PA1-920A

antibody from Invitrogen Antibodies

Targeting: SCNN1A ENaCalpha, SCNN1

Provider product page for PA1-920A

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [26]
Comments [0]
Validations
Immunocytochemistry [2]
Flow cytometry [1]
Other assay [20]

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Product number: PA1-920A - Provider product page
Provider: Invitrogen Antibodies
Product name: alpha-ENaC Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-920A detects alpha-epithelial sodium channel (alpha-ENaC) from human, mouse, and rat tissues and cells. PA1-920A has been successfully used in Western blot procedures. By Western blot, this antibody specifically detects a ~97 kDa band representing glycosylated alpha-ENaC from NIH-3T3 cells, and a ~75 kDa band representing the unglycosylated alpha-ENaC protein from human brain samples. The PA1-920A immunizing peptide is a synthetic peptide corresponding to residues 20-42 from human alpha-ENaC. This sequence is 82%, 73%, 64%, and 59% conserved in the rat and rabbit, mouse, guinea pig, and cow, respectively. PA1-920A immunizing peptide (Cat. # PEP-088) is available for use in neutralization and control experiments.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

SCNN1A protein structure - PA1-920A shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of alpha ENaC was performed using 70% confluent log phase HEK-293 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with alpha ENaC Rabbit Polyclonal Antibody (Product # PA1-920A) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of alpha ENaC was performed using 70% confluent log phase HEK 293 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with alpha ENaC Rabbit Polyclonal Antibody (Product # PA1-920A) at 2µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjµgate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Effect of formaldehyde on alpha-ENaC protein level in H441 cells. ( A ) Representative western blot of alpha-ENaC protein extracted from H441 cells exposed to 200 muM formaldehyde for 0-48 h. Blots were immunostained with antibody to alpha-ENaC, or to beta-actin as a loading control. ( B ) Graphical representation of data obtained from three sets of western blots and quantified using gray analysis (alpha-ENaC/beta-actin). Data are shown as the mean +- SE, * P < 0.05, compared with control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 MiR-130b can be transferred into AT2 cells co-cultured with BMSCs and increases protein level alpha/gamma-ENaC in AT2 cells. a Transfected miR-130b labelled with Cy3 was localized in AT2 cells co-cultured with BMSCs for 24 h. BMSCs were transiently transfected with Cy3-miR-130b after which they were co-cultured with mouse AT2 cells and then fixed. Nuclei were counterstained by 4,6-diamidino-2-phenylindole (DAPI, blue; left panel), and the representative staining images were shown in CY3-miR-130b positive AT2 cells (red; middle panel). The merged images were seen (right panel). Scale bar, 10 mum. b The result of real-time PCR assays shows miR-130b level in AT2 cells treated with LPS for 12 h and/or co-cultured with BMSCs for 24 h. The relative level of miR-130b were calculated as miR-130b/U6 ratio. c Representative Western blot bands of alpha- and gamma-ENaC protein expression in AT2 cells transfected with miR-130b mimic/inhibitor for 72 h. Blots for beta-actin were used as internal controls. d , e Graphical representation of data obtained from Western blot assays for alpha- and gamma-ENaC subunits. Bands were quantified using gray analysis (alpha-ENaC/beta-actin and gamma-ENaC/beta-actin). ** P < 0.01, compared with control; & P < 0.05, compared with LPS; # P < 0.05, compared with miR-130b mimic negative control (NC); $ P < 0.05, compared with miR-130b inhibitor negative control (Inhibitor NC), n = 4-6

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 MiR-130b directly targets PTEN and BMSCs transfected with miR-130b mimic/miR-130b inhibitor affect protein expressions of PTEN and alpha/gamma-ENaC in AT2 cells. a The potential binding sites for miR-130b on the 3'-UTRs of PTEN. b Dual luciferase assay for miR-130b binding with PTEN in H441 cells. H441 cells were co-transfected with miR-130b mimic negative control (NC) or miR-130b mimic (Mimic) together with pmirGLO-PTEN (WT or MT) for 24 h. c , e Representative Western blot bands of PTEN and alpha/gamma-ENaC protein expression in AT2 cells co-cultured with BMSCs that were transfected with miR-130b mimic/inhibitor. d , f and g Graphical representation of data obtained from Western blot assays for PTEN and alpha/gamma-ENaC. Bands were quantified using gray analysis (PTEN/beta-actin, alpha-ENaC/beta-actin and gamma-ENaC/beta-actin). * P < 0.05, ** P < 0.01, compared with miR-130b mimic negative control or plus wild-type pmirGLO-PTEN (NC or NC + WT); & P < 0.05, compared with miR-130b inhibitor negative control (Inhibitor NC), n = 4

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 PTEN gene knockdown enhanced the protein expression of alpha/gamma-ENaC in AT2 cells. a , c Representative Western blot bands of PTEN and alpha/gamma-ENaC protein expression in LPS-treated AT2 cells transfected with miR-130b mimic/inhibitor or siPTEN. b , d and e Graphical representation of data obtained from Western blot assays for PTEN and alpha/gamma-ENaC. Bands were quantified using gray analysis (PTEN/beta-actin, alpha-ENaC/beta-actin and gamma-ENaC/beta-actin). ** P < 0.01, compared with control; & P < 0.05, compared with LPS + miR-130b mimic negative control (NC); # P < 0.05, compared with LPS + miR-130b inhibitor negative control (Inhibitor NC); $ P < 0.05, compared with LPS + siPTEN, n = 4-6

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7 MiR-130b and PTEN regulate alpha/gamma-ENaC through PI3K/AKT pathway in AT2 cells. a Representative Western blot bands of p-AKT/AKT protein expression in AT2 cells that were transfected with miR-130b mimic/inhibitor. c Representative Western blot bands of alpha/gamma-ENaC protein expression in AT2 cells that were treated with 10 uM PI3K/AKT inhibitor (LY294002) and/or siPTEN for 30 min. b , d and e Graphical representation of data obtained from Western blot assays for p-AKT/AKT and alpha/gamma-ENaC. Bands were quantified using gray analysis (p-AKT/AKT, alpha-ENaC/beta-actin, and gamma-ENaC/beta-actin). ** P < 0.01, compared with Control or miR-130b mimic negative control (NC); && P < 0.01, compared with siPTEN group, n = 4

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4 Melatonin enhanced ENaC expression in CLP-induced ALI. The protein expressions of alpha-ENaC (A) , beta-ENaC (B) and gamma-ENaC (C) in ALI mouse were determined by western blot using GAPDH as a loading control. Results obtained by densitometry ratios of target proteins to GAPDH. The mRNA expressions of alpha-ENaC (D) , beta-ENaC (E) and gamma-ENaC (F) in ALI mouse were tested by real-time PCR. Gene expression was normalized with GAPDH as the housekeeping gene, and the relative expression of genes was calculated using the 2 -^^ Ct method. (G) The distribution and protein expression of alpha-ENaC was examined by immunofluorescence. All values are means +- SD ( n = 6). *** p < 0.001; ** p < 0.01; * p < 0.05 (one-way ANOVA followed by Turkey post hoc test).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 6 Melatonin up-regulated ENaC protein expression through SIRT1/SGK1/Nedd4-2 pathway in A549 cells. (A) A549 Cells were stimulated by LPS (1 mug/mL) with different concentrations of melatonin (10, 100 nM, 1 and 10 muM) for 24 h (B-G) A549 cells were treated with LPS in the presence or absence of melatonin (1 µM) for 24 h. Inhibitor of SIRT1, EX527 (1 µM) was administrated 1 h before melatonin. (B-E) The protein expression levels of alphaENaC, SIRT1, SGK1 and Nedd4-2 were measured by western blot using GAPDH as a loading control. Results obtained by densitometry ratios of target proteins to GAPDH. All values are means +- SD (n = 6). NS, no significance, *** p < 0.001; ** p < 0.01; * p < 0.05 (one-way ANOVA followed by Turkey post hoc test). (F, G) alphaENaC and SIRT1 were determined by immunofluorescence (H, I) Fluorescence intensity of SIRT1 and alpha-ENaC was determined by using ImageJ software (version 1.46, NIH, Bethesda, MD, USA, and all values were normalized to sham. All values are means +- SD ( n = 4). *** p < 0.001; ** p < 0.01; * p < 0.05 (one-way ANOVA followed by Turkey post hoc test).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 3 Mesenchymal stem cell-conditioned medium (MSC-CM) increases alpha-epithelial sodium channel (alpha-ENaC) protein level in H441 and alveolar type 2 epithelial (AT2) cells. A and C, Representative western blot measurement of alpha-ENaC from H441 and AT2 cells treated with LPS (15 mug/mL) for 12 hours and/or MSC-CM for 24 hours. B and D, Semi-quantitation of western blots (alpha-ENaC/beta-actin). n = 4-5. * P < .05; ** P < .01

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 5 Activation of epithelial sodium channel (ENaC) by mesenchymal stem cell-conditioned medium (MSC-CM) through regulating miR-214 levels. A and C, Representative western blot measurement of alpha-ENaC protein in H441 and AT2 cells transfected with miR-214 negative control (NC) or miR-214 mimic. B and D, Representative graphical representation of alpha-ENaC expression in H441 and AT2 cells, n = 4. E, Representative I sc traces recorded in H441 monolayers transfected with NC or miR-124 mimic. F, Average amiloride-sensitive I sc . n = 6. * P < .05; ** P < .01

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Effects of FA on the proteins and transcriptional expression level of ENaC in the trachea of mice. A C57BL/J male mice were treated intraperitoneally with normal saline or FA (100 mg/kg) 12 h before and 12 h after intratracheal administration of LPS (5 mg/kg), subsequently the proteins were extracted and analyzed by Western blot. The typical bands of alpha- and gamma-ENaC protein expression and beta-actin were used as internal controls. B and C Graphical representation of data obtained from Western blot assays for alpha- and gamma-ENaC subunits, which were quantified using gray analysis (alpha-ENaC/beta-actin and gamma-ENaC/beta-actin). D - F mRNA samples were isolated from mouse models of tracheal injury. Relative levels of mRNA were calculated as alpha- or beta- or gamma-ENaC/GAPDH ratios. *** P < 0.001, compared with Control group, & P < 0.05, compared with LPS group, n = 4-5

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP) deficiency enhances upregulation of renal alpha-subunit of the epithelial sodium channel (alphaENaC) protein expression by chronic angiotensin II (Ang II) infusion. Effects of Ang II (2000 ng/kg/min) infusion for 14 days on protein expression of the major sodium transporters ( a , sodium-proton antiporter 3 (NHE3); b , phosphorylated sodium-potassium-two chloride cotransporter (NKCC2) on Thr96; c , phosphorylated Na + -Cl - cotransporter (NCC) on Ser71; d , alphaENaC; e , betaENaC; and f , gammaENaC) in the kidneys of wild-type (WT) and ATRAP-knockout (KO) mice. Values are expressed as mean+-s.e. ( n =6-8 in each group). * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Characterization of pancreatic ductal epithelial cells. ( a - f , l : Characterization of organoids). a Characterization of pancreatic ductal organoids using epithelial cell markers, b cytokeratin 19 (KRT 19, c E-cadherin, e sodium transport channel (ENaC), and f ZO-1. d Hematoxylin and eosin (H&E) image shows the orientation of pancreatic ductal epithelial cells in spheroid of the organoid. ( g - k : Characterization of monolayer of pancreatic ductal epithelial cells (PDECs)). g Phase contrast and j H&E images show monolayer of PDECs formed from the organoids. Monolayer of PDECs showed positivity for tight junction h ZO-1, i F-actin and KRT 19, and k cystic fibrosis transmembrane conductance regulator (CFTR). l RNA-sequencing data was obtained from the pancreatic ductal organoids and verified the PDEC origin ( n = 4 sample preparation from the same patient). Data are mean +- SD. Scale bars: 10 um ( k ), 20 um ( b , c , e , f , h - j enlarge), 50 um ( d , g , j ), and 500 um ( a )

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Glass coverslips in six-well dishes were coated with Cultrex diluted 1:20 with PBS. The excess was removed, and H889 cells were plated on the coverslips. The following day, cells were fixed and subsequently analyzed by immunofluorescence with an antibody to alphaENaC. Phalloidin was used to identify the cell periphery and DAPI to locate nuclei. (Top) Three sets of images are shown without the neutralizing peptide, and (bottom) one set of images is shown with the peptide. The signal was specific also based on minimal background staining by omitting the primary antibody (not shown). Scale bars, 5 mum. Figure 3