Bicinchoninic acid protein assay

Distinct Liver samples (n = 3) were homogenized using bead crushers µT−12 (TAITEC) in RIPA buffer supplemented with a 1× protease inhibitor cocktail (both from Nacalai Tesque). Total proteins were quantitated using a bicinchoninic acid protein assay (Takara Bio Inc.), and 50 µg total protein was added to 1 M DTT and heated to 95 °C for 3 min for thermal denaturation. Proteins were separated on a 4–20% gradient SDS-PAGE gel and transferred to a nitrocellulose membrane using an iBlot Gel Transfer Device (Thermo Fisher Scientific). Membranes were blocked with Blocking One (Nacalai Tesque) at room temperature for 30 min and incubated with primary antibodies in blocking buffer at room temperature for 1 h or at 4 °C overnight. After washing thrice for 10 min with PBS-T, the membranes were incubated with secondary antibodies in blocking buffer at room temperature for 1 h. Then, membranes were washed thrice with PBS-T, and proteins were detected using enhanced chemiluminescence (Thermo Fisher Scientific). Images were collected using the Bio-Rad Molecular Imager ChemiDoc Touch (Supplementary Fig. 14).

Distinct Liver samples (n = 3) were homogenized using bead crushers µT−12 (TAITEC) in RIPA buffer supplemented with a 1× protease inhibitor cocktail (both from Nacalai Tesque). Total proteins were quantitated using a bicinchoninic acid protein assay (Takara Bio Inc.), and 50 µg total protein was added to 1 M DTT and heated to 95 °C for 3 min for thermal denaturation. Proteins were separated on a 4–20% gradient SDS-PAGE gel and transferred to a nitrocellulose membrane using an iBlot Gel Transfer Device (Thermo Fisher Scientific). Membranes were blocked with Blocking One (Nacalai Tesque) at room temperature for 30 min and incubated with primary antibodies in blocking buffer at room temperature for 1 h or at 4 °C overnight. After washing thrice for 10 min with PBS-T, the membranes were incubated with secondary antibodies in blocking buffer at room temperature for 1 h. Then, membranes were washed thrice with PBS-T, and proteins were detected using enhanced chemiluminescence (Thermo Fisher Scientific). Images were collected using the Bio-Rad Molecular Imager ChemiDoc Touch (Supplementary Fig. 14).