LMP1-induced RON expression is through its CTAR1 domain–mediated NF-κB activation. (A) EBV-negative Akata cells were infected with pSIN, LMP1, ΔCTAR1, ΔCTAR2, or ΔCTAR1/2-expressing lentiviruses at an MOI of 2 for 5 days. The RON transcripts were detected by RT-Q-PCR and the relative fold increase of RON was normalized to the amounts of RON transcripts in pSIN lentivirus-infected cells (top panel). The protein expression of RON, full-length LMP-1, CTAR1-deleted LMP1, CTAR2-deleted LMP1, CTAR1/2-deleted LMP1, and GAPDH was measured by Western blotting (bottom panel). (B) EBV-negative Akata cells were infected with pSIN or pSIN-LMP1 lentiviruses for 3 days and then cells were reseeded at a density of 1 × 10⁶ cells/mL. Cells were treated 2.5μM or 5μM BAY11-7082 for 48 hours. Total RNA and protein were harvested and the expression of RON transcripts was measured by RT-Q-PCR and the relative fold increase of RON was normalized to the amounts of RON transcripts in pSIN lentivirus-infected cells (top panel). The protein expression of RON, p-IκB-α, LMP1, and GAPDH was measured by Western blotting (bottom panel). (C) Cell lysates were harvested from vector or LMP1-expressed BJAB cells. The complexes of DNA and p65 were immunoprecipitated using anti-p65 Ab or rabbit IgG. RON promoter DNA and control GAPDH promoter DNA were detected in the immunoprecipitates by PCR. Total DNA was harvested from vector or LMP1-expressed BJAB cells and used as the input control (left panel). Vector and LMP1-expressing BJAB cells were harvested and total protein extracted. The expression of RON, LMP1, and GAPDH was measured by Western blotting (right panel).