Inhibitor cocktail solution

After being washed with PBS, cells were harvested and incubated with the RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing protease/phosphatase inhibitors (Inhibitor Cocktail solution; genDEPOT, Katy, TX, USA). The cells were lysed on ice for 30 min, and lysate was collected by centrifugation at 13,000 rpm for 10 min. Protein concentrations were determined using a BCA assay (Thermo Scientific, Waltham, MA, USA). Equal amounts of solubilized proteins were separated using SDS-PAGE, and were subsequently transferred to PVDF membranes (Mil-lipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk prepared in TBST (Tris-buffered saline containing 0.1% Tween 20). After washing (TBST), the membrane was incubated with primary antibodies. After washing again, the membrane was incubated with HRP-conjugated secondary antibody. Signals were visualized using the Clarity Western ECL substrate kit (Bio-Rad, Richmond, CA, USA). The membranes were re-probed with an anti-GAPDH or anti-tubulin antibody to verify that an equal amount of protein was loaded in each lane. Signal intensities were quantified using densitometry with the ImageJ software (version 1.52a) (NIH, Bethesda, MD, USA).

The cells were harvested in RIPA buffer (Thermo Scientific) containing protease/phosphatase inhibitors (Inhibitor Cocktail solution; genDEPOT, Katy, TX, USA) for 30 min at 4°C. Mouse liver samples were homogenized and lysed in RIPA buffer. Protein concentrations were determined using the BCA assay (Thermo Scientific). Equal amounts of solubilized proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were sequentially incubated in blocking buffer (5% skimmed milk prepared in Tris-buffered saline containing 0.1% Tween 20), primary antibodies, and then appropriate horseradish peroxidase-conjugated secondary antibodies. Signals were visualized using the Clarity™ Western ECL substrate kit (Bio-Rad, Richmond, CA, USA). The membranes were re-probed with an anti-GAPDH or anti-tubulin antibody to verify that an equal amount of protein had been loaded in each lane. Signal intensities were quantitated by densitometry using ImageJ software (version 1.52a) (NIH, Bethesda, MD, USA).