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: International Journal of Molecular Sciences

Article Title: The Diffusion Model of Intra-Golgi Transport Has Limited Power

doi: 10.3390/ijms24021375

Figure Lengend Snippet: Reagents.

Article Snippet: Rabbit polyclonal antibody against VAMP3 , ThermoFisher Scientific, Milan, Italy , PA5-116218.

Techniques: Recombinant, Transfection, Modification, Plasmid Preparation