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. 2016 May 5;11(5):e0154805. doi: 10.1371/journal.pone.0154805

Fig 6. Effect of endocytosis inhibitors on endosomal sorting of cystinosin-LKG.

Fig 6

HK-2 cells stably transfected with RFP-tagged cystinosin-LKG, after 48h serum starvation, were treated 30’ with 5 μg/ml chlorpromazine (CPZ) a clathrin-dependent endocytosis inhibitor or with 30 μg/ml methyl-β-cyclodextrin (MβCD) which affects clathrin-independent pathway. Qualitative analysis shows the presence of RFP-tagged cystinosin-LKG on the plasma membrane stained with WGA green, but the RFP signal accumulated more in CPZ treated cells (A). Scale bar = 20 μm. In the same experimental conditions, the uptake of Alexa Fluor® 488 transferrin (488-Tf) was assayed in order to confirm the inhibition of clathrin-dependent endocytosis (B). Scale bar = 20 μm. Quantitative analysis, achieved by protein surface biotinylation and SDS-PAGE, shows that CPZ treatment induces a significant increase of cystinosin-LKG presence on the plasma membrane (C). Means ± SEM of three experiments are shown.