Figure 2.

mCD40L-induced NORE1A expression is mediated by NFκB activation. (a) EJ cells were infected with 100 MOI of either RAdMock (AdM) or RAdnCD40L (AdnL), RAdnCD40L-infected cells were either treated with p-p38 inhbitor SB203580 (SB), the NFκB inhbitor SC-514 (SC), the MEK, the ERK kinase inhbitor PD98059 (PD), the AKT pathway inhbitor LY294002 (LY) and the JNK pathway inhbitor SP600125 (SP) at the following concentrations 20, 35, 50, 30, 50 μM, respectively, or left untreated as control for 26 h. Cells were lysed and total protein lysates were prepared and examined for the expression of mCD40L, NORE1A, p-p38, IκBα, p-ERK, p-AKT, p-JNK and β-actin as a loading control. (b) EJ cells were infected with 100 MOI of either AdM or AdnL or left untreated as a negative control, AdnL-infected cells were either treated with the NFκB inhbitor SC-514 (SC; 35 μM) or the BMS-345541 (BMS; 8 μM) or left untreated as a control for 21 h. Cells were lysed and total cell lysates were examined for the expression of p-p65 and p-IκBα, IκBα, NORE1A and the β-actin as a loading control. (c) EJ cells were infected with 100 MOI of either AdM or AdnL or left untreated as a negative control or treated with sCD40L (1 μg/ml) for the indicated time, ChIP assays were performed and one-tenth of the volume of the chromatin obtained was used for PCR as input, and the remaining volume was immunoprecipitated with anti-c-Rel (α-c-Rel) or anti-RNA polymerase II (α-Pol II) or Rabbit isotype (isotype) antibodies as described in materials and methods. Precipitated DNA spanning the NORE1A gene promoter was evaluated by PCR under the following conditions (95 °C 5 min, (93 °C 30 s, 64.3 °C 30 s, 72 °C 1 min) × 35, 72 °C 5 min), the PCR products were resolved by 2% agarose gel electrophoresis. The c-Rel-binding site within the NORE1A gene promoter and the sites where the primers for amplification of NORE1A gene promoter spanning the c-Rel-binding sequence were designed are indicated in the schematic diagram