Western Blot Analysis of Chondrocyte Proteins

Expression of proteins of interest in chondrocytes and articular cartilage tissues were explored through western blot analysis. Briefly, cultured chondrocytes and previously ground tissues powder (100 mg) were lysed for 1 h using ice cold 1 × RIPA lysis buffer (Beyotime) supplemented with phenylmethanesulfonyl fluoride (PMSF, 1 mM, Beyotime). The lysates were centrifugated for 10 min at 4 °C and 12,000 rpm. The supernatant was transferred to a new tube for protein quantification. A standard curve was prepared with gradient concentration of BSA according to the instructions of a BCA quantification kit (Beyotime, China). For western blotting assay, the protein samples were mixed with 5 × loading buffer and heated for 10 min at 98 °C. Thereafter, 30 ug protein samples were separated by 10%–12.5% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Bio-Rad, USA). The membrane was rinsed with TBS-T, following with 5% nonfat milk for 1.5 h. Then, the membranes were incubated with primary antibodies specific to HBEGF (1:200, Abcam, Cat# ab92620), MMP3 (1:1000, Abcam, Cat# ab137659), MMP13 (1:1000, Abcam, Cat# ab84594), ADAMTS4 (1:1000, Abcam, Cat# ab84792), COL2A1 (1:1000, Abcam, Cat# ab34712), Aggrecan (1:100, ABclonal, Cat# A8536), or β-actin (1:2000) (Cell Signaling Technology, Cat# 4970) at 4 °C overnight. The blots were then rinsed and probed with secondary HRP-conjugated anti-rabbit or anti-mouse antibodies (Beyotime Institute of Biotechnology, Nantong, China), and proteins were detected using an ECL kit (Santa Cruz Biotechnology, TX, USA).