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Fig. 5. Cholesterol extraction inhibits the agonist-induced endocytosis of LPA₁ but not M₁ mAChRs. (A) HeLa cells were transiently transfected with plasmids encoding either LPA₁ or M₁ mAChRs and were incubated in the absence (Untreated) or presence of agonist (10 µM LPA or 1 mM carbachol, respectively) and 50 µg/ml Alexa 594-Tfn for 30 minutes and then processed for indirect immunofluorescence localization of the transfected receptors. Bar, 10 µm. (B and C) HeLa cells were transfected as described above and were pre-incubated for 1 hour in the absence (Control) or presence of 5 mM MßCD or MßCD and 10 mM cholesterol/MßCD complexes prior to a subsequent incubation in the presence or absence of 10 µM LPA and 50 µg/ml Alexa 594-Tfn. The cells were fixed and processed for immunofluorescence localization of the transfected receptors. The extent of colocalization between LPA₁ (B) or M₁ mAChRs (C) and the internalized Alexa 594-Tfn was quantified using Metamorph image analysis as described in Materials and Methods. The data are expressed as the mean±s.e.m. of 20 cells/condition from a representative experiment that was performed three times with similar results. ^**P<0.01 compared with control, LPA-treated cells.

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