LMP1 induces all 3 pathways of the UPR. (A) BJAB/HA-LMP1 cells were untreated, treated with 10 ng/mL tetracycline for 48 hours, or treated with 2.5 μg/mL tunicamycin for 4 hours. The 721 cell line was sorted for low, medium, and high levels of LMP1. RNA was isolated from these cells and analyzed by RT-PCR using primers specific for both XBP-1 and GAPDH. To determine whether splicing of XBP-1 had occurred, the PCR fragments were digested with PstI. The ratios of spliced XBP-1 to GAPDH were calculated. The numbers represent the fold-difference relative to the ratio of untreated BJAB/HA-LMP1 cells, which was set at 1. *P (1-sided) <.05, 1-sided alternative: low, mid, high in increasing order, Jonckheere-Terpstra test. A gel representative of 3 independent experiments is shown. Production of Ig was measured by Western blotting the culture supernatant of EBV-positive strain cells (B) and 721 cells (C) that were sorted for low, medium, and high levels of LMP1 by staining with lysotracker red as described.⁶⁰  For Ig heavy chain detection, 1 μL supernatant was analyzed, and for Ig light chain detection, 10 μL supernatant was analyzed, because the antibody preferentially detects Ig heavy chain. The cells were harvested and analyzed by Western blotting using antibodies to detect LMP1 and tubulin. A representative gel of duplicates of 2 independent experiments is shown. (D) BJAB/HALMP1 was electroporated with p3xFLAG-ATF6. Cells were either left untreated or treated with 10 mM DTT for 1.5 hours and 10 ng/mL tetracycline for 48 hours to induce LMP1. The cells were harvested and analyzed by Western blotting using antibodies to detect LMP1, tubulin, and Flag. The numbers represent the fold-difference of ratios of nuclear N-terminus ATF6 to tubulin. *P (2-sided) less than .05 (Wilcoxon rank sum test). A representative example of 3 independent experiments is shown.