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. 2015 Jun 1;26(11):2030–2043. doi: 10.1091/mbc.E14-12-1597

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© 2015 Singh et al. 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 3: — PDZ domain–mediated interaction of SNX27 maintains plasma membrane localization and activity of NHE3. (A) Western blot analysis of total cell lysate prepared from HEK293-HA-NHE3 cells infected with lentivirus control shRNA or SNX27 shRNA. GAPDH was used as internal control. (B) Densitometric analysis of total protein expression from control shRNA and SNX27 shRNA–infected lysates showed that SNX27 protein expression was significantly reduced in HEK293-HA-NHE3 cells. Results are means ± SE of four separate studies. p values are comparison between control shRNA and SNX27 shRNA. (C) Na⁺/H⁺ exchange was measured in HEK293-HA-NHE3 cells expressing SNX27 shRNA or control shRNA with or without reconstitution of SNX27 by cotransfection of rat SNX27-GFP or PDZ mutant SNX27-GFP-GAGA. Results are means ± SE of four separate studies. p values are comparison between control shRNA and SNX27 shRNA or GFP-SNX27 and GFP-SNX27-GAGA. (D) Western blot analysis of NHE3 surface expression using biotinylation in HEK-HA-NHE3 cells expressing SNX27 shRNA or control shRNA with or without replacement by cotransfection of rat SNX27-GFP or its PDZ mutant SNX27-GFP-GAGA. A representative blot from three independent experiments with similar results. (E) GST, GST-SNX27-PDZ, or GST-SNX27-PDZ-GAGA was mixed with HEK-HA-NHE3 cell lysate and then subjected to pull down with GSH resin. Samples were analyzed by Western blot (IB) with antibodies against HA and GST. The experiment was repeated three times, and one representative result is shown. (F) Representative examples of fluorescence localization patterns of rat GFP-SNX27 or its PDZ mutant (GYGF → GAGA) versions of SNX27 relative to EEA1, verifying that its PDZ domain is not required for early endosomal localization of SNX27. Bar, 10 μm. Representative images.