Figure 6

Epigallocatechin-3-gallate inhibits activation and DNA binding of NF-κB in advanced glycation end product-BSA-stimulated osteoarthritis chondrocytes. Primary chondrocytes (70 to 80% confluent) were pretreated with epigallocatechin-3-gallate (EGCG) (25 and 75 μM) for 2 hours and were stimulated by advanced glycation end product (AGE)-BSA (600 μg/ml). (a) IκBα degradation and NF-κB translocation were analyzed by western immunoblotting using antibodies specific for the NF-κB p65 (Santa Cruz Biotech) (C-NF-κB, cytoplasmic NF-κB; N-NF-κB, nuclear NF-κB) and for IκBα (Santa Cruz Biotech). (b) Band intensities were obtained as described above. Data shown are cumulative of three experiments, and the optical density values (pixels/band) presented as mean ± standard deviation. (c) Activated NF-κB p65 in the nucleus was determined by the highly specific Transcription Factor ELISA kit (Panomics). The positive control nuclear extract supplied with the kit was used. Data are representative of two experiments and presented as mean ± standard deviation; data without a common letter differ, P < 0.05. (d) EGCG inhibited the IKKβ kinase activity in vitro. IKKβ kinase activity was determined in the absence or presence of EGCG (5 to 200 μM) using the HTScan^® IKKβ Kinase Assay Kit (Cell Signaling Technology). Each point is representative of three individual kinase assays and presented as mean ± standard deviation.