FIGURE 3.

Verification of direct actin connectivity and actin-dependent recycling of engineered receptors. A, anti-FLAG immunoblots of equal amounts (25 μg before clarification) of cell extracts prepared from mock-transfected HEK293 cells or cells transfected with either FLAG-tagged δOR or δOR-Abd fusion receptor. Bracket to the right of the figure indicates the region of the blot shown in B, and the band indicated by ^* indicates the species corresponding in electrophoretic mobility to the glycosylated receptor monomer. B, anti-FLAG immunoblot showing receptor (top panel) and Ponceau S stain showing actin (bottom panel) from actin co-sedimentation assay comparing δOR-Abd (left) and δOR (right) loaded in equal amount (as shown in A) and processed in parallel. C, background-corrected densitometry of the species corresponding to monomeric receptor. Data shown are representative of three independent experiments. D, flow cytometric analysis assessing the sensitivity of engineered receptor recycling to cytochalasin D. Recycling measured by flow cytometry in normal culture medium (gray bars) is displayed in comparison to that measured in the presence of vehicle (0.2% DMSO, black bars) or 2 μg/ml cytochalasin D (cytoD, white bars). ^* denotes p < 0.05 by Student's t test comparing vehicle-treated and cytochalasin D-treated cells, n = 4-5 experiments.