FIGURE 1.

AUP1 is involved in ER quality control. A, US11-expressing astrocytoma cells were transduced with shRNA specific against luciferase (shLUC), as a control, or one of two different constructs targeting AUP1 (A or B). Four days post-transduction, cells were pulse-labeled with ³⁵S-labeled cysteine and methionine. Samples were taken at the indicated chase times, and class I MHC heavy chain was recovered from the lysates. Immunoprecipitates (IP) were separated by SDS-PAGE and imaged by autoradiography. Amount of recovered protein was quantified by phosphorimagery and is shown as a percentage of glycosylated heavy chain compared with total heavy chain. Error bars represent standard deviation of three individual experiments. The level of AUP1 depletion was determined by immunoblotting (IB) with p97 as a loading control. The same experiment as described for A was performed using shRNA-transduced HeLa cells transfected with NHK (B) or RI₃₃₂-HA (C). NHK was immunoprecipitated using an anti-α1-antitrypsin antibody. RI₃₃₂-HA was immunoprecipitated using an anti-ribophorin I antibody that recovers both the full-length and the misfolded fragment of ribophorin I. Quantification in B and C shows the percentage of protein remaining compared with the amount recovered at the 0-min chase time.