Figure 6

Effect of Ras/Raf-1 modulation on transforming growth factor-beta-1 (TGF-β1)-induced expression of the Ank gene. Rat chondrocytes were electroporated with empty vector, wild-type, constitutively active, or dominant-negative plasmids for Ras (a) or Raf-1 (b) (2 μg/well of six-well plate) before stimulation with 10 ng/mL of TGF-β1 for 12 hours. Total RNA was extracted and subjected to real-time polymerase chain reaction analysis. The mRNA level of Ank was normalized to that of S29 mRNA and is expressed as mean percentages (± standard deviation) over control values from three independent experiments. Statistically significant differences from the control are indicated as *p < 0.05 and from TGF-β1-treated cells as #p < 0.05. (c) Effect of TGF-β1 on extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in electroporated cells with wild-type, constitutively active, or dominant-negative plasmids for Ras (2 μg/well of six-well plate). Total proteins were extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 for 15 minutes and subjected to Western blotting using anti-phospho- and anti-total-ERK 1/2 antibodies. The relative abundance of these proteins was normalized to that of β-actin protein.