Figure 5. IκBβ knock down results a significant reduction in IL-1β cytokine expression and secretion. (A) Silencing of IκBβ expression in RAW264.7 macrophages using siRNA. RAW264.7 macrophages were transfected with control or IκBβ-specific siRNAs or were left as an untransfected control. After 48 h, Western blot detected IκBβ expression. β-actin was used as loading control. (B) Measurement of IL-1β, MIP-2, and TNF cytokine secretion in control and IκBβ siRNA-transfected RAW264.7 cells. 48 h after transfection, cells were treated with LPS (100 ng/ml) and cytokine secretion was measured by ELISA. Data were obtained from three independent experiments performed in triplicate, and the results are presented as mean and SEM. (C) Schematic maps of different luciferase reporter genes containing IL-1β promoter luciferase reporter gene constructs. Three different luc-constructs encoding IL-1β promoter sequence starting from −518, −399, and −133, respectively, were used in transient transfection assays in RAW264.7 macrophages. The IL-1β–Luc construct IL-1B-518 includes two functional κB sites, whereas the IL-1B-399 contains only one functional κB site. No κB sites are present in the −133 IL-1β–Luc construct. In the mut-399-IL1B-luc construct, a mutation was introduced into the κB site by site-directed mutagenesis, as indicated. (D) RAW264.7 macrophages were transfected with 1 µg of the indicated IL-1β promoter luciferase reporter gene constructs. 48 h after the transfection, cells were stimulated with LPS (100 ng/ml) for 8 h and luciferase activity was determined. Data were obtained from three independent experiments performed in triplicate, and the results are presented as mean and SEM. (E) RAW264.7 macrophages were cotransfected using 1 µg of the indicated IL-1β promoter luciferase reporter gene constructs, a control siRNA, an IκBβ-specific siRNA, or they were left as an untransfected control. 48 h after the transfection, cells were stimulated with LPS (100 ng/ml) for 8 h and luciferase activity was determined. Data were obtained from three independent experiments performed in triplicate, and the results are presented as mean and SEM.
Figure 5.

IκBβ knock down results a significant reduction in IL-1β cytokine expression and secretion. (A) Silencing of IκBβ expression in RAW264.7 macrophages using siRNA. RAW264.7 macrophages were transfected with control or IκBβ-specific siRNAs or were left as an untransfected control. After 48 h, Western blot detected IκBβ expression. β-actin was used as loading control. (B) Measurement of IL-1β, MIP-2, and TNF cytokine secretion in control and IκBβ siRNA-transfected RAW264.7 cells. 48 h after transfection, cells were treated with LPS (100 ng/ml) and cytokine secretion was measured by ELISA. Data were obtained from three independent experiments performed in triplicate, and the results are presented as mean and SEM. (C) Schematic maps of different luciferase reporter genes containing IL-1β promoter luciferase reporter gene constructs. Three different luc-constructs encoding IL-1β promoter sequence starting from −518, −399, and −133, respectively, were used in transient transfection assays in RAW264.7 macrophages. The IL-1β–Luc construct IL-1B-518 includes two functional κB sites, whereas the IL-1B-399 contains only one functional κB site. No κB sites are present in the −133 IL-1β–Luc construct. In the mut-399-IL1B-luc construct, a mutation was introduced into the κB site by site-directed mutagenesis, as indicated. (D) RAW264.7 macrophages were transfected with 1 µg of the indicated IL-1β promoter luciferase reporter gene constructs. 48 h after the transfection, cells were stimulated with LPS (100 ng/ml) for 8 h and luciferase activity was determined. Data were obtained from three independent experiments performed in triplicate, and the results are presented as mean and SEM. (E) RAW264.7 macrophages were cotransfected using 1 µg of the indicated IL-1β promoter luciferase reporter gene constructs, a control siRNA, an IκBβ-specific siRNA, or they were left as an untransfected control. 48 h after the transfection, cells were stimulated with LPS (100 ng/ml) for 8 h and luciferase activity was determined. Data were obtained from three independent experiments performed in triplicate, and the results are presented as mean and SEM.

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