A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells
Abstract
:1. Introduction
2. Results and Discussion
2.1. Addition of DSP to living cells yields crosslinked complexes that will not enter a one-dimensional gel
2.2. Calmodulin, but not actin or other proposed interaction partners, co-immunoprecipitates with GAP-43 after addition of crosslinker to living cells
2.3. GAP-43 does not appear in the 100,000 × g pellet fractions
2.4. Polyclonal anti-GAP-43 yields co-immunoprecipitated calmodulin but not actin following crosslinker treatment of cells
2.5. Quantitation of GAP-43 and calmodulin in immunoprecipitates
2.6. Anti-actin does not yield co-immunoprecipitated GAP-43 when cells are treated with crosslinker
2.7. GAP-43 does not sediment with the membrane skeleton when DMSO- or DSP-treated cells are lysed in nonionic detergent
2.8. GAP-43 is crosslinked to calmodulin but not to actin if cells are lysed in nonionic detergent prior to the addition of crosslinker
3. Conclusions
4. Experimental Section
4.1. Materials
4.2. Cell culture and differentiation
4.3. Labeling of differentiated cells
4.4. Addition of crosslinker to living cells
4.5. Lysis of crosslinker-treated cells in SDS
4.6. Lysis of crosslinker-treated cells in nonionic detergent and isolation of the membrane skeleton
4.7. Addition of crosslinker to cell lysates
4.8. Immunoprecipitation of GAP-43, calmodulin, and actin
4.9. Gel electrophoresis
4.10. Determination of radioactivity in gels
Acknowledgments
References
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GAP43 | CaM | % | |
---|---|---|---|
Figure 2b DMSO | 2967 | 0 | 0 |
Figure 2d DSP | 1933 | 278 | 14 |
Figure 2e antiCaM | 322 | ||
Figure 4a DMSO | 791 | ||
Figure 4b DSP | 619 | ||
Figure 7b DSP | 1650 | 643 | 39 |
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Ollom, C.M.; Denny, J.B. A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells. Int. J. Mol. Sci. 2008, 9, 1753-1771. https://doi.org/10.3390/ijms9091753
Ollom CM, Denny JB. A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells. International Journal of Molecular Sciences. 2008; 9(9):1753-1771. https://doi.org/10.3390/ijms9091753
Chicago/Turabian StyleOllom, Callise M., and John B. Denny. 2008. "A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells" International Journal of Molecular Sciences 9, no. 9: 1753-1771. https://doi.org/10.3390/ijms9091753