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. Author manuscript; available in PMC: 2013 Sep 4.
Published in final edited form as: Traffic. 2008 Sep 13;9(12):2253–2264. doi: 10.1111/j.1600-0854.2008.00819.x

Figure 6. LY294002 inhibits rapid recycling in COS-1 cells.

Figure 6

A) COS-1 cells were incubated with 20 μg/ml Alexa568-Tf for 1 hour at 16°C, then chased in 37°C medium supplemented with unlabeled Tf for indicated times, all in the presence or absence of LY294002. Intracellular Tf levels were measured by flow cytometry. Rapid Tf recycling in the initial 5 to 10 min chase was markedly inhibited by LY294002. B) COS-1 cells transiently expressing YFP-GGA1 were treated as in (A), and images were acquired after 5 min chase (Video S11). A series of still images and the maximal image from a time stack of 50 continuous frames indicated that LY294002 significantly inhibited the loading of Tf into G-GGA/clathrin structures. In contrast, control cells were not affected (data not shown, but compare Figure 4D and Video S6). Boxed region includes several G-GGA structures. Bar, 1 μm. C) Quantitative analysis indicated about 85% inhibition of Tf loading of G-GGA structures after 5 min chase in the presence of LY294002.