Fig. 2

Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not Raf-1 phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE (n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA (P < 0.05). b Cells expressing control shRNA or Plk1 shRNA were treated with 20 ng/ml PDGF for 10 min, or they were not stimulated. Protein phosphorylation was then evaluated by immunoblotting. MEK1/2 phosphorylation induced by PDGF was significantly reduced in Plk1KD cells compared to cells treated with control shRNA (*P < 0.05). NS, basal MEK1/2 phosphorylation in Plk1 KD was not significantly different from cells expressing control shRNA (P > 0.05). Values are mean ± SE (n = 8). c PDGF-induced ERK1/2 phosphorylation was significantly reduced in Plk1 KD cells (*P < 0.05, n = 7). NS, basal ERK1/2 phosphorylation in Plk1 KD was not significantly different from cells expressing control shRNA (P > 0.05). d PDGF-stimulated Raf-1 phosphorylation at Ser-338 was comparable in Plk1 KD cells and cells expressing control shRNA. NS, Raf-1 phosphorylation in Plk1 KD was not significantly different from cells expressing control shRNA (P > 0.05, n = 8). Protein phosphorylation under various treatments was normalized to the levels in unstimulated cells expressing control shRNA