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Fig. 6. LPA₁ receptors localize to detergent-resistant cellular domains upon agonist stimulation. (A) LPA₁/HeLa cells were incubated with 10 µM LPA for different times and subsequently treated with 1% cold Triton X-100 on ice for 3 minutes, fixed and processed for indirect immunofluorescence localization of LPA₁. (B) Quantitative analysis of receptor expression after detergent extraction was performed by MetaMorph image analysis as described in Materials and Methods. Cells were either untreated, treated with 5 µM cytochalasin D (Cyto. D) for 30 minutes, or treated with 5 mM MßCD for 1 hour, prior to incubation with 10 µM LPA for the indicated times. The LPA₁ labeling in detergent-extracted cells was normalized to the amount of LPA₁ labeling observed in non-agonist-treated cells, which had not been subjected to detergent extraction. The data are presented as the mean±s.e.m. of five to six cells per time point and are from a representative experiment that was repeated twice with similar results. (C) LPA₁/HeLa cells were incubated with 10 µM LPA for different times and incubated with mouse anti-FLAG antibody on ice for 30 minutes prior to extraction with ice-cold 1% Triton X-100, to label surface LPA₁ receptors. Cells were then processed for indirect immunofluorescence localization of surface LPA₁. (D) Quantitative analysis of surface LPA₁ receptor expression after detergent extraction was performed by MetaMorph image analysis as described in Materials and Methods. The LPA₁ labeling in detergent-extracted cells was normalized to the amount of LPA₁ labeling observed in non-agonist treated cells, which had not been subjected to detergent extraction. The data are presented as the mean±s.e.m. of five to six cells per time point and are from a representative experiment that was repeated twice with similar results. ^**P<0.01, comparison of the amount of detergent-resistant surface LPA₁ staining after the indicated time of agonist treatment with that observed in unstimulated cells. Bar, 10 µm.

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