Bca protein assay reagent

The spore suspension was cultured in PDB medium at 25°C. After 24 h, a certain amount of canthin-6-one was added to the suspension, and the final concentration of canthin-6-one was 4.2 μg mL^-1 (EC₅₀). An equal amount of DMSO was added as a blank control. The mixed solution was then incubated at 25°C for 24 h. The mycelia were collected and washed three times with 1× PBS for protein extraction. Three biological replicates were used for each sample. Protein extraction and digestion were performed according to a previously described method [22 (link)]. Proteins were extracted using SDT buffer (4% sodium dodecyl sulfate, 0.1 M dithiothreitol, 100 mM Tris/HCl, pH 7.6), and then quantified using the BCA protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The filter-aided proteome preparation (FASP) method [23 (link)] was used to trypsinize (Promega, Madison, WI, USA), the appropriate amount of protein from each sample, and then the C18 cartridge (Empore™ SPE, bed I.D. 7 mm, volume 3 mL; Sigma, Kawasaki, Japan) was used to desalinize the hydrolyzed peptide. Dissolution buffer (40 μL, 0.1% trifluoroacetic acid) was added to the lyophilized peptides for reconstitution, and the peptide content was estimated using UV spectroscopy at 280 nm.

Whole-cell extracts were isolated using a RIPA lysis buffer supplemented with protease (Roche) and phosphatase inhibitors (Roche). The nuclei were broken with a 20 G needle. The concentration of the isolated proteins was determined using the BCA Protein Assay Reagent (BioRad). Fifteen to twenty micrograms of protein were separated on the BioRad gradient gels at 4% to 20% and transferred to nitrocellulose membranes (BioRad). The membranes were then incubated with the appropriate primary and secondary antibodies according to the manufacturer’s recommendations. The list of used antibodies are shown in Supplementary Data 2.

Valve interstitial cells (VICs) were isolated from AoV leaflets explants from 2-month old WT and Angptl2-KD mice, grown to confluence, sub-cultured and lysed in Laemmli sample buffer (Bio-Rad) for 10 min at 95 °C. HEK293 cells transfected with murine ANGPTL2 were lysed and the lysate clarified by centrifugation (15,000 rpm for 15 min at 4 °C). Protein concentration in the supernatant was determined using the BCA Protein Assay Reagent (Bio-rad). Then, 5–10 μg of proteins were loaded and separated on 10% acrylamide SDS-PAGE gels, transferred onto nitrocellulose membranes, and probed with antibodies against murine Angptl2 (1:1000, AF1444, R&D); VE-cadherin (1:500, Santacruz, CSC-28644), Vimentin (1:500, VP-V683, Vector Laboratories), α-SMA (1:500, A5228, Sigma-Aldrich). Membranes were then probed with secondary antibodies at a dilution factor of 1:5000. Chemiluminescence was used to detect protein expression (Western Lightning Plus ECL, Perkin Elmer).