Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel
Abstract The epithelial Na+ channel (ENaC) is traditionally composed of three subunits, although non‐canonical expression has been found in various tissues including the vasculature, brain, lung, and dendritic cells of the immune system. Studies of ENaC structure and function have largely relied on...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2023-01-01
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| Series: | Physiological Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.14814/phy2.15554 |
| _version_ | 1797397095603240960 |
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| author | Stephanie M. Mutchler Shujie Shi Sarah Christine M. Whelan Thomas R. Kleyman |
| author_facet | Stephanie M. Mutchler Shujie Shi Sarah Christine M. Whelan Thomas R. Kleyman |
| author_sort | Stephanie M. Mutchler |
| collection | DOAJ |
| description | Abstract The epithelial Na+ channel (ENaC) is traditionally composed of three subunits, although non‐canonical expression has been found in various tissues including the vasculature, brain, lung, and dendritic cells of the immune system. Studies of ENaC structure and function have largely relied on heterologous expression systems, often with epitope‐tagged channel subunits. Relevant in vivo physiological studies have used ENaC inhibitors, mice with global or tissue specific knockout of subunits, and anti‐ENaC subunit antibodies generated by investigators or by commercial sources. Availability of well‐characterized, specific antibodies is imperative as we move forward in understanding the role of ENaC in non‐epithelial tissues where expression, subunit organization, and electrophysiological characteristics may differ from epithelial tissues. We report that a commonly used commercial anti‐α subunit antibody recognizes an intense non‐specific band on mouse whole kidney and lung immunoblots, which migrates adjacent to a less intense, aldosterone‐induced full length α‐subunit. This antibody localizes to the basolateral membrane of aquaporin 2 negative cells in kidney medulla. We validated antibodies against the β‐ and γ‐subunits from the same commercial source. Our work illustrates the importance of validation studies when using popular, commercially available anti‐ENaC antibodies. |
| first_indexed | 2024-03-09T01:06:02Z |
| format | Article |
| id | doaj.art-c069e62a4cf34f8c876b037ad15f1779 |
| institution | Directory Open Access Journal |
| issn | 2051-817X |
| language | English |
| last_indexed | 2024-03-09T01:06:02Z |
| publishDate | 2023-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Physiological Reports |
| spelling | doaj.art-c069e62a4cf34f8c876b037ad15f17792023-12-11T10:10:45ZengWileyPhysiological Reports2051-817X2023-01-01111n/an/a10.14814/phy2.15554Validation of commercially available antibodies directed against subunits of the epithelial Na+ channelStephanie M. Mutchler0Shujie Shi1Sarah Christine M. Whelan2Thomas R. Kleyman3Department of Medicine University of Pittsburgh Pittsburgh Pennsylvania USADepartment of Medicine University of Pittsburgh Pittsburgh Pennsylvania USADepartment of Medicine University of Pittsburgh Pittsburgh Pennsylvania USADepartment of Medicine University of Pittsburgh Pittsburgh Pennsylvania USAAbstract The epithelial Na+ channel (ENaC) is traditionally composed of three subunits, although non‐canonical expression has been found in various tissues including the vasculature, brain, lung, and dendritic cells of the immune system. Studies of ENaC structure and function have largely relied on heterologous expression systems, often with epitope‐tagged channel subunits. Relevant in vivo physiological studies have used ENaC inhibitors, mice with global or tissue specific knockout of subunits, and anti‐ENaC subunit antibodies generated by investigators or by commercial sources. Availability of well‐characterized, specific antibodies is imperative as we move forward in understanding the role of ENaC in non‐epithelial tissues where expression, subunit organization, and electrophysiological characteristics may differ from epithelial tissues. We report that a commonly used commercial anti‐α subunit antibody recognizes an intense non‐specific band on mouse whole kidney and lung immunoblots, which migrates adjacent to a less intense, aldosterone‐induced full length α‐subunit. This antibody localizes to the basolateral membrane of aquaporin 2 negative cells in kidney medulla. We validated antibodies against the β‐ and γ‐subunits from the same commercial source. Our work illustrates the importance of validation studies when using popular, commercially available anti‐ENaC antibodies.https://doi.org/10.14814/phy2.15554antibodyENaCepithelial biology |
| spellingShingle | Stephanie M. Mutchler Shujie Shi Sarah Christine M. Whelan Thomas R. Kleyman Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel Physiological Reports antibody ENaC epithelial biology |
| title | Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel |
| title_full | Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel |
| title_fullStr | Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel |
| title_full_unstemmed | Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel |
| title_short | Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel |
| title_sort | validation of commercially available antibodies directed against subunits of the epithelial na channel |
| topic | antibody ENaC epithelial biology |
| url | https://doi.org/10.14814/phy2.15554 |
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