Figure 5
Gi/o-mediates MCP1-induced Fyn and cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation.
(a) HASMCs that were transfected with siControl, siGαq or siGα11 and growth-arrested were treated with vehicle or MCP1 for 8 hrs and cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for p21Cip1 levels and normalized to β-tubulin. Equal amounts of protein from the same cell extracts were also analyzed by Western blotting for Gαq and Gα11 levels to show the efficacy of the siRNA on the target molecule level. (b) Growth-arrested cells were treated with vehicle or MCP1 in the presence and absence of PT (50 ng/ml) for 8 hrs and the cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for p21Cip1 levels and normalized to β-tubulin. (c) All the conditions were same as in panel b except that equal amounts of protein from control and each treatment were analyzed by Western blotting for pFyn levels using anti-pFyn antibodies and normalized to Fyn. Equal amounts of protein from the same cell extracts were also immunoprecipitated with anti-cortactin antibodies and the immunocomplexes were analyzed by Western blotting with anti-pTyr antibodies and normalized to cortactin. (d) All the conditions were the same as in panel b except that cells were treated with vehicle or MCP1 for 6 hrs and co-immunostained for p21Cip1 and 20Sα/β using their specific antibodies. (e) All the conditions were the same as in panel b except that cells were subjected to MCP1-induced DNA synthesis. The bar graphs in panels a,b,d and e represent Mean ± SD values of three independent experiments. *p < 0.05 vs vehicle control or siControl; **p < 0.05 vs MCP1 or siControl + MCP1.
