(A) Phosphoproteomic heatmap showing differential phosphorylation events in WT and ULK1 knockdown (KD) cultured hepatocytes. (B) Co-immunoprecipitation and immunoblot analyses demonstrating interaction of transfected myc-tagged ULK1 and NCOA3 in hepa1c1c7 cells. (C) In vitro kinase assay confirming direct ULK1-dependent phosphorylation of NCOA3. (D) Heatmap depicting repression of NCOA3 phosphorylation at S544, S847, and S850 in hepatocyte-specific ULK1 KO mice (UKO) fed NCD or HFD. Color scale represents log2 fold change, with red indicating increased phosphorylation and blue indicating decreased phosphorylation. (E) Protein levels of lipogenic regulators after combined silencing using siULK1 (50 nM) and siNCOA3 (50 nM) in hepa1c1c7 cells. (F) Hepa1c1c7 cells were transfected with siULK1 (50 nM), NCOA3 (300 ng), and a phosphorylation defective NCOA3 mutation (mut) (300 ng), and then protein levels of lipogenic regulators were measured. (G) Lipogenic regulators in hepa1c1c7 cells transfected with NCOA3 (300 ng), siNCOA3 (50 nM), and ULK1 M92A (300 ng). (H) CRE consensus sequence and expected CREB binding sites on the promoter region of SREBP1c. (I) Luciferase activity in hepa1c1c7 cells transfected with siULK1 (50 nM), siNCOA3 (50 nM), and luc-CRE (200 ng). (J) Lipogenic regulators in hepa1c1c7 cells transfected with siULK1 (50 nM), siNCOA3 (50 nM), siCREB (50 nM), and NCOA3 (300 ng). All data represent the mean ± SEM. Data were analyzed by 1-way ANOVA followed by Tukey’s post hoc test versus the control group (E–G, I, and J). Actual P values are shown; in F, *P < 0.05 versus control or NCOA3 WT. FC, fold change; HFDUKO, ULK1 KO mice fed an HFD; HFDWT, WT mice fed an HFD; MBP, myelin basic protein; NCDUKO, ULK1 KO mice fed an NCD; NCDWT, WT mice fed an NCD.