Fig. 9. Dominant-inhibitory GFP-Rab5a S34N inhibits LPA₁ internalization. (A) LPA₁-expressing HeLa cells were incubated for 30 minutes in the absence (Untreated) or presence of 10 µM LPA prior to indirect immunofluorescence localization of LPA₁. (B) Stable LPA₁ transfectants were transiently transfected with plasmids encoding either WT GFP-Rab5a or dominant-inhibitory GFP-Rab5a S34N. The cells were then incubated with 10 µM LPA for 30 minutes, fixed and processed for indirect immunofluorescence localization of LPA₁ using mouse anti-FLAG antibodies followed by Alexa594-labeled goat anti-mouse IgG. Bar, 10 µm. (C) Quantitation of inhibitory phenotype of dominant-negative dynamin2 and Rab5 mutants on LPA₁ internalization. Stable LPA₁ transfectants that were transiently transfected with no plasmid, Dyn2-GFP K44A, or GFP-Rab5a S34N were then incubated with 10 µM LPA for 30 minutes. The cells were fixed and processed for indirect immunofluorescence localization of LPA₁. Two hundred cells per sample were scored for the presence of endocytic vesicles that contained LPA₁ in an experiment. The data from three independent experiments were expressed as the mean ± s.d. of the percentage of cells that contained LPA₁⁺ endosomal structures under each transfection condition (n=3).