Role of NFκB in PEL cell lines (A) Constitutive expression of NFkB in PEL cells.

<p>Nuclear extracts from BC1, BC3, BCBL1 and HBL6 cell lines were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039945#s2" target="_blank">material and Methods</a> and electrophoretic mobility shift assay (EMSA) was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039945#s2" target="_blank">Materials and Methods</a>. Briefly, 5×10⁶ cells were washed with cold PBS and suspended in 0.4 mL hypotonic lysis buffer containing protease inhibitors for 30 minutes. The cells were then lysed with 10% Nonidet P-40. (<b>B</b>) <b>Bay11-7085 inhibits constitutive nuclear NFkB in PEL cells.</b> BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Nuclear extracts were prepared and EMSA was performed. (<b>C</b>) <b>Effect of Bay11-7085 on IκBα phosphorylation in PEL cells.</b> BC1 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against phospho-IκBα and Beta actin as indicated. (<b>D</b>) <b>Bay11-7085 treatment causes down-regulation of expression of down-stream targets of p65.</b> BC1 and BC3 cells were treated with 5 and 10 µM Bay11-7085 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against IκBα, Bcl-2, Bcl-Xl, XIAP, Survivin and Beta-actin. (<b>E</b>) <b>Transcriptional down-regulation of p65 causes decreased expression of p65 targets in PEL cells.</b> BC1 and BC3 cells were transfected with siRNA against p65 for 48 hours. Following transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against IκBα, Bcl-2, Bcl-Xl, XIAP, Survivin and Beta-actin.