1. Src tethers Cas to adhesion complexes.
Src tethers Cas to adhesion complexes. (A) SYF cells, with or without the restoration of Src expression, were subjected to combinations of co-infection with retroviruses that mediated the expression of either GFP–CasWT or GFP–Cas15YF. Immunoblotting was conducted to analyze the expression and phosphorylation of Cas proteins (GFP–CasWT or GFP–Cas15YF) (upper two panels). The expression of exogenous Src and actin was also analyzed (lower two panels). Note the phospho-Cas (pCas-165) blot showing Src-dependent CasSD phosphorylation of GFP–CasWT (lane 2) and its reduction by treatment with CGP77675 (lane 3), neither of which was seen in cells expressing GFP–Cas15YF (lanes 5, 6). (B) FRAP analysis of GFP–CasWT expressed in SYF cells (circle, refer to lane 1 in A) and those expressing exogenous Src in the presence (square) or absence (diamond) of 10 µM CGP77675 (refer to lanes 3 and 2 in A, respectively). (C) The mobile fraction was determined from the results in B. (D) FRAP analysis of GFP–Cas15YF expressed in SYF cells (circle, refer to lane 4 in A) and those expressing exogenous Src in the presence (square) or absence (diamond) of 10 µM CGP77675 (refer to lanes 6 and 5 in A, respectively). (E) The mobile fraction was determined from the results in D. All statistical analyses were performed between the control condition [black bar, SYF cells expressing GFP–CasWT (C) or SYF cells expressing GFP–Cas15YF without exogenous Src expression and CGP77675 treatment (E)] and another condition. Data show the mean±s.e.m.; **P<0.01 (n>50); Student's t-test.
Hiroaki Machiyama et al. J Cell Sci 2014;127:
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