Journal: Frontiers in Physiology

Article Title: Antiproteinuric and Hyperkalemic Mechanisms Activated by Dual Versus Single Blockade of the RAS in Renovascular Hypertensive Rats

doi: 10.3389/fphys.2021.656460

Figure Lengend Snippet: Effect of dual versus single RAS blockade on the expression of total and cleaved ENaC subunits in the non-clipped kidney of renovascular hypertensive rats. Samples isolated from the non-clipped kidney of renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) containing equal amounts of membrane proteins (75 μg for α-ENaC, β-ENaC, and γ-ENaC and 20 μg for actin) were subjected to SDS-PAGE, after which the proteins were transferred to a PVDF membrane and incubated with primary antibodies. Actin was used as a loading control. Representative immunoblots of electrophoresed renal membrane proteins and graphical representation of the relative expression of (A) Full-length (FL) α-ENaC, (B) cleaved α-ENaC, (C) β-ENaC, (D) cleaved γ-ENaC. The values represent individual measurements and the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs. 2K; ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. 2K-1C; &&& P < 0.001 and &&&& P < 0.0001 vs. 2K-1C + E; $$ P < 0.01 and $$$$ P < 0.0001 vs. 2K-1C + L.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% non-fat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and the following specific primary antibodies overnight (4°C): polyclonal antibody against podocin (1:1,000; Santa Cruz Biotech, sc-21009), polyclonal antibody against nephrin (1:1,000; Abcam, Cambridge, MA, Ab 58968), polyclonal antibody against megalin (1:50,000; a gift from Dr. Daniel Biemesderfer) , polyclonal antibody against cubilin (1:1,000; Santa Cruz Biotech, sc-20609), polyclonal antibody against the ClC-5, H + /Cl – exchange transporter 5 (1:1,000; Alpha Diagnostic International, CLC51-A, San Antonio, TX, United States) , polyclonal antibody against α-ENaC (1:1,000; Alomone Labs, ASC-030, Jerusalem, Israel), polyclonal antibody against β-ENaC (1:1,000; StressMarq Biosciences, SPC-404, Victoria, British Columbia, Canada), polyclonal antibody against cleaved γ-ENaC (1:1,000; Alomone Labs, ASC-011), or actin (1:5,000; Abcam ab179467).

Techniques: Expressing, Isolation, SDS Page, Incubation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: NBCe1-A is required for the renal ammonia and K + response to hypokalemia

doi: 10.1152/ajprenal.00481.2019

Figure Lengend Snippet: Antibodies used (alphabetical order)

Article Snippet: The specific antibodies, sources, and dilutions used are shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Source Dilutions Used ENaCα StressMarq Biosciences (SPC-403, Victoria, BC, Canada) WB, 1:5,000 ENaCβ StressMarq Biosciences (SPC-404, Victoria, BC, Canada) WB, 1:5,000 ENaCγ StressMarq Biosciences (SPC-405, Victoria, BC, Canada) WB, 1:2,500 Glutamine synthetase Abcam (ab73593, Cambridge, MA) IHC, 1:10,000; WB, 1:1,500 H + -ATPase, a4 subunit Dr. Fiona Karet (Cambridge Institute for Medical Research, Cambridge, UK) IHC, 1:50,000 Na + -Cl − cotransporter Dr. David Ellison (Oregon Health Sciences University, Portland, OR) IHC, 1:20,000; WB, 1:1,000 NBCe1-A Michael F. Romero, Ph.D (α-333) IHC, 1:2,000; WB, 1:1,000 Pan-NBCe1 ProteinTech IHC, 1:1,000; WB, 1:2,000 Phosphate-dependent glutaminase Dr. Norman Curthoys (Colorado State University) WB, 1:3,000 Phospho enol pyruvate carboxykinase Cayman Chemical Co. (no. 10004943, Ann Arbor, MI) IHC, 1:5,000; WB, 1:2,000 Phospho-NCC PhosphoSolutions (no. p1311-53, Aurora, CO) IHC, 1:100,000; WB, 1:1,000 Rhbg Generated in our laboratory IHC, 1:20,000 Rhcg Generated in our laboratory IHC, 1:10,000 Open in a separate window ENaC, epithelial Na + channel; NBCe1-A, Na + -bicarbonate cotransporter electrogenic, isoform 1, splice variant A; Rh, Rhesus; WB, Western blot; IHC, immunohistochemistry.

Techniques: Generated

Journal: American Journal of Physiology - Renal Physiology

Article Title: NBCe1-A is required for the renal ammonia and K + response to hypokalemia

doi: 10.1152/ajprenal.00481.2019

Figure Lengend Snippet: Effects of Na+-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) deletion and diet-induced hypokalemia on epithelial Na+ channel (ENaC). A–C: ENaCα, -β, and -γ expressions, respectively, in wild-type (WT) and NBCe1-A knockout (KO) mice on K+-free diet. ENaCα expression was slightly but significantly decreased in KO versus WT mice; ENaCβ expression was slightly but significantly increased in KO versus WT mice; and ENaCγ expression did not differ significantly between WT and KO mice. Thus, there was no consistent effect of NBCe1-A deletion on ENaC subunit expression; n = 6 for each genotype.

Article Snippet: The specific antibodies, sources, and dilutions used are shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Source Dilutions Used ENaCα StressMarq Biosciences (SPC-403, Victoria, BC, Canada) WB, 1:5,000 ENaCβ StressMarq Biosciences (SPC-404, Victoria, BC, Canada) WB, 1:5,000 ENaCγ StressMarq Biosciences (SPC-405, Victoria, BC, Canada) WB, 1:2,500 Glutamine synthetase Abcam (ab73593, Cambridge, MA) IHC, 1:10,000; WB, 1:1,500 H + -ATPase, a4 subunit Dr. Fiona Karet (Cambridge Institute for Medical Research, Cambridge, UK) IHC, 1:50,000 Na + -Cl − cotransporter Dr. David Ellison (Oregon Health Sciences University, Portland, OR) IHC, 1:20,000; WB, 1:1,000 NBCe1-A Michael F. Romero, Ph.D (α-333) IHC, 1:2,000; WB, 1:1,000 Pan-NBCe1 ProteinTech IHC, 1:1,000; WB, 1:2,000 Phosphate-dependent glutaminase Dr. Norman Curthoys (Colorado State University) WB, 1:3,000 Phospho enol pyruvate carboxykinase Cayman Chemical Co. (no. 10004943, Ann Arbor, MI) IHC, 1:5,000; WB, 1:2,000 Phospho-NCC PhosphoSolutions (no. p1311-53, Aurora, CO) IHC, 1:100,000; WB, 1:1,000 Rhbg Generated in our laboratory IHC, 1:20,000 Rhcg Generated in our laboratory IHC, 1:10,000 Open in a separate window ENaC, epithelial Na + channel; NBCe1-A, Na + -bicarbonate cotransporter electrogenic, isoform 1, splice variant A; Rh, Rhesus; WB, Western blot; IHC, immunohistochemistry.

Techniques: Variant Assay, Knock-Out, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: NBCe1-A is required for the renal ammonia and K + response to hypokalemia

doi: 10.1152/ajprenal.00481.2019

Figure Lengend Snippet: Correlation of epithelial Na+ channel (ENaC) expression with plasma K+ in wild-type (WT) and Na+-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) knockout (KO) mice on K+-free diet. A: ENaCα expression plotted as a function of plasma K+ for WT and KO mice on K+ control diet and K+-free diet. Both plasma K+ and genotype were independently correlated with ENaCα expression (P < 0.001 for each by ANOVA). B: ENaCβ expression relative to plasma K+ in WT and NBCe1-A KO mice. Plasma K+ correlated significantly with ENaCβ expression (P < 0.001 by ANOVA), but there was no independent effect of genotype. C: ENaCγ expression relative to plasma K+. Both plasma K+ and genotype were independently correlated with ENaCγ expression (P < 0.001 for each by ANOVA).

Article Snippet: The specific antibodies, sources, and dilutions used are shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Source Dilutions Used ENaCα StressMarq Biosciences (SPC-403, Victoria, BC, Canada) WB, 1:5,000 ENaCβ StressMarq Biosciences (SPC-404, Victoria, BC, Canada) WB, 1:5,000 ENaCγ StressMarq Biosciences (SPC-405, Victoria, BC, Canada) WB, 1:2,500 Glutamine synthetase Abcam (ab73593, Cambridge, MA) IHC, 1:10,000; WB, 1:1,500 H + -ATPase, a4 subunit Dr. Fiona Karet (Cambridge Institute for Medical Research, Cambridge, UK) IHC, 1:50,000 Na + -Cl − cotransporter Dr. David Ellison (Oregon Health Sciences University, Portland, OR) IHC, 1:20,000; WB, 1:1,000 NBCe1-A Michael F. Romero, Ph.D (α-333) IHC, 1:2,000; WB, 1:1,000 Pan-NBCe1 ProteinTech IHC, 1:1,000; WB, 1:2,000 Phosphate-dependent glutaminase Dr. Norman Curthoys (Colorado State University) WB, 1:3,000 Phospho enol pyruvate carboxykinase Cayman Chemical Co. (no. 10004943, Ann Arbor, MI) IHC, 1:5,000; WB, 1:2,000 Phospho-NCC PhosphoSolutions (no. p1311-53, Aurora, CO) IHC, 1:100,000; WB, 1:1,000 Rhbg Generated in our laboratory IHC, 1:20,000 Rhcg Generated in our laboratory IHC, 1:10,000 Open in a separate window ENaC, epithelial Na + channel; NBCe1-A, Na + -bicarbonate cotransporter electrogenic, isoform 1, splice variant A; Rh, Rhesus; WB, Western blot; IHC, immunohistochemistry.

Techniques: Expressing, Variant Assay, Knock-Out

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Immunohistochemistry analysis of ENaC beta and MARCKS protein expression in Hamp -/- (KO) mice or wild-type (WT) mice. ( A ) ENaC beta (red) protein expression and MARCKS (green) protein expression in WT controls. ( B ) ENaC beta (red) protein expression and MARCKS (green) protein expression in iron overloaded Hamp -/- mice. Data is representative of 3 mice in each group.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Immunohistochemistry, Expressing

Journal: American Journal of Translational Research

Article Title: Tempol treatment normalizes membrane expression of epithelial transport proteins in the kidney of salt-loaded hypertensive diabetic db/db mice

doi:

Figure Lengend Snippet: Sources of antibodies used in this study

Article Snippet: ENaC alpha , StressMarq , SMC-242D.

Techniques:

Journal: bioRxiv

Article Title: Water and Electrolyte Homeostasis in a Mouse Model with Reduced ENaC Gamma Subunit Expression

doi: 10.1101/2021.01.26.428168

Figure Lengend Snippet: Immunoblots of kidney and lung tissue lysates were probed with antibodies against the β or γ subunit of ENaC, then stripped and probed with antibodies against GAPDH. Arrowheads on the left of each blot show molecular weight marker positions. Carrots on the right of each blot indicate approximate region taken for signal quantification. Dot plots show protein normalized to mean signal from γ +/+ mice. Squares represent males, circles represent females. Open or shaded symbols represent data from 129sv or C57BL/6 mice, respectively. γ subunit protein levels in γ mt/mt mouse kidneys were reduced from 100 ± 10 % (mean ± SD, N = 5 mice) to 8 ± 4 % (N = 6). ***: p < 0.0001 (two-tailed Student’s t-test). In lung, the γ subunit was reduced from 100 ± 43 % (N = 6) to 16 ± 4% (N = 4) **: p < 0.01 (two-tailed Student’s t-test). The kidney β subunit point estimate decreased from 100 ± 60 % (N = 6) to 62 ± 9% (N = 6), but the difference was not significant. Lung β subunit protein signal also did not differ significantly between groups: 100 ± 49 % (N = 6) for γ +/+ vs. 144 ± 132 % for γ mt/mt (N = 5).

Article Snippet: Primary antibodies included anti-γ subunit (StressMarq; catalogue number SPC-405; 0.5 μg/mL), anti-β subunit (StressMarq; catalogue number SMC-241; 1 μg/mL), and anti-GAPDH (ProteinTech, 0.1 μg/mL).

Techniques: Western Blot, Molecular Weight, Marker, Two Tailed Test

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: ENaC expression in kidney lysates prepared at different temperatures. Kidney homogenate was prepared in Laemmli buffer with beta‐mercaptoethanol either at room temperature (RT) for 10 min, 37°C for 30 min, or 97°C for 3 or 10 min (denoted for each lane). Three separate replicates of these conditions were run and the subsequent membranes were probed with Stressmarq antibodies directed against each ENaC subunit: (a) α, (b) β, or (c) γ. Asterisks denote the expected α‐ and β‐subunits on their respective membranes. The γ‐subunit displays both a full‐length band and a cleaved product. Blots are representative of results from at least three separate validations.

Article Snippet: We aimed to validate commonly used commercial ENaC antibodies from StressMarq to ensure the integrity of our experimental results.

Techniques: Expressing

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Mouse kidney, but not lung, demonstrate differences in ENaC α‐ and γ‐subunit expression with aldosterone. Lysate from mouse kidney and lung collected from animals on a high salt diet (HS) or with an aldosterone infusion (Aldo) were blotted for the presence of each ENaC subunit. (a) The Stressmarq anti‐α‐subunit antibody revealed an intense 80 kDa nonspecific band, denoted by ** and a 95 kDa full length α‐subunit, denoted by *. The top panel was exposed for a longer period (~7 min) to show the 95 kDa band and the bottom panel shows a quick initial exposure (~5 s) before saturation of the 80 kDa band occurred. (b) The blot was stripped and reprobed with a previously characterized antibody, produced in the Loffing laboratory. The panel shows two exposures, separated by a dashed line, to reveal both the full‐length 95 kDa α‐subunit, denoted by *, and a 30 kDa α‐subunit N‐terminal cleavage product. (c) The Stressmarq antibody directed against the β‐subunit shows the presence of a band at 90 kDa (denoted by *). (d) The Stressmarq antibody directed against the γ‐subunit revealed bands corresponding to a full‐length 80 kDa γ‐subunit and 70 kDa cleavage products, as indicated. (e) Signal from the kidney samples was quantified by densitometry, with each band normalized to total protein. Quantification is shown as a fold change from the average HS signal with p values shown for relationships that were significant ( p < 0.05) as assessed by multiple t ‐tests. (f) Mouse lung lysate was first probed with the Stressmarq α antibody, followed by a light chain only secondary antibody (LC‐only HRP). The blot was then stripped and reprobed again with the Stressmarq α antibody but followed by a whole IgG secondary antibody (H + L HRP). The blot was then stripped again and reprobed with the Loffing α antibody, followed by a whole IgG secondary antibody (H + L HRP). The Stressmarq antibody revealed an intense nonspecific band of ~80 kDa (denoted by **) and a full length α‐subunit migrating ~95 kDa (denoted by *). The Loffing antibody reveal both the full‐length 95 kDa α‐subunit and a 30 kDa N‐terminal α‐subunit cleavage product. Blots are representative of results from at least three separate experiments.

Article Snippet: We aimed to validate commonly used commercial ENaC antibodies from StressMarq to ensure the integrity of our experimental results.

Techniques: Expressing, Produced

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Immunoprecipitation eliminates the non‐specific α‐ subunit band in mouse lung tissue and FRT cells. (a) Mouse lungs were homogenized and incubated with the α‐subunit StressMarq antibody, followed by immunoprecipitation with protein G beads. Both the pulldown (left) and the lysate (right) were run together on a gel and blotted for the α‐subunit with the same StressMarq antibody as utilized for the IP. (b) Mouse kidneys collected from animals on a high salt diet (HS) or with an aldosterone infusion (Aldo) were immunoprecipitated with the StressMarq α antibody and protein G beads. Both the IP (left) and the kidney lysate (right) were run next to each other on an 8%–16% gel to probe for the α‐subunit with the Loffing α antibody. (c) Lysate from FRT cells either mock transfected or transfected with the three ENaC subunits were run for comparison (first two lanes). The lysate was incubated with either StressMarq (SM) anti‐α‐subunit antibody, beads alone, or V5‐tagged beads and subsequent pulldown was performed. The product was run on two separate gels simultaneously, with one being probed with the StressMarq anti‐α‐subunit antibody while the other was probed with an antibody directed against the HA tag. In all panels * illustrates the α‐subunit band while ** indicates to the non‐specific band. Results are representative of three separate experiments.

Article Snippet: We aimed to validate commonly used commercial ENaC antibodies from StressMarq to ensure the integrity of our experimental results.

Techniques: Immunoprecipitation, Incubation, Transfection

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: The α‐subunit antibody demonstrates minimal signal in AQP2‐positive cells and is mislocalized in kidney medulla. Kidney sections from mice kept on control diet (a, top) or 4 days of high K + diet (a, middle) were labeled with the StressMarq anti‐α‐subunit antibody (red on left and converted to grayscale in second column). AQP2 (green) was used as a marker of the apical lumen of principal cells. The basolateral surfaces of tubules are denoted by solid lines and the apical surface by dashed lines. A no primary control is shown for comparison (a, bottom). (b) Positive staining for the α‐subunit (red) was only observed in the inner medulla of the kidneys, with representative images displayed here. While AQP2 positive cells within the medulla (green) did show expression of ENaC (denoted by *), the majority of the signal was localized to the basolateral side of both AQP2 positive and negative tubules (shown by arrows). Scale bar represents 20 μm in all images and images are representative of three separate regions examined in three mice of each treatment.

Article Snippet: We aimed to validate commonly used commercial ENaC antibodies from StressMarq to ensure the integrity of our experimental results.

Techniques: Labeling, Marker, Staining, Expressing