FIG. 5.

Sucrose density gradient analysis of 3AB proteins. (A and B) Translation reactions were programmed with FLAG epitope-tagged wild-type 3AB and cytochrome b₅ in the absence (A) or presence (B) of canine pancreas microsomal membranes. Products were sedimented through discontinuous sucrose gradients, and fractions were collected and immunoprecipitated with anti-FLAG antibody before analysis on SDS-PAGE gels. A mixture of individually translated FLAG epitope-tagged wild-type 3AB, cytochrome b₅, and β-globin proteins served asmarkers for the gel (lane M); lane T in panel B represents a sample of the translation reaction not subjected to gradient sedimentation. (C) Radioactivity in each fraction of the sucrose gradients containing proteins from translation reactions programmed with cytochrome b₅ mRNA and either wild-type 3AB, 3AB-M79T, or β-globin mRNAs, in the presence of canine pancreas microsomal membranes, was quantitated by phosphorimager. The percentage of total radioactivity cosedimenting with membranes (fractions 2 to 6) was calculated for each protein and plotted.