Figure 4
Fyn mediates MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation.
(a) Growth-arrested cells were treated with vehicle or MCP1 in the presence and absence of PP1 (10 μM), PP2 (10 μM), or PP3 (10 μM) for 6 hrs and the cell extracts were prepared. An equal amount of protein from control and each treatment was immunoprecipitated with anti-cortactin antibodies and the immunocomplexes were analyzed by immunoblotting with anti-pTyr antibodies and normalized for cortactin. Equal amounts of protein from the same cell extracts were also analyzed by Western blotting for p21Cip1 levels and normalized to β-tubulin. (b) All the conditions were same as in panel a except that cells were subjected to MCP1-induced DNA synthesis. (c) Equal amounts of protein from control and various time periods of MCP1-treated HASMCs were analyzed by Western blotting for phosphorylation levels of the indicated SFKs using their phosphospecific antibodies and normalized to their total levels. (d) All the conditions were the same as panel a except that an equal amount of protein from control and each treatment was analyzed by Western blotting for pFyn levels and normalized to total Fyn levels. (e) HASMCs that were transfected with siControl or siFyn and growth-arrested were treated with vehicle or MCP1 for 6 hrs, cell extracts prepared and an equal amount of protein from control and each treatment was immunoprecipitated with anti-cortactin antibodies and the immunocomplexes were analyzed by Western blotting with anti-pTyr antibodies and normalized for cortactin. Equal amounts of protein from the same cell extracts were also analyzed by Western blotting for p21Cip1 or Fyn levels and normalized to β-tubulin. (f) All the conditions were same as in panel e except that cells were treated with vehicle or MCP1 for 6 hrs and co-immunostained for p21Cip1 and 20Sα/β. (g) All the conditions were the same as in panel e except that cells were subjected to MCP1-induced DNA synthesis. The bar graphs in panels a–e and g represent Mean ± SD values of three independent experiments. *p < 0.05 vs vehicle control or siControl; **p < 0.05 vs MCP1 or siControl + MCP1.
