israel acc 034 ao 5 gapdh santa cruz biotechnology sc  (Santa Cruz Biotechnology)

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Single-Cell RNA-Seq Reveals Coronary Heterogeneity and Identifies CD133 ⁺ TRPV4 ^high Endothelial Subpopulation in Regulating Flow-Induced Vascular Tone in Mice

doi: 10.1161/atvbaha.123.319516

Figure Lengend Snippet: Figure 3. Identification of mice CD133+TRPV4high coronary artery endothelial cells (ECs; CAECs). A, Left, T-distributed stochastic neighbor embedding (t-SNE) of CAECs from all coronary arterial branches, cluster 3 (CD133+TRPV4high) CAECs colored in red; Middle, t-SNE of CAECs in each coronary artery branch. Right, Summarizes cluster 3 (CD133+TRPV4high) CAEC composition in a bar graph. B, Heatmap of the expression of GPCRs (G-protein-coupled receptors) and flow-related genes in cluster 3 (CD133+TRPV4high) CAECs. C, Dot plots of the top 10 enriched Kyoto Encyclopedia of Genes and Genomes pathways of significantly changed marker genes in the cluster 3 CAECs subpopulation. Dot size, member of enriched genes in each pathway; color, enrichment P value. D, Violin plots showing the distribution of expression of Trpv4 and Prom1 in CD133+TRPV4high and TRPV4low ECs. E, Validation of the presence of TRPV4-eGFP (transient receptor potential vanilloid 4-enhanced green fluorescent protein) expression by en face immunofluorescence in coronary branches. CDH5, EC marker. Scale bar, 50 µm. n=4 for each branch. F, Representative figure showing the results of CD133 sorting of CAECs, CD144 was used as an EC sorting marker. G, Western blots of TRPV4 expression in CD133+ and CD133− CAECs from wild-type (WT) and endothelium-specific TRPV4 knockout (TRPV4EC −/−) mice. CAECs represent CAECs before cell sorting; CD133+ and CD133− represent CD133-sorted CAECs. CAECs were obtained from 6 WT or TRPV4EC −/− mice and then applied in a Western blot as 6 replicates using 1-way ANOVA. *P<0.001 vs CAECs, #P<0.001 vs CD133+. Ordinary 1-way ANOVA was performed, and P values were adjusted with Dunnett test. Data are expressed as the mean±SEM. cAMP indicates cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; Cir-LCA, circumflex left coronary artery; Des-LCA, descending left coronary artery; FC, fold change; GPCR, G-protein- coupled receptor; PKG, protein kinase G; RCA, right coronary artery; and TRP, transient receptor potential.

Article Snippet: Primary antibodies for TRPV4 (No. ACC-034-AO; Alomone Labs, Israel, 1:200), GAPDH (No. sc-365062; Santa Cruz Biotechnology, 1:500), and corresponding secondary antibodies to each primary antibody were used.

Techniques: Expressing, Marker, Biomarker Discovery, Immunofluorescence, Western Blot, Knock-Out, FACS

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Single-Cell RNA-Seq Reveals Coronary Heterogeneity and Identifies CD133 ⁺ TRPV4 ^high Endothelial Subpopulation in Regulating Flow-Induced Vascular Tone in Mice

doi: 10.1161/atvbaha.123.319516

Figure Lengend Snippet: Figure 4. The function of CD133+TRPV4high coronary artery endothelial cells (ECs; CAECs). A, Schematic of the functional studies of CD133+TRPV4high ECs in vitro and in vivo. B, Representative traces and summary of cell-attached patch clamp recording of TRPV4 (transient receptor potential vanilloid 4) activity induced by GSK1016790A (GSK) and HC067047 in primary CAECs from wild-type (WT) mice. CD133+ and CD133− represent CD133-sorted CAECs; n=6 for each type of mouse. C, Representative traces and summary of whole-cell patch clamp recording in primary CAECs from WT, endothelium-specific TRPV4 knockout (TRPV4EC −/−) or endothelium-specific TRPV4 knock-in (TRPV4EC +/+) mice. CD133+ and CD133− represent CD133-sorted CAECs; ordinary 1-way ANOVA was performed and P values were adjusted with the Dunnett test. *P<0.001 vs WT CD133+. Data are expressed as the mean±SEM. D, Flow- induced Ca2+ influx in primary CAECs. Welch 1-way ANOVA was performed, and P values were adjusted with the Dunnett T3 test. *P<0.001 vs WT CD133+. Data are expressed as the mean±SEM. E, Representative traces and summary of dilation induced by flow in freshly isolated coronary arterial branches from right coronary artery (RCA), descending left coronary artery (Des-LCA), and circumflex left coronary artery (Cir-LCA). Multiple comparisons in 2-way ANOVA were performed in each shear stress between WT RCAs vs TRPV4EC +/+ or EC −/− RCAs, WT Cir-LCAs vs TRPV4EC +/+ or EC −/− Cir-LCAs, and WT Des-LCAs vs TRPV4EC +/+ or EC −/− Des-LCAs. P values were adjusted with the Tukey test. *P<0.001 vs WT branches. n=5 for each treatment. Data are expressed as the mean±SEM. F, Representative Doppler color flow images and summary data for the coronary artery flow spectrum of WT, TRPV4EC −/− and TRPV4EC +/+ mice. At least 8 data were collected for each branch of each type of mouse. Multiple comparisons in 2-way ANOVA were performed. P values were adjusted with the Tukey test. *P<0.05 vs WT branches. Data are expressed as the mean±SEM. SSC indicates side scatter.

Article Snippet: Primary antibodies for TRPV4 (No. ACC-034-AO; Alomone Labs, Israel, 1:200), GAPDH (No. sc-365062; Santa Cruz Biotechnology, 1:500), and corresponding secondary antibodies to each primary antibody were used.

Techniques: Functional Assay, In Vitro, In Vivo, Patch Clamp, Activity Assay, Knock-Out, Knock-In, Isolation, Shear

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Detection of TRPV4 expression in gastric adenocarcinoma tissues and adjacent noncancerous tissues by IHC staining of tissue microarrays. ( A ) Representative images of TRPV4 expression in gastric adenocarcinoma tissues and adjacent noncancerous tissues. (a, b) Weakly focal positive TRPV4 expression in adjacent noncancerous gastric epithelia. (c, d) Strongly positive TRPV4 expression in a case of gastric adenocarcinoma. ( B ) Scatterplots of IHC scores of TRPV4 expression in gastric adenocarcinoma tissues and adjacent noncancerous tissues. ( C ) Percents of tissue samples with high TRPV4 expression and low TRPV4 expression based on the cut-off value of IHC scores in gastric adenocarcinoma tissues and adjacent noncancerous tissues (***P<0.001). Magnification: (a,c) ×50 and (b,d) ×400. Abbreviations: IHC, immunohistochemical; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Association of TRPV4 Expression with Clinicopathological Characteristics in Patients with Gastric Cancer

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Representation images of TRPV4, E-cadherin and vimentin expression in gastric cancer tissues by immunohistochemical staining. Magnification, ×400. ( A – C ) High TRPV4 expression in a case of poor differentiated gastric adenocarcinoma with loss expression of E-cadherin and positive vimentin expression. ( D – F ) Low TRPV4 expression in a case of signet ring cell carcinoma with high E-cadherin expression and negative Vimentin expression. Abbreviation: TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Correlation Between Expression of TRPV4, E-Cadherin and Vimentin in Gastric Cancer

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Survival curves using the Kaplan–Meier method by Log rank test. ( A and B ) The patients with high TRPV4 expression had shorter overall survival and disease-free survival than those with low TRPV4 expression. ( C and D ) The patients with low E-cadherin expression had shorter overall survival and disease-free survival than those with high E-cadherin expression. ( E and F ) The patients with high vimentin expression had shorter overall survival and disease-free survival than those with low vimentin expression. ( G ) Overall survival analysis of TRPV4 gene in gastric cancer obtained from Kaplan–Meier Plotter online ( http://kmplot.com/analysis/ ). *P<0.05, **P<0.01, ***P<0.001. Abbreviations: Cum, cumulative; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analyses of Overall Survival in Patients with Gastric Cancer

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analyses of Disease-Free Survival in Patients with Gastric Cancer

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: The protein expression of TRPV4 in gastric cell lines and down-regulation of TPRV4 inhibited proliferation of gastric cancer cells. ( A ) The protein expression levels of TPRV4 were detected in gastric cancer cell lines (HGC-27 and MGC-803) and normal gastric mucosa cell GES-1 by Western blot. **P<0.01, ***P<0.001. ( B ) Western blot analyses of the expression levels of TRPV4 protein in gastric cancer cell lines following transfection with shTRPV4 or shCtrl. ***P<0.001. ( C ) Proliferation capacities of gastric cancer cells lines were assayed at 24 h, 48 h and 72 h by CCK-8 following transfection with shTRPV4 or shCtrl. ***P<0.001. ( D ) Colony formation assays were performed in gastric cancer cell lines following transfection with shTRPV4 or shCtrl. *P<0.05. Abbreviations: CCK-8, Cell Counting Kit-8; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing, Western Blot, Transfection, CCK-8 Assay, Cell Counting

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Transwell assays demonstrated that TRPV4 down-regulation greatly inhibited invasion abilities of gastric cancer cells. ( A and B ) Representative images of invasive cells following transfection with shTRPV4 or shCtrl. Magnification, ×200. ( C and D ) The column diagram of the numbers of invasive cells following transfection with shTRPV4 or shCtrl. **P<0.01, ***P<0.001. Abbreviation: TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Transfection

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Down-regulation of TPRV4 regulated the expression levels of EMT-related proteins in gastric cancer cell lines ( A ) The expression levels of EMT-related proteins (E-cadherin, vimentin, N-Cadherin, Snail, Slug and Twist) were detected by Western blot assays following transfection with shTRPV4 or shCtrl. ( B ) The column diagram of the expression levels of EMT-related proteins in HGC-27 following transfection with shTRPV4 or shCtrl. ( C ) The column diagram of the expression levels of EMT-related proteins in MGC-803 following transfection with shTRPV4 or shCtrl. *P<0.05, ***P<0.001. Abbreviations: ns, non-significant; EMT, epithelial–mesenchymal transition; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing, Western Blot, Transfection

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitation (IP) analysis of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in mCCDcl1 cells and mouse kidney. A: immunoblots of mCCDcl1 cell homogenates show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). B: immunoblots of mouse kidney homogenates also show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously ((20), Fig. 1). CAV-1 (22 kD) protein band is demonstrated in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunoprecipitation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitaton (IP) analysis of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney. Immunoblots of mCCDcl1 cell homogenates show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (A). Immunoblots of mouse kidney homogenates also show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (B). Endogenous immunoprecipitaton of IK1 (C) or BKα (D) followed by blotting for CAV-1, TRPV4, and each of the KCa channels, in mCCDcl1 cells, showed enrichment of CAV-1, TRPV4, BKα, SK3, and BKα or IK1. Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously [(20) Fig. 1]. CAV-1 (22 kD) was verified in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Western Blot, Immunoprecipitation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Effect of caveolin-1 (CAV-1) knockdown on transient receptor potential cation channel subfamily V member 4 (TRPV4)-mediated (GSK101) intracellular Ca2+ ([Ca2+]i) response to blockers of TRPV4 and KCa channels. The normal peak [Ca2+]i response to TRPV4 activation (GSK101) is markedly reduced from 597 ± 45 nM (n = 70) to 231 ± 47 nM (n = 85) following CAV-1 siRNA treatment (A and B). Furthermore, the apparent initial rate of TRPV4-mediated Ca2+ influx was markedly depressed from 108 ± 27 nM/min (n = 70) in normal conditions to 42 ± 16 nM/min (n = 85) in the presence of CAV-1 siRNA (C), reflecting a markedly depressed level of TRPV4 activation. The subsequent panels demonstrate that the typical normal peak elevation in [Ca2+]i following TRPV4 activation is markedly reduced upon inhibition of each KCa channel following CAV-1 knockdown. Iberiotoxin (IbTX) treatment reduces the peak [Ca2+]i for BK inhibition from 227 ± 34 nM (n = 73) to 84 ± 33 nM (n = 59) after CAV-1 siRNA treatment (D and E). TRAM-34 treatment reduces the peak [Ca2+]i for IK1 inhibition from 312 ± 31 nM (n = 90) to 81 ± 12 nM (n = 95) after CAV-1 siRNA treatment (F and G). Apamin treatment reduces the peak [Ca2+]i for SK3 inhibition from 414 ± 60 nM (n = 72) to 196 ± 23 nM (n = 60) after CAV-1 siRNA treatment (H and I). Therefore, after CAV-1 siRNA treatment the response for each of the KCa channels to TRPV4 activation is markedly depressed. ***P < 0.001. BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Activation Assay, Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Representative immunofluorescence images show colocalization of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in collecting duct mCCDcl1 cells in mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for TRPV4 (TRPV4, green) and SK3 (SK3-ATTO-594, red) show strong colocalization as apparent in the merged image (Merged, yellow) (A). Similarly, immunostaining for TRPV4 (red) and BKα (green) (B) and for TRPV4 (red) and IK1 (green) (C) demonstrated noted colocalization in the merged images (Merged, yellow). Mouse kidney CCD immunostained for TRPV4 (TRPV4, green) and SK3 (SK3, ATTO-594, red) display noted colocalization in the merged images (Merged, yellow) (D). In a similar manner, immunostaining for TRPV4 (TRPV4, red) and BKα (BKα, green) (E) and for TRPV4 (TRPV4, red) and IK1 (IK1, green) (F) likewise demonstrated apparent colocalization in the merged images (Merged, yellow). Areas of more diffuse staining are also apparent in some cases, especially for IK1 (see F). Note that we verified the primary antibodies using blocking peptides in an earlier publication [(20) Figs. 1 and ​and4].4]. All the immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunofluorescence, Immunostaining, Staining, Blocking Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Immunofluorescence images demonstrates colocalization of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) showed strong colocalization in the merged image (Merged, yellow) (A). Similarly, immunostaining for CAV-1 with each of the KCa channel demonstrated robust colocalization in the merged images as shown for CAV-1 (green) and SK3 (red) (B), CAV-1 (red) and BKα (green) (C), and CAV-1 (red) and IK1 (green) (D). Diffuse localization was also apparent, especially for CAV-1 and IK1 (D). Mouse kidney CCD immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) also displayed robust colocalization as shown in the merged image (Merged, yellow) (E). Similarly, immunostaining for CAV-1 and each of the KCa channels displayed noted colocalization in the merged images (Merged, yellow) for CAV-1 (green) and SK3 (red) (F), for CAV-1 (red) and BKα (green) (G), and for CAV-1 (green) and IK1 (red) (H). Areas of more diffuse localization were also apparent, especially for BKα and IK1 (see merged images in G and H). Immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunofluorescence, Immunostaining