Rabbit anti p acc

Proteins were extracted from the hepatocytes and liver tissues using RIPA lysis buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein concentration was quantified using the BCA reagent (Thermo Fisher Scientific) following the manufacturer's protocol. An equal amount of protein from each sample was loaded into each lane for separation by SDS-PAGE and then transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking with 5% (w/v) skim milk powder dissolved in PBS containing Tween-20 (PBST) at room temperature for 2 hours, the membranes were incubated at 4 °C overnight with the primary antibodies: rabbit anti-Prkab1 (Proteintech Group), rabbit anti-Prkaa1 and Prkaa2 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pAMPK (Cell Signaling Technology), rabbit anti-ACC pan (Abcam), rabbit anti-pACC (Abcam), rabbit anti-CD36 (Abcam), rabbit anti-p65 (Abcam), rabbit anti-p-p65 (Cell Signaling Technology) and mouse anti-β-actin (Abcam). After washing with PBST, the membranes were incubated at room temperature for 1 hour with the horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Abcam). The density of each band was quantified by densitometric analysis with Image Lab 6.0 software.