Atg12

The protein was extracted from hippocampus tissues using RIPA lysis buffer (1:100; Beyotime, Shanghai, China). Equal amounts of protein were separated using the SDS–polyacrylamide gel (SDS–PAGE) gel preparation kit (Solarbio). Electrophoresis was carried out on SDS–PAGE using 20 μg of total protein in each lane. After electrophoresis, the protein was transferred to a PVDF membrane. Then, the membrane was incubated with corresponding primary antibodies (Sirt1, 1:2000; Bioworld; PI3K, 1:1000; Bioss; AKT, 1:2000; Cell Signaling; p‐AKT, 1:1000; Cell Signaling; Gabarapl1, 1:1000; Bioss; FoxO1, 1:1000; Bioss; ATG12, 1:1000; Abcam; acetyl‐FoxO1, 1:2000; Thermo Fisher; p‐FoxO1, 1:1000; Bioss; GAPDH, 1:2000; Zsbio; LaminB1, 1:1000; Bioss) at 4°C overnight and the secondary antibody (1:20,000; Abcam) for 1 h, respectively. Eventually, the targeted antigens were detected by standard chemical luminescence methods. Band intensities were measured with Image software. During the experiments, we first detected the protein expression levels of Sirt1, PI3K, AKT, p‐AKT, FoxO1, ATG12, acetyl‐FoxO1, p‐FoxO1, and Gabarapl1 and then detected the protein expression levels of p‐FoxO1 in the cytoplasm and FoxO1 in the nucleus.

Western blot analysis was performed as previously described^{17 (link),39 (link)}. Murine liver tissues and HL-7702 cells were lysed in a lysis buffer (KeyGEN, China) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMSF, KeyGEN, China) at 4 °C for 30 min followed by centrifugation to remove cell debris. Protein concentration was measured using BCA protein assay kit (Beyotime Institute of Biotechnology). The protein was boiled and subjected to western blot with antibodies against CFTR, DNMT1, DNMT3a, DNMT3b, EZH2, p62, BECN1, microtubule-associated protein 1 light chain 3 (LC3), Atg12 and β-actin (all from Abcam Inc., Cambridge, MA, USA) respectively. Optical densities of the bands were analysed with Bio-Rad image analysis (Bio-Rad, Hercules, CA, USA).

Western blot was performed in accordance to standard protocol. The primary antibodies and dilutions used in this study were described as following: HOXA9 (Abcam, ab140631, 1:2000) and RELA (SellckChem, A5075, 1:2000); BCL-XL (SellckChem, Houston, TX, USA, A5091, 1:2000), ULK1 (Cell Signaling Technology, Danvers, MA, USA, #8054, 1:2000), ATG3 (SellckChem, A5304, 1:2000), ATG12 (SelleckChem, A5565, 1:2000) and GAPDH (Santa Cruz Biotechnology, sc-25778,1:5000). Anti-mouse IgG-horseradish peroxidase (HRP) and anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA) were used as secondary antibodies. Luminata Forte Western HRP substrate (Merck Millipore, Burlington, MA, USA) was used to detect the bound antibodies. Densimetric quantification of the Western bands was performed using Quantity One software (Bio-Rad, Philadelphia, PA, USA).
Xenograft tumors were formalin-fixed, paraffin-embedded and sectioned for IHC staining. The following antibodies were used: HOXA9 (Abcam; 1:100), RELA (1:100), BCL-XL (1:100), ULK1 (1:100), ATG3 (1:100) or ATG12 (1:100). Stained sections were imaged using BX53 microscope (Olympus) to get representative images for statistical analysis.

Cells were lysed using ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Then, cells were centrifuged for 15 min at 4 °C at the speed of 12,000 r.p.m. and cell supernatants containing proteins were collected. Next, protein concentration was determined through Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, 30 μg of protein samples in each lane were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). After the blockage of nonspecific sites using 5% nonfat milk, the membranes were incubated overnight at 4 °C with primary antibodies against ATG12 (Abcam, Cambridge, UK), LC3B (Abcam), Bcl-2 (Abcam), Bax (Abcam), cleaved caspase 3 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Abcam). Next, the membranes were probed for 1 h at room temperature with secondary antibody labeled with horseradish peroxidase (HRP) (Abcam). Finally, protein signals were visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories) and quantified using Quantity One software Version 4.1.1 (Bio-Rad Laboratories).

Cells were rinsed with ice-cold lysis buffer (50 mM, Tris, 10 mM EDTA, 1 % v/v Triton-X100), supplemented with protease inhibitor cocktail set III (80 µM aprotinin, 5 mM bestatin, 1.5 mM leupeptin, 1 mM pepstatin; Calbiochem, San Diego, CA, USA), 2 mM phenylmethylsulfonyl fluoride and 1 mM Na₃VO₄, then sonicated and centrifuged at 13,000 g for 10 min at 4 ^oC. 20 µg protein extracts were subjected to SDS-PAGE and probed with antibodies directed against ATG5, ATG7, ATG12, beclin, p62, LC3, GRP78, ATF6, IRE-1α and PERK (all from Abcam, Cambridge, UK), followed by incubation with a peroxidase-conjugated secondary antibody (Bio-Rad Laboratories). Next, the membranes were washed with Tris-buffered saline-Tween 0.1 % v/v solution, and proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). To check for equal loading, the samples were probed with an anti-β-tubulin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Briefly, the total cellular protein was isolated with RIPA cell lysis buffer supplemented with protease inhibitors. Cytosolic protein was isolated using the Mitochondrial and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Rockford, IL). Protein content was determined by the Bradford assay. Equal amounts (30-50 μg) of proteins were separated by 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a PVDF Immobilon-P membrane (Millipore, MA). After blocking with 5% skim milk, the membrane was then incubated with indicated primary antibodies and secondary antibodies conjugated to horseradish peroxidase. Antibody-bound proteins were detected by ECL (enhanced chemiluminescence) Western Blotting Substrate (Pierce, Rockford, IL). The band intensity of the western blots and the normalization was analyzed using the ImageJ program (National Institutes of Health, Bethesda, MD). The primary antibodies used include rabbit polyclonal anti-human LC3B (1:500, Abcam), p62 (1:500, Abcam), ATG12 (1:800, Abcam), rabbit monoclonal anti-human caspase-3 (1:500, Abcam), caspase-9 (1:500, Abcam), rabbit monoclonal anti-human cytochrome C (1:500, Epitomics), EZH2 (1:500, Epitomics) and rabbit polyclonal anti-human Actin (1:4,000, Abcam). HRP-conjugated goat anti-rabbit IgG antibody (Abcam) was used as the secondary antibody.