FIG. 5.
Expression of US11 triggers the UPR. (A) Tet-On cells were incubated for 48 h in the presence or absence of DOX (1 μg/ml). Total RNA was extracted, and XBP-1 splicing was analyzed by RT-PCR. PCR products were analyzed by 11% PAGE and visualized by ethidium bromide staining. (B) Tet-On cells and U373 cells that express US2 or US11 constitutively were pulse-labeled with [35S]methionine for 20 min. Cells were lysed in 1% SDS, and the lysate was then diluted to 0.07% SDS with NP-40 lysis mix. Samples were divided into three parts. One portion was immunoprecipitated with αKDEL monoclonal antibodies, the second portion was immunoprecipitated with anti-β-actin, and the third portion was immunoprecipitated with anti-US11 or anti-US2 antibodies. Loading was normalized to equal the number of trichloroacetic acid (TCA)-precipitable counts. (C) Autoradiograms were quantitated as mentioned above, and the ratio of Bip to β-actin was calculated.