Las 3000 imaging system

MCF-7 cells were seeded in a 6-well-plate (1–2×10⁶ cells) and lysed in Cell Lysis Buffer (Cell Signaling Technology, Inc.). Supernatants were harvested, and total protein was assessed using a BCA assay (KeyGen Biotech Co., Ltd.). Total protein (40–80 µg) was subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blotted with primary antibodies against Bax (cat. no. sc-6236; 1:500), p53 (cat. no. sc-6243; 1:500), p21 (cat. no. sc-397; 1:500), β-catenin (cat. no. sc-7199; 1:500), cyclin D1 (cat. no. sc-717; 1:500) and GADPH (cat. no. sc-25778; 1:500; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. Incubation then followed with secondary antibody goat anti-rabbit IgG-HRP (cat. no. sc-2004; 1:5000; Santa Cruz Biotechnology, Inc.). The proteins were detected using ECL Plus Western Blotting Detection System (GE Healthcare Life Sciences, Chalfont, UK). A protein blank was scanned with a Fujifilm LAS-3000 Imaging System and analyzed using MultiGauge Software (Fujifilm, Brookvale, Australia).

The proteins were extracted using the M-PER Mammalian Protein Extraction Reagent (Thermo Scientific). For each sample, 10 μg of total proteins was resolved by SDS-PAGE (10%) and transferred to nitrocellulose membranes. The membranes were incubated with the following primary antibodies overnight at 4°C: anti-DNMT1 (#sc-10219; Santa Cruz Biotechnology), anti-DNMT3A (#ab71424; Abcam), anti-DNMT3B (#ab119282; Abcam), and anti-Albumin (#ab10241; Abcam). The DNMT and ALB antibodies were used at dilutions of 1/500 and 1/1,000, respectively. A 1/1,500 dilution of the anti-β-Tubulin antibody (#T4026; Sigma) was used as a loading control. The antigen-antibody complexes were visualized by chemiluminescence using the ECL Plus western blotting detection system (GE Healthcare) and scanned with the Fujifilm LAS-3000 imaging system (Fujifilm).

Total protein was extracted from the cells as described above. The proteins (50 µg) were separated using 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBST for 1 h at 37°C and incubated with primary antibodies against Bax (sc-6236; 1:10,000), PI3K (sc-293172; 1:2,000), phosphorylated (p)-Akt (sc-7985-R; 1:1,000), Akt (sc-135829; 1:1,000), p-mTOR (sc-293133; 1:1,000), mTOR (sc-1549; 1:1,000), and GADPH (sc-47724; 1:1,000; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. The membranes were subsequently incubated with goat anti-rabbit IgG-horseradish peroxidase secondary antibodies (sc-2004; 1:5,000; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h and developed using an enhanced chemiluminescent detection system (Beyotime Institute of Biotechnology). The protein bands were scanned using a Fujifilm LAS-3000 Imaging system (Fujifilm Corporation, Tokyo, Japan) and analyzed with Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Western blotting was performed essentially as previously described.^{25 (link)} 15μg of protein sample was mixed with sample buffer (1% SDS, 10% glycerol, 0.5% DTT, 0.1% bromphenol blue, final concentration) and subjected to SDS–polyacrylamide gel electrophoresis before being transferred to nitrocellulose (Hybond ECL, GE Healthcare, Rydalmere, NSW, Australia). Nitrocellulose membranes were stained with Ponceau S (0.5% Ponceau in 1% acetic acid) to assess the efficacy of the transfer. The membranes were then washed in TBST (Tris-buffered saline with Tween) (150 mM sodium chloride, 10 mM Tris, 0.075% Tween-20, pH 7.5) and blocked in 5% skimmed milk powder in TBST for 1 h at 25 °C. The membranes were washed in TBST and incubated with anti-Arc (1:2000 Synaptic Systems #156002), overnight at 4 °C. The membranes were washed in TBST and incubated with horse-radish peroxidase-linked anti-IgG secondary specific antibodies for 1 h at 25 °C. The membranes were visualized on Fujifilm Las-3000 imaging system (Fuji, Stamford, CT, USA) using Luminata Forte Western HRP substrate (Millipore, Billerica, MA, USA). The density of bands was measured using a MultiGauge V3.0 (Fuji). Arc protein levels were normalized to β-actin. All the results are expressed as a fold change relative to the addiction/relapse resilient group.