Clx imager

Livers were homogenized in RIPA lysis buffer and 50 μg of protein lysate was resolved on Tris-glycine SDS-PAGE gels. Antibodies against Total STAT3 (1:1000), pSTAT3 (1:2000), Total AKT (1:1000), pAKT (1:2000), GAPDH (1:1000) (all from Cell Signaling) were used for Western blotting overnight at 4°C. Secondary antibodies (Donkey anti-rabbit (1:30,000) and Donkey anti-mouse (1:20,000) (Li-Cor)) were used for 1 hour at room temperature. Membranes were scanned using Li-Cor CLx Imager.

The Seahorse XF_P instrument (Agilent) was used to measure O₂ consumption and extracellular acidification, with procedures slightly modified from the manufacturer’s protocols. Briefly, XF_P miniplates were coated with 2 μg collagen (100 ug/mL) and incubated at room temperature for 30 minutes. Cells were seeded at 1.8 × 10⁴ cells/well, and cultured in a CO₂ incubator at 37 °C for 18 hours. The assay was performed following the Real-Time ATP Rate Assay kit instructions (Agilent 103591), with the exception that 1.5 mM oligomycin (Cayman 11342) and 0.5 mM rotenone/antimycin A (Cayman 13995/Sigma A8674) stock solutions were prepared in-house. The assay was performed with cells incubated in a DMEM medium containing 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine, pH 7.4, using the solutions supplied by the manufacturer. Raw data were normalized to in-cell Western signals. Briefly, cells in the XF_P miniplate were fixed with 12% formaldehyde and permeabilized with 0.1% Triton X-100. After incubating with anti-actin primary antibody (Santa Cruz 69879, 1:200) and goat-anti mouse 680 secondary antibody (LI-COR 926-68070, 1:800), Western signals were obtained using the LI-COR CLx imager. Seahorse data were analyzed using the Agilent Analytics online server.