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An in vivo system for analysis of stable complex formation between Src and AFAP-110

  • Cell Biology
  • Published:
Methods in Cell Science

Abstract

The actin filament-associated protein, AFAP-110, forms a stable complex with activated pp60src (Src) as both an SH2 and SH3 binding partner. AFAP-110 contains two putative SH3 binding motifs and six putative SH2 binding motifs, indicating that interactions with Src may be complex. In order to evaluate the mechanism by which AFAP-110 and Src form a stable complex, an in vivo expression system was utilized that permits co-expression of each protein from the same plasmid construct subsequent to transient transfection of Cos-1 cells. Sixty hours post-transfection, western blot analysis indicated that both Src and AFAP-110 were expressed efficiently. Furthermore, Src phosphorylated AFAP-110 on tyrosine and was in stable complex with this protein. These results reflect observations detected in chick embryo fibroblast (CEF) cells. This system has several advantages in that (a) it is rapid, (b) high levels of protein can be overexpressed, stable complex formation occurs, and all transfected cells have the potential of expressing both protein products, (c) proper protein folding and post-translational modifications are likely to occur, (d) mAbs directed against Src and AFAP-110 are specific for these avian cDNA gene products, and (e) the plasmid constructs are amenable to site-directed mutagenesis, allowing for rapid analysis of the mechanism of stable complex formation.

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Abbreviations

CEF:

chick embryo fibroblasts

Src:

activated pp60527F

cSrc:

repressed pp60c-src

AFAP-110:

actin filament-associated protein, 110 kDa

CMV:

cytomegalovirus

SV40:

simian virus 40

PVDF:

polyvinylidene fluoride

Ab F1:

anti-AFAP-110 polyclonal rabbit

mAb 4C3:

anti-AFAP-110 monoclonal antibody

EC10:

anti-Src monoclonal antibody

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Authors and Affiliations

  1. The Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, USA

    Anne C. Guappone, Yong Qian, Tracy Weimer & Daniel C. Flynn

  2. the Department of Microbiology and Immunology, West Virginia University, Morgantown, West Virginia, USA

    Anne C. Guappone, Yong Qian, Tracy Weimer & Daniel C. Flynn

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  1. Anne C. Guappone
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  2. Yong Qian
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  3. Tracy Weimer
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  4. Daniel C. Flynn
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Guappone, A.C., Qian, Y., Weimer, T. et al. An in vivo system for analysis of stable complex formation between Src and AFAP-110. Methods Cell Sci 18, 55–65 (1996). https://doi.org/10.1007/BF00123524

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