A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function.
A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function.
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C57 BL/6小鼠NKCC 2中的5个氨基酸缺失影响NKCC 2磷酸化的分析，但不影响肾功能。
DOI：
10.1111/apha.13705
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发表时间：
2021-09
期刊：
影响因子：
--
通讯作者：
Loffing J
中科院分区：
文献类型：
--
作者：
Moser S;Sugano Y;Wengi A;Fisi V;Lindtoft Rosenbaek L;Mariniello M;Loffing-Cueni D;McCormick JA;Fenton RA;Loffing J
厚升支（TAL）中呋塞米敏感性Na+-K+-2Cl−协同转运蛋白（NKCC 2）的磷酸化水平被用作NKCC 2激活和TAL功能的替代标志物。然而，在小鼠中，用磷酸化苏氨酸96和101抗体（抗pT 96/pT 101）分析NKCC 2磷酸化得到的结果不一致。我们的目的是（a）阐明这些不一致性和（B）开发一种磷酸化特异性抗体，以确保可靠地检测小鼠中的NKCC 2磷酸化。使用遗传信息、分子生物学、生物化学技术和小鼠表型分析研究了两种常用小鼠品系（即129 Sv和C57 BL/6小鼠）的NKCC 2和肾功能。此外，开发并表征了一种新的磷酸化特异性小鼠NKCC 2抗体。氨基酸序列比对显示，C57 BL/6小鼠在NKCC 2中存在品系特异性5个氨基酸缺失（ΔF97-T101），这减少了使用先前开发的pT 96/pT 101 NKCC 2抗体对NKCC 2磷酸化的检测。相反，抗体与磷酸化噻嗪敏感性NaCl协同转运蛋白（NCC）发生交叉反应，这可能会模糊结果的解释。有趣的是，NKCC 2中的缺失不影响肾功能和/或肾离子转运蛋白的表达，如通过杂交129 Sv和C57 BL/6小鼠的F2代的分析所指示的。新开发的pT 96 NKCC 2抗体可检测两种小鼠品系中的pNKCC 2，且与磷酸化NCC无交叉反应性。我们的工作揭示了一个迄今未被重视的，但必要的，在小鼠NKCC 2的氨基酸序列，需要考虑在小鼠中分析NKCC 2磷酸化的菌株差异。新的pNKCC 2抗体规避了这一技术警告。
The phosphorylation level of the furosemide‐sensitive Na+‐K+‐2Cl− cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti‐pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform‐specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice. Genetic information, molecular biology, biochemical techniques and mouse phenotyping was used to study NKCC2 and kidney function in two commonly used mouse strains (ie 129Sv and in C57BL/6 mice). Moreover, a new phosphoform‐specific mouse NKCC2 antibody was developed and characterized. Amino acids sequence alignment revealed that C57BL/6 mice have a strain‐specific five amino acids deletion (ΔF97‐T101) in NKCC2 that diminishes the detection of NKCC2 phosphorylation with previously developed pT96/pT101 NKCC2 antibodies. Instead, the antibodies cross‐react with the phosphorylated thiazide‐sensitive NaCl cotransporter (NCC), which can obscure interpretation of results. Interestingly, the deletion in NKCC2 does not impact on kidney function and/or expression of renal ion transport proteins as indicated by the analysis of the F2 generation of crossbred 129Sv and C57BL/6 mice. A newly developed pT96 NKCC2 antibody detects pNKCC2 in both mouse strains and shows no cross‐reactivity with phosphorylated NCC. Our work reveals a hitherto unappreciated, but essential, strain difference in the amino acids sequence of mouse NKCC2 that needs to be considered when analysing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat.
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影响因子：
4.5
作者：
Gamba, Gerardo;Friedman, Peter A.
通讯作者：
Friedman, Peter A.
影响因子：
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作者：
Ares, Gustavo R.;Haque, Mohammed Z.;Ortiz, Pablo A.
通讯作者：
Ortiz, Pablo A.
DOI：
10.1152/ajprenal.00316.2006
发表时间：
2007-09-01
影响因子：
4.2
作者：
Fenton, Robert A.;Brond, Lone;Praetorius, Jeppe
通讯作者：
Praetorius, Jeppe
影响因子：
15.9
作者：
Gonzalez-Villalobos, Romer A.;Janjoulia, Tea;McDonough, Alicia A.
通讯作者：
McDonough, Alicia A.
DOI：
10.1074/jbc.m109.060004
发表时间：
2009-12-18
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hannemann A;Christie JK;Flatman PW
通讯作者：
Flatman PW