Fig. 7. Overexpression of EGFP-rab11WT decreases ß₂AR trafficking to lysosomes and inhibits receptor degradation. (A) 12ß6 cells transiently overexpressing empty vector (a,a') or EGFP-rab11WT (b,b') were treated with ISO for 6 hours in the presence of leupeptin. ß₂ARs were identified using an anti-C terminal antibody and are visualized in red, whereas labeling with anti-LAMP-2 antibody is visualized in green. Areas of colocalization of ß₂ARs and LAMP-2 appear yellow and are indicated by arrowheads in panel a. Arrows in panel b indicate areas of colocalization of ß₂ARs and EGF-rab11WT. Scale bar, 10 µm. (B,C) EcR293: ß₂AR cells transfected with EGFP-rab11WT were treated with either vehicle () or ponasterone () for 48 hours to induce rab11 overexpression, then surface receptors were biotinylated by exposure to EZ-link sulfo-NHS-biotin. Cells were washed and treated with ISO (5 µM) for the indicated times. Following incubation with agonist, the cells were lysed and receptors retrieved from the lysates for immunoblot analysis as described in Materials and Methods. Panel B, representative immunoblot of ß₂ARs following indicated times of exposure to agonist, showing receptors migrating at 43 kDa. Panel C, quantification of ß₂ARs remaining after varying times of exposure to agonist. Results are shown as the mean±s.d. of 3 separate experiments. * P<0.05 for the indicated time point.