Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway

For surface staining of ERGIC-53 and mutants the cells were cooled to 4°C for 20 min followed by an incubation with G1/93 or 9E10.2 at a 1:10 dilution in 250 txl culture medium for 30 rain. After six washes with culture medium the cells were fixed with 3% paraformaldehyde, pH 8.3, for 30 min. Thereafter the cells were rewarmed to room temperature, fixed for another 30 min, washed twice with PBS and twice with PBS containing 20 mM glycine, and then permeabilized for 20 min with PBS-glycine containing 0.1% saponine. The cells were then subjected to indirect immunofluorescence microscopy as described below. For differential staining of the cell surface and intraceUular structures of CD4 and CD4/ERGIC-53 chimeric proteins the following double-immunofluorescence protocol was applied. The cells were cooled to 4°C for 20 min followed by an incubation with mAb 6D10 at a dilution of 1:10 in culture medium for 30 min. After six washes with culture medium the cells were reincubated with IgGl-specific FITC-labeled affinity-purified goat anti-mouse secondary antibody (Cappel, West Chester, PA) for 30 rain, washed again six times with culture medium, and fixed with 3% paraformaldehyde, pH 8.3, for 30 min. Thereafter the cells were rewarmed to room temperature, fixed for another 30 rain, washed twice with PBS and twice with PBS containing 20 mM glycine, and then permeabilized for 20 min with PBS-glycine containing 0.1% saponine. The cells were then incubated with HP2/6.1 at a 1:150 dilution in PBS-saponine for 30 min, washed four times with PBS-saponine, and incubated with rhodamine (TRITC)-labeled IgG2a-specific affinity-purified goat anti-mouse secondary antibody (Cappel) for 30 min. After four final washes with PBS-saponine the cells were mounted in 90% glycerol, 10% PBS, and 0.1 mg/ml phenylenediamine. The cells were analyzed by a Reichert Polyvar immunofluorescence microscope.