Fig. 10. Behavior of SEAP, Cab₃₀₈Myc and Cab45 after saponin permeabilizing intracellular membranes and sedimenting the resultant extract. Both non-aggregative (N-A.; neutral pH, low calcium) and aggregative (Agg.; low pH, high calcium) conditions were used for extraction as described in the Materials and Methods. (A) Western blotting of SEAP and Cab₃₀₈Myc after saponin permeabilization of organelle membranes. SEAP, which enters secretory granules, appears largely (>90%) soluble under both non-aggregative and aggregative conditions, whereas Cab₃₀₈Myc is almost completely (>95%) sedimentable under these conditions. (B) Immunoprecipitation of newly synthesized Cab₃₀₈Myc after saponin permeabilization or Triton X-100 solubilization, followed by sedimenting the resultant extracts. INS-Cab₃₀₈Myc cells were pulse labeled with ³⁵S-amino acids for 100 minutes without chase. Organellar membranes from these cells were saponin permeabilized under non-aggregative conditions (control, lanes 1 and 2), or dissolved with 1.5% Triton X-100 (lanes 3 and 4). After centrifugation, the supernatants (S) and solubilized pellets (P) were immunoprecipitated with anti-Myc and analyzed by SDS-PAGE and fluorography. (C) Western blot of Cab45 as in A, using non-aggregative conditions in the absence (No Deterg.) or presence of detergents as shown. The data shown are representative of three independent experiments. (D) INS-Cab₃₀₈Myc cells were pulse labeled as in panel B in the presence of brefeldin A (BFA, 10 µg/ml). Organellar membranes from these cells were saponin permeabilized under non-aggregative conditions, or dissolved with 1.5 % Triton X-100 (TX100), or mock treated without detergent (No deterg.). After centrifugation, the supernatants (S) and solubilized pellets (P) were immunoprecipitated with anti-Myc and analyzed by SDS-PAGE and fluorography.