BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells

Figure 3

CREB sites within the IL-1β promoter partially mediate enhanced IL-1β mRNA expression in BRAT cells. (a) Cells stably expressing BRAT were transiently transfected with the indicated IL-1β deletion reporter constructs and a Renilla constitutive luciferase reporter plasmid for normalization. Lysates were collected, subjected to two freeze-thaw cycles, and assayed in triplicate on a manual luminometer using Promega’s Dual Luciferase Assay Kit. Luciferase activity was normalized to Renilla luciferase activity for each triplicate, averaged, and results are expressed as mean reporter activity ± standard error. (b) Stable BRAT (white bars) and pcDNA3.1 (grey bars) cells were analyzed by western immunoblot for comparison of cellular levels of phospho-CREB, CREB, and actin. The results are expressed in relative densitometric units. (c) Cells stably expressing BRAT cells were cotransfected with nontargeting control siRNA (siCon) or siRNA targeting CREB, collected, and assayed similarly to (a). Protein lysates were collected in parallel for knockdown analyses and CREB protein silencing demonstrated by western blot (inset). Symbol () denotes statistical significance ≤ 0.03 confidence level.

(a)

(b)

(c)