Identification of several signaling pathways in transforming growth factor-beta-1 (TGF-β1)-induced expression of the Ank gene. (a) Kinetics of signaling events induced by TGF-β1. Total proteins were extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 for 10 to 180 minutes and subjected to Western blotting using anti-phospho-ERK 1/2, anti-phospho-p38-MAPK, anti-phospho-pan-PKC, or anti-phospho-Smad 3/1. The relative abundance of these proteins was normalized to that of β-actin protein. (b) Effect of specific signaling inhibitors on TGF-β1-induced expression of Ank mRNA. Total RNA was extracted from rat chondrocytes stimulated with 10 ng/mL of TGF-β1 in the presence of 10 μM RcAMP (PKA inhibitor) or 10 μM SB203580 (a selective p38-MAPK inhibitor) or 10 μM PD98059 (a MEK-1 inhibitor) added 1 hour before TGF-β1. The mRNA level of Ank obtained from real-time polymerase chain reaction analysis 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. ERK, extracellular signal-regulated kinase; MEK-1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1; p38-MAPK, p38-mitogen-activated protein kinase; PKA, protein kinase A; PKC, protein kinase C.