REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 9
Identification of α2 AR and REEP expression in permeabilized and non-permeabilized cells.
HEK293A cells were transfected with HA-α2A AR, HA-α2C AR, Flag-REEP1, Flag-REEP2, or Flag–REEP6 cDNA. Forty-eight hrs post-transfections, cells were fixed with 4% PFA, under permeabilized and non-permeabilized conditions, and examined by wide field immunofluorescent microscopy. HA-α2 ARs were labeled with 16B12-conjugated FITC (anti-HA) antisera and Flag-REEPs were identified by M2-conjugated Alexa 594 (anti-Flag) antisera. Permeabilized: α2 AR staining of permeabilized cells demonstrated plasma membrane and intracellular fluorescence. Note predominant plasma membrane staining, compared to intracellular staining, consistent with efficient plasma membrane trafficking of α2A ARs. α2C AR staining revealed predominant intracellular staining with a perinuclear shadow, due their predominant localization within the ER when expressed in HEK293A cells (25). REEP staining of permeabilized cells showed strong intracellular localization, due to their ER localization. Non-permeabilized: α2A ARs demonstrated extensive staining of the plasma membrane, however, α2C ARs showed staining only of the plasma membrane, demonstrating that the extracellular HA epitope was not accessible by anti-HA antibody under non-permeabilized conditions. Intracellular REEP staining was similar when performed under permeabilized and non-permeabilized conditions, demonstrating that the carboxyl terminal Flag epitope was accessible by anti-Flag antibody under either condition, due to its cytoplasmic localization. REEP staining of non-permeabilized cells revealed intracellular staining, due to the ability of antibodies to gain intracellular entry following PFA fixation [27]. Representative of three separate transfections. Scale bars: 10 µm.