These authors contributed equally to this study.
Control of neurite outgrowth by RhoA inactivation
Article first published online: 24 NOV 2011
© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry
Journal of Neurochemistry
Volume 120, Issue 5, pages 684–698, March 2012
How to Cite
Jeon, C.-Y., Moon, M.-Y., Kim, J.-H., Kim, H.-J., Kim, J.-G., Li, Y., Jin, J.-K., Kim, P.-H., Kim, H.-C., Meier, K. E., Kim, Y.-S. and Park, J.-B. (2012), Control of neurite outgrowth by RhoA inactivation. Journal of Neurochemistry, 120: 684–698. doi: 10.1111/j.1471-4159.2011.07564.x
- Issue published online: 10 FEB 2012
- Article first published online: 24 NOV 2011
- Accepted manuscript online: 29 OCT 2011 09:55AM EST
- Received April 12, 2011; revised manuscript received October 19, 2011; accepted October 20, 2011.
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 (***p < 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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