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KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts
We recently reported potassium–chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium–chloride cotransport (KCC) isoforms by reverse transcriptase–polymerase chain re...
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| Published in: | Experimental eye research 2006-11, Vol.83 (5), p.1287-1294 |
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| container_title | Experimental eye research |
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| creator | Misri, Sandeep Chimote, Ameet A. Adragna, Norma C. Warwar, Ronald Brown, Thomas L. Lauf, Peter K. |
| description | We recently reported potassium–chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium–chloride cotransport (KCC) isoforms by reverse transcriptase–polymerase chain reaction (RT–PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT–PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150
kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105
kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112
kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100
kDa band in HLE-B3 cells. In the human lens tissues, a 115
kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100
kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4. |
| doi_str_mv | 10.1016/j.exer.2006.07.006 |
| format | article |
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kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105
kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112
kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100
kDa band in HLE-B3 cells. In the human lens tissues, a 115
kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100
kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.</description><identifier>ISSN: 0014-4835</identifier><identifier>EISSN: 1096-0007</identifier><identifier>DOI: 10.1016/j.exer.2006.07.006</identifier><identifier>PMID: 16949074</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>Blotting, Western - methods ; cataract ; cation–chloride cotransporter ; Cell Line ; Chlorides - metabolism ; Eye Proteins - analysis ; Eye Proteins - metabolism ; human lens ; human lens epithelial B3 cell line ; Humans ; Immunochemistry - methods ; immunofluorescence ; Immunohistochemistry - methods ; Isomerism ; K Cl- Cotransporters ; KCC isoform ; Lens, Crystalline - chemistry ; Membrane Proteins - metabolism ; Microscopy, Confocal ; Microscopy, Fluorescence - methods ; NKCC1 isoform ; Potassium - metabolism ; Reverse Transcriptase Polymerase Chain Reaction - methods ; reverse transcriptase–polymerase chain reaction ; RNA, Messenger - analysis ; Symporters - analysis ; Symporters - metabolism ; Tissue Extracts - chemistry ; Western blot</subject><ispartof>Experimental eye research, 2006-11, Vol.83 (5), p.1287-1294</ispartof><rights>2006 Elsevier Ltd</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c354t-a0c8cbe921b156eae1f3b25302af6f3fac7a483e3a5d1405a3acfe2d7c31bb0b3</citedby><cites>FETCH-LOGICAL-c354t-a0c8cbe921b156eae1f3b25302af6f3fac7a483e3a5d1405a3acfe2d7c31bb0b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,783,787,27936,27937</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16949074$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Misri, Sandeep</creatorcontrib><creatorcontrib>Chimote, Ameet A.</creatorcontrib><creatorcontrib>Adragna, Norma C.</creatorcontrib><creatorcontrib>Warwar, Ronald</creatorcontrib><creatorcontrib>Brown, Thomas L.</creatorcontrib><creatorcontrib>Lauf, Peter K.</creatorcontrib><title>KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts</title><title>Experimental eye research</title><addtitle>Exp Eye Res</addtitle><description>We recently reported potassium–chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium–chloride cotransport (KCC) isoforms by reverse transcriptase–polymerase chain reaction (RT–PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT–PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150
kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105
kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112
kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100
kDa band in HLE-B3 cells. In the human lens tissues, a 115
kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100
kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.</description><subject>Blotting, Western - methods</subject><subject>cataract</subject><subject>cation–chloride cotransporter</subject><subject>Cell Line</subject><subject>Chlorides - metabolism</subject><subject>Eye Proteins - analysis</subject><subject>Eye Proteins - metabolism</subject><subject>human lens</subject><subject>human lens epithelial B3 cell line</subject><subject>Humans</subject><subject>Immunochemistry - methods</subject><subject>immunofluorescence</subject><subject>Immunohistochemistry - methods</subject><subject>Isomerism</subject><subject>K Cl- Cotransporters</subject><subject>KCC isoform</subject><subject>Lens, Crystalline - chemistry</subject><subject>Membrane Proteins - metabolism</subject><subject>Microscopy, Confocal</subject><subject>Microscopy, Fluorescence - methods</subject><subject>NKCC1 isoform</subject><subject>Potassium - metabolism</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - methods</subject><subject>reverse transcriptase–polymerase chain reaction</subject><subject>RNA, Messenger - analysis</subject><subject>Symporters - analysis</subject><subject>Symporters - metabolism</subject><subject>Tissue Extracts - chemistry</subject><subject>Western blot</subject><issn>0014-4835</issn><issn>1096-0007</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><recordid>eNp9kEtPwzAQhC0EoqXwBzggnxAcEtaxE6cSF6h4iUq9wNlynI3qKo9iJ6j8e1ylEjdOI62-Gc0OIZcMYgYsu9vEuEMXJwBZDDIOckSmDOZZBADymEwBmIhEztMJOfN-E65cSHFKJiybizlIMSWr98WCWt9VnWs8tS3VdD00uqU1tp7i1vZrrK2uqcG6prVtkd488luq23JEeuv9gBR3vdOm9-fkpNK1x4uDzsjn89PH4jVarl7eFg_LyPBU9JEGk5sC5wkrWJqhRlbxIkk5JLrKKl5pI3UojlynJROQaq5NhUkpDWdFAQWfkesxd-u6rwF9rxrr9x11i93gVZbnUgohApiMoHGd9w4rtXW20e5HMVD7GdVG7WdU-xkVSBUkmK4O6UPRYPlnOewWgPsRwPDjtw12byy2Bkvr0PSq7Ox_-b_pRYOY</recordid><startdate>20061101</startdate><enddate>20061101</enddate><creator>Misri, Sandeep</creator><creator>Chimote, Ameet A.</creator><creator>Adragna, Norma C.</creator><creator>Warwar, Ronald</creator><creator>Brown, Thomas L.</creator><creator>Lauf, Peter K.</creator><general>Elsevier Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20061101</creationdate><title>KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts</title><author>Misri, Sandeep ; Chimote, Ameet A. ; Adragna, Norma C. ; Warwar, Ronald ; Brown, Thomas L. ; Lauf, Peter K.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c354t-a0c8cbe921b156eae1f3b25302af6f3fac7a483e3a5d1405a3acfe2d7c31bb0b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Blotting, Western - methods</topic><topic>cataract</topic><topic>cation–chloride cotransporter</topic><topic>Cell Line</topic><topic>Chlorides - metabolism</topic><topic>Eye Proteins - analysis</topic><topic>Eye Proteins - metabolism</topic><topic>human lens</topic><topic>human lens epithelial B3 cell line</topic><topic>Humans</topic><topic>Immunochemistry - methods</topic><topic>immunofluorescence</topic><topic>Immunohistochemistry - methods</topic><topic>Isomerism</topic><topic>K Cl- Cotransporters</topic><topic>KCC isoform</topic><topic>Lens, Crystalline - chemistry</topic><topic>Membrane Proteins - metabolism</topic><topic>Microscopy, Confocal</topic><topic>Microscopy, Fluorescence - methods</topic><topic>NKCC1 isoform</topic><topic>Potassium - metabolism</topic><topic>Reverse Transcriptase Polymerase Chain Reaction - methods</topic><topic>reverse transcriptase–polymerase chain reaction</topic><topic>RNA, Messenger - analysis</topic><topic>Symporters - analysis</topic><topic>Symporters - metabolism</topic><topic>Tissue Extracts - chemistry</topic><topic>Western blot</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Misri, Sandeep</creatorcontrib><creatorcontrib>Chimote, Ameet A.</creatorcontrib><creatorcontrib>Adragna, Norma C.</creatorcontrib><creatorcontrib>Warwar, Ronald</creatorcontrib><creatorcontrib>Brown, Thomas L.</creatorcontrib><creatorcontrib>Lauf, Peter K.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Experimental eye research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Misri, Sandeep</au><au>Chimote, Ameet A.</au><au>Adragna, Norma C.</au><au>Warwar, Ronald</au><au>Brown, Thomas L.</au><au>Lauf, Peter K.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts</atitle><jtitle>Experimental eye research</jtitle><addtitle>Exp Eye Res</addtitle><date>2006-11-01</date><risdate>2006</risdate><volume>83</volume><issue>5</issue><spage>1287</spage><epage>1294</epage><pages>1287-1294</pages><issn>0014-4835</issn><eissn>1096-0007</eissn><abstract>We recently reported potassium–chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium–chloride cotransport (KCC) isoforms by reverse transcriptase–polymerase chain reaction (RT–PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT–PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150
kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105
kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112
kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100
kDa band in HLE-B3 cells. In the human lens tissues, a 115
kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100
kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>16949074</pmid><doi>10.1016/j.exer.2006.07.006</doi><tpages>8</tpages></addata></record> |
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| subjects | Blotting, Western - methods cataract cation–chloride cotransporter Cell Line Chlorides - metabolism Eye Proteins - analysis Eye Proteins - metabolism human lens human lens epithelial B3 cell line Humans Immunochemistry - methods immunofluorescence Immunohistochemistry - methods Isomerism K Cl- Cotransporters KCC isoform Lens, Crystalline - chemistry Membrane Proteins - metabolism Microscopy, Confocal Microscopy, Fluorescence - methods NKCC1 isoform Potassium - metabolism Reverse Transcriptase Polymerase Chain Reaction - methods reverse transcriptase–polymerase chain reaction RNA, Messenger - analysis Symporters - analysis Symporters - metabolism Tissue Extracts - chemistry Western blot |
| title | KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts |
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