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. 2022 Sep 7;13:971251. doi: 10.3389/fphys.2022.971251

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Copyright © 2022 Rosenbaek, Petrillo, van Bemmelen, Staub, Murali and Fenton.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Nedd4-2 deficiency does not change the constitutive internalization of NCC from the apical plasma membrane in MDCKI-hNCC cells. (A) Semi-quantitative assessment of the percentage of internalized NCC (steady-state surface levels equals 100%) at 15 and 30 min. Data are means ± S.E.M. (n = 12). Significance is assessed with 2way ANOVA followed by Tukey’s multiple comparisons test. (B) Representative blots showing NCC internalization. Following biotinylation, MDCKI-hNCC Ctrl and Nedd4-2 (N4-2) KD cells were incubated for 15 or 30 min at 37˚C to allow membrane protein internalization before treatment with the reducing agent MesNa (stripping biotin). Lane 1-3 show expression of steady-state surface NCC. Lane 4-6 show surface NCC after treatment with the stripping agent MesNa. Internalized NCC was isolated, detected by western blot, and quantified by densitometry.