Processing of HShh is dependent on disulfide bridge formation and reduction. (A) HShh-HA was stably expressed in 293T cells. Protein synthesis was inhibited with cycloheximide (CHX), and the fate of the protein was followed by SDS-PAGE and immunoblotting with anti-HA antibodies. Immunoblotting for p97 was used as a loading control. (B) As in A, but with HShh-HA containing a mutation in the catalytic cysteine (C198S). (C) As in A, but with HShh-HA containing a mutation in the conserved noncatalytic cysteine (C363A). (D) HA-tagged HShh was stably expressed in 293T cells. The cells were pulsed with [³⁵S]methionine for 3 min and chase incubated with unlabeled methionine for the indicated times. 200 µM diamide or 0.5 mM DTT was added 10 min before the pulse and was present during the pulse and chase. The proteasome inhibitor epoxomicin (1 µM) was present, beginning at 1 h before the pulse. The samples were analyzed by immunoprecipitation with HA antibodies followed by reducing SDS-PAGE and fluorography. An equal number of cells were processed for each condition. (E) As in D, except that, where indicated, 10 µM brefeldin A and 1 µM epoxomicin were present, which were added 1 h before the pulse. The samples were analyzed as in D. Molecular masses are given in kilodaltons.