Figure 3

Phosphorylation of p38 mitogen-activated protein (MAP) kinase, Akt, p44/42 and stress-activated protein kinase/Jun-N-terminal kinase (SAP/JNK) by endothelin-1 (ET-1) in human osteoarthritis (OA) chondrocytes. (a) Western immunoblot of p38 MAP kinase. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 10 or 45 min and the cell extracts were prepared as described in Materials and methods. Western immunoblots used antiserum against activated (phospho-p38) and total p38 MAP kinase (p38 T). Representative result of three different experiments. (b) Western immunoblot of Akt. Cells were incubated for 2, 5, 15, 30, 45 or 60 min in the presence of ET-1 (10 nM) and cell extracts were prepared as described in Materials and methods. Western immunoblot was carried out using an antiserum specific for phospho Ser 473 of Akt. Representative result of three different experiments. (c) Western immunoblot of p44/42. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 0, 5, 15 or 60 min and cell extracts were prepared as described in Materials and methods. (d) Western immunoblot of SAP/JNK protein kinase. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 0, 5, 30, 45 and 60 min, and cell extracts were prepared as described in Materials and methods. Actin detection was used as a control of the level of proteins loaded. Representative blot of three independent experiments.