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

Immunoprecipitation: Cell monolayers were rinsed in ice-cold phosphate buffered saline (PBS), harvested, and lysed in buffer containing 1% each of deoxycholate and Triton X-100, 0.1% SDS, and 0.15 M NaCI in 25 mM Tris-HCl, pH 8.0, containing 100 U/ml Traysylol. The nuclei were pelleted (34) after the addition of more detergent. Standard procedures (3), with some modification, were used for immunopreeipitation of proteins from the postnuclear supernatant by rabbit polyclonal antiserum to VP7 and protein Aconjugated Sepharose C1AB (PAS) (Pharmacia Fine Chemicals). Beads were swollen in distilled water and added to transfected cell lysates before incubation with antibody. PAS beads were pelleted by centrifugation and discarded (12) in an attempt to eliminate any nonspecifically adherent proteins. Polyclonal anti-VP7 serum was then added to lysates which were incubated at 4"C overnight before the addition of PAS beads. These had been preincubated for several hours in a solution containing 10 mg/ml bovine serum albumin and a postnuclear cell lysate of unlabeled, untransfected COS 7 cells.