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. 2013 Sep 1;24(17):2689–2702. doi: 10.1091/mbc.E12-12-0854

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© 2013 Solis 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 2: — Reggie-1 head (SPFH) domain is necessary for its localization at tubules in HeLa cells. (A) Schematic representation of the C-terminal, EGFP-tagged reggie-1 constructs used in this study. Reggie-1 full-length (R1) and its membrane-associated deletion constructs lacking the tail (flotillin) domain (R1NT), the head domain (R1MCT), or both domains (R1WTSH). The EGFP tags are depicted as green ovals. (B–D) Confocal images of HeLa cells expressing wild-type reggie-1-mRFP (R1-mRFP) and reggie-1 deletion mutants revealed that R1NT-EGFP (B) and R1MCT-EGFP (C) decorate reggie-tubules, whereas the R1WTSH-EGFP (D) construct was not observed at tubules. Boxed areas are enlarged in insets. (E) Expression levels of reggie-1 (R1), reggie-2 (R2), Rab11a, SNX4, TfR, and glyceraldehyde-3-phosphate dehydrogenase as loading control were analyzed by Western blots (WBs) from extracts of shRNA stably transfected and untransfected HeLa cells. shRNA against reggie-1 (shR1) strongly reduced reggie-1 and reggie-2 expression compared with control transfected shRNA (shLuc) and HeLa cells, whereas no effects were observed on the levels of Rab11a and SNX4. Biotinylation analysis showed that TfR surface expression was not affected in shR1 cells. Total TfR expression level was significantly reduced in shR1 cells compared with shLuc and HeLa cells. This effect was rescued by blocking lysosomal degradation with 50 μM chloroquine (n = 4, **p < 0.01, one-way ANOVA, mean ± SEM). (F) Expression of the reggie-1 deletion constructs in shR1 HeLa cells revealed the formation of reggie-tubules by the construct containing the head domain (R1NT-EGFP). The reggie-1 deletion mutants lacking this domain (R1MCT and R1WTSH) were not observed at tubules. Scale bars, 10 μm.