Skimmed milk

Protein extraction and blotting were performed as previously described (35 (link)). Proteins were extracted using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Following the determination of protein concentration by a Protein Determination kit (cat. no. 704002; Cayman Chemical Company, Inc., Ann Arbor, MI, USA), equal amounts of protein samples (20 µg loaded per lane) were size-fractioned using SDS-PAGE (8% gels), electrotransferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% skimmed milk (BD Biosciences), and then were hybridized with primary antibodies overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies (1:10,000; cat. no. #W4011; Promega Corporation, Madison, WI, USA) for 2 h at room temperature. Finally, the enhanced chemiluminescent reagents (MILLIPORE, WBKLS0500) was used to treat the membranes. Densitometric quantification of bands was performed using the ImageJ software (version 1.50; National Institutes of Health, Bethesda, MD, USA). The following primary antibodies were used: Anti-GAPDH (cat. no. ab9485; 1:2,500), anti-IL-4 (cat. no. ab9811), anti-TNF-α (cat. no. ab6671) and anti-TGF-β1 (cat. no. ab92486; all 1:1,000; all Abcam).

Cells were harvested and lysed in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na^{3 (link)}VO^{4 (link)}, 2 mM EDTA, 1mM PMSF and protease inhibitors (cat. no. HY-K0010; MedChemExpress)) as previously described.^{30 (link)} Briefly, samples were loaded and separated by SDSPAGE, transferred to PVDF membranes (EMD Millipore) and blocked with 5% skimmed milk (BD Bioscience) at room temperature for 1 h. Subsequently, membranes were probed overnight at 4°C with the following primary antibodies: mouse anti-spastin (1:100, cat. no. sc-374068; Santa Cruz Biotechnology, Inc.), mouse anti-GAPDH (1:5,000, cat. no. ab8245; Abcam), rabbit anti-GFP (1:1,000, cat. no. ab290; Abcam), rabbit anti-mCherry (1:1,000, cat. no. ab125096; Abcam), mouse anti-Flag (1:1,000, cat. no. F1802; Sigma-Aldrich; Merck KGaA). HRP-conjugated secondary antibodies (cat. nos. AS038 and AS003; ABclonal Biotech Co, Ltd.) were used for protein signal detection with an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).

H9c2 cardiomyocytes were lysed in cold radioimmunoprecipitation assay lysis buffer (Fermentas; Thermo Fisher Scientific, Inc.). The concentration of the extracted protein was determined using a BCA Protein Quantification Kit (AS1086; Aspen Biotechnology). A total of 50 µg protein was loaded per lane. Proteins were then separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (MilliporeSigma), followed by the addition of Tris-buffered saline and 0.1% Tween 20 (ASPEN) containing 5% skimmed milk (BD Biosciences) for 1 h at room temperature to block the membranes. Subsequently, the membranes were incubated with primary antibodies overnight at 4˚C (Table SI). Then, the membranes were then washed thoroughly three times with phosphate-buffered saline containing 0.1% Tween-20 (PBST) and then incubated with goat anti-rabbit secondary antibodies (1:10,000; Abcam) for 1 h at room temperature. After washing three times with PBST, chemiluminescence detection was performed with ECL chemiluminescence detection kit (ASPEN). Relative protein expression was analyzed with Image-Pro Plus software 6.0 (Media Cybernetics, Inc.) and normalized to GAPDH.