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. 2019 Jul 1;30(14):1716–1728. doi: 10.1091/mbc.E18-12-0811

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© 2019 Gravotta et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 2: — Clathrin silencing disrupts the apical sorting of megalin. MDCK cells stably expressing HA-mMeg were transfected with siRNA targeting clathrin heavy chain and analyzed 4 d after culture on Transwells. (A) Western blot analysis of clathrin knockdown. (B) Domain-selective biotinylation (top panels) and quantification (bottom panels) showing steady-state localization of megalin, transferrin receptor (TfR), and claudin-2 in control and clathrin knockdown MDCK cells. Note the decreased polarity of HA-mMeg and TfR in clathrin KD cells. (C) Surface immunofluorescence showing steady-state HA-mMeg localization in control (left) and clathrin knockdown (right) HA-mMeg MDCK cells. Cells were incubated apically with AF488 (green) labeled anti-HA antibody or basally with AF647 (red) labeled anti-HA antibody. Right panel, integrated fluorescence quantification of images in C, in entire confocal stacks and on a pixel by pixel basis, according to Perez Bay et al. (2016).