2.5. Western Blotting

Whole cell lysates were prepared using ice-cold RIPA buffer (50 mMTris-HCl pH 8.0, 150 mMNaCl, 0.1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). The protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Equal amounts (20 μg) of protein were separated on a 12% Bis–TrisNuPage precast gel (Thermo Fisher Scientific) and transferred to a Hybond-C membrane (GE Healthcare Life Sciences, Marlborough, MA, USA) using the iBlot dry blotting system (Thermo Fisher Scientific). The membranes were immunoblotted with appropriate primary antibodies at 4 °C and subsequently with HRP-conjugated secondary antibody (ab6721, Abcam, Cambridge, UK) for 1 h atRT. The following primary antibodies were used: anti-APOD (RD181118100, BioVendor, Brno, Czech Republic), anti-C1QL1 (ab68528, Abcam), and anti-CTSL (GTX26314, GeneTex, Irvine, CA, USA). Specific bands were visualized using WESTAR ETA C 2.0 (XLS070,0250) (Cyanagen, Bologna, Italy) and the G-Box chemiluminescence imaging station (Syngene, Cambridge, UK).