P acc1

For protein expression analyses, frozen liver tissue (∼25 mg) was homogenized by the Qiagen TissueLyser II in a sucrose buffer (250 mM sucrose, 50 mM Tris–Cl, pH 7.4) with 1:100 protease inhibitor (Sigma–Aldrich). The tissue lysates were denatured by boiling, separated on a 4 to 15% SDS/polyacrylamide gel electrophoresis gel (Bio-Rad), and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with TBS-T containing 5% milk for 1 h at room temperature and incubated with primary antibodies; ACLY, ACSS2, FASN, SCD1, ACC1, p-ACC1, GAPDH, β-actin purchased from Cell Signaling, or SREBP1c antibody purchased from Millipore. The blot was washed in TBS-T for an hour, incubated at room temperature with corresponding second antibody at room temperature for 30 min, washed again, and incubated with ECL (PerkinElmer) and visualized with the ChemiDox XRS+ image-forming system.

Protein lysates were prepared from olfactory bulbs and hippocampi of 5 months old WT and KO female mice using RIPA buffer with protease inhibitors followed by sonication by ultrasound and clearing via centrifugation at maximum speed using Eppendorf tabletop centrifuge. Protein concentration was quantified using BCA reagent (Thermo Fisher Scientific, Rockford, IL). Protein extracts (40 μg/well) were subjected to Western immunoblot analysis. We used antibodies against the following proteins: PRDX1 (Sigma), ACC1, pACC1, AMPKα, pAMPKα, NGFR, TrkB, Bcl-xL, Rack1 (all from Cell Signaling Technology), Calretinin (Millipore), LC3 (Abcam). The band intensity was determined using ImageJ software, normalized to loading control (β-actin). The data are presented as a relative intensity of WT bands over KO bands. Phospho-index was determined as a ratio of phosphorylated/total protein intensities.