REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 10
Representative FACS histograms and REEP gating strategy.
HEK293A cells were co-transfected with HA-α2A or -α2C ARs and control vector, Flag-REEP1, -REEP2, or –REEP6 cDNAs. Forty-eight hrs post-transfection, relative expression levels of each receptor were determined in non-permeabilized (Surface) and permeabilized (Total) cells by using a FACS assay. α2 ARs and REEPs were labeled with FITC-conjugated anti-HA and Cy3-conjugated anti-Flag (M2) antibodies respectively. A. Representative α2A AR FACS fluorescence distributions under non-permeabilized (Top) and permeabilized (Bottom) conditions (UT = untransfected). B. Representative α2C AR FACS fluorescence distributions under non-permeabilized (Top) and permeabilized (Bottom) conditions (UT = untransfected). Note the shift to higher median fluorescence upon permeabilization, which is greater for α2C vs. α2A ARs due to the larger pool of intracellular α2C ARs [25]. C. Representative gating strategy for FACS analysis of co-expressed α2 ARs and REEPs. Background staining of non-transfected HEK293A cells with FITC-conjugated anti-HA and Cy3-conjugated anti-Flag (M2) antibodies was determined (Top) and used to set the FACS gating thresholds for background fluorescence (Q4). Representative α2A AR and REEP1 FACS data set demonstrating co-expression of both proteins is shown (Bottom). All cells contained in quadrants Q1-3 were analyzed for calculation of REEP effects on co-expressed α2 AR surface and intracellular expression (see Table 4A). Data summarized from between five and eight different transfections for each combination of α2 AR and REEP with a minimum of 1000 cells analyzed for each transfection.