P ampk α 2535

Protein extraction was performed in liver for Western Blot analysis, as previously described [26 (link),27 (link)]. About 30 μg of proteins were loaded and specific antibodies were used to evaluate PPARα (ab61182), SREBP-1 (ab28481) (1:250 and 1:1000 dilution respectively; Abcam, Cambridge, MA, USA), p-AMPK-α (2535), and β-actin (4970) (1:1000 dilution each; Cell Signaling, Beverly, MA, USA) which was used as loading control. The images were obtained using specific software (C-DiGit Blot Scanner with Image Studio 4.0 software, LI-Cor, Lincoln, NE, USA), and densitometric data were expressed as integrated intensity, following evaluation of a region of interest (ROI) identical for all samples within the same blot.

Antibodies were obtained from venders as follows: HIF-1α (Novus biologicals. 10771 E Easter Ave, Centennial, CO, USA. NB100-479), Raptor (Proteintech. 5500 Pearl St # 400, Rosemont, IL, USA. 20984-1-AP), anti-pimonidazole (Hypoxyprobe. 121 Middlesex Turnpike, Burlington, MA, USA. 4.3.11.3 mouse MAb), septin-2 (Novus biologicals, NBP2-75660), septin-5 (Proteintech, 11631-1-AP), septin-11 (Proteintech, 14672-1-AP). Other septin antibodies were purchased from Sigma Aldrich (septin-6, HPA005665; septin-7, HPA029524; septin-9, HPA042564; septin-10, HPA056304). All other antibodies were purchased from Cell Signaling Technology, (3 Trask Lane, Danvers, MA, USA): AMPKα (5831), p-AMPKα (2535), p-Raptor (2083), ACC (3676), p-ACC (11818), α-tubulin (2144), acetyl-α-tubulin (5335), β-catenin (8480), HER2 (4290), p-HER2 (2247), Erk1/2 (9102), p-Erk1/2 (4370), cleaved PARP (5625), cleaved caspase 7 (9491), and PD-L1 (13684).