Western Blot Analysis of Tumor Lysates

Tumor lysates were analyzed by western blotting, as described previously [43 (link)]. Tumor lysate protein was transferred onto Immobilon-P membranes (Millipore; Bedford, MA, USA). Blots were incubated with Glut4 (1F8) Mouse mAb, AceCS1(D19C6) Rabbit mAb, caspase-3 antibody, cleaved-PARP, or LC3B antibody. GAPDH was used as a loading control. (All above antibodies from Cell Signaling, Beverly, MA, USA). The membranes were then incubated with the anti-rabbit secondary antibody (GE Healthcare, Little Chalfont, UK). Specific-binding antibody–target protein interactions were detected using enhanced chemiluminescence plus reagents (Amersham Biosciences, Chalfont St Giles, UK) and exposure to either Hyperfilm ECL (Amersham) or X-OMAT Kodak (Kodak, Rochester, NY, USA) autoradiography film. Tumors with 3 days post-irradiation in the irradiated and non-irradiated groups were removed, frozen and embedded, sliced and stained with hematoxylin and eosin stain [43 (link)]. In a separate group of mice, 3 tumors of each group were included.