EphA4 is downregulated by LMP1. (A-H) EBV⁻ TW01 cells were transfected with plasmids harboring the EBV viral genes LMP1, Zta, EBNA2, EBER1, EBER2, and BARF0. Total RNAs and protein lysates were obtained from each transfectant at day 3 posttransfection. (A, C, E, G) EphA4 transcripts were detected by RT-Q-PCR. EphA4 mRNA-relative folds were normalized to internal control GAPDH and then standardized with vector controls. (Top panels of B, D, F, H) Total proteins were harvested from the vector control and each transfectant. EphA4 protein-relative folds were normalized to internal control β-actin and standardized with vector controls. Expression levels of LMP1, Zta, EBNA2, and β-actin protein were estimated by western blotting. (Bottom panels of F and H) The EBER1, EBER2, BARF0, and β-actin transcripts were analyzed by RT-PCR. (I-J) EBV⁻ BJAB cells were infected with LMP1-expressing lentivirus at MOI 0.5 or 1. The expression levels of EphA4 mRNA were determined by RT-Q-PCR. EphA4 mRNA-relative folds were normalized to internal control GAPDH and then standardized with the pSIN vector control. (J) EphA4, LMP1, and β-actin were detected by western blotting. EphA4 protein-relative folds were normalized to internal control β-actin and then standardized with pSIN vector control. (K-L) Knockdown of LMP1 in LCLs was performed by lentiviral transduction at the MOI of 1 for 5 days and infected cells were selected with 2 µg/mL puromycin for 2 days. (K) Expression levels of EphA4 mRNA were measured by RT-Q-PCR. EphA4 mRNA-relative folds were normalized to internal control GAPDH and then standardized with vector control shLuc. (L) EphA4, LMP1, and β-actin were determined by western blotting. EphA4 protein-relative folds were normalized to β-actin and then standardized with vector control shLuc (*P < .05; **P < .01; ***P < .001, Student t test).