Loading buffer reagents

Western blotting was performed as reported previously (80 (link), 81 (link)). In brief, whole-cell lysates were prepared using loading buffer reagents (Santa Cruz Biotechnology, Inc) without trypsin treatment. Equal amounts of total protein were electrophoresed on a 10% SDS–polyacrylamide gel. The proteins were transferred to polyvinylidene difluoride membranes (ATTO). The membranes were blocked with blocking solution (5% skimmed milk with 0.1% Tween-20 dissolved in Tris-buffered saline [pH 7.5]), incubated with the first antibody for PGC-1α (Calbiochem), C/EBPβ (Santa Cruz Biotechnology), and β-tubulin (Sigma), which were diluted in blocking solution, incubated with the peroxidase-conjugated second antibody diluted in blocking solution, visualized with the ECL-Western blotting detection system (Amersham) according to the manufacturer's protocol, and used to expose hyperfilm-ECL (Amersham). To reuse the blot, the membranes were stripped in Restore Western stripping buffer (Pierce). Western blot bands were quantified by ImageJ (U. S. National Institutes of Health), and the quantification values of three independent experiments are provided in Table S2 of the revised manuscript. The uncropped images of immunoblots are shown in Fig. S1.

Western blotting was performed as reported previously (53 (link), 54 (link)). In brief, whole cell lysates were prepared using loading buffer reagents (Santa Cruz Biotechnology, Inc) without trypsin treatment. Equal amounts of total protein were electrophoresed on a 10% SDS-polyacrylamide gel. The proteins were transferred to polyvinylidene difluoride membranes (ATTO). The membranes were blocked with blocking solution [5% skimmed milk with 0.1% Tween-20 dissolved in Tris-buffered saline (pH 7.5)], incubated with the first antibody for GLUT1 (Abcam), C/EBPβ (Santa Cruz Biotechnology), WT1(Abcam), and β-tubulin (Sigma), which were diluted in blocking solution, incubated with the peroxidase-conjugated second antibody diluted in blocking solution, visualized with the ECL-Western blotting detection system (Amersham) according to the manufacturer's protocol, and used to expose hyperfilm-ECL (Amersham). To reuse the blot, the membranes were stripped in Restore Western stripping buffer (Pierce). Western blot bands were quantified by ImageJ. The uncropped images of immunoblots are shown in Fig. S2.