mouse anti vapa mab5820  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

doi: 10.1073/pnas.1805757115

Figure Lengend Snippet: Kv2 channels impact localization of VAPA and VAPB in HEK cells. (A) VAPA-GFP expressed alone displays uniform localization across the ER. (B) VAPA-GFP expressed with Kv2.1-loopBAD redistributes to Kv2.1-induced ER/PM junctions. (C) VAPA expressed with the ER/PM junction forming protein, JPH4, does not redistribute to junctions. (D) Kv2.2 coexpressed with VAPB-GFP redistributes this VAP to the induced ER/PM junctions. (E) VAPA(K87D/M89D) has a reduced ability to redistribute to Kv2.1-induced ER/PM junctions. (Scale bars: 5 µm.) (F) Bar graph summarizing VAP redistribution by calculating the ratio of fluorescence at ER/PM junctions to that at ER deeper within the cell when junctions are formed using Kv2 and/or JPH4 as indicated. Only Kv2.1 and Kv2.2 increase the ratio of VAP fluorescence. For analysis, a log transformation was used to satisfy the homogeneity of variance condition and a one-way ANOVA was performed, F(8, 36) = 25.699, P = 1.106 × 10−12 with post hoc pairwise Tukey’s tests. *P < 0.0001, significant difference relative to the dsRedER control,. #P < 0.01 significance. Error bars represent SEM. Twenty-five ROIs from five cells were examined in each case.

Article Snippet: Incubation with anti-VAPA and -VAPB mouse antibodies (1:1,000 and 1:2,000 dilutions, MAB5820 and MAB58551, respectively; R&D Systems) followed by HRP-conjugated goat anti-mouse antibody and detection with SuperSignal West Dura (product 34075; Thermo Scientific) were used to assess VAP expression in the presence of various siRNAs.

Techniques: Fluorescence, Transformation Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

doi: 10.1073/pnas.1805757115

Figure Lengend Snippet: FRET between Kv2.1 and both VAPs in transfected HEK cells. (A) Representative images of donor, acceptor, and FRET efficiency between the indicated constructs. FRET efficiency magnitude is illustrated by the representative heat maps. (Scale bars: 5 μm.) (B) Quantified FRET efficiency. Here the FRET signals were standardized to that obtained with the linked Clover-Ruby2 positive control. Positive controls are indicated by the black bars, negative controls are in light gray, and the Kv2.1/VAP interactions are in darker gray. A one-way ANOVA was performed, F(5, 481) = 195.7, P = 1.81 × 10−133 with post hoc Tukey’s tests to examine significance. *P < 0.000001, significant difference relative to the unlinked negative control. Error bars represent SEM. n = 109 linked, 104 VAPA, 76 VAPA (mutant), 48 VAPB, 75 Kv2.1, and 58 unlinked cells. Each cell had 15 ROIs examined.

Article Snippet: Incubation with anti-VAPA and -VAPB mouse antibodies (1:1,000 and 1:2,000 dilutions, MAB5820 and MAB58551, respectively; R&D Systems) followed by HRP-conjugated goat anti-mouse antibody and detection with SuperSignal West Dura (product 34075; Thermo Scientific) were used to assess VAP expression in the presence of various siRNAs.

Techniques: Transfection, Construct, Positive Control, Negative Control, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

doi: 10.1073/pnas.1805757115

Figure Lengend Snippet: Effect of siRNA-mediated knockdown of VAPA and VAPB on Kv2.1 clustering. (A) Western blot demonstrating efficacy of VAPA and VAPB siRNA. Protein blot was probed with anti-VAPB antibody which cross-reacts with VAPA. (B) Representative image of GFP-Kv2.1-loopBAD clustering in the presence of scrambled or VAPA and VAPB siRNA taken via spinning-disk microscopy. z-stack maximum intensity projections are shown. In Upper Right image all three cells (a–c) were scored as having clustered Kv2.1. In the Lower Right image only cell i was scored as having clusters. Note that this image is presented simply to illustrate how clustering was defined as opposed to being quantitative with respect to the effect of the siRNA treatment. (Scale bars: 5 µm.) (C) Quantification of the percentage of cells displaying Kv2.1 clustering after various siRNA treatments. Eighty-six cells receiving the scrambled siRNA, 90 cells receiving VAPA siRNA, 61 cells receiving VAPB siRNA, and 144 cells receiving both VAPA and VAPB siRNA were examined within 26, 27, 21, and 41 images, respectively. Error bars represent SEM. For analysis, a one-way ANOVA was performed, F(3, 111) = 13.61, P = 1.27 × 10−7, with post hoc Tukey’s tests. *P < 0.01 significance.

Article Snippet: Incubation with anti-VAPA and -VAPB mouse antibodies (1:1,000 and 1:2,000 dilutions, MAB5820 and MAB58551, respectively; R&D Systems) followed by HRP-conjugated goat anti-mouse antibody and detection with SuperSignal West Dura (product 34075; Thermo Scientific) were used to assess VAP expression in the presence of various siRNAs.

Techniques: Knockdown, Western Blot, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

doi: 10.1073/pnas.1805757115

Figure Lengend Snippet: Kv2 interaction with VAPs in rat hippocampal neurons. (A) Coexpression of Kv2.1-loopBAD and VAPA-GFP. Surface Kv2.1-loopBAD was visualized with CF640-conjugated streptavidin. (B) Coexpression of GFP-Kv2.2 and VAPB-mRuby2. (C–E) Colocalization of Kv2.1-loopBAD, GFP-Kv2.2, and the CD4-Kv2.1:573–589 chimera with VAPA-GFP, VAPB-mRuby2, and VAPA-GFP, respectively, within the AIS. The AIS was confirmed with anti-neurofascin antibody staining as illustrated in SI Appendix, Fig. S2. (Scale bars: 5 μm.) (F) Summary of the percentage of neurons concentrating VAPs at induced ER/PM junctions. Error bars indicate SEM. P values comparing the interaction of the first three CD4-Kv2.1 chimeras with either VAPA or VAPB to WT Kv2.1 were not significant, with Kruskal–Wallis ANOVA values of P = 0.37 and P = 0.1, respectively.

Article Snippet: Incubation with anti-VAPA and -VAPB mouse antibodies (1:1,000 and 1:2,000 dilutions, MAB5820 and MAB58551, respectively; R&D Systems) followed by HRP-conjugated goat anti-mouse antibody and detection with SuperSignal West Dura (product 34075; Thermo Scientific) were used to assess VAP expression in the presence of various siRNAs.

Techniques: Staining

Journal: European journal of neurology

Article Title: Expression of vesicle-associated membrane-protein-associated protein B cleavage products in peripheral blood leukocytes and cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis.

doi: 10.1111/ene.12334

Figure Lengend Snippet: Figure 2 Expression of VAPB and VAPA proteins in PBL and CSF from healthy (HC) and neurological (NC) controls. (a), (b), (d) Representative immunoblots showing the expression pattern of VAPB (a), (b) and VAPA (d) identified in PBL and CSF. (c) Peptide competition experiments to verify specificity of the anti- VAPB antibody. Western blots were carried out with (a) 100 lg or (b)–(d) 65 lg proteins of extracts. Differences in the molecu- lar sizes of VAPB and VAPA bands are reported in leucocytes. VAPB and VAPA exhibit two close bands that approximate the molecular size of 17 kDa. In CSF, only the 14 kDa band of VAPB was identified in most samples examined, and no VAPA product was detected; in rare instances, a 24 kDa band of VAPB (asterisk) was additionally detected.

Article Snippet: In some experiments, a VAPA mouse anti-human monoclonal antibody (1–132 amino acids, aa) from R&D Systems (Minneapolis, MN, USA; Clone # 604101; 1:400) was used.

Techniques: Expressing, Western Blot