[PMC free article] [PubMed] [Google Scholar]Chen HJ, Yuan J, Lobel P. domain. INTRODUCTION The function of membrane-bounded organelles requires correct targeting of resident membrane proteins to each organelle and in many cases selective cycling of membrane proteins between organelles. Selective localization and targeting of membrane proteins to their appropriate destinations depend on structural features of these proteins, termed sorting determinants. Sorting determinants are recognized by sorting machinery that functions to concentrate proteins bearing the appropriate sorting determinants into specific transport vesicles, which can then carry their Xantocillin cargo vectorially to the correct destination. A number of sorting Xantocillin determinants have been characterized, including those that mediate localization to clathrin-coated pits in the trans-Golgi network (TGN)11 or at the cell surface, sorting to the basolateral cell surface in polarized epithelial cells, and selective transport from endosomes to lysosomes (Sandoval and Bakke, 1994 ; Mellman, 1996 ; Kirchhausen (1989) . CJ236 cells were used to recover uridine-substituted single-stranded Bluescript plasmid using M13K07 helper phage (Stratagene, La Jolla, CA). DH10- cells (Life TechnologiesCBethesda Research Laboratories, Gaithersburg, MD) were Xantocillin used for growing all other plasmids. pBSLLP (Green (1990) . CHO cells were transfected with 6 g of pCB6 plasmids containing LLP constructs. NRK cells and CHO cells were cotransfected with pSV2neo and with pCDL-SRalpha containing cDNA inserts, using these plasmids at a ratio of 1 1:120, with a total of 6 g of plasmid DNA. Transfected cells were passaged into selection medium containing 400 g/ml G418 3 d after transfection. Clones were picked directly from the plates using 200-l pipets after washing once with HBSS lacking divalent cations and containing 1 mM EDTA and 5 mM HEPES. CHO transfectants expressing LLP constructs were screened by immunofluorescence microscopy using the C7 anti-LDL receptor mAb. CHO cells expressing the LLP chimeric protein were subcloned to obtain clones with uniform expression levels. NRK cells or CHO cells expressing P-selectin and mutants thereof were screened by immunofluorescence microscopy using a mixture of ascites containing S12, G5, and 2B8 monoclonal antibodies. Immunofluorescence Labeling and Microscopy Cells were processed for immunofluorescence microscopy at room temperature. Cells grown on 12-mm glass coverslips coated with poly-d-lysine were washed with PBS and fixed in 3% formaldehyde and 100 mM sodium phosphate, pH 7.4, for 15 min. After washing three times in PBS, cells were permeabilized in 2% BSA, 0.5% fish skin gelatin (Sigma, St. Louis, MO), 0.02% saponin (Sigma), 150 mM NaCl, and 10 mM HEPES, pH 7.4 (blocking buffer), for 30 min. Antibodies were applied for 1C1.5 h in blocking buffer. Ascites fluid and antisera were diluted 1:300, and purified antibodies were used at 20 g/ml. Secondary antibodies were diluted 1:300. For epifluorescence microscopy, mouse monoclonal antibodies were detected with FITC-conjugated secondary antibodies, and rabbit polyclonal antibodies were detected with Texas Red-conjugated secondary antibodies. Biotin-labeled antibodies were detected with Texas Red-Neutralite (Molecular Probes, Eugene, OR). Each antibody incubation was followed by three 5 min washes in blocking buffer. The cells were then washed three times in PBS and once in distilled water and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Samples were viewed and photographed on Kodak T-Max 400 film using a Axioplan epifluorescence microscope with a 63 oil immersion planapochromatic objective lens. Negatives were scanned on a Nikon LS3510AF Film Scanner. Images were processed using Adobe Photoshop and were printed on a Kodak XLS 8600 printer. Metabolic Labeling For pulse metabolic labeling with [35S]amino acids, cells were washed with PBS and then incubated for 20 min in DME lacking methionine and cysteine and containing 10% dialyzed FBS or LDL-depleted serum. Cells were labeled in the same medium containing 300C800 Ci/ml [35S]amino acid mixture (EXPRE35S35S Protein Labeling Mix; New England Nuclear, Boston, MA). Chase incubations were performed by washing the cells once in complete growth medium and then culturing in growth medium supplemented with 3 mM methionine and 3 mM cysteine. Cell Surface Biotinylation For irreversible cell surface biotinylation to measure turnover of cell surface proteins, cells were grown to 70C90% confluence on 6-cm Rabbit Polyclonal to ARHGEF11 dishes. Cells were washed three times with ice-cold PBS containing 1 mg/ml.