Radioimmunoprecipitation assay lysis buffer

The total proteins were extracted from cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), and subsequently quantified using the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA, USA). In total, 20 µg protein/well was loaded and separated by SDS-PAGE (10% gel) and then transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). Subsequent to blocking by 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA), membranes were incubated with the following primary antibodies at 4°C overnight: Rabbit monoclonal Lin28B antibody diluted at 1:1,000 (Abcam) and polyclonal rabbit anti-GAPDH antibody diluted at 1:5,000 (ab115698; Abcam). The polyvinylidene fluoride membranes were then incubated with peroxidase-conjugated goat anti-rabbit secondary antibody (1:4,000; ab6721; Abcam) for 1 h after washing with PBST (Phosphate Buffered Saline containing 1% Tween-20). Finally, proteins were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.).

Cells or homogenized tissues were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The concentration of total protein was quantified using a BCA Protein Assay Reagent kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Equal amounts of total protein were separated using a 10% SDS-PAGE gel and then transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA).
Subsequent to blocking at room temperature with 5% fat-free dried milk diluted in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h, the membranes were incubated with the primary antibodies overnight at 4 °C. The primary antibodies used in this study were as follows: rabbit anti-human FOXP1 antibody (1:1,000, cat. no. ab196978) and rabbit anti-human GAPDH antibody (1:1,000, cat. no. ab181603, both from Abcam, Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. ab205718, Abcam, Cambridge, UK) was used at a dilution of 1:5,000 for 2 h at room temperature and the protein signals were detected using a Pierce ECL Western Blotting Substrate (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The total protein was extracted in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China), and the concentration of protein was quantified using a bicinchoninic acid protein assay kit (Beyotime). Equal amounts (20 µg/lane) of protein were fractionated using SDS-PAGE and transferred to polyvinylidene difluoride membranes. These membranes were then incubated with anti-Bax, anti-Caspase-3, anti-Cleaved Caspase-3, anti-Bcl-2, anti-cyclin B1, anti-cyclin D1, anti-P21, anti-CDC21, anti-PI3K, anti-p-mTOR, anti-mTOR, anti-p-EKR1/2, anti-EKR1/2, anti-pAKT, anti-AKT, and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA). Next the membranes were incubated with peroxidase-conjugated secondary antibodies. The Western blot bands were visualized using electrochemi-luminescence kits in accordance with the manufacturer’s instructions (Abcam, Cambridge, UK). GAPDH was used for normalization of protein loading.

Heart tissues were homogenized in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The homogenates were centrifuged at 1,600 × g for 10 min at 4°C. A bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) was used for protein quantification. A total of 20 µg protein was electrophoresed on 10–15% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were subsequently blocked with 5% (w/v) non-fat milk in TBS containing 0.1% (v/v) Tween-20 and incubated overnight at 4°C with anti-PPARα (cat no. WL00978; 1:1,000; Wanleibio, Co., Ltd., Shanghai, China), anti-Desmin (cat no. ab32362; 1:8,000; Abcam, Cambridge, UK), anti-GRP78 (cat no. WL00621; 1:1,000; Wanleibio, Co., Ltd.), and anti-GADPH (cat no. ab181602; 1:8,000; Abcam). Following washing, bound antibodies were detected following incubation for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit IgG (cat no. ZB-2301; 1:10,000; OriGene Technologies, Inc., Beijing, China). Blots were developed using Western Lightning BeyoECL Plus reagent (Beyotime Institute of Biotechnology) and were quantified using ImageJ software (version 2.1.4.7; National Institutes of Health, Bethesda, MD, USA).