Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

CV-1 or NRK cells were plated at a density of 3 x 10 ~ cells/100-mm culture dish and grown overnight at 37~ 3 h before transfection, cells were fed with 10 ml of fresh complete medium. Cells were incubated 16 to 20 h with 20 t~g recombinant plasmid DNA and 2 #g pFNeo (Saito et al., 1987) precipitated by calcium phosphate (Graham and van der Eb, 1973 ; as modified by Gorman et al., 1983) . Cells were then washed with PBS, treated with 10% DMSO in ice-cold PBS for 5 rain, washed with PBS, and then incubated in complete medium for 24 h before selection in 1 mg/ml (for CV-1 cells) or 2 mg/ml (for NRK cells) active geneticin (G418; GIBCO/BRL, Gaithersburg, MD) in complete medium. Stably transfected clones were identified by immunofluorescence microscopy as below.