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. 2014 Jul 11;289(34):23653–23661. doi: 10.1074/jbc.M113.531145

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© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Inability to ubiquitinate ZnT2 in response to PRL impaired ZnT2 trafficking, zinc accumulation, and secretion. A, representative confocal images of ZnT2-HA (green) and K4R/K6R-HA (green) and Rab3A (red) in MECs treated with (+PRL) or without prolactin (−PRL) for 2 h. Nucleus was stained with TOPRO and pseudocolored blue. Pearson's coefficient (R) was determined by correlation analysis using an Olympus FluoView viewer. Shown are values (−1 (no overlap of pixels) and +1 (100% overlap)) from five fields of view from two independent experiments. B, representative confocal images of FluoZin-3 (green) in cells expressing ZnT2-HA or K4R/K6R-HA in MECs treated with or without prolactin for 2 h. C, representative immunoblots of ZnT2-HA and K4R/K6R-HA detected at the cell surface by cell surface biotinylation (+) treated with and without prolactin over 8 h. Infrared images were normalized across immunoblots according to the manufacturer's instructions. D, zinc secretion from MECs expressing ZnT2-HA or K4R/K6R-HA treated with or without PRL. Data represent mean pmol of zinc/μg of protein ± S.D. (error bars) (n = 4 samples/time point). Experiments were repeated two times.