primary antibodies against ncx1  (Alomone Labs)

Journal: Biological Research

Article Title: Inhibition of forward and reverse transport of Ca 2+ via Na + /Ca 2+ exchangers (NCX) prevents sperm capacitation

doi: 10.1186/s40659-024-00535-9

Figure Lengend Snippet: Identification of NCX isoforms. Identification of NCX1, NCX2 and NCX3 in porcine sperm. Representative immunoblots using sperm samples from different boars with A ) anti-NCX1, ( B ) anti-NCX2 and ( C ) anti-NCX3 antibodies, and their respective blocking peptides

Article Snippet: ; Basel, Switzerland) at room temperature and agitation for 1 h. Membranes were subsequently incubated with specific primary antibodies against NCX1 (SLC8A1), NCX2 (SLC8A2), or NCX3 (SLC8A3) (Alomone Labs, Jerusalem, Israel), which were previously diluted in blocking solution at 1:2,000 (v: v), at 4 °C overnight under agitation.

Techniques: Western Blot, Blocking Assay

Journal: Biological Research

Article Title: Inhibition of forward and reverse transport of Ca 2+ via Na + /Ca 2+ exchangers (NCX) prevents sperm capacitation

doi: 10.1186/s40659-024-00535-9

Figure Lengend Snippet: Immunolocalization of NCX1 isoform. Localization of NCX1 in the plasma membrane of porcine sperm ( A – C ), and after the peptide competition assay ( D – F ). NCX1 appears stained in green (Alexa Fluor 488) and nuclei in blue (DAPI; 4′6′-diamidion-2-phenylindole). Scale bar: 15 μm

Article Snippet: ; Basel, Switzerland) at room temperature and agitation for 1 h. Membranes were subsequently incubated with specific primary antibodies against NCX1 (SLC8A1), NCX2 (SLC8A2), or NCX3 (SLC8A3) (Alomone Labs, Jerusalem, Israel), which were previously diluted in blocking solution at 1:2,000 (v: v), at 4 °C overnight under agitation.

Techniques: Clinical Proteomics, Membrane, Competitive Binding Assay, Staining

Journal: Journal of the American Heart Association

Article Title: Sodium‐Glucose Cotransporter 2 Inhibitor Canagliflozin Antagonizes Salt‐Sensitive Hypertension Through Modifying Transient Receptor Potential Channels 3 Mediated Vascular Calcium Handling

doi: 10.1161/jaha.121.025328

Figure Lengend Snippet: Figure 3. Canagliflozin inhibited the vascular contraction and calcium channel-related protein pathways. A, Venn diagram in groups between upregulated proteins of ND vs HS and downregulated proteins of HS vs HS+canagliflozin. B, The KEGG pathways enriched by differentially expressed proteins in groups between upregulated proteins of ND vs HS and downregulated proteins of HS vs HS+canagliflozin. C and D, The vascular-associated biological process terms (GO) enriched by differentially expressed proteins proteins in groups between upregulated proteins of ND vs HS and down-regulated proteins of HS vs HS+canagliflozin. E and F, Representative western blots of TRPC3, NCX1, CamkII, MYPT-1, MLC2, and phosphorylation of MYPT-1 and MLC2 (p-MYPT-1 and p-MLC2) in carotid artery from Dahl salt-sensitive and Dahl salt-insensitive rats. β-actin served as a loading control. n=8. Results are expressed as mean±SEM. Statistical significance was determined by unpaired Student t test for comparisons between 2 groups and 1-way ANOVA followed by Bonferron’’s posttest for multiple comparisons. A 2-tailed Fishe’’s exact test was used in the GO KEGG enrichment analyses. *P<0.05, **P<0.01, ***P<0.001. ECM, extracellular matrix; GO, gene ontology; HS, high-salt diet; KEGG, Kyoto Encyclopedia of Genes and Genomes; NCX1, sodium-calcium exchanger 1; ND, normal diet; SS, Dahl salt-sensitive rats; SS-13BN, Dahl salt-insensitive rats; and TRPC3, transient receptor potential channels 3.

Article Snippet: Primary antibodies against NCX1 were purchased from ProteinTech Group (Chicago, IL) and the primary antibody against TRPC3 was purchased from Alomone (Alomone Labs).

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Journal of the American Heart Association

Article Title: Sodium‐Glucose Cotransporter 2 Inhibitor Canagliflozin Antagonizes Salt‐Sensitive Hypertension Through Modifying Transient Receptor Potential Channels 3 Mediated Vascular Calcium Handling

doi: 10.1161/jaha.121.025328

Figure Lengend Snippet: Figure 4. Canagliflozin regulated calcium influx in vascular smooth muscle cells mediated by TRPC3. A, Representative images of the immunofluorescence staining of TRPC3, NCX1, and DAPI using mesenteric arteries isolated from Dahl salt-sensitive and Dahl salt-insensitive rats. Bar represents 25 μm. Quantification of the intensities in each graph was presented in the right 2 panels. n=8. B, [Ca2+]cyt in ER Ca2+ release and Ca2+ uptake in mouse VSMCs transferred with TRPC3-nc, TRPC3-oe, and si-TRPC3 plasmids and treated with control, HS, and HS+canagliflozin. The quantitative results are shown on the right. n=12. C, Cytosolic calcium imaging of VSMCs transferred with TRPC3-nc, TRPC3-oe, and si-TRPC3 plasmids and treated with control, HS, and HS+canagliflozin, The relative fluorescence intensity of VSMCs were quantified. n=12. D, Effect of acute Pyr3 (TRPC3 inhibitor, 10 μmol/L) preincubation on PE- and U46619-induced vasoconstriction of the mesenteric artery rings from Dahl salt-sensitive fed with ND and HS. n=8. E, Representative western blots of TRPC3, CamkII, and phosphorylation of CamkII in mouse VSMCs transferred with TRPC3-nc, TRPC3-oe, and si-TRPC3 plasmids and treated with control (Con), HS, and HS+canagliflozin (HS+Cana). β-actin served as a loading control. n=8. Results are expressed as mean±SEM. Statistical significance was determined by unpaired Student t test for comparisons between 2 groups and 1-way ANOVA followed by Bonferroni’s posttest for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. ER indicates endoplasmic reticulum; HS, high-salt diet; NCX1, sodium-calcium exchanger 1; ND, normal diet; PE, phenylephrine; si-TRPC3, TRPC3 silence; SS, Dahl salt-sensitive rats; SS-13BN, Dahl salt-insensitive rats; TRPC3, transient receptor potential channels 3; TRPC3-nc, TRPC3 negative control; TRPC3-oe, TRPC3 overexpression; and VSMC, vascular smooth muscle cell.

Article Snippet: Primary antibodies against NCX1 were purchased from ProteinTech Group (Chicago, IL) and the primary antibody against TRPC3 was purchased from Alomone (Alomone Labs).

Techniques: Immunofluorescence, Staining, Isolation, Control, Imaging, Fluorescence, Western Blot, Phospho-proteomics, Negative Control, Over Expression

Journal: Journal of the American Heart Association

Article Title: Sodium‐Glucose Cotransporter 2 Inhibitor Canagliflozin Antagonizes Salt‐Sensitive Hypertension Through Modifying Transient Receptor Potential Channels 3 Mediated Vascular Calcium Handling

doi: 10.1161/jaha.121.025328

Figure Lengend Snippet: Figure 7. Schematic diagram of this work. TRPC3 and NCX1 maintain dynamic calcium balance and participate in vascular activities under physiological conditions. However, upregulation of TRPC3 by high salt leads to intracellular Na+ accumulation and then activated reverse mode of NCX1, which promotes sustained vasoconstriction caused by increased cytosolic calcium concentration. This might be an important reason for salt-sensitive hypertension. The antihypertensive effects of canagliflozin are mediated to decreasing cytosolic calcium concentration and vasoconstriction through inhibition of TRPC3. NCX1 indicates sodium-calcium exchanger 1; and TRPC3, transient receptor potential channels 3.

Article Snippet: Primary antibodies against NCX1 were purchased from ProteinTech Group (Chicago, IL) and the primary antibody against TRPC3 was purchased from Alomone (Alomone Labs).

Techniques: Concentration Assay, Inhibition