Journal: The Journal of General Physiology

Article Title: Proteomic and functional mapping of cardiac Na V 1.5 channel phosphorylation sites

doi: 10.1085/jgp.202012646

Figure Lengend Snippet: Phosphorylation sites, phosphopeptides, and site-discriminating ions identified in immunoprecipitated Na V 1.5 proteins from sham and TAC mouse left ventricles using MS

Article Snippet: Magnetic beads were then collected and washed rapidly four times with ice-cold lysis buffer, and isolated protein complexes were eluted from the beads in 1× SDS sample buffer (Bio-Rad Laboratories) at 60°C for 10 min. 99% of the immunoprecipitated mouse left ventricular Na V channel protein complexes were analyzed by MS, and the remaining 1% were used to verify IP yields by Western blotting using a rabbit polyclonal anti-Na V 1.5 antibody (RbαNa V 1.5, 1:1,000, ASC-005; Alomone Laboratories).

Techniques: Immunoprecipitation, Sequencing

Journal: The Journal of General Physiology

Article Title: Proteomic and functional mapping of cardiac Na V 1.5 channel phosphorylation sites

doi: 10.1085/jgp.202012646

Figure Lengend Snippet: Localization and quantification of 42 MS-identified Na V 1.5 phosphorylation sites in mαNa V PAN-IPs from sham and TAC mouse left ventricles (LVs). (A) Schematic representation of phosphorylation sites on the Na V 1.5 protein (UniProt reference sequence K3W4N7 ). Two phosphorylation site locations are possible at amino acids S1056-T1058. (B) The areas of extracted MS1 ion chromatograms, corresponding to MS2 spectra assigning phosphorylated (in red) and nonphosphorylated (in white) Na V 1.5 peptides at indicated phosphorylation site(s), in mαNa V PAN-IPs from sham and TAC LVs are indicated. No red color is visible for the phosphorylated peptide at position T1809, because this phosphopeptide area is very small (area = 80,291 arbitrary unit) relative to the areas of the nonphosphorylated peptides (areas = 80,060,220 arbitrary unit). (C) The areas of extracted MS1 ion chromatograms, corresponding to MS2 spectra assigning phosphorylated peptides at indicated phosphorylation site(s), in mαNa V PAN-IPs from sham and TAC LVs are indicated. The brackets indicate the subgroups of phosphorylation sites analyzed in B. Independent quantification of S459 and S460 phosphorylated peptides was not possible, because localization of the phosphorylation site in most of the phosphorylated peptides could not be discriminated. Similar to B, no red bar is visible for the phosphorylated peptide at position T1809, because this phosphopeptide area is very small (area = 80,291 arbitrary unit), relative to the areas of the other phosphorylated peptides. (D) Distributions and mean ± SEM relative abundances of individual Na V 1.5 phosphopeptides allowing assignments of indicated phosphorylation site(s), as well as of corresponding nonphosphorylated (NP) peptides, in TAC LV ( n = 5, in black) versus sham LV ( n = 4, in white) mαNa V PAN-IPs were obtained using TMT reporter ion intensities. The relative abundances of Na V 1.5 phosphopeptides exhibiting phosphorylation(s) on serine 671 (S671; n = 12 peptides) alone or in combination with serine 664 (S664 + S671; n = 9 peptides) or serine 667 (S667 + S671; n = 7 peptides) are increased (**, P < 0.01; ***, P < 0.001; Mann-Whitney test) in TAC LV versus sham LV mαNa V PAN-IPs. (E) Experimental workflow used in the study. Once immunoprecipitated using the mαNa V PAN antibodies, the Na V channel complexes from sham and TAC mouse LVs were labeled individually with different TMT 10 tags and combined in the same TMT set for multiplexed LC-MS/MS analysis. Na V 1.5 phosphorylation sites were identified, quantified, and analyzed by clusters in whole-cell voltage-clamp recordings in HEK-293 cells.

Article Snippet: Magnetic beads were then collected and washed rapidly four times with ice-cold lysis buffer, and isolated protein complexes were eluted from the beads in 1× SDS sample buffer (Bio-Rad Laboratories) at 60°C for 10 min. 99% of the immunoprecipitated mouse left ventricular Na V channel protein complexes were analyzed by MS, and the remaining 1% were used to verify IP yields by Western blotting using a rabbit polyclonal anti-Na V 1.5 antibody (RbαNa V 1.5, 1:1,000, ASC-005; Alomone Laboratories).

Techniques: Sequencing, MANN-WHITNEY, Immunoprecipitation, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of General Physiology

Article Title: Proteomic and functional mapping of cardiac Na V 1.5 channel phosphorylation sites

doi: 10.1085/jgp.202012646

Figure Lengend Snippet: Proteins identified in immunoprecipitated Na V channel complexes from sham and TAC mouse left ventricles using MS

Article Snippet: Magnetic beads were then collected and washed rapidly four times with ice-cold lysis buffer, and isolated protein complexes were eluted from the beads in 1× SDS sample buffer (Bio-Rad Laboratories) at 60°C for 10 min. 99% of the immunoprecipitated mouse left ventricular Na V channel protein complexes were analyzed by MS, and the remaining 1% were used to verify IP yields by Western blotting using a rabbit polyclonal anti-Na V 1.5 antibody (RbαNa V 1.5, 1:1,000, ASC-005; Alomone Laboratories).

Techniques: Immunoprecipitation, Sequencing

Journal: Basic Research in Cardiology

Article Title: Inhibition of Na V 1.8 prevents atrial arrhythmogenesis in human and mice

doi: 10.1007/s00395-020-0780-8

Figure Lengend Snippet: Expression of Na V 1.8 in the human atrium. Data are presented as scatter plot with mean ± SEM. P values were calculated using unpaired Student's t test. a Original Western blot for Na V 1.8 expression in atrial myocardium from patients with sinus rhythm compared to ventricular myocardium from non-failing donors (NF). b Normalized densitometry data comparing the protein expression of Na V 1.8 in human atria ( n = 6) and human ventricle ( n = 5). GAPDH was used as an internal loading control in all blots. c Normalized mRNA expression of Na V 1.8/GAPDH in human atrial myocardium ( n = 7 patients) compared to ventricular myocardium from healthy subjects ( n = 10 patients). d Original Western Blot for Na V 1.8 and Na V 1.5 protein in human atria from patients with sinus rhythm (SR) or atrial fibrillation (AF). e Normalized densitometry data from Western Blots using atrial myocardium from patients with SR or AF showing the protein expression of Na V 1.5 (SR: n = 14 patients, AF: n = 13) and ( f ) Na V 1.8 (SR: n = 14 patients, AF: n = 14). GAPDH was used as an internal loading control in all blots. g Normalized mRNA expression of Na V 1.5/GAPDH (SR: n = 8 patients, AF: n = 8) and Na V 1.5/GAPDH (SR: n = 8 patients, AF: n = 8) in human atrial myocardium from SR compared to AF

Article Snippet: Mouse monoclonal anti-Na V 1.8 antibodies (1:1,000, LSBio, LS-C109037), rabbit polyclonal anti-Na V 1.5 (1:2,000, Alomone labs, ASC-005), and mouse monoclonal anti-GAPDH (1:20,000, BIOTREND, BTMC-A473-9) were used.

Techniques: Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Cilostazol Ameliorates Peripheral Neuropathic Pain in Streptozotocin-Induced Type I Diabetic Rats

doi: 10.3389/fphar.2021.771271

Figure Lengend Snippet: Dose-dependent recovery of voltage-gated sodium (NaV) channel expression in dorsal root ganglia (DRG) of cilostazol-treated diabetic (DM) rats. Expressions of NaV-1.1, -1.2, -1.3, -1.6, -1.7, and -1.8 in DRG of naïve control, DM, and oral cilostazol (10, 30, and 100 mg/kg) treated DM rats were examined with (A) Western blots and (B) immunofluorescence assays. The western and immunofluorescence data revealed significant upregulation of NaV-1.1–1.7 expressions, and significant downregulation of NaV-1.8 in the DRGs of DM rats. Daily oral cilostazol treatments of 100 mg/kg, but not the lower dosages, for 6 weeks significantly reversed the DM-induced NaV dysregulation in the DRGs. Results are expressed as mean SEM for a minimum of five rats for each group. Statistical significances between the DM control and cilostazol DM treatment groups were calculated by one-way ANOVA analysis followed by the least significant difference test for multiple post hoc analyses. ** p < 0.01, * p < 0.05, *** p < 0.001.

Article Snippet: The filters were blocked with 5% milk in phosphate-buffered saline (PBS) with 0.1% Tween 20 for 1 h at room temperature and incubated for 24 h at 4 C with rabbit anti-rat Navs primary antibodies (Alomone Labs, Jerusalem, Israel) included Nav-1.1 (ASC-001), Nav-1.2 (ASC-002), Nav-1.3 (ASC-004), Nav-1.6 (ASC-009), Nav-1.7 (ASC-008), Nav-1.8 (ASC-016), and mouse anti-rat ß -actin (MilliporeSigma, MAB1501).

Techniques: Expressing, Western Blot, Immunofluorescence