Figure 6
CCR2 but not CCR4 activation is required for cortactin-mediated MCP1-induced p21Cip1 degradation.
(a) Growth-arrested HASMCs were treated with vehicle or MCP1 in the presence and absence of CCR2 antagonist (CCR2A) or CCR4 antagonist (CCR4A) for 6 hrs and the cell extracts were prepared. 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. (b) All the conditions were the same as in panel a except that equal amounts of protein from control and each treatment were analyzed by Western blotting for p21Cip1 levels using its specific antibodies and normalized to β-tubulin. (c) All the conditions were the same as in panel a except that cells were treated with vehicle or MCP1 for 6 hrs and co-immunostained for p21Cip1 and 20Sα/β using their specific antibodies. (d) All the conditions were the same as in panel a except that cells were subjected to MCP1-induced DNA synthesis. (e) Growth-arrested cells were treated with vehicle or MCP1 in the presence and absence of BAPTA-AM (10 μM) for 6 hrs and 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. The bar graphs in panels a,b,d and e represent Mean ± SD values of three independent experiments. *p < 0.05 vs vehicle control; **p < 0.05 vs MCP1.
