Ab128856

Total protein from hepatocytes and liver were extracted using RIPA lysis buffer (P0013B; Beyotime, Shanghai, China). The proteins were separated by SDS-PAGE gel, then transferred to PVDF membranes (Millipore, Darmstadt, Germany), and immunoblotted by primary antibodies according to the manufacturers’ manuals. The primary antibodies were detected by a HRP-conjugated secondary antibody from the appropriate species and reacted with Immobilon Western Chemiluminescent HRP Substrate (Millipore). Primary antibodies against the following proteins were used: AGT (IBL-America #JP28101, RRID: AB_2341481), SREBP1 (Abcam #ab28481, RRID: AB_778069), acetyl-CoA carboxylase (ACC) (Cell Signaling Technology #3676, RRID: AB_2219397), FASN (Abcam #ab128856, RRID: AB_11143234), phospho-protein kinase B (Akt) (Ser473) (Cell Signaling Technology #4060, RRID: AB_2315049), Akt (pan) (Cell Signaling Technology #4691, RRID: AB_915783), β-actin (KC-5A08, Aksomics Inc., Shanghai, China), cluster of differentiation 36 (CD36) (Abcam #ab133625, RRID: AB_2716564), LC3A/B (Cell Signaling Technology #4108, RRID: AB_2137703), P62 (Cell Signaling Technology #5114, RRID: AB_10624872), ATG7 (Cell Signaling Technology #8558, RRID: AB_10831194), and ATG12 (Cell Signaling Technology #4180, RRID: AB_1903898).

The tissue was fixed with 4% paraformaldehyde, embedded in paraffin and sectioned. The paraffin-embedded sections were dewaxed and rehydrated, and EDTA was used for antigen repair. At room temperature, sections were incubated with 3% H₂O₂ to quench endogenous peroxidase activity. Afterwards, samples were blocked with BSA and incubated overnight at 4℃ with specific primary antibodies, including anti-CD147 (ab212057, 1:200, Abcam, Cambridge, USA), anti-ACOX1 (ab184032, 1:200, Abcam), anti-FASN (ab128856, 1:200, Abcam). Later, samples were incubated for 1 h at 37°C with HRP-labeled goat anti-Rabbit secondary antibodies. The samples were then dyed with DAB and hematoxylin before sealing.