Ab93857

Western blot assay was proceeded based on the previous study.¹⁸ Simply put, total protein of LX‐2 cells was extracted with RIPA lysis buffer (E‐BC‐R327; Elabscience). After the protein concentrations were measured by BCA protein assay, 30 μg of the protein was separated by 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels, and transferred onto polyvinylidene fluoride membranes (3010040001; Roche). Then, the membranes were blocked, followed by the incubation with primary antibodies at 4°C overnight and a secondary antibody at 37°C for 1 h. The protein band was exposed with ECL buffer (R30199; Pierce), and the intensity analysis was achieved with iBright FL1500 Imaging System (A44115; Invitrogen). The primary antibodies used were those against G6Pase (1:1000; Rabbit; ab93857; abcam, 40 kDa), PEPCK (1:1000; Rabbit; No. ABIN2855891; antibodies‐online, 71 kDa), p‐PI3K (1:1000; Rabbit; #029801; Alamo Laboratories Inc, 85 kDa), PI3K (1:1000; Rabbit; #4249; Cell Signaling Technology (CST), 110 kDa), p‐Akt (1:2000; Rabbit; #4060; CST, 60 kDa), Akt (1:1000; Rabbit; #4685; CST, 60 kDa), insulin‐like growth factor‐1 receptor (IGF‐1R, 1:1000; Rabbit; ab182408; abcam, 156 kDa) and GAPDH (1:10000; Rabbit; ab181602; abcam, 36 kDa). The secondary antibody was goat anti‐rabbit antibody (1:2000, #14708; CST). GAPDH was served as a loading control.

Antibodies against G6Pase-α were quantified on Nunc Immuno Maxisorp plates (ThermoFisher, #442404) coated with 0.5 µg/mL recombinant G6Pase-α protein (Viva Biotech) in 50 mM Na₂CO₃ for 1 h at room temperature and blocked with SuperBlock (PBS) Blocking Buffer (ThermoFisher, #37515). Mouse serum diluted 1:20 dilution in PBS was incubated 1 h at room temperature and quantified with a standard curve using commercial rabbit anti-human G6Pase-α IgG (Abcam, #ab93857) at 0–2 µg/mL. Samples and standards were incubated 1 h at room temperature with goat anti-rabbit IgG-HRP (Abcam, #ab6721) or goat anti-mouse IgG H + L-HRP (Fitzgerald Laboratories, #43-GM30) secondary antibody at 1:100,000 dilution. ELISA was developed with 1-Step Ultra TMB-ELISA substrate (ThermoFisher, #34028) and Stop Solution (ThermoFisher, #SS04) before reading at 450 nm.