Polyvinylidene fluoride (pvdf)

Total protein was extracted from the controls and metformin-treated cells cultured in two different glucose conditions by RIPA (radioimmuno-precipitation assay) buffer (50 mM Tris, 0.15 M NaCl, 1 mM EGTA, 1% NP40, 0.25% SDS, pH 7.4). The cell lysate was quantified for protein content using Bradford assay. Equal amounts of the cell lysate (50 μg) were separated on 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (PVDF; Roche Diagnostics). The membrane was blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for one hour at room temperature. The membrane was then incubated with the appropriate antibody in TBS-T with 3% BSA for or overnight at 4°C. After washing in TBS-T, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. The proteins were detected using the ECL reagent. The used antibodies were diluted as follow: GAPDH antibody at 1:10,000 dilution, vimentin, β-catenin, E-cadherin antibodies at 1:500 dilutions, HRP-linked anti-mouse and anti-rabbit IgG at 1:2000 dilutions.

Protein from cells were mixed with RIPA buffer (Beyotime, China) containing 1mM PMSF. Protein extracts were obtained using a total and nuclear protein extraction kit (Thermo, USA). Equal amounts of protein were loaded onto 10% SDS-PAGE and proteins were transferred to PVDF (Roche). The membranes were then blocked with 0.05% Tween and 5% bovine serum albumin (BSA) (BBI life sciences Corporation, Canada) in Tris-buffered saline for 2 h at room temperature and incubated overnight at 4°C with primary antibodies against Nrf2, Notch1, HO-1, p21, Hes-1, c-myc, MMP-2, MMP-9, E-cadherin, N-cadherin (Abcam, USA). The preparative membranes were incubated with appropriate secondary antibodies conjugated to HRP. The Immunological complexes were visualized with electrochemiluminescence (Millipore, Germany). Band intensities were analyzed using Image J software.

H9c2 cells were prepared and lysed with cell lysis buffer solution and centrifuged at 12,000 g for 15 min at 4°C. Protein was extracted from whole-cell lysates, and their concentration was measured with BCA protein analyzing kit. Firstly, 30 μg protein samples were separated by 10% SDS-PAGE gel, subsequently; secondly, protein samples were shifted to polyvinylidene difluoride western blotting membranes (PVDF, Roche). Thirdly, PVDF membranes were probed with primary antibodies against NLK (1 : 1000; Santa Cruz), or β-actin (1 : 2,000) at 4°C overnight, afterward incubation with horseradish peroxidase-conjugated secondary antibodies (Goat anti-rabbit IgG, 1 : 1000; anti-Mouse IgG, 1 : 6000) at 25°C for 60 min.

Total proteins were harvested from cells with ice-cold RIPA buffer (Beyotime). Lysates were centrifuged at 12,000×g at 4 °C for 15 min, and then the protein samples were heated at 95 °C for 5 min. SDS-PAGE gels were utilized to separate the proteins, and then the proteins were transferred onto a polyvinylidene fluoride membrane (PVDF; Roche, Basel, Switzerland). The membranes were then blocked with 5% fat-free milk for 1 h at room temperature and incubated with the primary antibodies of interest overnight at 4 °C. The secondary antibodies were anti-rabbit IgG and anti-mouse IgG conjugated to HRP. Finally, the protein bands were developed by chemiluminescence detection (ECL; Thermo Fisher Scientific) and then analysed by band densitometry with ImageJ software. The relative levels of these proteins were normalized to β-actin. All antibodies are listed in Additional file 7: Table S1.

Western blot analysis was carried out for Ube3a^−/− and Ube3a^+/+ MEFs. The cells were washed with PBS, collected, and homogenized by sonication with an ice-cold lysis buffer containing the following (in mM): 10 HEPES pH 7.5, 150 NaCl, 50 NaF, 1 EDTA, 1 EGTA, 10 Na4P2O7 (EMD Millipore, Billerica, MA, USA), PMSF (Roche, Mannheim, Germany), and protease inhibitor cocktail (Roche, Mannheim, Germany). The samples (15 µg) were loaded on an SDS PAGE 4–20% gradient, followed by a transfer to polyvinylidene difluoride (PVDF; Roche, Mannheim, Germany) membranes and probed with primary antibodies using standard techniques. The primary antibodies and the dilutions for the Western blots were as follows: BAX 1:2000 (α-rabbit; Abcam #ab182733, Cambridge, UK), BCL-2 1:2000 (α-rabbit, Abcam #ab182858, Cambridge, UK), and UBE3A 1:1000 (α-mouse; Sigma-Aldrich #E8655, St. Louis, MO, USA). β-ACTIN 1:40,000 (α-mouse; MP Biomedicals #69100, Irvine, CA, USA) was used as a loading control. Secondary antibodies were used, respectively. The blots were developed and imaged using the Image Quant LAS 4000 system. All signals were normalized by the total protein and quantified using Image Studio Lite Ver 5.2 software.

The concentration of protein was quantified using BCA protein assay kit (Thermo Fisher Scientific). Proteins were extracted using a lysis buffer (RIPA, Beyotime, Shanghai, China) containing protease inhibitors. A total of 20 μg protein was separated on SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF, Roche, Mannheim, Germany) membrane that was blocked for 2 h at room temperature with 5% bovine serum albumin (BSA). Blots were probed with the following antibodies: CD 81 (1 : 1000, System Biosciences), HSP 70 (1 : 1000, System Biosciences), cyclin D1 (1 : 1000, Abcam), AKT, p-AKT (1 : 1000, Cell Signaling Technology (CST), Danvers, MA), and β-actin (1 : 1000, Abcam) at 4°C overnight. The next morning, membranes were incubated with anti-rabbit IgG antibody (1 : 5000, CST). The immunoreactive bands were detected with chemiluminescence detection kit (MilliporeSigma, Burlington, MA) and visualized using the FluorChem E imaging instrument (ProteinSimple, San Jose, CA).