Western Blot Analysis of Hsp70 and Hsp90

Protein samples were thawed and mixed using a spin column (Bio-Rad), and then separated on a 5% SDS-PAGE gel for Western blot analysis. After gel electrophoresis, proteins were transferred to nitrocellulose (NC) membranes (Millipore Corporation, Billerica, MA, USA). The membranes containing protein were blocked with 5% fat-free milk in TBST at room temperature (18~24 °C) for 2 h, and then hybridized using anti-Hsp70-and-Hsp90 antibody (1:1000, Abcam, Hong Kong) or rabbit polyclonal antibody (1:2000, Abcam, Hong Kong) at 4 °C overnight. The membrane was then washed four times with TBST and labeled with HRP-conjugated secondary antibody (1:4000,) for 2 h at room temperature (18~24 °C). After washing five times with 1× TBST, Hsp70 and Hsp90 were detected on the membrane with an ECL detection kit (Beyotime, Shanghai, China). The protein expression intensity was determined by optical density analysis. The intensities of β-actin bands were used for the single standard [20 (link)].