Infection, Expressing, Western Blot, Control

Journal: PLOS One

Article Title: Epsin bioactive coating reduced in-stent intimal hyperplasia by promoting early phase reendothelialization and inhibiting smooth muscle cell proliferation

doi: 10.1371/journal.pone.0318019

Figure Lengend Snippet: (A) MitoTracker Green staining showed the mitochondrial morphology of EC after 24 hours of treatment. (B) and (C) mitochondrial membrane potentials of EC were evaluated using JC-1 staining after treating for 24 h, J-aggregates (red) and monomers (green). (D) and (E) expression levels of Epsin1, Epsin2, Drp1 and Opa1 were determined by Western blotting using the appropriate antibodies. GAPDH served as a loading control. t-test, n = 3, in each group. * : P ＜ 0.05 vs. sinormal control (NC); #: P ＜ 0.05 vs. siEpsin1 + siEpsin2; &: P ＜ 0.05 vs. siNC+VEGF.

Article Snippet: Epsin1 antibody (purchased from Santa Cruz Biotechnology, USA).

Techniques: Staining, Membrane, Expressing, Western Blot, Control

Journal: PLOS One

Article Title: Epsin bioactive coating reduced in-stent intimal hyperplasia by promoting early phase reendothelialization and inhibiting smooth muscle cell proliferation

doi: 10.1371/journal.pone.0318019

Figure Lengend Snippet: (A) Representative Digital substraction angiography images after stents implantation in swine carotid arteries show no thrombus and stent displacement at perioperative period. AP: anteroposterior position, LP: lateral position. n = 3.(B),(C) and (D) H&E staining (×200) showed IH in the shnormal contrast (NC) stent group and the sh(Epsin1 + Epsin2) void vector group * : P ＜ 0.05 vs. shNC group; n = 3.

Article Snippet: Epsin1 antibody (purchased from Santa Cruz Biotechnology, USA).

Techniques: Staining, Plasmid Preparation

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: a , Schematic of Eps15 and Epsin1 functional domains. Eps15 consists of three EH domains at its N terminus followed by a coiled-coil domain and a long intrinsically disordered region followed by two ubiquitin interacting motifs (UIMs) at the C terminal end. Epsin1 consists of the N-terminus ENTH (Epsin1 N-Terminal Homology) domain followed by two UIMs. The central part includes the CLAP (Clathrin/AP2 binding) region and multiple DPW motifs for binding AP2 followed by the C-terminus containing 3 NPF (Asn-Pro-Phe) motifs that bind to EH domain-containing proteins like Eps15. The side cartoons depict domain organization of Eps15 in dimeric form and Epsin1 as monomer. b , Time course of fusion and re-rounding of Eps15 (12 μM) condensates (green) on contact. c, Varying concentration of Epsin1 (20 μM to 80 μM) in 3 weight% PEG8K. d , 40 μM Epsin1 in varying weight% of PEG8K (3 weight% to 10 weight%). e , Eps15 (16 μM) condensates (green) incubated with 1.6 μM of Epsin1 and Epsin1ΔNPF (red), respectively. Plots on the right depict the intensity profile of the Epsin1 / Epsin1ΔNPF channel along the white dashed line shown in the corresponding images. f , The distribution of the Epsin1 and Epsin1ΔNPF intensity ratio (Kapp) between the intensity inside the condensates and the solution. Partition of Amphyphysin was used as a negative control. In total, 100 condensates were analyzed under each condition. Data are mean ± standard deviation, calculated from n = 3 replicates. Statistical significance was tested using an unpaired, two-tailed student’s t test on GraphPad Prism. g , Representative time course of fusion events between condensates containing Eps15 and Epsin1. Inset in h shows the interaction between Eps15 and Epsin1 (the NPF motif of Epsin1 binds to the EH domains of Eps15). All droplet experiments were performed in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 with PEG8K, at room temperature, 24 °C. All scale bars equal 5 μm.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Functional Assay, Binding Assay, Concentration Assay, Incubation, Negative Control, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: a, Representative images of Eps15 condensates with varying stoichiometry of Epsin1. 16 μM Eps15 was used for all the experiments and Epsin1 was mixed with Eps15 from 8:1 to 1:1 molar ratio. The line profiles depict the intensity profile of green (Eps15) channels along the white dashed line. b, The distribution of the Eps15 intensity ratio (Kapp) between the intensity inside the condensates and the solution with varying molar ratio of Epsin1. In total 100 condensates were analyzed under each condition. Data are mean ± standard deviation, calculated from n = 3 replicates. Statistical significance was tested using an unpaired, two-tailed student’s t test on GraphPad Prism. c, Bar graph shows the melting temperature (Tm) of Eps15 and Eps15:Epsin1 mixed condensates, n = 3 replicates. The error bar represents ± 1°C of the melting temperature. d-e, Representative images of protein condensates at increasing temperatures. Plots show fluorescence intensity of Eps15 measured along dotted lines in each image. Condensates are formed from (d) 16 μM Eps15, (e) 16 μM Eps15 with 8 μM Epsin1. f, Eps15 (16 μM) condensates mixed with 16 μM (1:1) of Epsin1ΔNPF, the line profile shows the intensity of Eps15 across the white dashed line. g, Shows the cartoon of the Eps15 multimerization through intermolecular interaction between DPF motif and EH domains and how the addition of Epsin1 can destabilize Eps15 condensates. Epsin1 binds to the EH domain of Eps15 and destabilizes the condensates by preventing Eps15 multimerization. All condensate experiments were performed in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 with PEG8K, at room temperature, 24 °C. All scale bars equal 5 μm.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Standard Deviation, Two Tailed Test, Fluorescence

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Representative images of protein condensates composed of Eps15 to Epsin1 in 8:1 ratio at increasing temperatures. Plots show fluorescence intensity of Eps15 measured along dotted lines in each image. Condensates are formed from 16 μM Eps15 mixed with 2 μM Epsin1 in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 buffer with 3% PEG8K. n = 3 biological replicates, scale bars equal 5 μm.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Phase diagram of Eps15 condensates and Eps15:Epsin1 mixed condensates at 8:1, 4:1 and 2:1 ratio. Condensates mapped by Atto488-labelled Eps15 fluorescence intensity. Intensity was normalized based on the intensity of the solution. Dots on the right side are protein concentrations in condensates and dots on the left side are concentrations in solution. At least 20 condensates are analyzed under each temperature. Data are mean ± SD. Condensates are formed in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 buffer with 3% PEG8K. n = 3 biological replicates.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: a, Representative time course of fusion events between Eps15 (16 μM) condensates (green) with 1.6 μM of Epsin1 (red) and 0.1 μM K63 linkage TetraUb (magenta), respectively. b, Representative images of Eps15 condensates with varying molar ratio of Epsin1 (8:1 to 2:1) in presence of TetraUb (magenta). The corresponding line profiles depict the intensity profile of the TetraUb (magenta) channel along the white dashed line. c, The distribution of the TetraUb (magenta) intensity ratio (Kapp) between the intensity inside the condensates and the solution with varying molar ratio of Epsin1. In total 100 condensates were analyzed under each condition. Data are mean ± standard deviation, calculated from n = 3 replicates. Statistical significance was tested using an unpaired, two-tailed student’s t test on GraphPad Prism. d, Representative images of the condensates containing 16 µM of Eps15 (green) and 16 µM of Epsin1 (1:1 ratio) in addition to 0.12 µM and 0.25 µM of TetraUb, respectively. The corresponding line profile depicts intensity profile of green (Eps15) channel along the white dashed line, n = 3. e, The size distribution histogram in Gaussian fit of the condensates containing 16 µM of Eps15 and Epsin1 in presence of different (0.12 and 0.25 µM) TetraUb concentration. In total 200 condensates were analyzed under each condition from n = 3 replicates. f, The bar plot depicts the melting temperature (Tm) of condensates composed of Eps15, Eps15 mixed with Epsin1 (8:1), Eps15 with TetraUb and when all three present together, n = 3 replicates. The error bar represents ± 1°C of the melting temperature. f, Shows the cartoon of UIM mediated crosslinking between Eps15 and Epsin1 that can stabilize the condensates. All condensate experiments were performed in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 with PEG8K, at room temperature, 24°C. All scale bars equal 5 μm.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Standard Deviation, Two Tailed Test, Concentration Assay

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: TetraUb mediated cross-linking between Eps15 and Epsin1. a-f, Representative images showing the Epsin1 and Epsin1ΔUIM partition (red) in Eps15 condensates incubated with and without TetraUb and monoUb. Plots on the right depict intensity profiles of Epsin1 and Epsin1ΔUIM along the white dashed line shown in the corresponding images. Epsin1 partition in Eps15 condensates (a) . Epsin1 partition in Eps15 condensates in presence of 2 μM monoUb (b) . Epsin1 partition in Eps15 condensates in presence of 0.25 μM TetraUb (c) . Epsin1 partition in Eps15 condensates in presence of 0.5 μM TetraUb (d) . Epsin1ΔUIM partition in Eps15 condensates (e) . Epsin1ΔUIM partition in Eps15 condensates in presence of 0.5 μM TetraUb (f) . g , Plot shows Epsin1/Epsin1ΔUIM partition coefficient (Kapp) in Eps15 condensates in presence of monoTetraUb. All condensate experiments were performed by mixing 1.6 μM Epsin1 or Epsin1ΔUIM with 16 μM Eps15 (unlabeled) (1:10 ratio) in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 with 3% w/v PEG8K. The increased Kapp in Eps15 condensates in presence of TetraUb was found to be dependent on the UIM region of Epsin1. n = 3 biologically independent experiments with at least 100 total condensates were measured for each condition. Data are mean ± SD. An unpaired, two tailed student’s t test was used for statistical significance using GraphPad Prism. Imaging was carried out at room temperature, 24°C. All scale bars equal 5 μm.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Incubation, Two Tailed Test, Imaging

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: a, Representative image of a SUM cell expressing gene-edited AP2 σ2-HaloTag: JF646 (cyan) and Eps15-mCherry (red). The scale bars 10 μm. Large inset highlights three representative clathrin-coated structures shown in smaller insets. The scale bars 5 μm. b , Representative image of short-lived (blue), productive (yellow) and long-lived (green) structures lasting < 20 s, > 20 s and > 5 min, respectively. c, Histograms of lifetime distributions of clathrin-coated structures under different experimental groups. Lifetime shorter than 20 s is considered short-lived, lifetime between 20 and 180 s is labeled as productive and structures lasting longer than 180 s are long-lived. Data are mean ± S.E.M. WT represents SUM cells that have endogenous Eps15 and Epsin1 expression. Eps15 KO represents SUM cells that were CRISPR modified to knock out alleles of endogenous Eps15. Epsin1 KD represents Epsin1 knock down in WT cells using siRNA. Eps15-DUB represents Eps15 knock out cells transfected with Eps15 fused with DUB (to C terminal end of Eps15). Eps15-DUB Epsin1 KD represents Eps15 knock out cells transfected with Eps15 fused with DUB and knocked down for Epsin1. All three constructs have mCherry at their C terminus for visualization. d, Bar plot summarizing percentage of short-lived (abortive), productive and long-lived fraction for the four conditions. e, Bar plot of the productive fraction for all four groups. Data are mean ± S.E.M. f, Bar plot of the short-lived fraction for all four groups. Data are mean ± S.E.M. For WT, Epsin1 KD, Eps-DUB and Eps-DUB Epsin1 KD, n = 20, 20, 15 and 16 biologically independent cells, respectively, were imaged. In total > 10000 pits were analyzed for each group. An unpaired, two-tailed student’s t test was used for statistical significance using GraphPad prism, n.s. means no significant difference. p < 0.05 is considered significantly different. All cell images were collected at 37°C.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Expressing, Labeling, CRISPR, Modification, Knock-Out, Knockdown, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Phase diagram (Tm) of condensates consisting of Eps15 alone, Eps15:Epsin1 mixed at 8:1 ratio, Eps15 mixed with 0.12 μM TetraUb and Eps15:Epsin1 8:1 system incubated with 0.12 μM TetraUb. Condensates mapped by Atto488-labelled Eps15 fluorescence intensity. Intensity was normalized based on the intensity of the solution. Dots on the right side are protein concentrations in condensates and dots on the left side are concentrations in solution. Eps15 alone and Eps15:Epsin1 8:1 both melts at 32°C. Addition of TetraUb in Eps15 condensates increased the Tm to 36°C. Addition of Epsin1 in this system (Eps15 + TetraUb + Epsin1) increased the Tm further to 38°C. At least 20 condensates are analyzed under each temperature. Data are mean ± SD. Condensates are formed in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 buffer with 3% PEG8K. n = 3 biological replicates.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Incubation, Fluorescence

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Representative images of protein condensates composed of 16 μM Eps15 mixed with 2 μM Epsin1 and 0.12 μM TetraUb at increasing temperatures. Intensity plots show fluorescence intensity of Eps15 measured along dotted lines in each image, n = 3 replicates. All condensate experiments were performed in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 with PEG8K. All scale bars equal 5 μm.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Whole cell lysates from wild type SUM159/AP2-HaloTag (SUM WT) cells and SUM159/AP2-HaloTag Eps15 CRISPR knock out (SUM Eps15 KO) cells were separated by SDS-PAGE and immunoblotted for Epsin1 and β -Actin. Both cell types were transfected with siRNA against Epsin1 (twice, 24 hour interval) and were collected 24 hours post 2nd transfection.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: CRISPR, Knock-Out, SDS Page, Transfection

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Bar plot of the productive fraction (lifetime between 20 - 180 sec) for all four groups. Data are mean ± S.E.M. Eps15 KO represents SUM cells that were CRISPR modified to knock out alleles of endogenous Eps15. Eps15 rescue represents Eps15 KO cells expressing wild type Eps15. Eps15 KO Epsin1 KD represents Eps15 KO cells also knocked down for Epsin1 using siRNA. WT siRNA control represents wild type cells transfected with control siRNA. Eps15 KO siRNA control represents Eps15 KO cells transfected with control siRNA. Eps15-DUB dead represent Eps15 KO cells transfected with wild type Eps15 fused with catalytically inactive DUB (fused to C terminal end of Eps15). Eps15-DUB dead Epsin1 KD represent Eps15 KO cells transfected with wild type Eps15 fused with catalytically inactive DUB (fused to C terminal end of Eps15) and knocked down for Epsin1 using siRNA. All Eps15 constructs have mCherry at their C terminus for visualization. For Eps15 KO, Eps15 rescue, Eps15 KO Epsin1 KD, WT siRNA, Eps15 KO siRNA, Eps15-DUB dead and Eps15-DUB dead Epsin1 KD n = 19, 10, 16, 10, 14, 15 and 14 biologically independent cells, respectively, were used to collect data. In total > 10000 pits were analyzed for each group. An unpaired, two-tailed student’s t test was used for statistical significance using GraphPad prism, n.s. means no significant difference. p < 0.05 is considered significantly different. All cell images were collected at 37°C.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: CRISPR, Modification, Knock-Out, Expressing, Control, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Bar plot of the short-lived fraction (lifetime between < 20 sec) for all four groups. Data are mean ± S.E.M. Eps15 KO represents SUM cells that were CRISPR modified to knock out alleles of endogenous Eps15. Eps15 rescue represents Eps15 KO cells expressing wild type Eps15. Eps15 KO Epsin1 KD represents Eps15 KO cells also knocked down for Epsin1 using siRNA. WT siRNA control represents wild type cells transfected with control siRNA. Eps15 KO siRNA control represents Eps15 KO cells transfected with control siRNA. Eps15-DUB dead represent Eps15 KO cells transfected with wild type Eps15 fused with catalytically inactive DUB (fused to C terminal end of Eps15). Eps15-DUB dead Epsin1 KD represent Eps15 KO cells transfected with wild type Eps15 fused with catalytically inactive DUB (fused to C terminal end of Eps15) and knocked down for Epsin1 using siRNA. All Eps15 constructs have mCherry at their C terminus for visualization. For Eps15 KO, Eps15 rescue, Eps15 KO Epsin1 KD, WT siRNA, Eps15 KO siRNA, Eps15-DUB dead and Eps15-DUB dead Epsin1 KD n = 19, 10, 16, 10, 14, 15 and 14 biologically independent cells, respectively, were used to collect data. In total > 10000 pits were analyzed for each group. An unpaired, two-tailed student’s t test was used for statistical significance using GraphPad prism, n.s. means no significant difference. p < 0.05 is considered significantly different. All cell images were collected at 37°C.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: CRISPR, Modification, Knock-Out, Expressing, Control, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: a, Illustration showing TfR uptake at the plasma membrane. b, Flow cytometry histogram of the transferrin fluorescence intensity (green fluorescence) of cells in each condition. c, Bar graph represents transferrin fluorescence intensity measured by flow cytometry. Data are mean ± standard deviation, calculated from n = 3 biological replicates. WT represents SUM cells that have endogenous Eps15 and Epsin1 expression. Eps15 KO represents SUM cells that were CRISPR modified to knock out alleles of endogenous Eps15. Epsin1 KD represents Epsin1 knock down in WT cells using siRNA. Eps15-DUB represent Eps15 knock out cells transfected with wild type Eps15 fused with DUB (deubiquitylase fused to C terminal end of Eps15). Eps15-DUB Epsin1 KD represents Eps15 knock out cells transfected with wild type Eps15 fused with DUB and knocked down for Epsin1. All three constructs have mCherry at their C terminus for visualization. An unpaired, two tailed student’s t test was used for statistical significance using GraphPad Prism. P < 0.05 is considered significantly different. Flow cytometry runs were carried out at 24°C. d-e Schematic showing how polyubiquitin stabilizes the endocytic protein network by interacting with Eps15 and Epsin1 and how Epsin1 functions as a checkpoint for ubiquitylated cargo. (d) In presence of polyubiquitin Eps15 and Epsin1 forms a liquid-like initiator network resulting in productive clathrin-mediated endocytosis. (e) In absence of ubiquitylated cargo Epsin1 interacts with Eps15 and prevents the assembly of the liquid-like network of Eps15 and thereby acting as a checkpoint for ubiquitylated cargo to prevent unproductive endocytic events.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Membrane, Flow Cytometry, Fluorescence, Standard Deviation, Expressing, CRISPR, Modification, Knock-Out, Knockdown, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Flow cytometry histogram of the cells in absence of transferrin (Tf), n = 3 biological replicates. Wild type (WT) represents SUM cells that have endogenous Eps15 and Epsin1 expression. Eps15 KO represents SUM cells that were CRISPR modified to knock out alleles of endogenous Eps15. All flow cytometry runs were carried out at 24°C. The measured fluorescence reflects autofluorescence of the cells and background noise levels in the flow cytometry system. These values represent a small contribution (1-2%) of the values for cells exposed to fluorescent Tf ( and ).

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Flow Cytometry, Expressing, CRISPR, Modification, Knock-Out, Fluorescence

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Flow cytometry histogram of the transferrin fluorescence intensity (green fluorescence) of cells in each condition, n = 3 biological replicates. Eps15 KO represents SUM cells that were CRISPR modified to knock out alleles of endogenous Eps15. Eps15 rescue represents Eps15 KO cells expressing wild type Eps15. Eps15 KO Epsin1 KD represents Eps15 KO cells also knocked down for Epsin1 using siRNA. WT siRNA control represents wild type cells transfected with control siRNA. Eps15 KO siRNA control represents Eps15 KO cells transfected with control siRNA. Eps15-DUB dead represent Eps15 KO cells transfected with wild type Eps15 fused with catalytically inactive DUB (fused to C terminal end of Eps15). Eps15-DUB dead Epsin1 KD represent Eps15 KO cells transfected with wild type Eps15 fused with catalytically inactive DUB (fused to C terminal end of Eps15) and knocked down for Epsin1 using siRNA. All Eps15 constructs have mCherry at their C terminus for visualization. All flow cytometry runs were carried out at 24°C.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Flow Cytometry, Fluorescence, CRISPR, Modification, Knock-Out, Expressing, Control, Transfection, Construct

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Bar graph represents transferrin fluorescence intensity measured by flow cytometry for all groups. Data are mean ± standard deviation, n = 3 biological replicates. Eps15 KO represents SUM cells that were CRISPR modified to knock out alleles of endogenous Eps15. Eps15 rescue represents Eps15 KO cells expressing wild type Eps15. Eps15 KO Epsin1 KD represents Eps15 KO cells also knocked down for Epsin1 using siRNA. WT siRNA control represents wild type cells transfected with control siRNA. Eps15 KO siRNA control represents Eps15 KO cells transfected with control siRNA. Eps15-DUB dead represent Eps15 KO cells transfected with Eps15 fused with catalytically inactive DUB (fused to C terminal end of Eps15). Eps15-DUB dead Epsin1 KD represent Eps15 KO cells transfected with wild type Eps15 fused with catalytically inactive DUB (fused to C terminal end of Eps15) and knocked down for Epsin1 using siRNA. All Eps15 constructs have mCherry at their C terminus for visualization. flow cytometry runs were carried out at 24°C.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Fluorescence, Flow Cytometry, Standard Deviation, CRISPR, Modification, Knock-Out, Expressing, Control, Transfection, Construct

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: a, Schematic representation of model proteins with defined ubiquitin modifications; the 0-Ub model protein with no attached ubiquitin and the 1-Ub model protein with a single attached ubiquitin. All lysine residues in the cytoplasmic domain were mutated to arginines to prevent ubiquitylation. To further stabilize the attached ubiquitin moieties, all lysines within ubiquitin were mutated to arginines, and the C-terminal GG residues were altered to prevent intracellular deubiquitylation. b, Shows the illustration and recruitment of ubiquitylated and non-ubiquitylated model cargo proteins in the endocytic structure. c-d, spinning disk confocal images of the plasma membrane of cells transiently expressing the 0-Ub and 1-Ub model proteins. Green fluorescence (GFP) highlights the model proteins, while red fluorescence (AP2-JF647) marks endocytic structures. The scale bars are 500 nm (insets). e-f, The relative number of model proteins localized in clathrin-coated structures is shown against the relative concentration of model proteins on the plasma membrane around each structure. Data were collected from over 10000 endocytic sites in n = 30 independent cells, cells expressing 0-Ub and 1-Ub model protein. Each point reflects the average value from clathrin-coated structures binned by the local membrane concentration of the proteins. Data are mean ± standard deviation, while dashed lines represent model predictions based on the best-fit values. g, Bar plot shows the relative Csaturation (maximum local concentration of fusion proteins in clathrin-coated structures) values for the 0-Ub and 1-Ub in WT and Eps15-DUB Epsin1 KD cells. Each bar displays the mean ± standard deviation. An unpaired, two tailed student’s t test was used for statistical significance using GraphPad Prism. p < 0.05 is considered significantly different. h, Shows the schematic illustration of outcomes of cargo sorting at endocytic sites, in WT cells, ubiquitylated cargo (1-Ub) gets selectively enriched at the site over non-ubiquitylated cargo; in Eps15-DUB Epsin1 KD cells, the selective enrichment of 1-Ub at the endocytic site is lost.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Membrane, Expressing, Fluorescence, Concentration Assay, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

doi: 10.1101/2025.02.12.637885

Figure Lengend Snippet: Whole cell fluorescence images of the plasma membrane of SUM cells expressing the model proteins. a-b , Spinning disk confocal images of wild type (WT) whole cells expressing the 0-Ub model protein (a) and 1-Ub model protein (b) . c-d , Spinning disk confocal images of Eps15 KO Epsin1 KD whole cells expressing the 0-Ub model protein (c) and 1-Ub model protein (d) . Crops of these cells are shown in , respectively. Red fluorescence (AP2-JF646) marks endocytic sites and green fluorescence (GFP) highlights the model proteins. The scale bars are 5 µm.

Article Snippet: Proteins were transferred to a nitrocellulose membrane (BioRad), blocked for 1 hour at room temperature with LI-COR Block Buffer, and probed with an anti-Epsin1 antibody (mouse polyclonal, Santa Cruz) diluted 1:1000 in 1x TNET Wash Buffer (10 mM Tris, pH 7.5, 2.5 mM EDTA, pH 8.0, 50 mM NaCl, 0.1% Tween-20).

Techniques: Fluorescence, Membrane, Expressing