Fig. 4. Internalized LPA₁ co-localizes with the clathrin-dependent endosomal markers, EEA-1 and transferrin receptor (TfR). Stably transfected HeLa cells expressing LPA₁ were treated with 10 µM LPA for 30 minutes, fixed, processed for double-label indirect immunofluorescence and analyzed by confocal microscopy. The endosomal markers EEA-1 and TfR, and the lysosomal marker LAMP-2, were localized with mouse monoclonal antibodies followed by Alexa488-labeled goat anti-mouse secondary antibodies. LPA₁ was localized using rabbit anti-FLAG antibodies followed by Alexa594-labeled goat anti-rabbit secondary antibodies. The arrows in the upper panels indicate endosomal structures that contain both LPA₁ and TfR. The arrow in the bottom panel indicates a structure that is LAMP2⁺, but does not contain LPA₁. Bar, 10 µm.