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. 2005 Apr 21;24(9):1664–1673. doi: 10.1038/sj.emboj.7600655

Figure 5.

Figure 5

Cholesterol extraction inhibits NKA receptor internalization. HEK293 cells were transfected with GFP-tagged NK2r (A) or ETBr (B) and at t=0 s treated with monensin to prevent receptor recycling. Confocal images were acquired every 2–3 min and the ratio of membrane to cytosol fluorescence was determined by image analysis for each time point (see Materials and methods). The initial ratio was normalized to 1.0 to allow averaging of the results from 5 to 10 experiments for each condition. Mean and s.e. are plotted versus time. Scale bars, 5 μm. Note that receptors have not been exposed to agonists, and that all experiments have been performed in serum-free bicarbonate/HEPES-buffered saline (see Materials and methods). (C) Following treatment of cells with CD for 30 min, at t=0 monensin was added and cells were assayed for internalization of fluorescence. Note that tonic recycling of the NK2r is blocked by cholesterol extraction. (D) Diffusion of GFP-tagged NK2r at the plasma membrane was studied with FRAP in nontreated (black line) and CD treated (gray line) HEK293 cells. Cholesterol extraction dramatically lowers NK2r mobility.