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Fig. 6. Cytosolic CFTR fragments remain stably associated with proteasomes. (A) Supernatants from degradation reactions were collected after 2 hours in the presence of 100 µM MG132 and separated by glycerol gradient centrifugation. Fractions were collected top to bottom (1-14) and TCA-soluble ({circ}) and TCA-insoluble ({bullet}) material was quantified by scintillation counting. (B) Samples from gradients in panel A were analyzed directly by SDS-PAGE (12-17% gel) and autoradiography. Migration of molecular mass markers in the gradient are indicated at top of the gel. (C) Degradation was carried out as in A and membranes were pelleted through a 0.5 M sucrose cushion. Supernatant and pellet fractions were analyzed directly (lanes 1,4) or immunoprecipitated with preimmune sera (PIS) or antisera against the {alpha}3 proteasome subunit.