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

42 h after transfection the cells were washed once with PBS, incubated in labeling medium (PBS, 20% dialyzed FCS, 100 IU/ml penicillin, 100 g,g/ml streptomycin, 1% nonessential amino acids) for 15 min at 37°C and pulsed for 5 rain at 370C with 20 txCi [35S]methionine in 200 Ixl labeling medium per 35-mm dish. The cells were washed once with cold DMEM containing 10 mM methionine and then chased in DMEM containing 10 mM methionine at 37°C. The dishes were transferred to ice and all the subsequent steps were carried out on ice. The cells were washed twice with PBS, once with 100 mM sodium phosphate, 1% bovine serum albumin, pH 8, scraped off with a rubber policeman in 1 ml solubilization buffer (100 mM sodium phosphate, 1% Triton X-100, pH 8, and protease inhibitors), and passed five times through a 25-gauge needle. After incubation for 60 min the lysate was centrifuged at 100,000 g for 60 min, the resulting supernatant was incubated with either 2 ixl G1/93 ascites, 20 I~19E10.2, or 20 Ixl HP2/6.1 for 60 min followed by an incubation with 2 t~1 rabbit anti-mouse IgG (Cappel) for 30 min. Immunocomplexes were isolated by adding 10 Ixl protein A-Sepharose (Pharmacia, Uppsala, Sweden), rotated over night at 4°C, washed four times with solubilization buffer, once with 100 mM sodium phosphate, pH 8, and once with 10 mM sodium phosphate, pH 8. Proteins were eluted from the protein A beads by boiling in electrophoresis sample buffer. For nonreduced samples 4-10%, for reduced samples 7-10% SDS-polyacrylamide gradient slab gels (Laemmli, 1970) were run, salicylated, and visualized by fluorography using Kodak X-omat AR or Fuji RX films.