NF-κB activity is impaired in the MYC-transformed cells. (A) NF-κB EMSA using nuclear extracts of different murine lymphoma cells. WEHI231 cells show constitutive NF-κB activity and were used as control. Oct1 DNA binding served as a control for the integrity of the nuclear extracts. (B) EMSA using protein extracts from lymphoma cells treated with the standard NF-κB inducers TNFα (40 ng/mL), PMA (5 ng/mL) and ionomycin (1 μg/mL), or LPS (1 μg/mL) for the indicated time points. Jurkat cells treated with PMA/ionomycin for 1 hour were used as control for NF-κB binding. Sp1 DNA was used as a control for the integrity of the cell extracts. (C) NF-κB EMSA with nuclear extracts of B-cell lymphoma cell line 5522 treated for 2 hours with daunorubicin (5 μM) or okadaic acid (200 nM). Oct1 was used as control for the integrity of the extracts. (D) Immunoblot with cell extracts of B-cell lymphoma cell lines. Doxycycline treatment abrogates transgenic MYC expression. Cells were treated with doxycycline (2 μg/mL) for 12 hours or were left untreated. Expression of p65 was used as loading control. (E) EMSA using cell extracts of 2 lymphoma cell lines demonstrates induction of NF-κB activity in the absence of MYC expression. Immunoblot shows transient loss of MYC expression after doxycycline treatment. Cells were left untreated (lanes 1-2), pulse treated with doxycycline for 12 hours (lanes 3-4), and again left untreated for the consecutive days (lanes 5-6).