Anti pepck

To determine the effect of antidiabetic drugs on glucose and lipid metabolism in diabetic models, the protein expression levels of PEPCK and TGR5 were measured [36 (link)]. Total proteins (30 µg) prepared from tissue homogenates were separated through SDS/polyacrylamide gel electrophoresis (10% acrylamide gel) using the Bio-Rad Mini-Protein II System. Proteins were transferred to expanded polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). Following blocking, the membrane was probed with the primary antibodies. After removal of the primary antibody, the blots were incubated for 1 h at room temperature with the appropriate peroxidase-conjugated secondary antibody and then developed using the ECL-Western Blotting System (Amersham International, Buckinghamshire, UK). Antibodies used for immunoblotting were anti-PEPCK (62 kDa, Santa Cruz Biotechnology, Dallas, Tx, USA) and anti-TGR5 receptor protein (32 kDa, Abcam, Cambridge, MA, USA). Anti-β-actin (43 kDa, Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control.

Protein extracts from liver tissue or cells were lysed in RIPA buffer, and cytoplasmic and nuclear proteins were extracted from HepG2 cells or liver tissue using a commercial kit (Beyotime Biotechnology, Wuhan, China). The lysates were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight with primary antibodies at 4°C and then incubated with HRP-conjugated secondary antibodies for 2 hours. Immunoreactive protein was detected using an ECL Advance Western Blotting Detection kit (Amersham Bioscience, Piscataway, US). A quantitative analysis was performed using Image J software (V1.53k). The following primary antibodies were purchased: anti-CD36 (1:2000, Novus, cat# NB400-144), anti-Per1 (1:2000, LifeSpan, cat# LS-C807611), anti-AKT (1:1000, CST, cat# 4691S), anti-p-AKT (1:2000, CST, cat# 4060S), anti-PI3K (1:1000, CST, cat# 4257T), anti-p-PI3K (1:500, GeneTex, cat# GTX132597), anti-FoxO1 (1:1000, CST, cat# 2880S), anti-p-FoxO1 (1:1000, CST, cat# 9461T) anti-G6pase (1:2000, Santa Cruz, cat# sc-167939), anti-PEPCK (1:2000, Santa Cruz, cat# sc-32879) and anti-β-actin (1:5000, Bioss, cat# bs-0061R).

Western blot analysis was performed with primary antibodies obtained as follows: anti-c-Jun (Cell Signaling Technology, MA, USA; 1:1000), anti-UCP1, anti-FGF21 and anti-PEPCK (all from Santa Cruz Biotechnology, CA, USA; 1:500 for UCP1 and FGF21, 1:1000 for PEPCK), anti-G6PASE (Abcam, Cambridge, UK; 1:500). PEPCK and G6PASE activities were determined using a PEPCK activity kit (Solarbio Life Sciences, Beijing, China) and a G6PASE activity kit (Solarbio Life Sciences, Beijing, China), respectively. All of these assays were performed according to the manufacturer's instructions.