Immobilon western kit

Anti‐PARP, Anti‐cleaved‐PARP and anti‐phospho‐ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐β‐catenin was purchased from BD Biosciences (San Jose, CA, USA). Anti‐β‐actin, HRP‐linked anti‐mouse IgG, and HRP‐linked anti‐rabbit IgG were purchased from Sigma‐Aldrich. Cells were treated with compounds for 24 h. In the co‐treatment test using both 2‐DG and an uncoupler, cells were pre‐treated with 10 mM 2‐DG for 1 h before being treated with the uncoupler. Following treatment, cells were immediately harvested and lysed with RIPA buffer (25 mM HEPES, 1.5% TX‐100, 1% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.1 M Na₃VO₄, and cOmplete Protease Inhibitor Cocktail Tablets (Roche, Germany); pH 7.8). The lysates were centrifuged at 13,000 g for 15 min to remove the insoluble fraction. Equal amounts of total protein were subsequently separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and probed with the indicated antibodies. The chemiluminescence signal was detected using an Immobilon Western kit (Merck Millipore, Billerica, MA, USA) and ChemiDoc XRS+System (Bio‐Rad, Hercules, CA, USA).

MCF10A MII cells were seeded in 6-well-plates (2.5 × 10⁵ cells/well), and starved the following day for 16 h in 0.2% FBS, and cells were then stimulated with 5 ng/ml of TGFβ3 for indicated time-periods. Cells were lysed in 2× SDS Laemmli sampler buffer (5% SDS, 25% glycerol, 150 mM Tris–HCl pH 6.8, 0.01% bromophenol blue, 100 mM dithiothreitol (DTT)). Samples were separated by SDS-PAGE, blotted onto nitrocellulose membrane (Amersham Protran, GE Healthcare Life Science), and the chemiluminescent signal was detected using the Immobilon Western kit (Merck Millipore).

Cells were scraped and lysed in Laemmli 1 × solution. After sonication, equal amounts of protein extracts were loaded and separated on 10% SDS–polyacrylamide gel electrophoresis gels under denaturing conditions (4% β-mercaptoethanol). Proteins were transferred onto a nitrocellulose membrane (GE Healthcare, Mississauga, ON, Canada), followed by blocking of nonspecific binding sites with a 10% blotto/0.1% Tween solution. Membranes were then incubated overnight at 4 °C with the following primary antibodies: anti-human integrin α1/CD49a (1:2000, AF5676, R&D Systems, Minneapolis, MN, USA), anti-c-Myc (1:5000, Y69, ab32072, Abcam, Toronto, ON, Canada), anti-β1 (1:1000, BD Biosciences, Mississauga, ON, Canada) and anti-β-actin (1:80 000, C4, Millipore, Etobicoke, ON, Canada) as endogenous loading control. After washing three times with phosphate-buffered saline/Tween 0.1%, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (anti-sheep, 12-342, Millipore; anti-mouse NA931V, anti-rat NA935V and anti-rabbit NA934V, Amersham, Mississauga, ON, Canada). Chemiluminescence was developed using the Immobilon Western Kit (Millipore, WBKLS0100). All experiments were performed in triplicate and repeated three times.