1999), in addition to the endosomal sorting determinant (Straley et al. P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is usually its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors. peptide:N glycosidase F produced in (N-Glycanase) was purchased from Roche or from Glyko, Inc. -galactosidase was purchased from Roche or from Prozyme. Bovine milk galactosyltransferase was from Sigma-Aldrich. Antibodies mAbs S12, G1, G5, and Rabbit Polyclonal to HTR5B 2B8 recognizing P-selectin, and goat polyclonal antiserum recognizing P-selectin, were generously supplied by Rodger McEver (University of Oklahoma, Oklahoma City, OK). Antipeptide antiserum recognizing the COOH terminus of P-selectin was affinity-purified as described (Green et al. 1994). Purified antipeptide antibody was biotinylated by reaction with a 10-fold molar excess of sulfosuccinimidyl 6-(biotinamido) hexanoate (Pierce Chemical Co) for 30 min in PBS, followed by addition of a 20-fold molar excess of glycine to quench remaining reactive groups. S12 antibody was labeled with Alexa 488 (Molecular Probes) according to the manufacturer’s protocol. Polyclonal rabbit antiserum was K145 generated by a commercial support (Covance) against soluble P-selectin (Ushiyama et al. 1993), and showed specificity in immunofluorescence and immunoprecipitation experiments identical to that obtained with the mAbs. Polyclonal rabbit antibodies against rat or bovine CI-MPR were from William Brown (Cornell University, Ithaca, NY). Rabbit antiserum recognizing synaptophysin was from Regis Kelly (University of California, San Francisco, CA). Rabbit antiserum recognizing chromogranin A was from John Hutton (University of Colorado, Denver, CO). mAb H68.4 recognizing transferrin receptor was provided by Ian Trowbridge (Scripps, La Jolla, CA). Goat antiCrabbit IgG, rabbit antiCmouse IgG, Texas red-conjugated goat antiCrabbit IgG and FITC-conjugated goat antiCmouse IgG were from Cappel. Oregon green- and Texas red-conjugated deglycosylated egg avidin (Neutralite) were from Molecular Probes. Immunofluorescence Labeling Immunofluorescence labeling was performed as previously described (Straley et al. 1998). For screening, cells were incubated for 1 h with a mixture of ascites fluid made up of mAbs S12, G5, and 2B8. For double-labeling experiments, primary antibodies recognizing chromogranin A, synaptophysin, CI-MPR, and transferrin receptor were applied, followed by the appropriate secondary antibody. After labeling endogenous proteins, cells were incubated in preimmune rabbit serum (1:50) for 15 min, and were then labeled with biotinylated antiCP-selectin COOH-terminal peptide antibody diluted 1:200 in buffer made up of rabbit preimmune serum 1:50. After washing, samples were labeled with Oregon green or Texas red avidin 1:300 and washed again, with 10 g/ml free biotin included in the last IF buffer wash. Cells were then washed three times in PBS, rinsed in distilled water, and mounted in ProLong (Molecular Probes). As a positive control for colocalization, cells were labeled only with biotinylated antiCP-selectin COOH-terminal peptide antibody, followed by a mixture of Oregon green and Texas red avidin. Image K145 Collection and Analysis Immunofluorescence images were collected using a Zeiss Axioplan 2 microscope equipped with a 63 Apochromat objective lens, n.a. 1.4, a Hamamatsu C4742-95 CCD camera, in some cases fitted with a Zeiss 4 magnifying adapter (final pixel size, 0.0266 m) and OpenLab (Improvision) software. For PC12 cells, 30 conventional images were collected at 0.2-m intervals, in the Texas red and fluorescein channels sequentially, using an automation to drive the K145 microscope controls. Digital deconvolution of one image near the middle of each series was performed using the OpenLab constrained iteration (confocal) algorithm, using 10C12 neighbors (20C24 images) to deconvolve each image. Grayscale matching and merging of deconvolved images was performed using Adobe Photoshop. Several cells were analyzed for each labeling condition, and representative results are presented. Uptake of LDL and S12 Antibody CHO cells expressing native P-selectin or P-selectin?C1 were passaged onto coverslips and grown overnight. The medium was replaced with LDL-depleted medium and the cells were grown for an additional 16C18 h to increase surface expression of LDL receptor. After incubation for 10 min at 37C in PBS made up of 1 mg/ml glucose and 0.2% BSA (PBS/BSA), cells were incubated for 5 min at 37C in PBS/BSA.