Journal: Cardiovascular Research

Article Title: Loss-of-function mutation of the SCN3B -encoded sodium channel ?3 subunit associated with a case of idiopathic ventricular fibrillation

doi: 10.1093/cvr/cvp417

Figure Lengend Snippet: Navβ3-V54G reduced INa in HEK-293 cells (A) Whole-cell current traces from representative cells expressing SCN5A + vector, SCN5A co-expressed with β3-WT or Navβ3-V54G 24 h after transfection. INa was elicited from a step depolarization ...

Article Snippet: A rabbit polyclonal anti-Navβ3 antibody was generated against residues 65–83 of the Navβ3 (YenZym Antibodies, LLC, South San Francisco, CA, USA).

Techniques: Expressing, Plasmid Preparation, Transfection

Journal: Cardiovascular Research

Article Title: Loss-of-function mutation of the SCN3B -encoded sodium channel ?3 subunit associated with a case of idiopathic ventricular fibrillation

doi: 10.1093/cvr/cvp417

Figure Lengend Snippet: Navβ3-WT shifted inactivation kinetics, but Navβ3-V54G did not. (A) Peak current–voltage plot of summary INa normalized to the maximal INa in response to a series of depolarizations from a holding potential of −140 mV. ...

Article Snippet: A rabbit polyclonal anti-Navβ3 antibody was generated against residues 65–83 of the Navβ3 (YenZym Antibodies, LLC, South San Francisco, CA, USA).

Techniques:

Journal: Cardiovascular Research

Article Title: Loss-of-function mutation of the SCN3B -encoded sodium channel ?3 subunit associated with a case of idiopathic ventricular fibrillation

doi: 10.1093/cvr/cvp417

Figure Lengend Snippet: Co-IP of SCN5A and Navβ3 subunits. (A) Heart homogenates were obtained from adult or neonatal mouse hearts, and from cell lysates obtained from HEK-293 cells expressing SCN5A alone or cells co-expressing SCN5A and Navβ3. (B and C) Cell ...

Article Snippet: A rabbit polyclonal anti-Navβ3 antibody was generated against residues 65–83 of the Navβ3 (YenZym Antibodies, LLC, South San Francisco, CA, USA).

Techniques: Co-Immunoprecipitation Assay, Expressing

Journal: Cardiovascular Research

Article Title: Loss-of-function mutation of the SCN3B -encoded sodium channel ?3 subunit associated with a case of idiopathic ventricular fibrillation

doi: 10.1093/cvr/cvp417

Figure Lengend Snippet: Navβ3-V54G reduced INa in COS cells. Equal amounts of blank vector, Navβ3-WT or Navβ3-V54G, and in some cells Navβ1-WT were co-transfected with SCN5A in COS cells for a total amount of 1.5 µg DNA. (A) Whole-cell ...

Article Snippet: A rabbit polyclonal anti-Navβ3 antibody was generated against residues 65–83 of the Navβ3 (YenZym Antibodies, LLC, South San Francisco, CA, USA).

Techniques: Plasmid Preparation, Transfection

Journal: Cardiovascular Research

Article Title: Loss-of-function mutation of the SCN3B -encoded sodium channel ?3 subunit associated with a case of idiopathic ventricular fibrillation

doi: 10.1093/cvr/cvp417

Figure Lengend Snippet: (A) Left: Proband DNA sequence chromatogram showing a T > G substitution generating a valine (V) to glycine (G) substitution at residue 54 of Navβ3. Right: Wild-type DHPLC profile in blue colour, mutant, and aberrant profile in red colour. ...

Article Snippet: A rabbit polyclonal anti-Navβ3 antibody was generated against residues 65–83 of the Navβ3 (YenZym Antibodies, LLC, South San Francisco, CA, USA).

Techniques: Sequencing, Residue, Mutagenesis

Journal: PLoS Pathogens

Article Title: Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

doi: 10.1371/journal.ppat.1000857

Figure Lengend Snippet: ( A ) Sequence of prothrombin and overlapping peptides (indicated by numbers). In addition to the regular overlapping peptides, peptide regions of high net charge, and/or content of predicted helical regions (Agadir; http://www.embl-heidelberg.de/Services/serrano/agadir/agadir-start.html ) were selected. Peptides described in subsequent experiments are also indicated; GKY25, VFR17, and the major ∼11 kDa peptide (amino acids 527–622). ( B ) Overlapping peptides of prothrombin were analyzed for antimicrobial activities against E. coli . The inhibitory zones, relative hydrophobic moment (μHrel) as well as net charge of respective peptides (only active peptides are numbered) are indicated in the 3-D graph. Peptides showing antimicrobial activity are also indicated by red color in ( A ). For determination of antibacterial activities, E. coli (4×10 6 cfu) was inoculated in 0.1% TSB agarose gel. Each 4 mm-diameter well was loaded with 6 µl of peptide (at 100 µM). The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37°C for 18–24 h (mean values are presented, n = 3). ( C ) Helical wheel representation of the C-terminal peptide VFR17. The amino acids are indicated. ( D ) LPS-binding activity of the prothrombin-derived peptide sequences. Peptides (5 µg) were applied to nitrocellulose membranes followed by incubation in PBS (containing 2% bovine serum albumin) with iodinated ( 125 I)-LPS. Only peptides from the C-terminal part of prothrombin demonstrated significant binding to LPS. ( E ) Molecular model of thrombin. The peptides GKY25 (green-orange, indicated in ) and VFR17 (orange, peptide 48 in ) are indicated in the crystal structure of human thrombin (PDB code 1C5L). ( F ) Activities of prothrombin (PT), thrombin (T), GKY25 and VFR17 on E. coli ATCC 25922. In viable count assays GKY25 and VFR17 displayed significant antibacterial activities. 2×10 6 cfu/ml of bacteria were incubated in 50 µl with proteins and peptides at a concentration of 3 and 6 µM, respectively.

Article Snippet: In order to deplete acute wound fluids (AWF) of thrombin and fragments containing C-terminal peptides, AWF was diluted with an equal volume of PBS, and passaged 5 times through an affinity column (0.3 ml, Thermo Scientific) having coupled IgG antibodies specific for VFR17 (Innovagen AB).

Techniques: Sequencing, Activity Assay, Agarose Gel Electrophoresis, Incubation, Binding Assay, Derivative Assay, Concentration Assay

Journal: PLoS Pathogens

Article Title: Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

doi: 10.1371/journal.ppat.1000857

Figure Lengend Snippet: ( A ) RP-HPLC separation of thrombin digested with neutrophil elastase. The bars indicate the antibacterial activity of the fractions in low (gray) as well as high salt conditions (black). Fraction 30 (lower left) contained two peaks of masses 2034.78 and 2270.88, perfectly matching the indicated sequences obtained after ESI-MS/MS analysis. Fraction 38 was analyzed by MALDI-MS, and subsequently by ESI-MS. The ESI-MS analysis identified a dominant mass of 11041 corresponding to the 96-amino acid long peptide N527-E622 (indicated in ) with two intact disulphide bridges. A minor mass corresponding to V528-E622 was also detected by ESI-MS/MS. N- and C-terminal sequencing yelded NLPI and EGFQ, respectively. The rightmost insets illustrate the ∼11 kDa peptide analysed by SDS-PAGE and stained for protein (stain), or after immunoblot (blot), and below, the peptide (F38) was analyzed by gel-overlay for detection of antibacterial activity. The activity of F38 was identical to the major clearing zone generated by elastase-digested thrombin (T+NE). Right arrow indicates the position of clearing zone generated by the peptide GKY25. The gel was run top to bottom. Finally, peptides of fractions 20–21 were predicted using the FINDPEPT tool ( www.expasy.org/tools/findpept.html ) . ( B ) Degradation of thrombin by neutrophil supernatants generates antibacterial activity in RDA (upper inset). RDA was performed in low-salt conditions. E. coli (4×10 6 cfu) was used as test organism. Each 4 mm-diameter well was loaded with 6 µl of material (C, supernatant only; T, thrombin only; T+NS; thrombin incubated for 30 and 180 min respectively, with neutrophil supernatants). The digests were analysed by SDS-PAGE (16.5% Tris-Tricine gels) and immunoblotting with antibodies against VFR17 (lower panel). ( C ) Prothrombin was digested with the enzymes as indicated for 3 h, and analyzed by SDS-PAGE (16.5% Tris-Tricine gels) and immunoblotting using antibodies against VFR17 (NE, neutrophil elastase; CG, cathepsin G; PAE, P. aeruginosa elastase). ( D ) RDA results of prothrombin digested with cathepsin G (CG) and P. aeruginosa elastase (PAE) for different time periods. VFR17 and LL-37 (10 µM) are shown for comparison.

Article Snippet: In order to deplete acute wound fluids (AWF) of thrombin and fragments containing C-terminal peptides, AWF was diluted with an equal volume of PBS, and passaged 5 times through an affinity column (0.3 ml, Thermo Scientific) having coupled IgG antibodies specific for VFR17 (Innovagen AB).

Techniques: Activity Assay, Tandem Mass Spectroscopy, Sequencing, SDS Page, Staining, Western Blot, Generated, Incubation

Journal: PLoS Pathogens

Article Title: Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

doi: 10.1371/journal.ppat.1000857

Figure Lengend Snippet: ( A ) Fibrin clots were produced from human plasma and incubated with neutrophil elastase for the indicated time periods (Fibrin), or obtained from a patient with a venous, non-infected, chronic ulcer (PF), extracted, and analyzed by immunoblotting using polyclonal antibodies against the thrombin C-terminal peptide VFR17. ( B ) Human plasma, incubated with neutrophil elastase for the indicated time periods (Plasma, left panel), acute wound fluid (patients 1–2, AWF, middle panel), or wound fluid from patients with chronic ulcers (patients 1–6, CWF, right panel) was analysed by Western blot using polyclonal antibodies against the thrombin C-terminal peptide VFR17. ( C ) Flow cytometry analysis of binding of C-terminal thrombin epitopes to P. aeruginosa bacteria. Bacteria were incubated for 4 h with control plasma (P), human plasma depleted of prothrombin (DP), depleted plasma supplemented with the peptide GKY25, or, acute wound fluid (AWF). Binding of C-terminal epitopes to the bacteria was detected using primary antibodies against the C-terminal epitope VFR17 followed by addition of FITC-labeled secondary antibodies. ( D ) Visualization of binding and membrane damage by TCPs. P. aeruginosa bacteria were incubated ex vivo with human plasma (P), acute wound fluid (AWF), or wound fluid from a chronic leg ulcer (CWF), or visualized in vivo in fibrin slough (CWS) derived from a patient with a chronic ulcer infected by S. aureus . Arrows in P, AWF, and CWF point to damaged bacterial membranes. Coccoid bacteria (indicated by an arrow in CWS) show extensive binding of antibodies directed against the C-terminal peptide VFR17 (negative and positive bacterial controls, and additional material are found in and ). ( E ) TCPs inhibit bacterial growth in human plasma. Control plasma (P), plasma depleted of prothrombin (DP), depleted plasma supplemented with either prothrombin (DP+PT), or GKY25 (DP+GKY25) (PT and GKY25 at 1.5 µM), or control AWF or depleted AWF (D-AWF), were inoculated with P. aeruginosa bacteria under similar conditions as in ( C–D ). The multiplication factors at various time points are given. After incubation, CFUs were determined by plating. Experiments were repeated three times and a representative experiment is shown. ( F ) The thrombin C-terminal peptide GKY25 significantly increases survival. Mice were injected i.p. with P. aeruginosa bacteria, followed by subcutaneous injection of GKY25 or buffer only, after 1 h. The injections were repeated after 24 hours. Treatment with the peptide significantly increased survival (n = 10 for controls and treated, p = 0.002). ( G ) GKY25 suppresses bacterial dissemination to the spleen and kidney. Mice were infected as above, GKY25 was administrated subcutaneously after 1 h, and the cfu of P. aeruginosa in spleen and kidney was determined after a time period of 8 h (n = 10 for controls and treated, P<0.05 for spleen and kidney. Horizontal line indicates median value).

Article Snippet: In order to deplete acute wound fluids (AWF) of thrombin and fragments containing C-terminal peptides, AWF was diluted with an equal volume of PBS, and passaged 5 times through an affinity column (0.3 ml, Thermo Scientific) having coupled IgG antibodies specific for VFR17 (Innovagen AB).

Techniques: Produced, Incubation, Infection, Western Blot, Flow Cytometry, Binding Assay, Labeling, Ex Vivo, In Vivo, Derivative Assay, Injection

Journal: PLoS Pathogens

Article Title: Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

doi: 10.1371/journal.ppat.1000857

Figure Lengend Snippet: ( A ) Electron microscopy analysis. P. aeruginosa and S. aureus bacteria was incubated for 2 h at 37°C with 30 µM of GKY25 and LL-37 and analyzed with electron microscopy. Scale bar represents 1 µm. Control; Buffer control. ( B ) Permeabilizing effects of peptides on E. coli . Bacteria were incubated with the indicated peptides at 30 µM and permeabilization was assessed using the impermeant probe FITC. ( C ) Helical content of the thrombin-derived C-terminal peptides GKY25 and VFR17 in presence of negatively charged liposomes (PA). The two peptides showed a marked helix induction upon addition of the liposomes. ( D ) CD spectra of GKY25 and VFR17 in Tris-buffer and in presence of LPS. For control, CD spectra for buffer and LPS alone are also presented. ( E ) Effects of the indicated peptides on liposome leakage. The membrane permeabilizing effect was recorded by measuring fluorescence release of carboxyfluorescein from PA (negatively charged) liposomes. The experiments were performed in 10 mM Tris-buffer, in absence and presence of 0.15 M NaCl. Values represents mean of triplicate samples. ( F ) Activities of corresponding C-terminal peptides of the indicated coagulation factors. Peptides were tested in RDA against the indicated bacteria. Bacteria (4×10 6 cfu) were inoculated in 0.1% TSB agarose gels. Each 4 mm-diameter well was loaded with 6 µl of peptide at 100 µM. The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37°C for 18–24 h. ( G ) Overlay 3D-model showing the three coagulation factors thrombin, and factor X and IX. The C-terminal parts are indicated.

Article Snippet: In order to deplete acute wound fluids (AWF) of thrombin and fragments containing C-terminal peptides, AWF was diluted with an equal volume of PBS, and passaged 5 times through an affinity column (0.3 ml, Thermo Scientific) having coupled IgG antibodies specific for VFR17 (Innovagen AB).

Techniques: Electron Microscopy, Incubation, Derivative Assay, Fluorescence, Coagulation

Journal: PLoS ONE

Article Title: Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

doi: 10.1371/journal.pone.0074519

Figure Lengend Snippet: HeLa cells were treated with non-targeting control-, syndapin2- or EHBP1-siRNA for 72 h. Cells were then lysed, and equal amounts of proteins were separated by SDS-PAGE and subjected to immunoblotting with antibodies against syndapin2, EHBP1, EHD2, caveolin1 or actin (A). For confocal microscopy, cells growing on coverslips were fixed and stained with antibodies against EHD2 (B–D) or caveolin1 (E–G). Bar, 10 µm. The immunoblots and confocal micrographs are representative of three independent experiments.

Article Snippet: The following primary antibodies were used in this study: affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of EHD2 (VERGPDEAMEDGEEGSDDEA) (AnaSpec), rabbit anti-syndapin2 (ProteinTech, #10518-2-ap, 1∶500 for immunoblot), rabbit anti-EHBP1 (Novus, #nbp1-93614, 1∶700 for immunoblot), rabbit anti-caveolin1 (Cell Signaling Technology, #3238, 1∶300 for immunofluorescence, 1∶1000 for immunoblot), mouse anti-pan actin (Novus, #nb600-535, 1∶2500 for immunoblot), mouse anti-cytochrome C (BD Pharmingen, #556433, 1∶400 for immunoblot), rabbit anti-GST (Millipore, #ab3282, 1∶200 for immunofluorescence), rabbit anti-Myc (Santa Cruz Biotechnology, #sc-789, 1∶200 for immunofluorescence), mouse anti-Myc (Invitrogen, #13-2500, 1∶200 for immunofluorescence), and mouse anti-HA.11 (Covance, #mms-101p, 1∶500 for immunofluorescence).

Techniques: Control, SDS Page, Western Blot, Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

doi: 10.1371/journal.pone.0074519

Figure Lengend Snippet: (A) Schematic representation of EHD chimeras. The EHD2-EH1 chimera contains EHD2 amino acids 1–378 (shown in grey) and EHD1 amino acids 379–534 (encoding the EH domain, shown in white). Conversely, the EHD1-EH2 chimera contains EHD1 amino acids 1–378 (shown in white) and EHD2 amino acids 379–543 (encoding the EH domain, shown in grey). HeLa cells growing on coverslips were transfected with GFP-Myc-EHD2 (B), GFP-EHD2-EH1 (C), GFP-Myc-EHD1 (D), or GFP-EHD1-EH2 (E). After 24 h, the cells were fixed and analyzed directly for GFP-fusion protein expression by confocal microscopy. Bar, 10 µm. The confocal micrographs are representative of two independent experiments.

Article Snippet: The following primary antibodies were used in this study: affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of EHD2 (VERGPDEAMEDGEEGSDDEA) (AnaSpec), rabbit anti-syndapin2 (ProteinTech, #10518-2-ap, 1∶500 for immunoblot), rabbit anti-EHBP1 (Novus, #nbp1-93614, 1∶700 for immunoblot), rabbit anti-caveolin1 (Cell Signaling Technology, #3238, 1∶300 for immunofluorescence, 1∶1000 for immunoblot), mouse anti-pan actin (Novus, #nb600-535, 1∶2500 for immunoblot), mouse anti-cytochrome C (BD Pharmingen, #556433, 1∶400 for immunoblot), rabbit anti-GST (Millipore, #ab3282, 1∶200 for immunofluorescence), rabbit anti-Myc (Santa Cruz Biotechnology, #sc-789, 1∶200 for immunofluorescence), mouse anti-Myc (Invitrogen, #13-2500, 1∶200 for immunofluorescence), and mouse anti-HA.11 (Covance, #mms-101p, 1∶500 for immunofluorescence).

Techniques: Transfection, Expressing, Confocal Microscopy

Journal: PLoS ONE

Article Title: Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

doi: 10.1371/journal.pone.0074519

Figure Lengend Snippet: HeLa cells growing on coverslips were treated with DMSO or with 25 µM each of the PLD inhibitors CAY10593 and CAY10594 (CAY93/94) for 45 min. The cells were then fixed or washed 3X with DMEM and chased in complete growth medium for 2 h (CAY93/94+ washout). After fixation, the cells were stained with purified GST-PH-PLCδ1 to visualize PIP2 (A–C), or with antibodies against EHD2 (D–F) or caveolin1 (G–I), or with phalloidin-488 to visualize actin (J–L). After incubation with the appropriate primary and secondary antibodies, the cells were analyzed by confocal microscopy. The images showing PIP2 staining (A–C) are z-series stacks, while cells stained for EHD2, caveolin1 or actin (D–L) were imaged as single optical sections. The images shown in D–F and J–L are the same set of cells that were co-stained for EHD2 and actin. Bar, 10 µm. The confocal micrographs are representative of three independent experiments.

Article Snippet: The following primary antibodies were used in this study: affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of EHD2 (VERGPDEAMEDGEEGSDDEA) (AnaSpec), rabbit anti-syndapin2 (ProteinTech, #10518-2-ap, 1∶500 for immunoblot), rabbit anti-EHBP1 (Novus, #nbp1-93614, 1∶700 for immunoblot), rabbit anti-caveolin1 (Cell Signaling Technology, #3238, 1∶300 for immunofluorescence, 1∶1000 for immunoblot), mouse anti-pan actin (Novus, #nb600-535, 1∶2500 for immunoblot), mouse anti-cytochrome C (BD Pharmingen, #556433, 1∶400 for immunoblot), rabbit anti-GST (Millipore, #ab3282, 1∶200 for immunofluorescence), rabbit anti-Myc (Santa Cruz Biotechnology, #sc-789, 1∶200 for immunofluorescence), mouse anti-Myc (Invitrogen, #13-2500, 1∶200 for immunofluorescence), and mouse anti-HA.11 (Covance, #mms-101p, 1∶500 for immunofluorescence).

Techniques: Staining, Purification, Incubation, Confocal Microscopy

Journal: PLoS ONE

Article Title: Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

doi: 10.1371/journal.pone.0074519

Figure Lengend Snippet: (A) HeLa cells were treated with DMSO (control), or 15 µM each CAY10593 and CAY10594 for 4 h, or 25 µM each CAY10593 and CAY10594 for 45 min. The cells were then lysed, and equal amounts of proteins were separated by SDS-PAGE and subjected to immunoblotting with antibodies against EHD2, caveolin1 or actin. The immunoblots are representative of seven independent experiments. (B) HeLa cells were pre-treated with 100 µM leupeptin or with 10 µM lactacystin for 3 h. The cells were then treated with fresh 100 µM leupeptin or 10 µM lactacystin along with either DMSO, 15 µM each CAY10593 and CAY10594 (for 4 h), or 25 µM each CAY10593 and CAY10594 (for 45 min). Following treatment, the cells were lysed, and equal amounts of proteins were separated by SDS-PAGE and subjected to immunoblotting with antibodies against EHD2, caveolin1 or actin. The immunoblots are representative of three experiments conducted with leupeptin, and two experiments done with lactacystin. (C) HeLa cells were treated as in (A). The cells were then homogenized and post-nuclear supernatants were separated into cytosol and membrane fractions by ultracentrifugation. The samples were resolved by SDS-PAGE and immunoblotted with antibodies against EHD2, caveolin1 or cytochrome C (cytosolic fraction control). The immunoblots are representative of two independent experiments.

Article Snippet: The following primary antibodies were used in this study: affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of EHD2 (VERGPDEAMEDGEEGSDDEA) (AnaSpec), rabbit anti-syndapin2 (ProteinTech, #10518-2-ap, 1∶500 for immunoblot), rabbit anti-EHBP1 (Novus, #nbp1-93614, 1∶700 for immunoblot), rabbit anti-caveolin1 (Cell Signaling Technology, #3238, 1∶300 for immunofluorescence, 1∶1000 for immunoblot), mouse anti-pan actin (Novus, #nb600-535, 1∶2500 for immunoblot), mouse anti-cytochrome C (BD Pharmingen, #556433, 1∶400 for immunoblot), rabbit anti-GST (Millipore, #ab3282, 1∶200 for immunofluorescence), rabbit anti-Myc (Santa Cruz Biotechnology, #sc-789, 1∶200 for immunofluorescence), mouse anti-Myc (Invitrogen, #13-2500, 1∶200 for immunofluorescence), and mouse anti-HA.11 (Covance, #mms-101p, 1∶500 for immunofluorescence).

Techniques: Control, SDS Page, Western Blot, Membrane

Journal: PLoS ONE

Article Title: Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

doi: 10.1371/journal.pone.0074519

Figure Lengend Snippet: HeLa cells growing on coverslips were transfected with Myc-synaptojanin2 (A–B) or with Myc-Vam6 (C–D). After 24 h, the cells were fixed and stained with antibodies against Myc and EHD2. Following incubation with fluorochrome-conjugated secondary antibodies, the cells were analyzed by confocal microscopy. Bar, 10 µm. Transfected cells are marked with an asterisk. The confocal micrographs are representative of three independent experiments. (E) The number of untransfected, Myc-Vam6-transfected (control), or Myc-synaptojanin2-transfected cells that displayed EHD2 at the plasma membrane was counted. A total of at least 50 untransfected or transfected cells from 3 independent experiments were counted. The average percentage of cells displaying EHD2 at the plasma membrane was compared between the groups by performing a Tukey HSD test (*p<0.01).

Article Snippet: The following primary antibodies were used in this study: affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of EHD2 (VERGPDEAMEDGEEGSDDEA) (AnaSpec), rabbit anti-syndapin2 (ProteinTech, #10518-2-ap, 1∶500 for immunoblot), rabbit anti-EHBP1 (Novus, #nbp1-93614, 1∶700 for immunoblot), rabbit anti-caveolin1 (Cell Signaling Technology, #3238, 1∶300 for immunofluorescence, 1∶1000 for immunoblot), mouse anti-pan actin (Novus, #nb600-535, 1∶2500 for immunoblot), mouse anti-cytochrome C (BD Pharmingen, #556433, 1∶400 for immunoblot), rabbit anti-GST (Millipore, #ab3282, 1∶200 for immunofluorescence), rabbit anti-Myc (Santa Cruz Biotechnology, #sc-789, 1∶200 for immunofluorescence), mouse anti-Myc (Invitrogen, #13-2500, 1∶200 for immunofluorescence), and mouse anti-HA.11 (Covance, #mms-101p, 1∶500 for immunofluorescence).

Techniques: Transfection, Staining, Incubation, Confocal Microscopy, Control, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

doi: 10.1371/journal.pone.0074519

Figure Lengend Snippet: (A–C) HeLa cells growing on coverslips were transfected with HA-Arf6Q67L and Myc-EHD2 for 11 h. The cells were then fixed and stained with antibodies against HA and Myc. After addition of fluorochrome-conjugated secondary antibodies, the cells were analyzed by confocal microscopy. (D–E) HeLa cells growing on coverslips were transfected with HA-Arf6Q67L for 11 h. The cells were incubated in serum-free DMEM containing 0.5% bovine serum albumin during the last 3.5 h of transfection, and then pulsed with 1 µg/ml transferrin-488 for 20 min. The cells were then fixed and stained with antibody against HA. After incubation with fluorochrome-conjugated secondary antibody, the cells were analyzed by confocal microscopy. Insets are shown below each image. Bar, 10 µm. The confocal micrographs are representative of three independent experiments.

Article Snippet: The following primary antibodies were used in this study: affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of EHD2 (VERGPDEAMEDGEEGSDDEA) (AnaSpec), rabbit anti-syndapin2 (ProteinTech, #10518-2-ap, 1∶500 for immunoblot), rabbit anti-EHBP1 (Novus, #nbp1-93614, 1∶700 for immunoblot), rabbit anti-caveolin1 (Cell Signaling Technology, #3238, 1∶300 for immunofluorescence, 1∶1000 for immunoblot), mouse anti-pan actin (Novus, #nb600-535, 1∶2500 for immunoblot), mouse anti-cytochrome C (BD Pharmingen, #556433, 1∶400 for immunoblot), rabbit anti-GST (Millipore, #ab3282, 1∶200 for immunofluorescence), rabbit anti-Myc (Santa Cruz Biotechnology, #sc-789, 1∶200 for immunofluorescence), mouse anti-Myc (Invitrogen, #13-2500, 1∶200 for immunofluorescence), and mouse anti-HA.11 (Covance, #mms-101p, 1∶500 for immunofluorescence).

Techniques: Transfection, Staining, Confocal Microscopy, Incubation

Journal: PLoS ONE

Article Title: Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

doi: 10.1371/journal.pone.0074519

Figure Lengend Snippet: HeLa cells growing on coverslips were treated with DMSO, or with a low (0.3 µM) or high (5 µM) concentration of cytochalasin D for 45 min. The cells were then fixed and stained with antibodies against EHD2 (A–C) or caveolin1 (G–I) along with phaolloidin-488 to visualize actin (D–F, J–L). After incubation with fluorochrome-conjugated secondary antibody, the cells were analyzed by confocal microscopy. Insets depict the regions in the dashed boxes. Bar, 10 µm. The confocal micrographs are representative of three independent experiments with 0.3 µM cytochalasin D, and two independent experiments with 5 µM cytochalasin D.

Article Snippet: The following primary antibodies were used in this study: affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of EHD2 (VERGPDEAMEDGEEGSDDEA) (AnaSpec), rabbit anti-syndapin2 (ProteinTech, #10518-2-ap, 1∶500 for immunoblot), rabbit anti-EHBP1 (Novus, #nbp1-93614, 1∶700 for immunoblot), rabbit anti-caveolin1 (Cell Signaling Technology, #3238, 1∶300 for immunofluorescence, 1∶1000 for immunoblot), mouse anti-pan actin (Novus, #nb600-535, 1∶2500 for immunoblot), mouse anti-cytochrome C (BD Pharmingen, #556433, 1∶400 for immunoblot), rabbit anti-GST (Millipore, #ab3282, 1∶200 for immunofluorescence), rabbit anti-Myc (Santa Cruz Biotechnology, #sc-789, 1∶200 for immunofluorescence), mouse anti-Myc (Invitrogen, #13-2500, 1∶200 for immunofluorescence), and mouse anti-HA.11 (Covance, #mms-101p, 1∶500 for immunofluorescence).

Techniques: Concentration Assay, Staining, Incubation, Confocal Microscopy

Journal: PLoS ONE

Article Title: Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

doi: 10.1371/journal.pone.0074519

Figure Lengend Snippet: EHD2 localizes to the plasma membrane, and is predicted to form large oligomers at the neck of caveolae. PIP2, which is enriched in plasma membrane domains, including caveolae, has a crucial role in maintaining EHD2 at the cell surface. In the absence of caveolar invaginations, EHD2 maintains its plasma membrane association, likely through binding to PIP2 and, potentially, protein interactions mediated outside of the EH2 domain. EHD2 binds to the F-BAR domain-containing protein syndapin2, which facilitates membrane curvature, and also to EHBP1, which connects to the actin cytoskeleton. In contrast, localization of EHD1 to tubular recycling endosomes depends on binding of the EH1 domain to MICAL-L1 and sydnapin2, which require phosphatidic acid for their recruitment to the tubular recycling endosomes.

Article Snippet: The following primary antibodies were used in this study: affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of EHD2 (VERGPDEAMEDGEEGSDDEA) (AnaSpec), rabbit anti-syndapin2 (ProteinTech, #10518-2-ap, 1∶500 for immunoblot), rabbit anti-EHBP1 (Novus, #nbp1-93614, 1∶700 for immunoblot), rabbit anti-caveolin1 (Cell Signaling Technology, #3238, 1∶300 for immunofluorescence, 1∶1000 for immunoblot), mouse anti-pan actin (Novus, #nb600-535, 1∶2500 for immunoblot), mouse anti-cytochrome C (BD Pharmingen, #556433, 1∶400 for immunoblot), rabbit anti-GST (Millipore, #ab3282, 1∶200 for immunofluorescence), rabbit anti-Myc (Santa Cruz Biotechnology, #sc-789, 1∶200 for immunofluorescence), mouse anti-Myc (Invitrogen, #13-2500, 1∶200 for immunofluorescence), and mouse anti-HA.11 (Covance, #mms-101p, 1∶500 for immunofluorescence).

Techniques: Clinical Proteomics, Membrane, Binding Assay