REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 13
Immunoprecipitation of α2 ARs by REEPs.
HEK293A cells were co-transfected with HA-α2A or -α2C ARs and control vector, Flag-REEP1, -REEP2, or –REEP6. Eighteen hrs post-transfection, total cell lysates were isolated and analyzed by co-immunoprecipitation (co-IP) with M2 antibody (anti-Flag) and immunoblotting. Transferred proteins were probed with rabbit anti-HA Ab (A/C) or anti-M2 (B/D). Molecular weight markers (kDa) are shown to the left. Lanes representing total cell lysates (input), REEP co-IP, and α2 AR co-IP are labeled at the bottom of the blots. A. Note absence of α2A AR co-IP with any REEP tested (middle). B. Similar amounts of REEP1, REEP2, and REEP6 were present in REEP co-IP assays. C. REEP1, REEP2, and REEP6 could co-IP the REEP-enhanced, minimally glycosylated form of α2C AR (Figures 9 and 10). D. As seen with α2A AR/REEP co-IP assays, similar amounts of REEP1, REEP2, and REEP6 were present in REEP co-IP assays. Neither α2A nor α2C ARs could co-IP any REEP tested (B and D, right). IgG light chain artifacts are indicated by an arrow (far right).