Phosphatase inhibitor cocktail 78420

HEK293T cells were collected in ice-cold phosphate-buffered saline (PBS). Cells were centrifuged at 10,000 × g followed by removing PBS. Cells were lysed using a 22 G needle and syringe in mitochondrial isolation buffer MIB: (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM Hepes (pH 7.5), supplemented with Protease Inhibitor Cocktail P8340, Sigma and Phosphatase Inhibitor Cocktail 78420, Thermo Fisher Scientific). Lysates were centrifuged at 700 × g to remove the plasma membrane, and then centrifuged at 6000 × g to isolate mitochondria/heavy membrane fraction. Isolated mitochondrial/heavy membrane pellets were subsequently lysed in mitochondrial lysis buffer (MLB: 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl₂, 0.5% NP40, 10% glycerol, and supplemented with Protease Inhibitor Cocktail P8340, Sigma and Phosphatase Inhibitor Cocktail 78420, Thermo Fisher Scientific).

LPS (Escherichia coli 026:B6), adenosine 5′-triphosphate disodium salt hydrate (A2383; ATP), nigericin (N7143), and penicillin–streptomycin (Pen/Strep, P4333) were purchased from Sigma-Aldrich. Human IFN Beta (Beta 1a, Mammalian Expressed) (11415-1) and Human IFN Alpha Hybrid (Universal Type I IFN) (11200-1) were obtained from PBL Assay Science. Phosphatase inhibitor cocktail (78420) was sourced from Thermo Fisher Scientific, and FBS was obtained from Gibco.
Primary antibodies for Western blot analysis and their final concentrations were as follows: anti-human IL-1β (0.1 μg/ml, goat polyclonal, AF-201-NA; R&D Systems) and anti-β-actin HRP (0.2 μg/ml, mouse monoclonal, A3854; Sigma-Aldrich).