REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 7
In vivo biotinylation analysis of REEP plasma membrane expression.
To determine if REEPs were expressed at the plasma membrane, HEK293A cells were transfected with Flag-REEP1, Flag-REEP2, or Flag–REEP6 cDNA with or without co-transfected HA-α2A AR or HA-α2C AR cDNAs. Forty-eight hrs post-transfections, cells were treated with the biotinylating reagent EZ-Link Sulfo-NHS-SS-Biotin (Pierce), total cell lysates were isolated, and biotinylated proteins were precipitated by incubation with avidin-agarose. Avidin precipitated proteins and total cell lysates (Input) were then analyzed by SDS-PAGE and immunoblotting techniques. Transferred proteins were probed with monoclonal anti-HA or anti-M2 Ab. Molecular weight markers (kDa) are shown to the left. A. Top: Mature glycosylated α2A and α2C ARs (thick arrow) were predominantly precipitated by avidin, consistent with selective biotinylation of plasma membrane proteins. Aggregated α2 ARs can be seen at the very op of the blot (*). Bottom: No REEPs were precipitated by avidin, demonstrating that they were not present at the plasma membrane, when either expressed with α2 ARs or alone. B. Top: Analysis of total cell lysates for α2A and α2C ARs demonstrated the presence of both mature (thick arrow) and immature forms (thin arrow). Bottom: Immunoblotting of total cell lysates for REEPs is shown, demonstrating strong REEP expression. Representative of three experiments.