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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Cell Signal. 2008 Apr 15;20(8):1459–1470. doi: 10.1016/j.cellsig.2008.04.002

Figure 8. Effects of silencing macrophage Epac1 gene expression on 8-CPT-2-O-Me-cAMP-dependent events.

Figure 8

Panel A. Suppression of 8-CPT-2-O-Me-cAMP-upregulated protein synthesis in macrophages upon down regulation of Epac1 gene expression by RNAi. Protein synthesis is measured by cellular [3H]leucine uptake and is expressed as the mean ± SE from triplicate experiments. The data are expressed as pmol [3H]leucine incorporated mg protein. The bars are: (1) lipofectamine + buffer; (2) lipofectamine + 8-CPT-2-O-Me-cAMP (200 μM/30 min/37°C); (3) Epac1 dsRNA + 8-CPT-2-O-Me-cAMP; and (4) scrambled dsRNA + 8-CPT-2-O-Me-cAMP. Panel B. Silencing Epac1 gene expression inhibits 8-CPT-2-O-Me-cAMP induced DNA synthesis. DNA synthesis is measured as [3H]thymidine uptake and is expressed as the mean ±SE from triplicate experiments (pmol/mg protein). The bars are as in Panel A. Panel C. Down regulation of the Epac1 gene expression by RNAi causes inhibition of ERK1/2 (□) and B-Raf (■) activation. The immunoblot shown is represenative of two experiments. The lanes are: (1) lipofectamine + buffer; (2) lipofectamine + 8-CPT-2-O-Me-cAMP (200μM/30 min/30°C); and (3) Epac1 dsRNA + 8-CPT-2-O-Me-cAMP. The protein loading controls unphosphorylated ERK1/2 and B-Raf are also shown below the respective immunoblots. The changes in phospho-ERK1/2 and phospho-B-Raf are expressed as ratios of phosphorylated/unphosphorylated kinases and are shown as a bar diagram above the immunoblot. Panel D. Effect of silencing Epac1 gene expression on 8-CPT-2-O-Me-cAMP-induced upregulated protein levels of the p110α catalytic subunit of PI 3-kinase; phosphorylated-PDK1, and phosphorylated-p70s6k. Changes in protein levels of p-PDK1 and p-p70s6k are shown as ratios of protein/actin in a bar diagram above the immunoblot as in Panel C. In preliminary experiments transfection of cells with scrambled dsRNA showed negligible or little effect on various kinase proteins. Therefore, these controls were not performed in these experiments.