a crosslinking analysis of gap-43 interactions with other proteins in differentiated n1e-115 cellsgap-43交互与其他蛋白质的交联分析分化n1e - 115细胞.pdfVIP

Int. J. Mol. Sci. 2008, 9, 1753-1771; DOI: 10.3390/ijms9091753 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 /ijms/ Article A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells Callise M. Ollom and John B. Denny * Department of Ophthalmology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA. E-Mail: cdenny@ (C. O.) * Author to whom correspondence should be addressed; E-Mail: denny@ Received: 13 August 2008; in revised form: 3 September 2008 / Accepted: 13 September 2008 / Published: 16 September 2008 Abstract: It has been suggested that GAP-43 (growth-associated protein) binds to various proteins in growing neurons as part of its mechanism of action. To test this hypothesis in vivo, differentiated N1E-115 neuroblastoma cells were labeled with [35S]-amino acids and were treated with a cleavable crosslinking reagent. The cells were lysed in detergent and the lysates were centrifuged at 100,000 x g to isolate crosslinked complexes. Following cleavage of the crosslinks and analysis by two-dimensional gel electrophoresis, it was found that the crosslinker increased the level of various proteins, and particularly actin, in this pellet fraction. However, GAP-43 was not present, suggesting that GAP-43 was not extensively crosslinked to proteins of the cytoskeleton and membrane skeleton and did not