Perifosine and bortezomib inhibit NF-κB function in WM cells. (A) Chromatin immunoprecipitation (ChIP)-based assay. BCWM.1 cells were cultured with either perifosine (P, 10 μM), bortezomib (B, 10 nM), or the combination (B + P) overnight, then TNFα (10 ng/mL) was added for the last 30 minutes, and then anti–p65NF-κB immunoprecipitation was performed. Quantitative real-time PCR (Q-PCR) for IκB in the p65NF-κB-DNA immunoprecipitated fragments was assessed using SYBR. Experimental Q-PCR values were normalized against values obtained for 25 ng of input DNA with the same primer set. (B) NF-κB activity assay using Active Motif. BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (10 nM), or the combination for 6 hours, and then TNFα (10 ng/mL) was added for the last 30 minutes. NF-κBp65 transcription factor-binding to its consensus sequence on the plate-bound oligonucleotide was studied from nuclear extracts. WT and Mut are wild-type and mutated consensus competitor oligonucleotides, respectively. Data represent mean plus or minus SD of triplicate experiments. (C) BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (10 nM), or the combination for 6 hours, then TNFα (10 ng/mL) was added for the last 30 minutes, and then the effect on NF-κB pathway was studied using immunobloting on cell fractionation. Cytoplasmic and nuclear fractions were subjected to Western blotting using anti-IκBα, –NF-κBp50, –p-NF-κBp65, -nucleolin, and –α-tubulin antibodies.