REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 3
Confocal ER localization of REEPs with ER Tracker™ dye.
HEK293A cells were transfected with Flag-REEP1, -REEP2, or -REEP6. Forty-eight hrs post-transfection, cells were fixed with 4% PFA, permeabilized, and examined by confocal microscopy. The ER was identified by staining with the ER-specific dye ER Tracker™ Blue/White DPX, which is retained within the ER lumen, thus labeling the ER tubular network (29). REEPs were stained with M2-Alexa 488 antibody (anti-Flag). REEP1/2/6 staining (Left) delineated an intracellular reticular pattern that showed extensive overlap with the ER luminal network (Middle), as seen in merged images (Right). Areas of punctate REEP expression likely represent areas of focal accumulation within the ER and confocal cross-sections of ER tubules. REEP1 demonstrated focal accumulation near the nucleus (arrow), whereas REEP2 was not found in all ER Tracker™-labeled ER regions (arrow), suggesting the existence of possible REEP/ER subdomains. Representative of three separate transfections. Scale bars: 25 µm.