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. 2010 Nov 15;21(22):3985–3997. doi: 10.1091/mbc.E10-05-0456

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© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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Figure 7. — Analysis of NKCC2 and p-NKCC2 in control and MAL/VIP17-overexpressing transgenic mice. Kidneys from three control (CTR) and three transgenic (OVER-MAL/VIP17) mice were lysed in antiphosphatase buffer with 1% Triton X-100 and subjected to immunoblotting. (A) Western blotting was made with anti-NKCC2 (T4), anti-actin, and MAL/VIP17 antibodies. (A′) Densitometric analysis of the 130- to 160-kDa broad band revealed by T4 antibody, corresponding to the glycosylated form of NKCC2 (gly-NKCC2) in both strains of mice. Values were normalized for β-actin signal. (A″) Western blotting analysis was made with anti-NKCC (T4) in kidney lysates from MAL/VIP17-overexpressing transgenic mice after incubation without (−) or with (+) PNGase F. The Western blotting reported is representative of three independent experiments. (B) Western blotting was made with anti-phospho-NKCC (R5) or anti-actin antibodies. (B′) Densitometric analysis of the 160-kDa band revealed by R5 antibody, normalized for β-actin signal, in both strains of mice. The densitometric values are expressed as mean ± SE of three independent experiments. Student's t test, *p < 0.001. (C) Sections from paraformaldehyde-fixed kidneys of control or transgenic mice were subjected to immunofluorescence with R5 antibody. Images were collected with a confocal laser-scanning microscope from kidneys of three control and three transgenic mice. Two representative pictures are presented. The staining with R5 was present in cells lining the cysts (C) as well as in normal tubules (T) in MAL/VIP17-transgenic mice (OVER-MAL/VIP17). The staining is absent in kidneys from control mice.