Journal: The Journal of Biological Chemistry

Article Title: Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells *

doi: 10.1074/jbc.M111.225839

Figure Lengend Snippet: Quantitative PCR primer sequence and amplicon sizes for SNAREs Primers were designed using Beacon Designer 3.01 (PREMIERE Biosoft International) to span introns and yield amplification fragments of 100–200 bp.

Article Snippet: Membranes were incubated first in blocking buffer containing 50 m m Tris, 500 m m NaCl, 0.1% Tween 20 (TBS-T), and 5% nonfat dried milk followed by primary antibody (mouse VAMP2 1/2000 or rabbit VAMP3 1/4000; Synaptic Systems) ( – ).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Amplification

Journal: The Journal of Biological Chemistry

Article Title: Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells *

doi: 10.1074/jbc.M111.225839

Figure Lengend Snippet: Representative image of quantitative PCR products for VAMP2 and VAMP3. PCR products were resolved in an agarose gel stained with ethidium bromide ( n = 3). Lane 1 is molecular weight ladder. Lanes 2–5 are PCR products from total kidney homogenate illustrating B2M, renin, VAMP2, and VAMP3, respectively. Lanes 6–9 correspond to PCR products from freshly isolated JG cells B2M, renin, VAMP2, and VAMP3, respectively. Some lanes not relevant to the current study have been intentionally cut out of the picture.

Article Snippet: Membranes were incubated first in blocking buffer containing 50 m m Tris, 500 m m NaCl, 0.1% Tween 20 (TBS-T), and 5% nonfat dried milk followed by primary antibody (mouse VAMP2 1/2000 or rabbit VAMP3 1/4000; Synaptic Systems) ( – ).

Techniques: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Molecular Weight, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells *

doi: 10.1074/jbc.M111.225839

Figure Lengend Snippet: Real-time PCR of SNAREs in JG cells ΔΔCt was calculated as ((ΔCt in JG cells) − (ΔCt in total kidney homogenates)), where ΔCt is the threshold value cycle (Ct) of the gene studied − Ct from the internal control B2M. –Fold enrichment factor of genes in JG cells versus total kidney was calculated as 2 −ΔΔCt as previously reported ( 51 ). SNAP25 was not included in the table, as no amplification was detected. Values are the mean ± S.E.; n = 3. Negative ΔΔCt values (Syntaxin-3 and Munc18b) represent a -fold enrichment of <1, indicating a higher abundance of these genes in total kidney versus JG cells.

Article Snippet: Membranes were incubated first in blocking buffer containing 50 m m Tris, 500 m m NaCl, 0.1% Tween 20 (TBS-T), and 5% nonfat dried milk followed by primary antibody (mouse VAMP2 1/2000 or rabbit VAMP3 1/4000; Synaptic Systems) ( – ).

Techniques: Real-time Polymerase Chain Reaction, Control, Amplification

Journal: The Journal of Biological Chemistry

Article Title: Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells *

doi: 10.1074/jbc.M111.225839

Figure Lengend Snippet: Expression and subcellular localization of VAMP2 and VAMP3 in JG cells. A , a representative Western blot shows expression of VAMP2 (18 kDa) and VAMP3 (11 kDa) in a JG cell lysate. The top panel is VAMP2, and the bottom panel is VAMP3. Lane 1 is brain homogenate (2.5 μg) used as a positive control, and lane 2 is JG cell lysate (7.5 μg) ( n = 4). B , immunofluorescence and confocal microscopy of VAMP2 and renin on a single mouse JG cell are shown. The left panel shows a representative image from five preparations of a single JG cell labeled with an antibody against renin ( green ); 32 confocal slices (z-step 0.3 μm) were stacked into one image projection. Large renin-containing granules that range in size from 0.8 to 1.5 μm can be observed. The middle panel shows VAMP2 labeling ( red ) in the same cell. The right panel shows a merged image illustrating co-localization of renin with VAMP2 as illustrated by a yellow-orange color ( n = 5 different preparations). Bar , 3 μm. C , immunofluorescence and confocal microscopy of VAMP3 and renin on a single mouse JG cell are shown. The left panel shows an individual JG cell immunolabeled with renin ( green ); the middle panel ( red ) is the same JG cell labeled with VAMP3 antibody; the right panel is the merged image. No co-localization was observed between small VAMP3-labeled vesicles and renin granules.

Article Snippet: Membranes were incubated first in blocking buffer containing 50 m m Tris, 500 m m NaCl, 0.1% Tween 20 (TBS-T), and 5% nonfat dried milk followed by primary antibody (mouse VAMP2 1/2000 or rabbit VAMP3 1/4000; Synaptic Systems) ( – ).

Techniques: Expressing, Western Blot, Positive Control, Immunofluorescence, Confocal Microscopy, Labeling, Immunolabeling

Journal: The Journal of Biological Chemistry

Article Title: Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells *

doi: 10.1074/jbc.M111.225839

Figure Lengend Snippet: Internalization and cleavage of VAMP2 and VAMP3 by tetanus toxin. A , tetanus toxin is efficiently internalized in intact JG cells. JG cells were incubated for 19 h with either vehicle ( Control ; left panel ) or FITC-labeled receptor binding domain of tetanus toxin (C-fragment; List Biological Laboratories) ( right panel ). After fixation and mounting, incorporated fluorescence was analyzed by confocal fluorescence imaging. B , representative Western blots show efficient cleavage of VAMP2 and VAMP3 by tetanus toxin. The left panel is VAMP2, and the right panel is VAMP3 protein expression. JG cells were incubated with either vehicle ( left lane in both panels) or tetanus toxin ( right lane in both panels). After treatment, JG cells were lysed, and SDS-PAGE was resolved in 12% polyacrylamide gel and transferred to PVDF membranes, which were subsequently blotted with VAMP2 or VAMP3 antibodies. Note that a decrease in VAMP2 and VAMP3 band intensity reflects efficient cleavage by tetanus toxin. Membranes were re-blotted with an antibody against GAPDH as an internal loading control ( M r ∼ 35). GAPDH signal was not different between groups ( p = ns).

Article Snippet: Membranes were incubated first in blocking buffer containing 50 m m Tris, 500 m m NaCl, 0.1% Tween 20 (TBS-T), and 5% nonfat dried milk followed by primary antibody (mouse VAMP2 1/2000 or rabbit VAMP3 1/4000; Synaptic Systems) ( – ).

Techniques: Incubation, Control, Labeling, Binding Assay, Fluorescence, Imaging, Western Blot, Expressing, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells *

doi: 10.1074/jbc.M111.225839

Figure Lengend Snippet: Effective adenoviral delivery and specific knockdown of VAMP2 and VAMP3 in JG cells. A, left panel , transduction efficiency in JG cells is shown. JG cells were transduced for 24 h with an adenovirus encoding GFP under the control of cytomegalovirus promoter ( Ad-CMV-GFP ). No fluorescence was detected in the control (non-transduced) cells. Right panel , shown is a comparison of cAMP-stimulated response in either non-transduced ( black bar ) or JG cells transduced with Ad-CMV-GFP ( gray bar ). After transduction of JG cells for up to 48 h, cells were serum-starved for 2 h and treated for 1 h with forskolin plus IBMX (10 μ m /0.5 m m ) or vehicle according to description under “Experimental Procedures.” Data show stimulated renin release (forskolin/IBMX stimulated renin release − basal (vehicle-treated) renin release) expressed as a percentage of renin content. Basal renin release values for non-transduced and transduced JG cells were 1.0 ± 0.1 and 0.9 ± 0.3% of renin content, respectively ( n = 4; p = ns). Data are expressed as the mean ± S.E. B , shown is a representative Western blot illustrating effective silencing of VAMP2 ( left panel ). JG cells were transduced with adenoviral particles encoding shRNA for either a scrambled sequence or VAMP2. 28 h after transduction, JG cells were lysed, and SDS-PAGE was resolved in 12% polyacrylamide gel and transferred to PVDF membranes, which were subsequently blotted with VAMP2 antibody. Membranes were reblotted with an antibody against GAPDH as an internal loading control ( M r = ∼35 kDa). The right panel shows quantification of VAMP2 protein expression expressed as a ratio of GAPDH signal ( n = 4). Scrambled shRNA was arbitrarily set to 1. C , shown is a representative Western blot illustrating effective silencing of VAMP3 ( left panel, third lane ) versus scrambled shRNA ( first lane ). Lane 2 shows JG cells transduced with shRNA for VAMP2, which does not decrease VAMP3 expression. The right panel is a quantification of VAMP3 protein expressed as a ratio of GAPDH signal ( n = 3; p < 0.01).

Article Snippet: Membranes were incubated first in blocking buffer containing 50 m m Tris, 500 m m NaCl, 0.1% Tween 20 (TBS-T), and 5% nonfat dried milk followed by primary antibody (mouse VAMP2 1/2000 or rabbit VAMP3 1/4000; Synaptic Systems) ( – ).

Techniques: Knockdown, Transduction, Control, Fluorescence, Comparison, Western Blot, shRNA, Sequencing, SDS Page, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells *

doi: 10.1074/jbc.M111.225839

Figure Lengend Snippet: Effect of adenoviral delivery of short hairpin silencing for VAMP2 and VAMP3 on cAMP-stimulated renin release and total renin content in JG cells. A , forskolin plus IBMX-stimulated renin release is shown in JG cells transduced with scrambled ( black bar ), VAMP2 shRNA ( gray bar ) and VAMP3 shRNA ( striped bar ). After transduction of JG cells for 28 h, cells were serum-starved for 2 h and treated for 1 h with forskolin plus IBMX (10 μ m /0.5 m m ) or vehicle according to the description under “Experimental Procedures.” Data show stimulated renin release (forskolin/IBMX-stimulated renin release − vehicle-treated (basal) renin release) expressed as percentage of renin content. Basal renin release values were not significant different: scrambled shRNA = 0.6 ± 0.1; VAMP2 shRNA = 0.8 ± 0.2; VAMP3 shRNA = 0.7 ± 0.3; ( p = ns). Data are expressed as the mean ± S.E. B , VAMP2 and VAMP3 knockdown does not affect renin content. Total renin content values are corrected by protein concentration (ng of ANGI/h of incubation/mg of protein). Renin content from scrambled shRNA was arbitrarily set to 100. Data are expressed as the mean ± S.E. ( n = 4; p = ns).

Article Snippet: Membranes were incubated first in blocking buffer containing 50 m m Tris, 500 m m NaCl, 0.1% Tween 20 (TBS-T), and 5% nonfat dried milk followed by primary antibody (mouse VAMP2 1/2000 or rabbit VAMP3 1/4000; Synaptic Systems) ( – ).

Techniques: Transduction, shRNA, Knockdown, Protein Concentration, Incubation

Journal: Cellular and Molecular Immunology

Article Title: VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

doi: 10.1038/cmi.2016.46

Figure Lengend Snippet: Expression of VAMP proteins in human antibody-secreting cells. Immunoblots revealing protein levels of vesicle associated membrane protein (VAMP) isoforms in U266 and IM9 cell lines and PCs isolated from human tonsils or from the bone marrow of patients diagnosed with multiple myeloma are shown. Pellets corresponding to 3 × 105 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies. As a positive control, 20 μg of total protein lysate from human brain was used. For band quantifications, see Supplementary Table S1.

Article Snippet: Pellets corresponding to 3 × 10 5 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies.

Techniques: Expressing, Western Blot, Isolation, Positive Control

Journal: Cellular and Molecular Immunology

Article Title: VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

doi: 10.1038/cmi.2016.46

Figure Lengend Snippet: Subcellular co-localization of vesicles containing Igs with VAMP2. (a) U266 cells were examined for co-localization of VAMP2 with IgE using IF confocal microscopy as described in ‘Materials and methods’ section. For VAMP2 detection, a mouse anti-human VAMP2 (1:100) as a primary antibody and a goat anti-mouse Alexa Fluor647 (1:500) as a secondary antibody were used. For IgE detection, direct IF with an anti-human IgE FITC-conjugated antibody (1:200) was used. A merged image with differential interference contrast (DIC) and a magnified image are shown. (b) U266 cells were examined for co-localization of VAMP2 with several subcellular markers. For LAMP1 (1:200), Rab9 (1:100) and catalase (1.800), rabbit primary antibodies and a goat anti-rabbit Alexa Fluor488-conjugated (1:500) as a secondary antibody were used. Overlapping images (merge) and DIC are shown in the right panels. Scale bars (10 μm) are included.

Article Snippet: Pellets corresponding to 3 × 10 5 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies.

Techniques: Confocal Microscopy

Journal: Cellular and Molecular Immunology

Article Title: VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

doi: 10.1038/cmi.2016.46

Figure Lengend Snippet: Subcellular localization of vesicle associated membrane protein (VAMP) isoforms. U266, IM9 and MMPC cells were analyzed by indirect IF with anti-VAMP2 (R&D, 0.5 μg/μl), anti-VAMP3 (Abcam, 1:100), anti-VAMP4 (SySy 1:50), anti-VAMP5 (Sigma-Aldrich 1:100), anti-VAMP7 (Covalab 1:100) and anti-VAMP8 (SySy 1:50) antibodies. As secondary antibodies, an appropriated anti-mouse or an anti-rabbit Alexa-488 conjugated antibody was used. A representative image of the cellular distribution of each VAMP isoform is shown. Differential interference contrast merged images are shown. All images were obtained with a Leica TCS-SP confocal microscope.

Article Snippet: Pellets corresponding to 3 × 10 5 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies.

Techniques: Microscopy

Journal: Cellular and Molecular Immunology

Article Title: VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

doi: 10.1038/cmi.2016.46

Figure Lengend Snippet: Effects of gene silencing of vesicle associated membrane proteins (VAMPs) on the secretion of IgE by PCs. U266 cells were transiently nucleofected with 100 μM siRNA against each VAMP isoform and small interfering RNA (siRNA) controls as described in ‘Material and methods’ section. Two days after transfection, an equal number of cells (5 × 105) for each condition were placed in a 24-well plate, cultured for 24 h and then the pellet and the supernatant were collected and analyzed by WB and ELISA, respectively. (a) The efficiency of VAMP knockdown was determined using assays examining the cell pellets by WB followed by specific band quantification. The numbers under the bands indicate the mean±s.e.m. (n=4) of the remaining protein compared with the control siRNA used. (b) Quantification of IgE secretion from U266 cells. The IgE content in the supernatants from cultured VAMP siRNA-silenced cells after transfection with targeting siRNA or siControl was measured by ELISA. The relative level of IgE secretion for each targeting siRNA was compared with the non-targeting siRNA control. The number of independent experiments performed for each condition was n=12 for siControl and VAMP2, n=5 for VAMP5, n=4 for STX4 and n=9 for VAMP3, −4, −7 and −8. ANOVA and the Tukey post-hoc test were applied. Data represent the mean±s.e.m., and the asterisk denotes *P<0.05.

Article Snippet: Pellets corresponding to 3 × 10 5 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies.

Techniques: Small Interfering RNA, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

doi: 10.1038/cmi.2016.46

Figure Lengend Snippet: In vitro co-localization of VAMP2 with vesicles containing IgM in RPMI-1788 cells. (A) Supernatants or purified vesicle fractions from the IgM-secreting cells, RPMI-1788, were subjected to dot-blot assays. An IgM gradient (1-100 μg) was used as a positive control. (B) IgM is contained in vesicles. Osmotically unlysed (b) or lysed (c) vesicle fractions were laid on poly-lysine treated coverslips, probed with a goat anti-human IgM antibody and analyzed with a rabbit anti-goat Dyligth-800 antibody followed by a goat anti-rabbit tertiary antibody, Alexa-568, and visualized by IF microscopy. As a negative control (a), no primary antibody was added to the reaction. The results show that IgM could be only detected in the lysed vesicles. (C) Purified and lysed vesicle fractions were laid on poly-lysine coverslips and tested for co-labeling of VAMP2 and IgM. For VAMP2, (a) a mouse anti-human VAMP2 primary antibody and a goat anti-mouse Alexa Fluor488 conjugate as a secondary antibody was used. For IgM detection (b), goat anti-human IgM as a primary antibody and a rabbit anti-goat Dyligth-800 antibody as a secondary antibody followed by goat anti-rabbit Alexa-568 was used. For the negative control (c), no primary antibody was added to the reaction. For co-localization (d,e), a single coverslip preparation was incubated with both primary and secondary/tertiary antibody pairs described in a and b. An overlay image is shown (f), and arrows indicate some of the colocalizations. All images were acquired with a 10 × objective fluorescence microscope (Zeiss ISC 25).

Article Snippet: Pellets corresponding to 3 × 10 5 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies.

Techniques: In Vitro, Purification, Dot Blot, Positive Control, Microscopy, Negative Control, Labeling, Incubation, Fluorescence

Journal: Cellular and Molecular Immunology

Article Title: VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

doi: 10.1038/cmi.2016.46

Figure Lengend Snippet: Effect on IgE secretion using Tetanus toxin light chain overexpression. Positive transfected U266 cells with an empty pIRES2-EGFP or with TeTN-LC-pIRES2-EGFP constructs were isolated by FAC sorting. (a) Similar numbers (5x105) of isolated cells were then cultured for 24 h, and the level of IgE secreted into the medium was measured by ELISA. Empty vector transfected cells were used as a control. Data represent the mean±s.e.m. (n=4), Mann–Whitney test, *P<0.05. (b) Cell pellets were used to confirm that TeTN-LC was functional and proteolyzed the VAMP2 and VAMP3 isoforms. VAMP7 and β-actin served as TeTN-LC-insensitive and loading controls, respectively. The right panel shows an overexposure of the left panel with arrows indicating residual VAMP2 and VAMP3 bands. (c) IgE content in the supernatants or in the cell pellet (d) from each cell transfected condition (empty vector=pIRES2-EGFP, STX4=siRNA STX4, Tx=TeTN-LC-pIRES2-EGFP, and combinations of Tx+siRNA indicated) were measured by ELISA. Data represent the mean±s.e.m. (n=4), Mann–Whitney test, *P<0.05.

Article Snippet: Pellets corresponding to 3 × 10 5 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies.

Techniques: Over Expression, Transfection, Construct, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, MANN-WHITNEY, Functional Assay

Journal: Cellular and Molecular Immunology

Article Title: VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

doi: 10.1038/cmi.2016.46

Figure Lengend Snippet: Effect on IgE secretion using VAMP2 dominant-negative overexpression. Positive transfected U266 cells with an empty vector expressing mRUBY, VAMP2-mRUBY or VAMP2-ΔTMD-mRUBY were isolated by FAC sorting. (a) Representative confocal microscopy images of the subcellular distribution of the overexpressed proteins. (b) Similar numbers of isolated cells (5 × 105) where then cultured for 24 h, and the level of IgE secreted into the medium was measured by ELISA. Ruby transfected cells were used as a control. Data represent the mean±s.e.m. (n=7), Mann–Whitney test, *P<0.05. (c) Transfected cells were analyzed after intracellular staining for IgE. A representative histogram showing the mean fluorescence intensity (MFI) for each transfected construct is shown. Data represent the mean±s.e.m. (n=4), U-Mann–Whitney test, *P<0.05.

Article Snippet: Pellets corresponding to 3 × 10 5 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies.

Techniques: Dominant Negative Mutation, Over Expression, Transfection, Plasmid Preparation, Expressing, Isolation, Confocal Microscopy, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining, Fluorescence, Construct

Journal: Cellular and Molecular Immunology

Article Title: VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

doi: 10.1038/cmi.2016.46

Figure Lengend Snippet: VAMP2 interactions with STX4 and SNAP23. (a) Duolink in situ proximity ligation assay was performed on U266 cells. Rabbit anti-human STX4 and mouse anti-human VAMP2 were used as primary antibodies to detect complexes. Omission of the primary antibodies served as a nonspecific negative control. As a positive control, rabbit and mouse anti-VAMP2 antibodies were used with the same preparation. Cells were visualized using differential interference contrast microscopy (left column), confocal fluorescence microscopy (IF, middle column), and the merge of both (right column). Arrows indicate spots located in plasma membranes. (b) Quantification of spots/cell obtained from 10 cells for each condition. U-Mann–Whitney test, *P<0.05. (c) Co-IP studies showing the interactions between VAMP2, SNAP23 and STX4. Total U266 cell lysates were immunoprecipitated with a nonspecific antibody isotype as a negative control, a specific antibody against VAMP2 or against STX4 bound to protein G-magnetic beads. Total lysates before (LB) and after (LA) immunoprecipitation and bound eluted (BE) samples were resolved by SDS–PAGE and analyzed by western blotting (WB) with the indicated antibodies.

Article Snippet: Pellets corresponding to 3 × 10 5 cell per lane were resolved and analyzed with anti-β-actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti-VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies.

Techniques: In Situ, Proximity Ligation Assay, Negative Control, Positive Control, Microscopy, Fluorescence, MANN-WHITNEY, Co-Immunoprecipitation Assay, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot

Journal: Journal of Alzheimer's disease : JAD

Article Title: Methionine sulfoxide reductase-B3 (MsrB3) protein associates with synaptic vesicles and its expression changes in the hippocampi of Alzheimer’s disease patients

doi: 10.3233/JAD-170459

Figure Lengend Snippet: MsrB3 immuno-gold labeled synaptic vesicles in rat CA3 (A) and CA1 (B). In rat CA3 immunohistofluorescence (C) MsrB3 punctate signal outlined pyramidal somata and co-localized with vesicle associated membrane protein 2 (VAMP2) (D-F) and with vesicular glutamate transporter type 1 (VGLUT1) (G-I). Scale bar A, B = 300 nm, C = 800 μm, F, I = 30 μm.

Article Snippet: Immunohistofluorescence (IF) We examined MsrB3 localization in comparison to synaptic markers and hippocampal transmitters using mouse anti-VAMP2 monoclonal antibody (1:50-1:100, AM05628PU-S, Acris Antibodies) and mouse anti-VGLUT1 monoclonal antibody (1:50, #135311, Synaptic Systems) in hippocampal sections from a well-perfused rat brain.

Techniques: Labeling, Immunohistofluorescence