Anti hsp90 αβ

Western blot (WB) analysis was performed according to standard protocols. Briefly, 50 µg of total protein isolated from EVs were separated by SDS-PAGE gel electrophoresis. Then, proteins were transferred to a PVDF membrane and primary antibodies Anti-HSP90-αβ (1:500; SC13119; Santa Cruz Biotechnology, Dallas, TX, USA), Anti-Alix (1:500; SC53540; Santa Cruz Biotechnology), and Anti-Flot-1 (1:500; BD 610821; BD Biosciences, Franklin Lakes, NJ, USA) were incubated overnight at 4 °C. Protein expression was visualized after membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies (1:5000; SC-516102; Santa Cruz Biotechnology), and then protein spots were revealed by using 1-Step Ultra TMB-Blotting reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Cells were lysed for 30 min on ice in 1% Triton-X100 containing buffer (1,5 mM MgCl₂, 150 mM NaCl, 1 mM EDTA and 5 mM HEPES pH 7,4) supplemented with 1 × protease inhibitor cocktail from Roche. Cell lysates were cleared by centrifugation for 20 min at 14,000 × g and 4 °C. Equal amounts of proteins (50 µg) from each sample assessed by bicinchoninic acid (BCA) were separated by SDS-PAGE in 8% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in 10% nonfat dry milk and then probed with primary rabbit polyclonal TRPA1 (PA1-46159, Thermo Fisher Scientific) antibody, which was diluted (1:1000) in 5% milk dissolved in phosphate buffered saline (PBS), and incubated overnight at 4 °C. The protein was then detected with goat anti-rabbit (sc-2004) coupled with horseradish peroxidase enzyme (HRP) and diluted (1:10,000) in the same buffer as the primary antibody for 1 h at RT. Subsequently, membranes were soaked in mouse anti-tubulin (1:1000, T6199_Sigma) or anti-HSP 90α/β (sc-13119, Santa Cruz Biotechnology) antibodies as the loading control. The results were viewed by chemiluminescent reaction using the Pierce ECL substrate (32106, Thermo Fisher Scientific). Band intensity was quantifed using NIH ImageJ software and the results were then normalised to the non-tumoral HPDE cells.