To quantitate relative AP2 membrane staining, random fields were visualized by epifluorescence and digitized. From micrographs, membrane fluorescence was traced using the segmented line tool, and intracellular staining

regions of interest (ROI) were measured using the ImageJ Measure ROI tool. The averaged background pixel intensity was subtracted from both the averaged membrane and intracellular intensities, and the ratio of basolateral versus intracellular fluorescence intensity was determined. The K+ depletion/repletion assays were performed as previously described.22 For ASGP-R antibody trafficking studies, K+-depleted cells Smoothened Agonist cell line were surface labeled with anti-ASGP-R antibodies (1:25) for 20 minutes on ice. Cells were reincubated in prewarmed medium supplemented with 10 mM of KCl, and the ASGP-R antibody-antigen complexes

were allowed to traffic for desired times at 37°C. Cells were fixed, permeabilized, and the trafficked ASGP-R antibodies were labeled with secondary antibodies. Cells were stained as described above and mounted in Tris-buffered saline (pH 10.5) containing 5% glycerol and 4 mg/mL of phenylenediamine. Fluorophores were excited with a 2.5-W Kr/Ar laser Lapatinib mouse (Spectra Physics, Irvine, CA) and visualized using an Olympus 1X 71 inverted microscope and total internal reflection fluorescence (TIRF) illuminator (Olympus, Center Valley, PA). Images were collected using a Photometrics Evolve EM-CCD (charge-coupled

device) camera (Photometrics, Tuscon, AZ) and Metamorph software (Molecular Devices, Sunnyvale, CA). Puncta were counted using the FociPicker three-dimensional ImageJ plugin. Fully covered 10,000 px2 ROIs were selected from random images. In general, five images/experiment were acquired and two to five fields/image were counted. For transmission electron microscopy (TEM), cells were fixed and processed using standard Epon embedding techniques. Ultrathin sections were cut and stained with uranyl acetate, followed by lead citrate. Grids were viewed on a Hitachi 7600 transmission electron microscope (Hitachi, Tokyo, Japan), and images were captured with an AMT CCD camera (Advanced Microscopy Techniques, Woburn, from MA). We previously determined that ethanol exposure led to the dramatic redistribution of ASGP-R from intracellular endosomes to the basolateral membrane in WIF-B cells.15 Closer examination using confocal microscopy revealed that the membrane-associated ASGP-R in ethanol-treated cells was present in discrete puncta (Fig. 1A). Because these puncta resembled clathrin-coated pits visualized at the light level, we examined the distributions of core clathrin-coat proteins. In control cells, CHC localized primarily to an intracellular compartment (Fig. 1A). As observed for ASGP-R, CHC redistributed to the basolateral membrane in discrete puncta in ethanol-treated cells.