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Open Access Highly Accessed Research article

c-Jun N-terminal kinase in synergistic neurite outgrowth in PC12 cells mediated through P90RSK

Kok Huei Seow1, Lihan Zhou12, Gregory Stephanopoulos13 and Heng-Phon Too12*

Author Affiliations

1 Chemical & Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore

2 Department of Biochemistry, National University of Singapore, 14 Medical Drive, Singapore, Singapore

3 Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, USA

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BMC Neuroscience 2013, 14:153  doi:10.1186/1471-2202-14-153

Published: 12 December 2013

Additional files

Additional file 1: Figure S1:

Time-course profiles of activations of kinases upon PACAP, NGF, and NP treatments. Fold changes of (a) pErk, (b) pJNK, (c) pP38, and (d) pAkt from 0-1 hour.

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Additional file 2: Figure S2:

Non-synergistic phosphorylation of P38 and Akt upon combinatorial NGF (0-50 ng/ml) and PACAP (0-100 ng/ml) treatments. (a) Time-course of P38 and Akt phosphorylations at 20 and 60 minutes following NGF-PACAP treatments. Phosphorylation levels of the proteins were analyzed by western blotting, and normalized to the levels of actin. Fold changes of (b) pP38, and (c) pAkt under (i) uni-ligand treatments, (ii) bi-ligand treatments at 10 ng/ml of NGF, and (iii) bi-ligand treatments at 50 ng/ml NGF. Significant differences between combinatorial experimental treatment of NGF-PACAP and summation of their individual effects were calculated using the paired Student’s t-test. A value of p<0.05 was considered significant.

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Open Data

Additional file 3: Figure S3:

Synergistic phosphorylation of Erk and JNK upon combinatorial NGF (0-50 ng/ml) and PACAP (0-100 ng/ml) treatments. Fold changes of (a) pErk, and (b) pJNK under (i) uni-ligand treatments, (ii) bi-ligand treatments at 10ng/ml of NGF, and (iii) bi-ligand treatments at 50 ng/ml NGF. Significant differences between combinatorial experimental treatment of NGF-PACAP and summation of their individual effects were calculated using the paired Student’s t-test. A value of p<0.05 was considered significant (**p<0.01).

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Open Data

Additional file 4: Figure S4:

Non-Synergistic phosphorylation of P38 and Akt upon FP and EP treatments. Time-course of quantified P38, and Akt phosphorylations at 20 and 60 minutes following (a) FGFb (50 ng/ml)-PACAP (100 ng/ml), and (b) EGF (50 ng/ml)-PACAP (100 ng/ml) treatment. Fold changes of (i) pP38, and (ii) pAkt were quantified by densitometry and normalized to the levels of actin. Significant differences between combinatorial experimental treatment of growth factor-PACAP and summation of their individual effects were calculated using the paired Student’s t-test. A value of p<0.05 was considered significant.

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Open Data

Additional file 5: Figure S5:

Total levels of Erk, JNK, P90RSK, Akt and P38 were not changed following treatments with ligands. The total protein levels were assayed at 20 and 60 minutes post-stimulation. The same control (C, at t=0 minutes) was used for both time-points.

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Additional file 6: Figure S6:

Representative images of cells treated with growth factors-PACAP in the presence of inhibitors in the three systems. NP, FP and EP.

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Additional file 7: Figure S7:

Positive controls for the kinase inhibitors following treatment with NGF (50 ng/ml). (a) Inhibition of Erk phosphorylation in the presence of U0126. (b) Inhibition of c-Jun phosphorylation in the presence of SP600125. (c) Inhibition of P38 phosphorylation in the presence of SB203580. (d) Inhibition of Akt phosphorylation in the presence of LY294002.

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Open Data

Additional file 8: Figure S8:

Synergistic phosphorylation of P90RSK upon combinatorial NGF (0-50 ng/ml) and PACAP (0-100 ng/ml) treatments. (a) Fold changes of pP90RSK under (i) uni-ligand treatments, (ii) bi-ligand treatments at 10 ng/ml of NGF, and (iii) bi-ligand treatments at 50 ng/ml NGF. (b) Representative images of cells treated with growth factors (50 ng/ml)-PACAP (100 ng/ml) in the presence of BRD7389 in the three systems. Significant differences between combinatorial experimental treatment of NGF-PACAP and summation of their individual effects were calculated using the paired Student’s t-test. A value of p<0.05 was considered significant (**p<0.01).

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Open Data

Additional file 9: Figure S9:

Total levels of Erk, JNK, and P90RSK were not changed following treatments with inhibitors. The total protein levels were assay at 20 minutes post-stimulation. A normalizer (NGF-PACAP co-treated cells) in each blot served as a control to normalize between different blots. U=U0126 (20 μM) and S=SP600125 (10 μM).

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Open Data