Radioimmunoprecipitation assay lysis and extraction buffer

Total protein was extracted using radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher, MA, US, 89900), supplemented with a protease inhibitor cocktail (Sigma-Aldrich, P8340) and a phosphatase inhibitor cocktail (Sigma-Aldrich, P0044). The nuclear extraction kit (Sigma-Aldrich, NXTRACT) was used for extracting the nuclear fraction per the manufacturer's instructions. The extracted proteins were quantified and fractionated, and transferred onto a membrane for antigen–antibody reaction. The protein was visualized using the enhanced chemiluminescence (ECL) solution (Thermo Fisher, 32109). The following antibodies were used: anti-LC3B (1:300, Novus Biologicals, CO, US, NB100-2220), anti-Beclin 1 (1:300, Novus Biologicals, NB110-87318), anti-GAPDH (1:1000, Santa Cruz Biotechnology, TX, US, sc-32233), anti-lamin A (1:1000, Santa Cruz Biotechnology, sc-518013), and anti-β-catenin (1:500, Cell Signaling, MA, US, 8480T). Mouse IgG kappa binding protein conjugated to horse radish peroxidase (HRP) (1:5000, Santa Cruz Biotechnology, sc-516102) and mouse anti-rabbit IgG-HRP (1:2000, Santa Cruz Biotechnology, sc-2357) were used as secondary antibodies.

Total protein from the gastrocnemius muscle was isolated using Radioimmunoprecipitation Assay Lysis and Extraction Buffer (ThermoFisher; cat. number: 89900) according to the manufacturers protocol. Homogenates were separated on 4% to 12% Bis‐Tris gels (ThermoFisher; cat. number: NP0326BOX), and transferred to nitrocellulose membranes (BioRad; cat number: 1620094).⁶, ⁷, ⁹ ADAM12 protein bands were detected by anti‐ADAM12 antibody (1:1000 dilution; cat. number: MBS9128704; MyBio Source), using a secondary antibody conjugated to an infrared dye (1:10 000 dilution IRDye 800CW; LI‐COR Biosciences; cat. number: 926–32 211). Signals were recorded using iBright imager (ThermoFisher; Model 1500). Bands from Western blots were quantified using the Image Studio Lite software (LI‐COR). Intensity of protein band signals in different lanes were normalized to the total protein content in each lane on Ponceau S–stained membranes. During analysis, we have found that expression of most housekeeping genes are altered in ischemia (unpublished personal observation), and moreover, studies have shown total protein staining to be more accurate than use of a house keeping protein as a loading control.¹², ¹³

Treated cells were harvested and lysed using ice-cold radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher Scientific, Inc.) containing a protein inhibitor cocktail (cat. no. P2714; Sigma-Aldrich; Merck KGaA). The protein concentration was determined using a Pierce Rapid Gold Bicinchoninic Acid Protein assay kit (Thermo Fisher Scientific, Inc.) and a NanoDrop 2000 instrument (Thermo Fisher Scientific, Inc.). The proteins (30 µg) were separated by SDS-PAGE (12.5% gel) and transferred to a polyvinylidene difluoride membrane. Subsequently, the membrane was blocked with 5% fat-free milk in Tris-buffered saline and Tween-20, and sequentially incubated overnight at 4°C with 1:500 diluted primary antibodies and 2 h at room temperature with 1:2,000 diluted horseradish peroxidase-conjugated secondary antibodies (ab6721; Abcam, Cambridge, UK). The signals were detected with the SuperSignal enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.) and recorded with the Gel Documentation 2000 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Rabbit polyclonal primary antibodies against human methyl CpG-binding protein 2 (MECP2; ab2828), DNMT1 (ab19905), DNMT3a (ab2850), DNMT3b (ab16049) and β-actin (ab8227) were purchased from Abcam.

Xenograft tumor tissues were lysed with radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher Scientific, Inc.). Proteins (20 µg/lane) were separated by 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore) by electroblotting. Membranes were blocked with 5% BSA in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% Tween-20) for 1–1.5 h, and then incubated with the following primary antibodies (Abcam) overnight at 4°C: THBS2 (1:500 dilution; cat. no. ab84469), MMP2 (1:5,000 dilution; cat. no. ab37150), MMP9 (1:5,000 dilution; cat. no. ab38898), ECM components (1:1,000 dilution; cat. no. ab130585) and β-actin (1:5,000 dilution; cat. no. ab228001). Subsequent to washing, the blots were incubated with goat anti-rabbit immunoglobulin G (H+L)-horseradish peroxidase secondary antibodies (1:5,000 dilution; cat. no. BS13278; Bioworld Technology, Inc.) and visualized using super enhanced chemiluminescence detection reagent (Amersham; GE Healthcare). Band intensity was scanned using Bio-Rad Universal Hood II (Bio-Rad Laboratories, Inc.) and analyzed using the Image Lab™ software (version 5.1; Bio-Rad Laboratories, Inc.) and normalized to the expression of β-actin.