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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Leukemia. 2008 Nov 6;23(2):365–374. doi: 10.1038/leu.2008.297

Figure 3. C/EBPα and a C/EBPαLZ oncoprotein depend on NF-κB p50 for binding to the bcl-2 promoter.

Figure 3

(a) Splenocytes from mice with the indicated genotype were exposed to 200 cGy and cultured for 0, 7, or 24 hours. Total cellular proteins extracts were obtained and subjected to Western blotting for bcl-2 and β-tubulin. The ratio of bcl-2:tubulin in each sample is shown. (b) Total bone marrow cells extracted from nfkb1−/− or wild-type control mice were subjected to ChIP using C/EBPα (α) or NF-κB p50 (p50) antisera or IgG control and primers specific for the bcl-2 P2 or neutrophil elastase (NE) promoters. (c) RNA isolated from total bone marrow cells from the hind limbs of age, sex, and strain-matched nfkb1−/− or wild-type (WT) mice were subjected to quantitative RT-PCR analysis of bcl-2 or NE expression, normalized to mS16. Relative mRNA expression between WT and nfkb1−/− mice is shown, with expression in nfkb1−/− marrow set to 1. Data from four comparisons are shown. (d) F9 cells were transiently co-transfected with 1.5 µg of P2-LUC or its variant harboring clustered point mutations in the −170 κB site (mκB), with 100 ng of CMV, CMV-C/EBPα, or CMV-F3901, and 5 with ng of CMV-β-galactosidase as an internal control. Activation of the wild type and mutant promoters by C/EBPα or F3901 was analyzed 48 after transfection. Fold-activation compared to the empty CMV vector was determined after adjustment for β-galactosidase activity. The mean of four independent experiments is presented. Also shown is the average ratio of P2-LUC:P2-LUCmκB induction by C/EBPα or F3901. The p-values shown compare these induction ratios to the null hypothesis value of 1.0.