Western Blot Protein Analysis Protocol

Briefly, for western blot analysis, 50 μg of proteins were loaded onto a 12% poly-acrylamide gel Mini- PROTEAN^® TGXTM (BIO-RAD, Milan, Italy). Electro-transfer to nitrocellulose membrane was obtained through Trans- Blot^® TurboTM (BIO-RAD), using Trans-Blot^® SE Semi-Dry Transfer Cell (BIO-RAD) [20 (link), 21 (link)]. Membranes were blocked in Odyssey Blocking Buffer (Licor, Milan, Italy), according to the manufacturer’s protocol. After blocking, membranes were washed three times in PBS for 5 min and incubated with primary antibodies against HSP90 (heat shock protein 90) (1:500, ab203085, ABCAM), HSP60 (heat shock protein 60) (1:500, ab190828, ABCAM), CLPP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) (1:500, Cat #PA5-52722, Invitrogen) and HO-1 (heme oxygenase 1) (1:1000, ab52947, ABCAM), overnight at 4 ℃. The next day, membranes were washed three times in PBS (Phosphate buffered saline) for 5 min and incubated with anti-rabbit IRDye700CW secondary antibodies (1:5000, Licor) in PBS/0.5% Tween-20 for 1 h at room temperature. All the antibodies were diluted in Odyssey Blocking Buffer. The obtained blots were visualized by Odyssey Infrared Imaging Scanner (Licor, Milan, Italy). Densitometric analysis was used for protein levels quantification, normalizing data to protein levels of β-actin (1:2000, ab8229, ABCAM).