Neurofibromin interacts with CRMP-2 and CRMP-4 in rat brain
Section snippets
Materials and methods
Antibodies. Neurofibromin antibodies NF1(N) and NF1(D) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Myc antibody 9B11 and CRMP-4 antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA) and BD Biosciences (San Jose, CA), respectively. Rabbit polyclonal pan-CRMP antibodies were generated using a GST-tagged full length CRMP-2 recombinant protein, and the resulting pan-CRMP antibodies were purified by His-tagged-CRMP-2 conjugated to an AminoLink® Plus column
Results and discussion
To identify neurofibromin-associated proteins, proteins from homogenized rat brains were co-immunoprecipitated using the neurofibromin NF1(D) antibody and separated by 2D electrophoresis. Two sets of phosphorylated proteins, around 60 and 70 kDa, were precipitated by the neurofibromin antibody but not by control IgG (Fig. 1A). Mass spectrometry analysis identified the phosphorylated proteins at ∼60 kDa as collapsin response mediator protein-2 (CRMP-2) (Fig. 1B and C) and the phosphorylated
Acknowledgments
We thank Dr. Li-Huei Tsai, Massachusetts Institute of Technology, USA, for the CDK5 and p35 constructs, and the staff at the Proteomic Core Facility at the Institute of Biological Chemistry, Academia Sinica for technical assistance. This work was supported by grants from Academia Sinica, the National Science Council (NSC 96-2321-B-001-005 to Y.-P. H.), and the National Health Research Institute (NHRI-EX96-9403NI to Y.-P. H.).
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