Regulation of CCL2 Gene Expression overexpressed protein), FLAG (only detects virally-produced form of the p65 protein) and tubulin (as a control for protein loading). The blot shown is representative of two independent experiments. B–G. Rat islets (E) and 832/13 cells (B, C, D, F, G) were treated for 24 h with the indicated adenoviruses. B, D. 4 h following adenoviral transduction cells were transfected with either a 5X NF-kB- Luc promoter (B) or the 3.6-Luc promoter (D) for 24 h; promoter activity was normalized to protein content. B. *p,0.05 vs. bGAL; **p,0.01 vs. bGAL; #p,0.001 vs. bGAL, D,**p,0.01 vs. p65 S276A. C, E and F. Total RNA was isolated and gene expression monitored via real-time PCR. C. *p,0.05 vs. p65 S276A; **p,0.01, E. *p,0.05, F. n.s. = not significant. G. CCL2 secretion into the cell culture media was measured via ELISA and the data normalized to total intracellular protein content. *p,0.05 vs. bGAL; **p,0.01 vs. bGAL. RNA abundance, promoter luciferase activity, and ELISA data are represented as means 6 SEM from 3–4 individual experiments. The dashed line in B. and C. represents the induction by IL-1b exposure. doi:10.1371/journal.pone.0046986.g003 the ERK inhibitor did blunt the c-IFN-mediated induction of a multimerized GAS element driving the luciferase gene (Figure S4). While IL-1b induced an 8-fold increase (set at 100%) in the expression of the COX2 gene, this induction was unaffected by inhibition of the p38 MAPK (Figure 5D). In addition to the results observed in Figures 5B & 5C, the transcriptional activity of the CCL2 promoter in response to IL-1b was also diminished by pharmacological inhibition of p38 (Figure 5E). Moreover, the IL- Figure 4. IKKb drives the expression of the CCL2 gene. A. 832/13 cells were pre-treated for 1 h with 0.5, 1 or 2 mM TPCA, followed by a 6 h stimulation with 1 ng/mL IL-1b. #p,0.001 vs. DMSO. B. Cells were transduced with either bGAL or three increasing concentrations of IKKb S177E/ S181E (CA IKKb) overnight, or 1 ng/mL IL-1b for 15 mins (dashed line indicates separation between cytokine-stimulated IkBa degradation from that induced by CA-IKKb). Whole cell lysates were harvested and immunoblot analysis was performed using antibodies to detect IKKb, IkBa and b Actin. C. Cells were transduced with the indicated adenoviruses and subsequently transfected with 5X NF-kB-Luc; relative promoter activity was normalized to total cellular protein. *p,0.05 vs. bGAL. D. Cells were treated with either CA-IKKb adenovirus alone or in the presence of two increasing concentrations of IkBa SR. Suppression by the IkBa SR is shown in the grey bars. *p,0.05 vs. bGAL; #p,0.001 vs. respective controls. A, D. Total RNA was isolated and CCL2 gene expression monitored via real-time PCR. Promoter luciferase activity and RNA abundance data represent means6 SEM from 3–4 individual experiments. doi:10.1371/journal.pone.0046986.g004 PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e46986