Western Blot Analysis of SOX9 and HSP90

Cells were lysed in EBC buffer and 2×SDS loading buffer to collect proteins. The protein samples were boiled at 100°C for 5 min and then electrophoresed in the Bio-Rad Mini PROTEAN Tetra System (Bio-Rad, USA). After electrophoresis, the proteins were transferred to the PVDF membrane under ice bath conditions, and then the membrane was washed twice with 1×TBST. The membrane was blocked with 5% free-fat milk for 2 h at room temperature. The primary antibodies to SOX9 (67439-1-Ig, Proteintech) and HSP90 (ab203126, Abcam) were diluted with 2% free-fat milk at ratios of 1:4,000 and 1: 10,000, respectively, and then incubated overnight at 4°C. Membrane was washed thrice for 15 min and incubated the secondary antibodies for 2 h at room temperature. After another three times washing, the bands were solarized and imaged using the Syngene G: BOX Imaging System (Cambridge, UK) (19 (link), 31 (link)).