Mouse t5168

Total protein extracts were prepared as follows: Cells were trypsined and washed with cold DPBS 1X (ThermoFisher) and then were centrifuged at 200 × g for 10 min at 4 °C. Cell pellets were suspended in 50 µL of ice-cold lysis buffer [100 mM Tris-HCl, pH 7.4, 1% Triton, 0.25% sodium deoxycholate, 0.1% SDS, 300 mM NaCl, 1 mM EDTA, 1X PhosSTOP (Sigma), 1X Protease Inhibitor Cocktail (Sigma)]. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (ThermoFisher). Migration of an equal mass of total protein and of 5 µL PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa (ThermoFisher) was carried out on an 8% acrylamide denaturing gel. Proteins were transferred onto a nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad, city, state abbreviation if USA or Canada, country). The primary antibodies used against NRF1, p-AMPK, p-ACC and tubulin were mouse ab55744 (Abcam, Cambridge, United Kingdom), rabbit #07-681 (Merck), rabbit #11818 (Cell Signaling, Danvers, MA, USA), and mouse T5168 (Sigma), respectively. Incubation of the membrane with the primary antibodies was performed according to the manufacturer’s protocol. The secondary antibodies used were goat anti-rabbit #65-6120 and goat anti-mouse #62-6520 (Invitrogen). Detection of proteins was performed by chemiluminescence using Clarity Western ECL Blotting Substrate (Bio-Rad).