Figure 1
Cortactin mediates MCP1-induced p21Cip1 degradation and HASMC proliferation.
(a) HASMCs that were transfected with siControl or siCortactin (100 nM) and growth-arrested were treated with vehicle or MCP1 (50 ng/ml) for 36 hrs and DNA synthesis was measured by [3H]-thymidine incorporation. (b) Equal amounts of protein from control and various time periods of MCP1-treated HASMCs were analyzed by Western blotting for the indicated CDKIs and cyclins using their specific antibodies and normalized to β-tubulin. (c) All the conditions were the same as in panel a except that after growth-arrest cells were treated with and without 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, cylcin A2 and cyclin D1 levels. Equal amounts of protein from the same cell extracts were also analyzed by Western blotting for cortactin and β-tubulin levels to show the efficacy of the siRNA on its target and off target molecules levels. (d) Growth-arrested cells were treated with vehicle or MCP1 in the presence and absence of MG132 (10 μM) for 8 hrs, cell extracts were prepared and an equal amount of protein from control and each treatment was analyzed by Western blotting for p21Cip1 levels and normalized to β-tubulin. (e) All the conditions were the same as in panel d except that cells were treated with vehicle or MCP1 for 36 hrs and DNA synthesis was measured. (f) All the conditions were the same as in panel a except that cells were treated with vehicle or MCP1 for 6 hrs and immunostained for p21Cip1 and 20Sα/β using their specific antibodies. The bar graphs in panels a–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.
