ONX0912 leads to inhibition of the canonical and noncanonical NF-κB pathway. (A) BCWM.1 cells were cultured with ONX0912 (0-100nM) for 4 hours, and then TNF-α (10 ng/mL) was added for the last 20 minutes. NF-κBp65 transcription factor binding to its consensus sequence on the plate-bound oligonucleotide was studied from nuclear extracts. Wild-type and mutant are wild-type and mutated consensus competitor oligonucleotides, respectively. All results represent means ± SD of triplicate experiments. (B) BCWM.1 cells were cultured with ONX0912 (0-100nM) for 4 hours, and TNF-α (10 ng/mL) was added for the last 20 minutes. Nuclear extracts were subjected to Western blotting using anti–p-NF-κBp65, -p65, -p105, -p50, -p100, -p52, -RelB, and -nucleolin antibodies. (C) BCWM.1 cells were cultured with ONX0912 (0-100nM) for 4 hours, and TNF-α (100 ng/mL) was added for the last 20 minutes. Cytoplasmic extracts were subjected to Western blotting using anti–p-IκB and -actin antibodies.