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. 2012 Apr 15;23(8):1533–1545. doi: 10.1091/mbc.E11-06-0553

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© 2012 Jović 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: — Regulation of GBA transport by PI4Ks. (A) Following cotransfection of HA-GBA with LIMP-2-GFP for 24 h, COS-7 cells were fixed, permeabilized, and incubated with rabbit polyclonal antibody against the HA epitope. HA-GBA was found to colocalize with LIMP-2-GFP in LE/lysosomes, although a significant fraction of the enzyme also localizes to the ER and Golgi under the overexpression conditions. Scale bar: 10 μm. (B) COS-7 cells were transfected with either control siRNA duplexes or duplexes directed against LIMP-2 or PI4KIIα. After 2 d, cells were lysed and the efficiency of depletion was determined by Western blot analysis using a goat anti–LIMP-2, rabbit anti-PI4KIIα, or mouse anti-actin antibody. (C) Cells transfected with HA-GBA for 1 d were washed prior to addition of fresh media. After 6 h, the culture medium was collected and activity of secreted GBA on its fluorogenic substrate PFB-FDGlu was measured at pH 5.0, as described in Materials and Methods. GBA activity in untreated medium was compared with that of the medium incubated with GBA inhibitor CondB or with GlcCer. Bar graphs represent mean values ± SEM. (D and E) Cells were treated for 2 d with control or PI4KIIα siRNA oligos prior to transfection with HA-GBA. On the following day, cells were incubated for 6 h, either in fresh medium (D) or fresh medium containing 100 nM PIK93 (E). Activity of secreted GBA in collected medium was measured in three independent samples from three separate experiments and represented as mean fluorescence values ± SEM. (F) After a 2-d treatment with siRNA oligos (control or PI4KIIα), COS-7 cells were transfected with LIMP-2-GFP for 24 h; this was followed by preincubation with 100 nM LysoTracker Green for 10 min. Scale bars: 10 μm. (G) Following a 2-d treatment with siRNA oligos, cells were transfected with HA-GBA together with HA-PI4KIIα wild-type, KD, or LL mutant for 24 h. Cells were then washed and incubated for 6 h in fresh medium and assayed for the activity of secreted GBA in the collected medium. Shown are the mean fluorescence values ± SEM from eight independent experiments. Rescue efficiency of individual PI4KIIα mutants was assessed in comparison with the secretion recorded for the PI4KIIα knockdown sample (taken as 100%). Stars denote statistically significant differences relative to the PI4KIIα knockdown sample, as determined by one-way ANOVA (p < 0.05).