Ab133495

Protein concentration was measured using a BCA Protein Assay Kit following the manufacturer’s protocol (Thermo Fisher Scientific). Proteins (25 μg/sample) were separated on a 4–12% SDS-PAGE gel and transferred onto nitrocellulose membrane blots using semi-dry blotting system (Bio-Rad) following the manufacturer’s protocol. To ensure the equal protein loading in each lane, the blots were stained Ponceau S (Sigma) and imaged on a ChemiDoc™ imaging system (Bio-Rad). Blots were then incubated with primary monoclonal/polyclonal antibodies including CFD (R&D systems, AF1824, 1:2500), GPX3 (R&D systems AF4199, 1:200), CST3 (Abcam ab133495, 1:13000), PON1 (Abcam, ab92466, 1:5000), MRC1 (Abcam ab195193, 1:1000) and COMP (Abcam, ab74524, 1 :200), followed by respective HRP-conjugated secondary antibodies. Blots were imaged using a Li-Cor Odyssey Blot imager (LI-COR Biosciences). Quantitation of signal intensity of the bands in Western blots was performed using Image lab software version 5.0 (Bio-Rad) and Image Studio Lite version 5.2 (LI-COR Biosciences).

Total proteins were harvested using a RIPA buffer (Merck, KGaA, Darmstadt, Germany). Prior to electrophoresis, the samples were diluted 1:1 in a Laemmli buffer (Merck, KGaA, Darmstadt, Germany) and heated at 95 °C for 5 min. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using TGX FastCast 10% acrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to the nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was processed and stained using the iBind Western system (Thermo Fisher Scientific, Waltham, MA, USA), and the antibodies specific for CstC (1:1000 dilution of #ab133495, Abcam, Cambridge, UK), β-tubulin (1:2000 dilution of #ab6046, Abcam, Cambridge, UK), and horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution of #1706515, Bio-Rad Laboratories, Hercules, CA, USA). The bands were visualized via chemiluminescence using an NZY Supreme ECL HRP substrate (NZYTech, Lisbon, Portugal) in an iBright CL1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). The quantification of band intensity was performed in Fiji software.