Sc 9013

Tissues were homogenized as described (Brüning et al., 1998 (link)). Western blot analyses of insulin signalling proteins were performed on liver homogenates as previously described (Valverde et al., 1998 (link)). For immunoprecipitation, after protein content determination, equal amounts of protein (400 μg) were immunoprecipitated overnight at 4°C with anti-GLUT-2 antibodies (C-19) (1:500; sc-7580, Santa Cruz Biotechnology, Dallas, TX). The immune complexes were collected on protein A-agarose beads and submitted to SDS-PAGE. For western blot, the antibodies used were anti-insulin receptor β subunit (1:1000; sc-711), anti-GLUT-2 (H-67) (1:500; sc-9117) and anti-IGF-I (1:200; sc-9013) (all from Santa Cruz Biotechnology) and anti-β-actin (1:5000; A2228, Sigma-Aldrich, St. Louis, MO). Rabbit and mouse primary antibodies were immunodetected using horseradish peroxidase-conjugated polyclonal anti-rabbit or mouse antibodies, respectively (1:5000; NA931V and NA934V, GE Healthcare, Buckinghamshire, UK). Loading was normalized by β-actin. The band intensities were quantified using ImageJ software (http://rsb.info.nih.gov/ij).