Analysis of NF-κB signaling in CYLD^ex7/8 BMDCs. (A) Differentiated day 6 WT BMDCs were stimulated (100 ng/mL LPS, 12 hours) or left untreated, and quantitative real-time–polymerase chain reaction (qRT-PCR) was performed to detect mRNA levels of Bcl-3. mRNA levels were normalized by HPRT levels and expressed as fold change. (B) Nuclear BMDC extracts from unstimulated and stimulated (100 ng/mL LPS, 12 hours) were examined by Western blotting using anti–Bcl-3. Shown are lysates from WT, CYLD^ko, and CYLD^ex7/8 mice. HDAC1 antibody was used as a loading control. (C) CYLD^ko mouse embryonic fibroblasts (MEFs) cells were transfected with empty vector or with sCYLD vector. Comparison of Bcl-3 up-regulation when sCYLD is expressed in CYLD^ko MEFs compared with mock-transfected CYLD^ko (bar graph). Gel represents the qRT-PCR products obtained after performance of qRT-PCR on sCYLD-transfected or mock-transfected MEFs as indicated in figure. Vertical line has been inserted to indicate a repositioned gel lane. (D) NF-κB luciferase reporter activity of BMDCs transfected with a NF-κB luciferase reporter construct untreated or stimulated (LPS 100 ng/mL, 12 hours). Cells were lysed after 24 hours, and luciferase activity was measured by a luminometer (Berthold Technologies, Bad Wildbad, Germany) using the dual luciferase reporter assay system from Promega. Data were standardized according to the Renilla luciferase activity and normalized to represent fold differences. *P < .05 using 1-way ANOVA between unstimulated samples; **P < .05 using 1-way ANOVA between LPS-stimulated samples. In Panels A, C and D: Data represent mean values with standard mean error bars. (E) EMSA was performed with nuclear extracts from BMDCs that were unstimulated or stimulated with 100 ng/mL LPS for 12 hours and subsequently incubated with NF-κB–specific labeled probe and p50 antibody to perform supershift, and separated by native polyacrylamide gel electrophoresis (PAGE). (F) Nuclear BMDC extracts from unstimulated and stimulated (100 ng/mL LPS, 12 hours) were examined by Western blotting using anti-p50 and anti-p65. Shown are lysates from WT, CYLD^ko, and CYLD^ex7/8 mice. HDAC1 antibody was used as a loading control.