trans-Golgi retention of a plasma membrane protein: mutations in the cytoplasmic domain of the asialoglycoprotein receptor subunit H1 result in trans-Golgi retention

For indirect immunofluorescence staining, cells were grown on 14-ram glass coverslips. Cells were fixed in I% paraformaldehyde for 20 rain at room temperature for surface staining and with cold methanol for 6 mill at -20~C for internal staining. After fixation the coverslips were washed in PBS and incubated in 50 mM NH4C1 in PBS for 10 min at room temperature. Nonspecific antibody binding was blocked by incubation for 30 min with PBS containing 0.2% gelatine (PBSG). Coverslips were placed onto drops of primary antibody diluted in PBSG for 1 h. After washing three times with PBSG, cells were similarly incubated with secondary antibody for 30 min. After additional washes with PBSG, PBS, and water, the coverslips were mounted in Mowiol 4-88 (HOECHST), containing 2.5% 1,4-diazobicyclo-[2,2,2]-octane and analyzed using a Zeiss Axiophot microscope. Staining of cells with antibodies AC17 and Nll was done essentially as described by Nabi et al. (1991) and Ktistakis et al. (1991) , respectively. Internalization of fluorescein-conjugated dextran (FITC-dextran; Sigma) was done by preincubating the cells on ice for 10 rain, adding 10 mg/ml FITC-dextran in medium on ice to the cells, incubating for 10 rain on ice and for 5-15 rain at 37°C. After several washes in PBS, the cells were fixed in 3% paraformaldehyde and mounted in Mowiol.