A20 silencing in EB-LCLs induces constitutive NF-κB activation. (A) Repression efficiency of A20 when using siRNA was determined by immunobloting in whole-cell extracts of CB33 expressing A20, luciferase, and GFP siRNA. Vertical line represents a repositioned gel lane. (B) CB33 cells were cotransduced with a lentiviral vecter encoding κB sequence–firefly luciferase and Renilla luciferase reporter vector. The data shown represent the mean relative luciferase activity normalized against Renilla luciferase activity ± SD. The experiments were performed in triplicate. IKKβ_SS/EE served as positive control for NF-κB activation. (C) Immunoblots for the canonical and noncanonical NF-κB protein from the indicated cell lines. DLBCL cell lines with (OCI-LY8) and without (SUDHL6) A20 deletion were included in each blot. A20 deletion and A20 silencing are involved in p50 nuclear translocation, whereas A20 had little effect on p100 processing. NE indicates nuclear extract; and WCL, whole-cell lysate. (D) Effect of A20 knockdown on the nuclear DNA-binding activity of the NF-κB p50 and p52 subunits. Binding to an oligonucleotide containing the NF-κB consensus sequence was measured in nuclear extracts from LCLs expressing siRNA for A20 and luciferase. DNA-binding activity was quantified by colorimetry. The findings of triplicate experiments are presented as mean ± SD. NC indicates negative control; VC, vector-only control; and Luc, firefly luciferase. (E) Expression of cancer-related NF-κB target genes in A20 knockdown CB33. Relative expression level of the 4 NF-κB target genes (Cyclin D2, IRF4, BCL-xL, and c-FLIP) is determined by real-time PCR and normalized on the basis of the corresponding β-actin content. The findings of triplicate experiments are presented as mean ± SD.