rabbit anti na v 1 9 antibody  (Alomone Labs)

Journal: Pain

Article Title: Protein arginine methyltransferase 7 modulates neuronal excitability by interacting with Na V 1.9

doi: 10.1097/j.pain.0000000000002421

Figure Lengend Snippet: Yeast two-hybrid screen to identify proteins that interacted with Na V 1.9. (A) Schematic structure of the Na V 1.9 channel. Residues 402 to 570 of mNa V 1.9 were used as bait for the yeast two-hybrid (Y2H) screen. (B) Location of the repeat-independent clones of PRMT7 identified by Y2H (blue lines, residues 594-692). The 2 domains (pink/green) and degrees of homology between full-length mouse and human PRMT7 are shown. (C) Residues 594 to 692 of mouse PRMT7 were purified from yeast clones for direct Y2H to confirm the interaction on QDO medium. (D) Amino acid sequence comparison of hLoop1 and mLoop1 using ClustalW (EMBL–European Bioinformatics Institute). Potential PRMT7 methylation motif, RXR, is boxed. PRMT7, protein arginine methyltransferase 7; QDO, quadruple dropout.

Article Snippet: Proteins were detected using specific antibodies: mouse anti-GST antibody (AE001, ABclonal, Wuhan, China), mouse anti-HA antibody (AE008, ABclonal), mouse anti-GFP antibody (AE012, ABclonal), rabbit anti-PRMT7 (A12159, ABclonal), mouse anti-FLAG antibody (M185-3 L, MBL, Tokyo, Japan), rabbit anti-Na V 1.9 antibody (ASC-017, Alomone Labs, Jerusalem, Israel), rabbit anti-methyl (mono) arginine antibody (ICP0801, ImmuneChem, Burnaby, Canada), control mouse immunoglobulin G (IgG), and rabbit IgG (B900620, 30000-0-AP, ProteinTech, Wuhan, China).

Techniques: Two Hybrid Screening, Clone Assay, Purification, Sequencing, Methylation

Journal: Pain

Article Title: Protein arginine methyltransferase 7 modulates neuronal excitability by interacting with Na V 1.9

doi: 10.1097/j.pain.0000000000002421

Figure Lengend Snippet: Interaction and coexpression of Na V 1.9 with PRMT7. Interaction between hLoop1 and full-length hPRMT7 was analysed by GST pull-down assays (A) and reciprocal coimmunoprecipitation assays (B). (C) Interaction between the intracellular domains of hNa V 1.9 and hPRMT7 was examined by coimmunoprecipitation assays. (D) Interaction of mPRMT7 with mNa V 1.9 in mouse DRG tissues was assessed. Scn11a +/+ or Scn11a −/− mouse DRG tissue lysates were immunoprecipitated using anti-mPRMT7 antibodies and control IgG. The precipitates were immunoblotted with the indicated antibodies. (E) Immunohistochemical analysis of mPRMT7/mNa V 1.9 expression in mouse DRG sections. The sections were stained for mPRMT7 (red) and mNa V 1.9 (green) and DAPI (blue). mPRMT7 showed considerable colocalization with mNa V 1.9 in mouse DRG neurons. Scale bars, 50 μm. (F) Percentages of mPRMT7-positive and mNa V 1.9-positive neurons are shown. DAPI, 4,6-diamidino-2-phenylindole; DRG, dorsal root ganglion; GST, glutathione S-transferase; IgG, immunoglobulin G; hNa V 1.9, human hNa V 1.9; PRMT7, protein arginine methyltransferase 7.

Article Snippet: Proteins were detected using specific antibodies: mouse anti-GST antibody (AE001, ABclonal, Wuhan, China), mouse anti-HA antibody (AE008, ABclonal), mouse anti-GFP antibody (AE012, ABclonal), rabbit anti-PRMT7 (A12159, ABclonal), mouse anti-FLAG antibody (M185-3 L, MBL, Tokyo, Japan), rabbit anti-Na V 1.9 antibody (ASC-017, Alomone Labs, Jerusalem, Israel), rabbit anti-methyl (mono) arginine antibody (ICP0801, ImmuneChem, Burnaby, Canada), control mouse immunoglobulin G (IgG), and rabbit IgG (B900620, 30000-0-AP, ProteinTech, Wuhan, China).

Techniques: Immunoprecipitation, Immunohistochemical staining, Expressing, Staining

Journal: Pain

Article Title: Protein arginine methyltransferase 7 modulates neuronal excitability by interacting with Na V 1.9

doi: 10.1097/j.pain.0000000000002421

Figure Lengend Snippet: Protein arginine methyltransferase 7 affects the current density of Na V 1.9 by promoting its accumulation on the cell membrane. (A) Representative whole-cell sodium currents evoked by voltage from Scn11a −/− mouse DRG neurons electroporated with SCN11A or SCN11A and PRMT7 . (B) Current–voltage relationships in the indicated experimental groups. The peak current density (normalized by membrane capacitance) was statistically analysed (mock, n = 15; hNa V 1.9, n = 25; and hNa V 1.9 + hPRMT7, n = 23), and significant differences were tested by two-way ANOVA, followed by a post hoc Bonferroni test; infection × voltage: F(32, 948) = 5.846, P < 0.0001; infection: F(2, 948) = 66.28, P < 0.0001; voltage: F(15, 948) = 19.39, P <0.0001; * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the mock group. (C) Representative whole-cell mNa V 1.9 currents evoked by voltage from Scn11a +/+ mouse small DRG neurons treated without (control) or with DS-437. (D) Quantification of the peak mNa V 1.9 current density in the experimental and control groups (control, n = 16; 10 μM DS-437, n = 13; 100 μM DS-437, n = 19). Significant differences were tested by two-way ANOVA, followed by a post hoc Bonferroni test; DS-437 × voltage: F(30, 693) = 3.237, P < 0.0001; DS-437: F(2, 693) = 27.49, P < 0.0001; voltage: F(15, 693) = 79.98, P < 0.0001; *** P < 0.001 compared with the control. (E) Schematic representation of the chimeric proteins. (F) HEK293T cells transiently expressing HA-CD4-hLoop1 and GFP-hPRMT7 or mock-transfected GFP cells were harvested and lysed, the whole-cell lysates (WCLs) were cleared of debris, and the postnuclear supernatant was fractionated into the membrane (Mem) and cytosolic (Cyto) fractions with a kit. Aliquots from all stages of fractionation were analysed by immunoblotting with the indicated antibodies. Na + K + -ATPase was used as a cell-surface–protein control. Quantification of Western blotting data showed that hPRMT7 significantly increased the amount of chimeras in plasma membranes (G) but did not affect the total expression level of the chimeric proteins (H). Data were statistically analysed by the unpaired Student t test; * P < 0.05 compared with GFP. All studies were repeated at least 3 times. DRG, dorsal root ganglion; hNa V 1.9, human Na V 1.9; PRMT7, protein arginine methyltransferase 7.

Article Snippet: Proteins were detected using specific antibodies: mouse anti-GST antibody (AE001, ABclonal, Wuhan, China), mouse anti-HA antibody (AE008, ABclonal), mouse anti-GFP antibody (AE012, ABclonal), rabbit anti-PRMT7 (A12159, ABclonal), mouse anti-FLAG antibody (M185-3 L, MBL, Tokyo, Japan), rabbit anti-Na V 1.9 antibody (ASC-017, Alomone Labs, Jerusalem, Israel), rabbit anti-methyl (mono) arginine antibody (ICP0801, ImmuneChem, Burnaby, Canada), control mouse immunoglobulin G (IgG), and rabbit IgG (B900620, 30000-0-AP, ProteinTech, Wuhan, China).

Techniques: Infection, Expressing, Transfection, Fractionation, Western Blot

Journal: Pain

Article Title: Protein arginine methyltransferase 7 modulates neuronal excitability by interacting with Na V 1.9

doi: 10.1097/j.pain.0000000000002421

Figure Lengend Snippet: Protein arginine methyltransferase 7 modulates Na V 1.9 currents by binding and methylating hLoop1. (A) Schematic representation of one deletion mutant and 4 single-alanine–scanning constructs spanning the region between residues 563 and 572 used in the Y2H assay. The percentage of yeast transformant growth on QDO medium was analysed correspondingly by one-way ANOVA (n = 3). *** P < 0.001, n.s., no significance. (B) Immunoblotting analysis showing the enhanced monomethylation of hLoop1 in hPRMT7-overexpressing cells with the indicated antibodies. (C) Immunoblotting with the indicated antibodies showed decreased MMA levels of hLoop1 in hPRMT7-knockdown cells and increased MMA levels of hLoop1 in hPRMT7-overexpressing cells. (D) Immunoprecipitation of FLAG-tagged wild-type (WT) R519A, R521A, or R519A/R521A hLoop1 proteins expressed in HEK293T cells using anti-FLAG antibodies. The methylation signals and target bands were immunoblotted with the indicated antibodies. (E) Representative whole-cell hNaV1.9 currents evoked by voltage from Scn11a −/− mouse small DRG neurons electroporated with the indicated groups. (F) Current–voltage relationships of the indicated experimental groups. The peak current density (normalized by membrane capacitance) was statistically analysed (hNa V 1.9-4A, n = 14; hNa V 1.9-4A+hPRMT7, n = 12; hNa V 1.9-R519A, n = 10; hNa V 1.9-R519A+hPRMT7, n = 9), and significant differences were tested by two-way ANOVA. ANOVA, analysis of variance; DRG, dorsal root ganglion; hNa V 1.9, human Na V 1.9; MMA, monomethylarginine; PRMT7, protein arginine methyltransferase 7; QDO, quadruple dropout; WCLs, whole-cell lysates; Y2H, yeast two-hybrid.

Article Snippet: Proteins were detected using specific antibodies: mouse anti-GST antibody (AE001, ABclonal, Wuhan, China), mouse anti-HA antibody (AE008, ABclonal), mouse anti-GFP antibody (AE012, ABclonal), rabbit anti-PRMT7 (A12159, ABclonal), mouse anti-FLAG antibody (M185-3 L, MBL, Tokyo, Japan), rabbit anti-Na V 1.9 antibody (ASC-017, Alomone Labs, Jerusalem, Israel), rabbit anti-methyl (mono) arginine antibody (ICP0801, ImmuneChem, Burnaby, Canada), control mouse immunoglobulin G (IgG), and rabbit IgG (B900620, 30000-0-AP, ProteinTech, Wuhan, China).

Techniques: Binding Assay, Mutagenesis, Construct, Y2H Assay, Western Blot, Immunoprecipitation, Methylation

Journal: Pain

Article Title: Protein arginine methyltransferase 7 modulates neuronal excitability by interacting with Na V 1.9

doi: 10.1097/j.pain.0000000000002421

Figure Lengend Snippet: DS-437 reduced DRG neuronal excitability and relieved pain hypersensitivity in Scn11a A796G/A796G mice. (A) Current-clamp responses to a 200-ms depolarizing current of 200 pA in representative Scn11a +/+ mouse DRG neurons and Scn11a A796G/A796G mouse DRG neurons with DS-437 or control. (B) Rheobase, (C) resting membrane potential (RMP), and (D) V threshold (the threshold at which AP takeoff occurs) showed no significant changes in DRG neurons treated with or without DS-437. Data were statistically analysed by one-way ANOVA; * P < 0.05 compared with the control. (E) Comparison of the average spike numbers of repetitive action potentials (APs) fired in response to the 200-ms current injection ranging from 0 to 225 pA in DRG neurons (Na V 1.9-WT + control, n = 21; Na V 1.9-WT + 100 μM DS-437, n = 22; Na V 1.9-KI + control, n = 30; Na V 1.9-KI + 10 μM DS-437, n = 22; Na V 1.9-KI + 100 μM DS-437, n = 24). Significant differences were tested by two-way ANOVA, followed by a post hoc Bonferroni test; DS-437 × current: F(36, 1041) = 2.049, P = 0.0003; DS-437: F(3, 1041) = 50.86, P < 0.0001; current: F(9, 1041) = 74.58, P < 0.0001; ** P < 0.01 and *** P < 0.001 compared with the control. (F) The duration of licking and lifting behaviours in Na V 1.9-KI mice and Na V 1.9-WT in the 45 minutes after intraplantar administration of formalin to the hind paws binned at 5-min intervals. (G) Data from 2 phases of the formalin test are summarized. Phase I: 0 to 10 minutes. Phase II: 10 to 45 minutes (saline, n = 6; DS-437, n = 6). Significant differences were tested by one-way ANOVA; * P < 0.05 and ** P < 0.01 compared with saline. (H) Heat threshold was assessed using the hot-plate test. (I) Mechanical withdrawal threshold was tested by applying von Frey filaments (saline, n = 6; DS-437, n = 6). Significant differences were tested by two-way ANOVA; * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with saline. ANOVA, analysis of variance; DRG, dorsal root ganglion.

Article Snippet: Proteins were detected using specific antibodies: mouse anti-GST antibody (AE001, ABclonal, Wuhan, China), mouse anti-HA antibody (AE008, ABclonal), mouse anti-GFP antibody (AE012, ABclonal), rabbit anti-PRMT7 (A12159, ABclonal), mouse anti-FLAG antibody (M185-3 L, MBL, Tokyo, Japan), rabbit anti-Na V 1.9 antibody (ASC-017, Alomone Labs, Jerusalem, Israel), rabbit anti-methyl (mono) arginine antibody (ICP0801, ImmuneChem, Burnaby, Canada), control mouse immunoglobulin G (IgG), and rabbit IgG (B900620, 30000-0-AP, ProteinTech, Wuhan, China).

Techniques: Injection, Hot Plate Test

Journal: Pain

Article Title: Protein arginine methyltransferase 7 modulates neuronal excitability by interacting with Na V 1.9

doi: 10.1097/j.pain.0000000000002421

Figure Lengend Snippet: Proposed working model of PRMT7-mediated Na V 1.9 trafficking and cellular excitability. The PRMT7 C-terminal domain interacts with residues 563 to 566 of hLoop1 and methylates arginine 519 (R519 me) in this loop using S-adenosyl-L-methionine (AdoMet) as a methyl donor to produce S-adenosylhomocysteine (AdoHcy). Human Na V 1.9; R519 me is involved in the regulation of Na V 1.9 trafficking to the cell surface through an undefined mechanism. Consequently, altered cell surface expression of Na V 1.9 increases sodium current density, leading to hyperexcitability of DRG neurons. DRG, dorsal root ganglion; PRMT7, protein arginine methyltransferase 7.

Article Snippet: Proteins were detected using specific antibodies: mouse anti-GST antibody (AE001, ABclonal, Wuhan, China), mouse anti-HA antibody (AE008, ABclonal), mouse anti-GFP antibody (AE012, ABclonal), rabbit anti-PRMT7 (A12159, ABclonal), mouse anti-FLAG antibody (M185-3 L, MBL, Tokyo, Japan), rabbit anti-Na V 1.9 antibody (ASC-017, Alomone Labs, Jerusalem, Israel), rabbit anti-methyl (mono) arginine antibody (ICP0801, ImmuneChem, Burnaby, Canada), control mouse immunoglobulin G (IgG), and rabbit IgG (B900620, 30000-0-AP, ProteinTech, Wuhan, China).

Techniques: Expressing

Journal: Nature Communications

Article Title: Spider venom-derived peptide induces hyperalgesia in Na v 1.7 knockout mice by activating Na v 1.9 channels

doi: 10.1038/s41467-020-16210-y

Figure Lengend Snippet: a – c Current-clamp recordings show that HpTx1 decreases the membrane excitability of small DRG neurons from Na v 1.9-KO mice. a Bars show no significant changes in RMP (left, n = 29) or AP amplitude (right, n = 25), but a significant increase in rheobase (middle, n = 25, nonparametric Wilcoxon matched-pair signed-rank two-tailed test: P = 0.008) in the presence of 0.75 μM HpTx1. b AP traces recorded from a representative small Na v 1.9-KO DRG neuron before (black) and after (red) application of 0.75 μM HpTx1. The dashed lines indicate 0 mV. c Statistics plots show significant decreases in AP spike number in the presence of 0.75 μM HpTx1 ( n = 25, two-way repeated measures ANOVA followed by Bonferroni’s multiple comparisons test, treatment × inject current: F (7,168) = 8.834, P < 0.0001; treatment: F (1,24) = 25.49, #### P < 0.0001; inject current: F (7,168) = 25.28, P < 0.0001). d Comparison of nocifensive behaviors (licking or biting) following intraplantar injection of vehicle (10 μl 0.9% saline, n = 6) versus HpTx1 (1 μM or 10 μM in 10 μl saline, n = 6) (two-way ANOVA followed by Tukey’s multiple comparisons test, treatment × genotype: F (2,30) = 8.551, P = 0.0012; treatment: F (2,30) = 11.04, P = 0.0003; genotype: F (1,30) = 24.37, P < 0.0001). e Mechanical response thresholds measured in paws in response to vehicle (black circles, n = 6), 1 μM HpTx1 (yellow squares, n = 6) or 10 μM HpTx1 (red triangles, n = 6) injections (two-way ANOVA followed by Tukey’s multiple comparisons test, treatment × genotype: F (2,30) = 18.68, P < 0.0001; treatment: F (2,30) = 0.0356, P = 0.9651; genotype: F (1,30) = 67.3, P < 0.0001). f Latency of WD to noxious heat stimuli measured after intraplantar injection of vehicle (black circles, n = 6), 1 μM HpTx1 (yellow squares, n = 6) or 10 μM HpTx1 (red triangles, n = 6) (two-way ANOVA followed by Tukey’s multiple comparisons test, treatment × genotype: F (2,30) = 44.54, P < 0.0001; treatment: F (2,30) = 9.701, P = 0.0006; genotype: F (1,30) = 113.5, P < 0.0001). All DRG neurons recorded were held at −53 ± 2 mV. Data are presented as the mean ± S.E.M. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P ( c – f ) are presented in Supplementary Data . Source data are provided as a . .

Article Snippet: The sections were permeabilized in PBS containing 0.5% TritonX-100) for 10 min, and were blocked with 10% goat serum for 1 h. The sections were incubated for 24 h at 4 °C with polyclonal rabbit anti-Na v 1.9 (1:200; alomone labs).

Techniques: Two Tailed Test, Injection

Journal: Nature Communications

Article Title: Spider venom-derived peptide induces hyperalgesia in Na v 1.7 knockout mice by activating Na v 1.9 channels

doi: 10.1038/s41467-020-16210-y

Figure Lengend Snippet: a Sequence alignments corresponding to the DIV s3b-s4 region of Na v subtypes. The highlighted sequences show the regions swapped between Na v 1.8 and Na v 1.9. b Representative current traces from Na v 1.9/1.8 DIV s3b-s4 P1 (top) and Na v 1.8/1.9 DIV s3b-s4 P1 (bottom) chimaera channels in the absence (black) and presence (red) of HpTx1. c Effects of HpTx1 on WT and mutant hNa v 1.9 channels. Dot plots display the effect of 0.75 μM HpTx1 on the peak current (top, n = 14 for WT; n = 4 for T1444L, M1445L, I1446F, and T1448A; n = 5 for L1449I and E1450L; n = 3 for N1451K) and the persistent current (bottom, n = 14 for WT; n = 4 for T1444L, I1446F, T1448A, and N1451K; n = 5 for M1445L, L1449I, and E1450L). Key residues involved in the interaction between HpTx1 and hNa v 1.9 are labeled (one-way ANOVA with Dunnett’s multiple comparison test, I 95 /I peak : F (7,35) = 17.72, P < 0.0001; I/I max : F (7,38) = 8.157, P < 0.0001). d (top) Sequence alignments corresponding to the DII s3b-s4 region of Na v subtypes. The highlighted sequences show the regions swapped between Na v 1.7 and Na v 1.8. Representative current traces from Na v 1.7/1.8 DII s3b-s4 (bottom left) and Na v 1.8/1.7 DII s3b-s4 (bottom right) chimaera channels in the absence (black) or presence of 5 μM HpTx1 (red). e Dose-dependent inhibitory curves show the effect of HpTx1 on WT ( n = 7) and mutant hNa v 1.7 channels ( n = 4 for F813S, n = 6 for L814A and A815S, n = 3 for D816K, n = 6 for V817K, n = 7 for E818G, n = 4 for E818R, n = 5 for G819S and n = 3 for Na v 1.7/1.8 DII s3b-s4) and the Na v 1.8/1.7 DII s3b-s4 chimaera channel ( n = 5). f Bars show the fold changes in IC 50 values of HpTx1 for mutant channels compared with that for the WT hNa v 1.7 channel. Data are presented as the mean ± S.E.M. Exact P ( c ) are presented in Supplementary Data . Source data are provided as a . .

Article Snippet: The sections were permeabilized in PBS containing 0.5% TritonX-100) for 10 min, and were blocked with 10% goat serum for 1 h. The sections were incubated for 24 h at 4 °C with polyclonal rabbit anti-Na v 1.9 (1:200; alomone labs).

Techniques: Sequencing, Mutagenesis, Labeling