Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

In an effort to reduce background bands from the media in other experiments, such as that examining the effect of tunieamycin, PAS beads were additionally pre-absorbed with media from COS 7 cell cultures. Beads were pelleted from all samples and washed extensively in buffers containing detergent and then in PBS (3). After boiling the beads in 0.05 M Tris-HC1, pH 6.7, containing 1% SDS (34) to remove the bound antibody-protein complexes, 0.2 M citratephosphate buffer, pH 5.0, was added prior to treatment of half of each sample with 0.041 U of endo-fl-N-acetylglucosaminidase H (endo-H) prepared as described (39), at 37"C for 1 h. A lysate of L-[35S]methionine-labeled SA1 linfected MA 104 cells was prepared and treated with endo-H for use as a marker. The reactions were stopped by addition of buffer containing 100 mM Tris, 5% SDS, 1 mM EDTA, 50 mM dithiothreitol, 10% glycerol, 0.1% bromophenol blue, 100 ug/ml soybean trypsin inhibitor, 200 U/ml Traysylol, 5 mM 8aminocaproic acid, 1 mM benzamidine, and 2 mM phenylmethylsulfonyl fluoride. Samples were boiled for 3 min and analyzed by SDS PAGE on 12% gels (25) run at constant voltage. Gels were fixed, fluorographed in Amplify (Amersham Corp., Arlington Heights, IL) for 20 min, dried, and then autoradiographed at -70"C using Kodak SB 5 film. Composites of photographs of gels were always assembled from equivalent or identical exposures with comparable markers.