Western blotting

Proteins from whole cell lysates were extracted using a radio-immunoprecipitation assay. Osteoblasts stimulated with rhHMGB1 were lysed in lysis buffer. Supernatants were prepared by centrifugation, electrophoresed on a 10% SDS/PAGE and blotted on to a polyvinylidene difluoride membrane. Immunoblotting was performed using antibodies specific to TLR2, TLR4 and GAPDH, followed by an horseradish peroxidase (HRP)-conjugated secondary antibody and developed using an ECL detection kit (Abcam). The relative amounts of protein bands on the blots were determined using IPP 6.0 software (Media Cybernetics Inc).