MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK
Section snippets
Experimental procedures
Plasmids. Plasmids were prepared using Endo Free plasmid purification kits (Qiagen). Oligonucleotides containing multicloning sites were inserted into pcCAG [9] to generate pCAG-MCS2. MAP1B [8], Cdk5 [14], p35 or p25 [15] cDNA was inserted into the pCAG-MCS2 vector. JNK expression vector was a generous gift from Dr. S. Tamura [16].
Antibodies and chemical regents. Primary antibodies used in this study were SMI31 (Sternberger Monoclonals), anti-MAP1B (Santa Cruz N-19), anti-phospho-Ser732 FAK
Results and discussion
E15 cerebral cortices were dissociated and after 1 day in culture, inhibitors or solvent was added for another 24 h. Cells were harvested and subjected to immunoblot analysis with SMI31 antibody which recognizes mode I phosphorylated MAP1B [19]. As previously reported [9], [20], treatment with a JNK inhibitor, SP600125, resulted in decreased mode I phosphorylation of MAP1B (Fig. 1A, SP25 and SP50). In contrast, treatment with a Cdk5 inhibitor, roscovitine, had no effect (Fig. 1A, R100), although
Acknowledgments
We thank A. Takashima, S. Tamura, L.H. Tsai, and Y. Uchida for providing plasmids. We also thank R. Yu and M. Sone for helpful comments and technical help. This work was supported by the center of excellence (COE) grant and Grant-in-Aid for Scientific Research on Priority Areas, “Elucidation of glia–neuron network mediated information processing systems (#16047220),” and “Membrane Traffic (#16044225)” from the Ministry of Education, Culture, Sports and Science and Technology, Japan, and by
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