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Figure S1. cAMP induces neurite outgrowth of PC12 cells. PC12 cells were cultured in RPMI 1640 media containing 5% heat-inactivated FBS and 10% heat-inactivated HS and changed to DMEM containing 1% HS, 0.5% FBS (low- serum media), antibiotics and 2 mg/mL bovine serum albumin for 12 h. NGF (100 ng/mL), 100 ng/mL bFGF, 1 mM db-cAMP, and 50 μM forskolin were added to the cells for 2 days to induce neurite. (a) K252a (100 nM), (b) AG1478 (250 nM), and (c) 20 μM SU5402 in DMSO were pre-treated for 30 min, and then neurite outgrowth was induced for 2 days in low-serum media, antibiotics and 2 mg/mL bovine serum albumin. The percentage of neurite-bearing cells was determined by containing at least 100 single cells (not aggregated)/3 arbitrary positions on dish. A cell was positive for the neurite extension if it had twofold diameters of the cell body. Cells were visualized using phase contrast (200-fold magnification) on microscope and GFP-positive cells were counted under an fluorescence microscope.

Figure S2. ERK1/2 activation is involved neurite outgrowth in response to db-cAMP. (a) PC12 cells were pre-treated with PD98059, an ERK inhibitor, for 30 min and then treated 1 mM db-cAMP for 2 days. Cells with neurites were assessed as described in Materials and methods. Data are represented as means ± SE of three independent experiments (***< 0.001, compared with control using Student’s t-test). (b) PC12 cells were treated with 1 mM db-cAMP or 50 μM forskolin for the indicated times and lysed. Proteins from each cell lysate were separated on SDS–PAGE and analyzed by western blotting using anti-phospho-ERK and anti-ERK antibodies. Data are representative of three independent experiments.

Figure S3. Phosphorylation of RhoGDI by cAMP. Phospho-Ser was immunoprecipitated from cell lysates of PC12 cells which was stimulated by db-cAMP, then RhoGDI were identified with western blotting.

Figure S4. Phosphorylation of Akt by cAMP. PC12 cells were treated with 1 mM db-cAMP for the indicated times and lysed. Proteins from each cell lysate were separated on SDS–PAGE and analyzed by western blotting using anti-phospho-Akt and anti-Akt antibodies.

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