Western blot analysis of COX-2 and iNOS

RAW264.7 cells were cultured in a 6-well plate at 1 × 10⁶/well for 4 h. The cells were pretreated with ChondroT, GHJTY, or celecoxib (20 μM) (Sigma Co., MO, USA) for 2 h and then LPS (500 ng/mL) (Sigma Co., MO, USA) was added to the cells for 24 h. Equal amounts of cell lysates (25 μg) were subjected to 10 % SDS-PAGE and electro-transferred onto polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA, USA). Western bot analysis was conducted using the above mentioned methods with polyclonal antibodies specific to COX-2 (Cell signaling Tech., Danvers, MA, USA), iNOS (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), or GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).