Abs20040

Total protein was extracted and separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (10–12% gels), and the separated protein bands were transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk diluted in Tris-buffered saline with 0.05% Tween-20 and incubated with primary antibodies (E-cadherin, 1 : 1000, abs130068; snail, 1 : 1000, abs151371; vimentin, 1 : 1000, abs131996; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 1 : 1000, abs132004; ING3, 1 : 1000, abs137556; Akt, 1 : 1000, abs131788; p-Akt, 1 : 1000, abs130889; mTOR, 1 : 1000, abs131824; p-mTOR, 1 : 1000, abs130933) overnight at 4°C. After rinsing, the membranes were probed with a goat antirabbit antibody (1 : 2000, abs20040, Absin Bioscience, Shanghai, China) for 2 h. Protein signals were visualised by chemiluminescence using an enhanced chemiluminescence kit (Millipore, USA) [17 (link)].

Cells were lysed in RIPA lysis buffer (PC101, Epizyme Biomedical Technology, Shanghai, China) with 1X protease Inhibitor cocktail (GRF101, Epizyme Biomedical Technology) and incubated on ice for 30 min. After centrifugation at 12,000 rpm for 30 min, the supernatant was mixed using 5× loading buffer. Subsequently, the supernatants were boiled at 95°C for 5 min. The samples were electrophoresed using 12% SDS-PAGE (PG113, Epizyme Biomedical Technology) and transfected into PVDF membranes (IPVH00010, Millipore, USA). After blocking with 5% milk powder (PS112L, Epizyme Biomedical Technology) for 1 h, the membranes were incubated with primary antibodies overnight at 4°C: MT-ND1(385032, ZENBIO, Chengdu, China), MT-ND4(PA5-97298, Invitrogen, Carlsbad, CA, USA), MT-CO IV (11967s, Cell Signaling Technology, Danvers, Massachusetts, USA), VDAC2 (11663-1-AP, Proteintech, Wuhan, China), cleaved-CASP3 (ab2302, abcam, London, UK) and β-actin (200068-8F10, ZENBIO). After washing thrice, the membranes were incubated with secondary antibodies, goat anti-mouse IgG-HRP (abs20039, Absin, Beijing, China), and goat anti-rabbit IgG-HRP (abs20040, Absin) for 1.5 h at room temperature. Finally, the membranes were exposed using a chemiluminescence apparatus (GelView6000Plus, Guangzhou, China).

Experiments were conducted using the following primary antibodies: Flag-tag antibody (Cell Signaling Technology, 8146 T, 1:1000 dilution), HA-tag antibody (Proteintech, 66006, 1:5000 dilution), caspase 3 antibody (Cell Signaling Technology, 9662 S, 1:1000 dilution), cleaved caspase 3 antibody (Cell Signaling Technology, 9661 S, 1:1000 dilution), cleaved PARP antibody (Cell Signaling Technology, 5625 S, 1:1000 dilution), β-actin antibody (Proteintech, 66009-1-Ig, 1:5000 dilution), Bax antibody (Cell Signaling Technology, 2772 S, 1:1000 dilution), BCL-2 antibody (Cell Signaling Technology, 15071, 1:1000 dilution), p53 antibody (Abcam, ab26, 1:1000 dilution) for p53-DBD, GST tag antibody (Cell Signaling Technology, 2625 S, 1:1000 dilution), and His-tag antibody (Abbkine, ABT2050, 1:5000 dilution). The secondary antibodies HRP-conjugated goat anti-mouse IgG (Abbkine, A21010, ATSDE1601, 1:2000 working dilution) and HRP-conjugated goat anti-rabbit IgG (Absin, abs20040, AS004, 1:2000 working dilution) were used. These antibodies were validated by western blotting according to the manufacturer’s website. Bands were visualized by enhanced chemiluminescence detection reagents (Vazyme, E411-04). Full blots have been included in the Source data file.