Horseradish peroxidase conjugated anti rabbit na931v

Western blot analyses were performed on liver homogenates as previously described (Escribano et al., 2003). The antibodies used were anti-IRβ (sc-711; 1:2000 in TTBS) from Santa Cruz Biotechnology (Dallas, TX, USA) and anti-β-actin (A2228; 1:5000 in TTBS) from Sigma-Aldrich (St. Louis, MO, USA). Rabbit and mouse primary antibodies were immunodetected using horseradish peroxidase-conjugated anti-rabbit (NA931V; 1:4000 in TTBS) or anti-mouse (NA934V; 1:5000 in TTBS) (GE Healthcare, Buckinghamshire, UK) antibody, respectively. Loading was normalized by β-actin. The band intensities were quantified using ImageJ v1.6 software (http://rsb.info.nih.gov/ij).

Western blot analyses were performed on liver homogenates and cultured cells as previously described (Escribano et al., 2003 (link)). The primary antibodies used are shown in Table S3 and all of them were diluted in TTBS. Rabbit and mouse primary antibodies were immunodetected using horseradish peroxidase-conjugated anti-rabbit (NA931V; 1:4000 in TTBS) or anti-mouse secondary antibody (NA934V; 1:5000 in TTBS) (GE Healthcare, Buckinghamshire, UK), respectively. When possible, phospho-proteins and their total expression were detected in the same gel, using Restore^TM Western Blot Stripping Buffer (Thermo Fisher Scientific) as per the manufacturer's instructions, blocking the membrane again before the incubation with the other antibody. Loading was normalized by α-tubulin or β-actin. The representative gels that share the same housekeeper were as follows: (1) at 8 weeks, ACC and p70S6K; (2) at 8 weeks, p85α, SIRT1 and MFN2; (3) at 8 weeks, PKCε, ULK1 and p-ULK1; (4) at 18 weeks, ACC and p-ULK1; (5) at 18 weeks, FAS, IRβ, p85α and SIRT1; (6) at 18 weeks, SCD1 and LC3. The band intensities were quantified using ImageJ v1.52k software (http://rsb.info.nih.gov/ij).