Heart tissue was pulverized in a glass douncer in NP40 lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 2 mM EDTA, 10 mM NaF, 10% glycerol, and 1% NP‐40) containing complete protease inhibitor cocktail (Roche), phosphatase inhibitor cocktails 2 and 3 (Sigma). Lysates sat on ice for 30 min with intermittent vortexing. Lysates were centrifuged (13k rpm, 10 min, 4°C). 50–100 μg of protein lysate was treated for 6–8 h with chondroitinase ABC (100 μU/mL; R&D Systems) before being resolved on 4%–12% bis‐tris gel (BioRad Criterion XT) by SDS/PAGE, transferred to nitrocellulose membrane (GE Life Sciences), and blocked in 5% nonfat milk (Blake, Parrish, et al., 2022 (link)). Samples were probed with mouse anti‐chondroitin‐4‐sulfate (1:1000; Millipore MAB2030), mouse anti‐chondroitin‐6‐sulfate (1:1000; Millipore MAB2035), rabbit anti‐NG2/CSPG4 (1:1000; Millipore AB5320), or rabbit anti‐CHST15 (1:1000; Proteintech 14298‐1‐AP). Blots were washed, incubated with goat anti‐mouse or anti‐rabbit HRP (1:10,000; Thermo 32430 & 32460, respectively), and bound antibody detected by chemiluminescence (Thermo). Protein expression was quantified with ImageJ densitometry and normalized to total protein as measured by Ponceau staining.
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