FIGURE 6.

US2-mediated retrotranslocation is independent of the Ufd1-Npl4 complex in vitro. a, the membrane fraction from digitonin-permeabilized astrocytoma cells was either untreated or treated with a buffer containing 350 mm salt. The membrane was then extracted with the Nonidet P-40 lysis buffer and the protein extract was analyzed by immunoblotting with the indicated antibodies. b, control CLC or Ufd1-Npl4-depleted CLC were analyzed by immunoblotting (IB) with the indicated antibodies. c, MG132-treated US11 cells were radiolabeled and permeabilized. The membrane pellet fraction was either untreated or salt treated and then incubated with the indicated CLCs. Samples taken at the indicated time points were analyzed by immunoprecipitation with αHC antibody. ctrl., control. d, MG132-treated US2 cells were radiolabeled and permeabilized. The membrane pellet fraction was incubated with the indicated CLCs to initiate retrotranslocation. Samples taken at the indicated time points were either analyzed directly by immunoprecipitation with αHC (T) or fractionated into membrane pellet (P) and supernatant (S) fractions before immunoprecipitation analysis. Shown is a representative gel from two independent experiments. The graph shows the quantification of the results. Error bars indicate the mean of two independent experiments.