Westar supernova substrate

Antibodies (anti-phospho-mTOR, mTOR, phospho-AKT, AKT, phospho-AMPK, AMPK) were acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-OPA1, Protein Phosphatase Inhibitor Cocktail IV, and Protein Protease Inhibitor Cocktail (EDTA-free) were purchased from Abcam (Cambridge, MA, USA). T-PER™ Tissue Protein Extraction Reagent, TRIzol™ Reagent, and M-MLV reverse transcriptase were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-GAPDH antibody was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Bradford's solution and PVDF membranes were from Bio-Rad Laboratories (Hercules, CA, USA). Secondary antibodies were obtained from Calbiochem (Burlington, ON, Canada). Organic and inorganic compounds, acids, and solvents were acquired from Merck (Darmstadt, Germany). Westar Supernova substrate was obtained from Cyanagen (Bologna, Italy). SensiFAST SYBR Hi-ROX was purchased from Bioline Meridian Biosciences (London, UK).

Proteins were resolved by 12.5% SDS-PAGE and transferred to 0.45 μm nitrocellulose membrane. Antibodies used were anti-FLAG M2-Peroxidase (A8592, Sigma), anti-FLAG M2 (F3165, Sigma), anti-IFIT1 (PA3-848, ThermoFisher), anti-IFIT2 (12604-1-AP, Proteintech), anti-IFIT3 (PA5-22230, ThermoFisher), anti-GAPDH (AM4300, ThermoFisher) and anti-penta-His (34660, Qiagen). For pull-down experiments FLAG-tagged proteins were detected by chemiluminescence using Westar Supernova substrate (Cyanagen) and visualized on Super RX-N film (Fujifilm). For all other western blot experiments an Odyssey CLx Imaging System (Li-Cor) was used. To normalize recombinant IFIT proteins and complexes, membranes were probed with anti-penta-His and quantified using ImageJ.