Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

doi: 10.1155/2017/6792694

Figure Lengend Snippet: Structural cores and blood markers of muscle damage (CK and LDH) at 4 months of age. (a–c) Representative histological images of normal fibers (a), fibers with unstructured cores (b), and fibers with contracture cores (c). (d) Percentage of EDL fibers presenting the features classified in (a–c) (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S1. (e and f) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). In (e) and (f), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d), n = number of fibers analyzed; in (e-f), n = number of mice. Scale bar: 10 μm .

Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

doi: 10.1155/2017/6792694

Figure Lengend Snippet: Fiber ultrastructure, calpain activity, and carbonyl protein content at 4 months of age. (a–f) Representative immunofluorescence and EM images showing fluorescent cross striation (a, c, and e) and myofibrillar/sarcomeric organization (b, d, and f) in EDL fibers. In (b) and (f), small arrows point at Z -lines, while in (c and d) large arrows point to areas in which cross striation and sarcomeric structure are compromised. (g) Calpain activity expressed in total hind limb muscle homogenates. (h) Carbonyl protein content in EDL muscle homogenates. In (g and h), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (g and h), n = number of mice. Scale bars in (a, c, and e) 10 μ m (insets 5 μ m); in (b, d, and f) 1 μ m.

Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Activity Assay, Immunofluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

doi: 10.1155/2017/6792694

Figure Lengend Snippet: In vivo grip strength and ex vivo specific force at 4 months of age. (a) Time-course of grip strength from 2 (beginning of short-term NAC treatment) to 4 months (end of short-term NAC treatment) of age expressed as force on body weight (N/g). (b) Change in grip strength from 2 to 4 months of age (shown as a percentage). (c and d) Force-frequency (1–140 Hz) relationship curves of specific force (c) and specific force during a single 2 s, 120 Hz stimulation train (d) recorded for the same EDL muscles. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (b), n = number of mice; in (d), n = number of EDL muscles.

Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: In Vivo, Ex Vivo

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

doi: 10.1155/2017/6792694

Figure Lengend Snippet: Mitochondrial damage, size, and volume at 4 months of age. (a–c) Representative EM images displaying apparently normal (a) and damaged mitochondria (b and c). (d) Percentage of damaged mitochondria. (e) Average number of damaged mitochondria/area of EM section. (f) Average size of apparently normal mitochondria (i.e., mitochondria not included in the quantitative analysis of (d and e)). (g) Percentage of fiber volume occupied by mitochondria. See also Table S2. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d and f), n = number of measurements; in (e and g), n = number of fibers analyzed. Scale bar: 0.1 μ m.

Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

doi: 10.1155/2017/6792694

Figure Lengend Snippet: Effects of long-term NAC treatment at 10 months of age. (a) Quantitative analysis of EDL fibers presenting the features classified in Figures , , and shown as percentage of fibers analyzed (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S4. (b and c) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). (d) Time course of grip strength from 4 (beginning of long-term NAC treatment) to 10 months (end of long-term NAC treatment) of age expressed as force on body weight (N/g). (e) Change in grip strength from 4 to 10 months of age (shown as a percentage). (f and g) Force-frequency (1–140 Hz) relationship curves of specific force (f) and specific force during a single 2 s, 120 Hz stimulation train (g) recorded for the same EDL muscles. In s (b–g), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (a), n = number of EDL fibers analyzed; in (b–e), n = number of mice; in (f and g), n = number of EDL muscles.

Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

doi: 10.1155/2017/6792694

Figure Lengend Snippet: Oxidative stress markers at 4 and 10 months of age following either short- or long-term treatment. (a and e) Representative immunoblots showing expression of 3-Nitrotyrosine (3-NT), SOD1, and SOD2 in total hind limb muscle homogenates. (b–d) and (f–h) Relative band densities normalized to GAPDH levels. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. n = number of mice.

Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Western Blot, Expressing

Journal: Disease Models & Mechanisms

Article Title: Characterization of a novel zebrafish model of SPEG -related centronuclear myopathy

doi: 10.1242/dmm.049437

Figure Lengend Snippet: Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) and desmin (DES; magenta; SPEG 2200-2960 a.a.) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) .

Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

Techniques: Activation Assay, Binding Assay

Journal: Disease Models & Mechanisms

Article Title: Characterization of a novel zebrafish model of SPEG -related centronuclear myopathy

doi: 10.1242/dmm.049437

Figure Lengend Snippet: Speg expression during zebrafish development. There are two Speg genes in zebrafish, spega and spegb . Each encodes a single transcript that is highly conserved with human SPEG . (A,B) RT-qPCR shows similar temporal expression patterns of spega (A) versus spegb (B) from 2 days post-fertilization (dpf) to 7 dpf. Both Speg mRNA transcripts are significantly upregulated in the heads (light blue bars; 20-fold for spega , 7-fold for spegb ), but stay at relatively similar levels in the skeletal-muscle-predominant tails (white bars). Each data point represents the average of technical triplicates, and three independent experiments are included. Columns and error bars represent mean±s.e.m. Unpaired two-tailed Student's t -test was performed: ** P <0.01; ns, not significant. (C) Whole-mount in situ hybridization using DIG-conjugated RNA probes in 1 dpf embryos shows distinct spatial expression patterns of spega versus spegb . spega is predominantly expressed in the developing brain (yellow arrowhead) and along the neural tube (red arrowheads), but absent from the notochord (cyan arrowheads). spegb staining, however, is predominantly detected at the chevron-shaped developing somites (black arrowheads). Scale bars: 200 µm (top and middle rows) or 100 µm (bottom row). (D,E) Confocal images showing 2 dpf (D) or 5 dpf (E) isolated wild-type (WT) skeletal myofibers double-stained with anti-RyR1 (34C, DSHB) and anti-SPEG (PA553875, Invitrogen). (D,D′) At 2 dpf, SPEG is predominantly localized at the sarcolemma (yellow arrows) and perinuclear regions (cyan arrows; nucleus, yellow asterisks), with weak expression in transverse striations (red arrow), whereas RyR1 is localized in transverse striations labeling the terminal SR. Little colocalization (white in Merge) of SPEG (green) and RyR1 (magenta) can be observed at this stage. (E,E′) At 5 dpf, SPEG expression becomes restricted in the transverse striations (red arrows) that overlap with RyR1. Scale bars: 10 µm (D,E); 5 µm (D′,E′).

Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, In Situ Hybridization, Staining, Isolation, Labeling

Journal: Disease Models & Mechanisms

Article Title: Characterization of a novel zebrafish model of SPEG -related centronuclear myopathy

doi: 10.1242/dmm.049437

Figure Lengend Snippet: spega/b deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. (A-D′) IF staining was performed on 2 dpf (A,B,C,D) and 5 dpf (A′,B′,C′,D′) isolated myofibers. Confocal images show disrupted transverse pattern of RyR1 (A,A′), DHPR (B,B′) and SERCA1a (D,D′) in speg -DKO starting from 2 dpf, while α-Actinin (D,D′) is not affected. Scale bars: 50 µm. (E-F′) Electron micrographs of 7 dpf WT and speg -DKO muscles. (E) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum (yellow arrowhead). (E′) Triads in speg -DKO appear structurally disrupted, losing the obvious terminal cisternae of the sarcoplasmic membrane (tSR)/T-tubule/tSR pattern (red arrowhead). (F,F′) More importantly, the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads (yellow arrows). Scale bars: 0.5 µm (E,E′); 1 µm (F,F′). (G) The total number of triads per 60 µm 2 (under an electron microscope) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P <0.05.

Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

Techniques: Staining, Isolation, Microscopy, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis

doi: 10.1074/jbc.RA119.007635

Figure Lengend Snippet: Antibody information

Article Snippet: RRID:AB_2722711 Tissue cultured supernatant (1:5) 6 Nir2 Recombinant fragment of human Nir2 aa 589–651 Rabbit pAb, Abcam catalog no. ab221381, RRID:AB_2801563 10 μg/ml ( A ), 1 μg/ml ( E ) S1 Nir2 Recombinant peptide of human Nir2 aa 365–444 Rabbit pAb, Novus Biologicals catalog no. NBP2–34132, RRID: AB_2801564 10 μg/ml ( A ) 6, S1 RyRs (34C) Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Developmental Studies Hybridoma Bank, RRID: AB_528457 1 μg/ml 6 Open in a separate window Antibody information Immunopurification and proteomic analyses Preparation of cross-linked mouse brain samples for immunopurification was as described previously ( 43 ).

Techniques: Concentration Assay, Affinity Purification, Purification, Western Blot, Cell Culture, 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: 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 Table 1. 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 Table 1. 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