Figure 7.

A pool of the hGLP-2R is associated with the cellular detergent insoluble fraction and transiently colocalizes with caveolin-1 in transfected cells. (A) DLD-1 cells transiently transfected with FLAG-hGLP-2R or pcDNA3.1 were lysed in a 1% Triton X-100 containing buffer as described in MATERIALS AND METHODS. After centrifugation, the supernatant (Triton X-100–soluble fraction) was recovered, and the pellet was solubilized in the same buffer also containing 0.5% SDS (Triton X-100–insoluble fraction). Aliquots of the Triton X-100–soluble and –insoluble fractions were deglycosylated, and analyzed by immunoblotting for EEA1, hGLP-2R, and caveolin-1. Antigen/antibody complexes were visualized by enhanced chemiluminescence after incubation with a horseradish peroxidase-conjugated secondary antibody. (B) BHK cells transiently expressing the FLAG-hGLP-2R were prelabeled on ice with anti-FLAG antibody and then incubated with media alone (control) or 10 nM hGLP-2R at 37°C for 0–60 min as described in MATERIALS AND METHODS. After fixation and permeabilization, cells were incubated with anti-caveolin-1-FITC. A Cy3-conjugated secondary antibody was used to visualize hGLP-2R/anti-FLAG antibody complexes. Confocal microscopy images are representative of two to three independent experiments.