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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.
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