FIGURE 3.
HA-CT A1-chain is rapidly degraded by the proteasome. A, cells were pretreated with lactacystin or vehicle alone before performing retrotranslocation experiments. Cytosolic fractions were analyzed by SDS-PAGE and immunoblotted (IB) using antibodies recognizing the A-subunit (CTA) and B-subunit (CTB) or HA tag. Lanes 1 and 2 are controls. B, in a time course of CT-induced Cl− secretion (Isc), T84 cells were incubated with WT CT (filled square) or HA-CT (filled triangle) or pretreated with lactacystin followed by WT CT (open square) or HA-CT (open triangle). The viability of monolayers was demonstrated by applying the cAMP agonist, forskolin. C, summarized data from two independent experiments, each done in triplicate, of maximal Isc for untreated or lactacystin (Lac) pretreated T84 cells is shown. Error bars in C and D indicate S.D.