Anti hsp70 antibody

Exosomes were lysed with a RIPA buffer, resuspended in the loading buffer, boiled at 95 °C for 5 min, and then electrophoresed on SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membrane, which was blocked with 5% non-fat dry milk in TBST. Immunodetection was performed with anti-HSP70 antibody(1:1000, Proteintech, China), anti-Flotillin-1 antibody (1:1000, Proteintech, China), anti-CD63 antibody (1:1000, Proteintech, China) and anti-calnexin antibody (1:1000, Proteintech, China) at a dilution of 1:1000 followed by incubation at 4 °C overnight. The next day, protein was incubated with appropriate HRP-conjugated secondary antibody (1:5,000, Abcam, USA). Bands were revealed using ECL Plus and then imaged on the electrophoresis gel imaging analysis system (D-Digital, USA) to analyze.

Primary antibodies utilized in our study: anti-HSP70 antibody (1 : 1000, Proteintech, Chicago, USA), anti-CD63 antibody (1 : 1000, ABclonal, Boston, USA), anti-CD81 antibody (1 : 1000, Abcam, MA, USA); secondary antibodies include HRP-labeled goat antirabbit IgG (1 : 1000, Beyotime, Shanghai, China). A chemiluminescence kit was purchased from Millipore (Darmstadt, Germany).