Tyrosine phosphatase inhibitor cocktail

Mouse liver homogenates were prepared using a RIPA buffer containing protease and tyrosine phosphatase inhibitor cocktail (Sigma). The protein concentration of the lysates was determined using the Bicinchoninic (BCA) Protein Assay Kit (Thermo Scientific, IL, USA), according to the manufacturer’s instructions. The isolated soluble proteins (20 mg) were separated on 8–15% SDS-polyacrylamide gels. The separated proteins were then electroblotted onto nitrocellulose transfer membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with 5% skim milk for 1 h and then probed with the following 1:1,000-diluted antibodies: anti-pAMPKa, anti-AMPKa, anti-pACC, anti-ACC, anti-CYP2E1, anti-CPT1a (Cell Signaling Technology), and anti-β-actin (Santa Cruz Biotechnology) for 18 h at 4°C. After 3 washes for 10 min each, polyclonal anti-rabbit or mouse HRP-conjugated secondary antibodies (Santa Cruz Biotechnology), which were linked to HRP-bound protein complexes and developed with a Pierce ECL Western Blot substrate (Thermo Fisher Scientific), were added to the mixture. Band densitometry was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA) and expressed as fold change relative to β-actin.

For enzymatic assays, B. subtilis strains were grown in LB with vigorous shaking in flasks at 37°C. Samples were harvested at the mid-exponential phase (OD₆₀₀ = 0.4). Cells were lysed by sonication. The lysis buffers (composition adapted for each assay) contained 1% tyrosine phosphatase inhibitor cocktail (Sigma) and 1 mg/ml lysozyme (Sigma). For the Ugd assay, the buffer contained 50 mm Tris-HCl pH 7.5, 50 mm NaCl and 10% glycerol. For the Asd assay, the buffer contained 50 mM 3-(N-morpholino) propanesulfonic acid (pH 7.0), 200 mM KCl, 0.1 mM EDTA, and 10 mM 2-mercaptoethanol. After centrifugation, the supernatant was desalted on PD-10 columns (GE healthcare), to remove all metabolites and cofactors. Total protein concentration was standardized using the Bradford assay. The Asd assay was performed with 100 μg of total protein, as described by Jers et al. (2010) (link). The only difference was that instead of adding commercially available aspartate kinase, the aspartate kinases present in the crude extract of B. subtilis (Graves and Switzer, 1990 (link); Roten et al., 1991 (link); Kobashi et al., 2001 (link)) were used to convert ATP and aspartate to aspartyl phosphate. The Ugd assay was performed with 100 μg of total protein, as described by Pagni et al. (1999) (link).