Kidney cortical lysates were prepared as follows. Renal cortexes were excised rapidly and snap frozen in liquid nitrogen. Lysates were prepared using a Dounce homogenizer in lysis buffer [containing 20 mM Tris-HCl pH 7.4, 50 mM NaCl, 50 mM NaF, 5 mM Na-pyrophosphate, 250 mM sucrose, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, and complete protease inhibitor cocktail (Roche)]. Homogenates were centrifuged at 16,000 × g for 30 min at 4°C, and the protein concentration in the supernatants was measured using the Bradford technique (Bio-Rad protein assay kit). Samples were separated by Nu-PAGE 4–12% gradient gels and electrically transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Membranes were cut horizontally at about 50 and 100 kDa to make three sections for probing. The top was probed for pACC. The middle was probed for pThr172-AMPKα. The bottom was probed for β-actin. The middle part was then stripped and reprobed for total AMPKα. We had similar results when we probed for AMPK-alpha first and then stripped and reprobed for pThr172-AMPKα (Li et al., 2015; Al-Bataineh et al., 2016). The membranes were blocked in 5% milk for 1 h and then incubated with primary antibodies in 5% BSA overnight at 4°C. After washes in TBS 0.05% Tween 20, the membrane was incubated with either goat anti-mouse or anti-rabbit secondary antibodies for 1 h at room temperature. Immunoreactive proteins were detected by the Versa-Doc imaging system (Bio-Rad). Quantification of Western blot bands was performed by densitometry using Quantity One software (Bio-Rad).