Sc1478

Total protein from each group was fractionated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and incubated with primary antibodies against α2A-adrenoreceptor (1:200, sc1478, Santa Cruz), β2-adrenoreceptor (1:500, ab137494, Abcam), aggrecan (1:500, ab36861, Abcam), MMP-3 (1:200, sc6839, Santa Cruz), MMP-13 (1:300, sc30073, Santa Cruz), RANKL (1:300, sc7628, Santa Cruz), β-actin (1:1000, 3700, Cell Signalling Technology, USA), Phospho-ERK1/2 (Thr202/Tyr204) (1:800, 4370, Cell Signalling Technology), total-ERK1/2 (1:1000, 4695, Cell Signalling Technology), phospho-p38 (Thr180/Tyr182) (1:800, 4511, Cell Signalling Technology), total-p38 (1:1000, 9212, Cell Signalling Technology), phospho-JNK (1:800, 4688, Cell Signalling Technology), total-JNK (1:1000, 9252, Cell Signalling Technology), phospho-Akt (Thr308) (1:800, 4056, Cell Signalling Technology) and total-Akt (1:1000, 4691, Cell Signalling Technology). Signals were revealed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:5000, Zhongshan Golden Bridge Biotechnology, China) and enhanced chemiluminescence detection22 (link)23 (link)24 (link)25 (link).

Tissue processing, section staining and counting of immune-positive cells were performed as reported previously21 (link)22 (link)23 (link)24 (link)25 (link). The primary antibodies were goat polyclonal α2A-adrenoreceptor (1:75; sc1478, Santa Cruz Biotechnology, Inc., USA), rabbit polyclonal β2-adrenoreceptor (1:100, ab137494; Abcam, Cambridge, United Kingdom), rabbit polyclonal aggrecan (1:200, ab36861, Abcam), goat polyclonal MMP-3 (1:50, sc6839, Santa Cruz), MMP-13 (1:50, sc30073, Santa Cruz), RANKL (1:50, sc7628, Santa Cruz). Six squares were applied at the quartering points of the central (each 0.15 mm × 0.15 mm) and posterior (each 0.2 mm × 0.2 mm) third of the condylar cartilage. Within the selected frames, the number of immune-positive cells and the percentage area of aggrecan-positive staining were determined. In the isotype control slides, isotype antibodies were substituted for the primary antibodies.