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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 5: — Down-regulation of reggie-1 impairs Tf recycling in HeLa cells. (A) Wild-type and shRNA stably transfected HeLa cells were pulsed with Tf-rhod for 5 min and then chased for 10 and 20 min. Reggie-depleted (shR1) cells showed no defects in Tf-rhod uptake (5-min pulse) and transport from early endosomes to the recycling compartment (10-min chase) compared with control transfected (shLuc) and untransfected HeLa cells. After a 20-min chase, however, the accumulation of Tf-rhod was retained at the perinuclear compartment in the majority of shR1 cells but reduced in shLuc and HeLa cells. (B, C) Quantification of the effect of reggie-1 down-regulation on Tf-rhod uptake (B) and recycling (C) in HeLa cells (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). (D) Western blot (WB) analysis of pulse-chase experiments in shLuc and shR1 cells using biotinylated Tf (Tf-biotin) confirmed that Tf recycling was delayed in reggie-depleted cells after a 20-min chase. No significant difference was observed in Tf-biotin uptake upon reggie down-regulation (n = 4, **p < 0.01, paired t test, mean ± SEM). α-Tubulin (α-tub) was used as loading control. (E) Expression of a shRNA-resistant reggie-1 construct (R1-EGFP rescue) rescued the Tf-rhod recycling defects observed after a 20-min chase in transfected (arrowheads) but not in untransfected shR1 cells. (F, G) Pulse-chase experiments were performed in shR1 (F) and control shLuc (G) cells expressing a Rab11a constitutively active (EGFP-Rab11a-CA) and dominant-negative (EGFP-Rab11a-DN) mutant, respectively. Whereas the Rab11a-CA construct was able to rescue the Tf-rhod recycling defects in shR1 cells without affecting its uptake (arrowheads; F), in shLuc cells the Rab11a-DN mutant impaired both Tf-rhod uptake and recycling (arrowheads; G). (H, I) Quantification of Tf-rhod perinuclear accumulation from pulse-chase experiments in E–G. A constitutively active mutant of Rab8a (EGFP-Rab8a-CA) was not able to rescue or mimic the effects of reggie depletion in shR1 or shLuc cells, respectively (n = 3, **p < 0.01, one-way ANOVA; error bars, SEM). Scale bars, 10 μm.