Figure 2

Cortactin tyrosine phosphorylation is required for MCP1-induced p21Cip1 degradation and HASMC proliferation.

(a) HASMCs that were transfected with WT, S405A, S418A or S405/418A mutant cortactin expression vector and treated with vehicle or MCP1 for 8 hrs were analyzed by immunoblotting for p21Cip1 levels and the blot was reprobed for GFP or β-tubulin to show the overexpression of WT or mutant cortactin or normalization. (b) All the conditions were the same as in panel a except that cells were subjected to MCP1-induced DNA synthesis. (c) Equal amounts of protein from control and the indicated treatments were immunoprecipitated with anti-acetyl lysine or anti-phosphotyrosine antibodies and the immunocomplexes were analyzed by immunoblotting for cortactin. The same cell extracts were also analyzed by immunoblotting for cortactin. (d) HASMCs were treated with vehicle or MCP1 in the presence and absence of HDAC6i or HDAC8i (10 μM) for 6 hrs and equal amounts of protein from control and each treatment were immunoprecipitated with anti-cortactin antibodies and the immunocomplexes were analyzed by immunoblotting with anti-acetyl lysine antibody and normalized to cortactin. (e) HASMCs were treated with vehicle or MCP1 in the presence and absence of HDAC6 inhibitor for 8 hrs and equal amounts of protein from control and each treatment were analyzed by immunoblotting for p21Cip1 and normalized to β-tubulin. (f) The cloning strategy for cortactin expression vectors is presented and the mutant amino acid is shown in red. (g) HASMCs were transfected with WT or Y5F mutant cortactin expression vector, treated with vehicle or MCP1 for 8 hrs and equal amounts of protein from control and each treatment were immunoprecipitated with anti-pTyr antibody and the immunocomplexes were analyzed by immunoblotting for GFP. Same cell extracts were also analyzed by immunoblotting for GFP-CTTN and p21Cip1 and normalized to β-tubulin. (h) All the conditions were the same as in panel g except that cells were subjected to MCP1-induced DNA synthesis. The bar graphs in (a–e,g,h) represent Mean ± SD values of three independent experiments. *p < 0.05 vs vehicle control or WT-CTTN; **p < 0.05 vs MCP1 or WT-CTTN + MCP1. HDAC6i, HDAC6 inhibitor; HDAC8i, HDAC8 inhibitor.