LMP2A downregulates the expression of E47 and PU.1 through the PI3K/Akt pathway. (A) BJAB cells were infected with LMP2A-, LMP2A-Δ74-85–, or LMP2A-Y112F–expressing lentiviruses. Expression of LMP2A, E47, PU.1, CD74, and MHC class II was analyzed by western blotting. β-actin was detected as an internal control. (B) BJAB cells were transfected with wild-type LMP2A or LMP2A with mutated PY motif expression plasmids, and the transfectants were subjected to western analysis of LMP2A, E47, and PU.1. β-actin was detected as an internal control. (C) LCLs were cultured in the presence of 25 μM Piceatannol or 10 μM PP2 at the indicated time. Expression of phospho-Akt (pAkt), total Akt, E47, and PU.1 was analyzed by western blotting. β-actin was detected as an internal control. (B-C) Experiments were performed twice, and 1 representative is shown. (D-E) BJAB cells were infected with LMP2A-expressing lentiviruses or vector control. After 5 days, the cells were cultured in the presence of 20 μM LY294002 (D) or 20 μM U0126 (E) paired to dimethylsulfoxide (DMSO) control for 48 hours. Cell lysates were subjected to western analysis of E47, PU.1 CD74, MHC class II, and LMP2A. β-actin was detected as an internal control. (D) Special detection of pAkt and total Akt and (E) detection of phosphoERK1/2 (pERK1/2) and total ERK. (F) LCLs were infected with sh-Luc or sh-PIK3CA expressing lentivirus and the cell lysates were subjected to western analysis of E47, PU.1, CD74, and MHC class II. β-actin was detected as an internal control. (G) BJAB cells were infected with LMP2A, PU.1, or forced-dimered-E47 (FD-E47) lentiviruses and transfectants were subjected to western analysis of FD-E47, endogenous E47 (endo-E47), PU.1, CD74, MHC class II, and LMP2A. β-actin was detected as an internal control. Each experiment was performed 3 times, and 1 representative is shown.