FIGURE 3.

Ubiquitin is required for US2-mediated retrotranslocation. a, a small amount of FITC-labeled ubiquitin was added to CLC to monitor the depletion efficiency. Fluorescence intensity in CLC was measured before (BD) and after (AD) depletion. b, MG132-treated US11-expressing cells were radiolabeled, permeabilized. The membrane pellet fraction was salt treated and incubated with either control (ctrl.) cytosol or ubiquitin-depleted CLC. Where indicated, bovine ubiquitin was added back to ubiquitin-depleted CLC. Samples taken at the indicated time points were analyzed directly by immunoprecipitation with αHC. c, as in b, except that US2 cells were used and samples taken at the indicated time points were fractionated into membrane pellet (P) and supernatant (S) fractions before immunoprecipitation analyses. The image shows a representative gel from two independent experiments.