Journal: The Journal of Neuroscience

Article Title: Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

doi: 10.1523/JNEUROSCI.0893-18.2018

Figure Lengend Snippet: Antibody information

Article Snippet: For the three VAPA-specific mAbs, N479/12, N479/22, and N479/24, we saw a reduction of the immunolabeling signal in the VAPA KO cells to levels approaching those of subclass-matched irrelevant primary and no primary control levels and, for the VAPA/B mAb N479/107, a smaller but still substantial reduction of signal in the VAPA KO cells. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Immunogen Manufacturer information Concentration used Figure(s) N479/12 Fusion protein aa 1-219 of rat VAPA Mouse IgG1 mAb, NeuroMab, RRID: AB_2722707 Tissue culture supernatant, 1:5 A–F , N479/22 Fusion protein aa 1-219 of rat VAPA Mouse IgG2a mAb, NeuroMab, RRID: AB_2722708 Tissue culture supernatant, neat , 1:5 , 1:10 ( ) G–J , N479/24 Fusion protein aa 1-219 of rat VAPA Mouse IgG2a mAb, RRID: AB_2722709 Tissue culture supernatant, 1:5 N479/107 Fusion protein aa 1-219 of rat VAPA Mouse IgG2b mAb, NeuroMab, RRID: AB_2722711 Tissue culture supernatant, 1:5 , KC Synthetic peptide aa 837-853 of rat Kv2.1 Rabbit pAb, in-house (Trimmer Laboratory), RRID: AB_2315767 Affinity purified, 1:150 , , K89/34 Synthetic peptide aa 837-853 of rat Kv2.1 Mouse IgG1 mAb, NeuroMab catalog #73-014, RRID: AB_10672253 Tissue culture supernatant, 1:10 34C Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Thermo Fisher catalog #MA3-925 RRID: AB_2254138 Purified, 1 μg/ml 34C Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Developmental Studies Hybridoma Bank, RRID: AB_528457 Concentrated tissue culture supernatant, 1 μg/ml N106/36 Full-length recombinant human ankG Mouse IgG2b mAb, NeuroMab catalog #75-147, RRID: AB_10675130 Tissue culture supernatant, 1:5 N372B/1 Fusion protein aa 717-907 of rat Kv2.2 long isoform Mouse IgG1 mAb, NeuroMab catalog #73-369, RRID: AB_2315869 Tissue culture supernatant, 1:2 , N372B/60 Fusion protein aa 717-907 of rat Kv2.2 long isoform Mouse IgG2a mAb, NeuroMab catalog #73-360, RRID: AB_2315867 Purified, 10 μg/ml , , Kv1.5E Synthetic peptide aa 271-284 of rat Kv1.5 Rabbit pAb, in-house (Trimmer Laboratory) RRID: AB_2722698 Affinity purified, 10 μg/ml 2–2.20.14, DyLight 488 HA peptide YPYDVPDYA Mouse IgG1 mAb, Thermo Fisher Scientific catalog #26183-D488; RRID: AB_2533051 Purified, 0.3 μg/ml Open in a separate window Antibody information fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A .

Techniques: Concentration Assay, Affinity Purification, Purification, Recombinant

Journal: The Journal of Neuroscience

Article Title: Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

doi: 10.1523/JNEUROSCI.0893-18.2018

Figure Lengend Snippet: CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A. Representative TIRF images of SEP-Kv2.1 expression in a live WT RAW264.7 cell expressing SEP-Kv2.1 (green is cell surface pHluorin fluorescence, magenta is total cellular mCherry fluorescence). Scale bar is 5 μm and holds for all panels in figure. B, Representative TIRF images of GFP-Kv2.1 (green) and BFP-SEC61β (magenta) in live WT (left) or VAPA KO (right) RAW264.7 cells coexpressing GFP-Kv2.1 and BFP-SEC61β. Note the reduction in clustered Kv2.1 expression in the VAPA KO cell. C, Line scan analysis of selection indicated in merged image of WT RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. D, Line scan analysis of selection indicated in merged image of VAPA KO RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. E, Representative TIRF image of live WT (left) or VAPA KO (right) RAW264.7 cells expressing GFP-Kv1.4. F, Summary graph of CV measurements of GFP-Kv2.1 in WT and VAPA KO RAW264.7 cells (****p = 6.648 × 10−9, n = 29 cells, two-tailed unpaired t test). G, Summary graph of CV measurement of GFP-Kv1.4 in WT and VAPA KO RAW264.7 cells (ns, 0.5603, n = 10 cells, two-tailed unpaired t test). H-K. Images of WT (left) or VAPA KO (right) RAW264.7 cells used for high-content analysis of immunolabeling (magenta) and with Hoechst labeling of nuclei (green). H, No primary (anti-IgG2b secondary antibody). I, Positive control anti-mortalin mAb N52A/42 (IgG1). J, Anti-VAPA mAb N479/22 (IgG2a). K, Anti-VAPA/B mAb N479/107 (IgG2b). Scale bar in H is 65 μm and holds for H–K. L, Mean immunolabeling intensity of all samples across 1300–2800 individual cells in each sample. See Materials and Methods for further details of these antibody validation results.

Article Snippet: For the three VAPA-specific mAbs, N479/12, N479/22, and N479/24, we saw a reduction of the immunolabeling signal in the VAPA KO cells to levels approaching those of subclass-matched irrelevant primary and no primary control levels and, for the VAPA/B mAb N479/107, a smaller but still substantial reduction of signal in the VAPA KO cells. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Immunogen Manufacturer information Concentration used Figure(s) N479/12 Fusion protein aa 1-219 of rat VAPA Mouse IgG1 mAb, NeuroMab, RRID: AB_2722707 Tissue culture supernatant, 1:5 A–F , N479/22 Fusion protein aa 1-219 of rat VAPA Mouse IgG2a mAb, NeuroMab, RRID: AB_2722708 Tissue culture supernatant, neat , 1:5 , 1:10 ( ) G–J , N479/24 Fusion protein aa 1-219 of rat VAPA Mouse IgG2a mAb, RRID: AB_2722709 Tissue culture supernatant, 1:5 N479/107 Fusion protein aa 1-219 of rat VAPA Mouse IgG2b mAb, NeuroMab, RRID: AB_2722711 Tissue culture supernatant, 1:5 , KC Synthetic peptide aa 837-853 of rat Kv2.1 Rabbit pAb, in-house (Trimmer Laboratory), RRID: AB_2315767 Affinity purified, 1:150 , , K89/34 Synthetic peptide aa 837-853 of rat Kv2.1 Mouse IgG1 mAb, NeuroMab catalog #73-014, RRID: AB_10672253 Tissue culture supernatant, 1:10 34C Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Thermo Fisher catalog #MA3-925 RRID: AB_2254138 Purified, 1 μg/ml 34C Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Developmental Studies Hybridoma Bank, RRID: AB_528457 Concentrated tissue culture supernatant, 1 μg/ml N106/36 Full-length recombinant human ankG Mouse IgG2b mAb, NeuroMab catalog #75-147, RRID: AB_10675130 Tissue culture supernatant, 1:5 N372B/1 Fusion protein aa 717-907 of rat Kv2.2 long isoform Mouse IgG1 mAb, NeuroMab catalog #73-369, RRID: AB_2315869 Tissue culture supernatant, 1:2 , N372B/60 Fusion protein aa 717-907 of rat Kv2.2 long isoform Mouse IgG2a mAb, NeuroMab catalog #73-360, RRID: AB_2315867 Purified, 10 μg/ml , , Kv1.5E Synthetic peptide aa 271-284 of rat Kv1.5 Rabbit pAb, in-house (Trimmer Laboratory) RRID: AB_2722698 Affinity purified, 10 μg/ml 2–2.20.14, DyLight 488 HA peptide YPYDVPDYA Mouse IgG1 mAb, Thermo Fisher Scientific catalog #26183-D488; RRID: AB_2533051 Purified, 0.3 μg/ml Open in a separate window Antibody information fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A .

Techniques: CRISPR, Expressing, Fluorescence, Selection, Two Tailed Test, High Content Screening, Immunolabeling, Labeling, Positive Control, Biomarker Discovery