Figure 1

Differential inhibition of IL-1β-induced activation of MKK3 and MKK6 by PE in human OA chondrocytes. (a) Specificity of anti-MKK3 and anti-MKK6 monoclonal antibodies used in these studies was determined by using in vitro translated MKK3 and MKK6 proteins. No cross reactivity with the non-target protein was observed. (b) Expression of MKK3 and MKK6 in human chondrocytes. Human OA chondrocytes were grown to 70 to 80% confluence, serum starved overnight and then stimulated with IL-1β (5 ng/ml) for 15 minutes while control cultures received only the fresh medium. Unstimulated and stimulated human OA chondrocytes were lyzed and lysates were used for the immunoprecipitation of MKK3 and MKK6 using monoclonal antibodies. Immunoprecipitated protein was resolved on a 4 to 20% gradient gel by SDS-PAGE and Western blots were probed with anti-phosphoMKK3/6 antibodies. (c) Effect of PE on MKK3 and MKK6 phosphorylation in IL-1β-stimulated human OA chondrocytes. After treatment with PE (6.25 to 100 μg/ml) for 2 h at 37°C, primary human OA chondrocytes (70 to 80% confluent) were incubated with IL-1β (10 ng/ml) for 30 minutes, and then the phosphorylation of MKK3 and MKK6 was determined by immunoblotting using anti-phospho-MKK3 (Ser189)/anti-phospho-MKK6 (Ser207) antibody. Band images were digitally captured and the band intensities (pixels/band) were obtained using the Un-Scan-It software and are expressed as average pixels/band. The data shown are cumulative of two experiments. Average pixel values are presented as Mean ± SD; data without a common letter differ, P < 0.05.