Pampk thr172

Total protein was extracted from the liver of each group using protein lysis buffer (Invitrogen, Carlsbad, CA) containing phosphatase inhibitor (GenDEPOT, Barker, TX, USA). Protein extracts were resolved by 10% or 12% SDS-PAGE gels and were later transferred onto PVDF membranes (Millipore, Billerica, MA). The protein bands were detected with antibodies against pAMPK (Thr172), AMPK, pACC, ACC, PPARα, SCD1, CPT1 (Abcam, Cambridge, UK), and GAPDH (Cell Signaling Technologies, Beverly, MA) using the appropriate secondary HRP-conjugated antibodies (Cell Signaling Technologies, Beverly, MA). The blots were developed and imaged using the MicroChemi 4.2 system (DNR Bio-imaging system, Israel).

NAD/NADH assay kit (Colorimetric) was purchased from Abcam. Bicinchoninic acid (BCA) protein detection kit, chemiluminescent substrate kit, and phosphocellulose units were from Pierce. Diaminobenzidine (DAB), and Hoechst 332621 were from Sigma-Aldrich. Lipofectamine 2000 was from Invitrogen. OPTIMUM and other reagents for cell culture were from Gibco. AICAR (5-aminoimidazole-4-carboxamide-1-β-riboside) was from Cell Signaling, and Compound C was from Millipore. Plasmid Flag-Nmnat2 was kindly gifted by Dr. Michael P. Coleman (The Babraham Institute, Babraham Research Campus, Cambridge, United Kingdom). The target sequences (GeneBanK NM_175460) for mouse Nmnat2 messenger RNA (mRNA) is 5′-GCACAAGACTGGAAGATTT-3′. The scrambled siRNA sequence is 5′-TTCTCCGAACGTGTCACGT-3′. The Nmnat2 siRNA and scrambled siRNA are inserted into pMagic4.1 vector to generate Nmnat2-siRNA-EGFP (siNmnat2) and scramble-siRNA-EGFP plasmids. Some antibodies are as follows: Nmnat2 (Abcam), 22C11 (Millipore), p-APP (Thr-668) (Biosource), ADAM10 (Abcam), G2-10 (Millipore), G2-11 (Millipore), 6E10 (Millipore), 4G8 (Millipore), Y188 (Abcam), AMPK (Sigma), p-AMPK (Thr172) (Abcam), DM1A (Abcam), β-actin (Abcam). See Table 1 for more details.