P sp1

The total protein of cultured cells was extracted and measured as described previously. To detect the phosphorylated protein, the phosphatase inhibitor was used with a working concentration of 1 mM in the total protein extraction. The proteins were transferred to Hybond-P polyvinylidene difluoride membranes in trans-buffer containing 25 mM Tris and 185 mM glycine (pH 8.3) together with 20 % methanol. After transfer, the membranes were blocked for 1 h in blocking buffer (TBS containing 0.1 % Tween-20, TBST, supplemented with 5 % non-fat dry milk) and the membranes were incubated overnight at 4 °C with antibodies (MUC1,MUC1-C and p-Sp1 were obtained from Abcam, UK; p-ERK, ERK, p-akt and akt were obtained from CST, USA; GAPDH and β-actin were obtained from Santa Cruz, USA) in blocking buffer. After being washed three times with TBST, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase (HRP) anti-mouse/rabbit IgG (Santa Cruz, USA) for 1 h at room temperature. The bands were visualized by an enhanced chemiluminescence (ECL) detection system (LAS-4000 MINI System, GE, California, USA).

Standard protocol for Western blotting was used^{19 (link)}. Protein was extracted from HL1 cardiomyocytes and heart tissue by using radio-immuno-precipitation assay lysis buffer (RIPA, cat # BP-115D, Boston BioProducts, USA), and quantified by using BCA protein assay kit (cat # 23227, Pierce, USA). Equal amounts (30 µg) of proteins were used for SDS-PAGE. The primary antibodies used were: β-MHC (cat # ab172967, Abcam, USA), ANP (cat # GTX 109255, Gentex, USA), CBS (cat # H00000875-M01, Abnova, USA), CTH (cat # H00001491-M02, Abnova, USA), β-actin (cat # sc-47778, Santa Cruz Biotechnology, USA), β-tubulin (cat # MA5-16308, Thermo Scientific Inc., USA), p-SP1 (cat # ab59257, Abcam, USA), and SP1 (cat # 07-645, Millipore, USA). The secondary antibodies used were: anti-mouse IgG-HRP, and anti-rabbit IgG-HRP (cat # sc-2005, and cat # sc-2054, respectively, Santa Cruz Biotechnology, USA). Restore™ PLUS Western Blot stripping buffer (cat # 46430, Thermo Scientific Inc., USA) was used for restriping and reprobing of blots. Western blot membrane was developed by a Clarity™ Western ECL Substrate (cat # 1705061, Bio-Rad Laboratories, USA), imaged by a Chemidoc (Bio-Rad Laboratories, USA), and band intensity was analyzed by a Chemidoc software Image Lab 4.1 (Bio-Rad Laboratories, USA).

Protein extracts of mouse liver tissue and L02 cells were prepared by a Whole Cell Lysis Assay Kit (Keygen Biotech, China), and the concentrations were determined by a BCA protein quantitative assay kit (Dingguo Changsheng Biotech, China). The same amounts of protein were separated by electrophoresis using 8%–12% SDS-PAGE gels and transferred to suitably sized nitrocellulose membranes (Pall Corp., USA). After blocking with 5% BSA or 5% skim milk in Tris-buffered saline (TBS), the membranes were incubated with primary antibodies at 4°C overnight, including PTP1B (Abcam, UK), SP1 (Abcam, UK), P-SP1 (Abcam, UK), SOD (Abcam, UK), Nrf2 (Abcam, UK), Keap1 (Abcam, UK), PP2A (Santa, USA), P-PP2A (Santa, USA), SREBP-1 (Santa, USA), and GAPDH (CST, USA). The next day, the membranes were washed three times with TBST and incubated with an anti-rabbit/mouse IgG secondary antibody (CST, USA). A LI-COR Odyssey system (LI-COR Biosciences, USA) was used for detection of the protein bands, which were quantified using Image Studio software (NIH, Bethesda, MD).