Antibody against gapdh

RIPA lysis buffer (Beyotime) was used to extract total protein. A BCA Protein Quantitative Kit (Beyotime) was adopted to detect protein concentrations. Protein was separated by SDS-PAGE (10% gel) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). For lectin blotting, the membranes were blocked with a carbo-free blocking solution (Vector Labs) and subsequently incubated with biotin-labeled LCA. For western blotting, the membranes were blocked with 5% non-fat milk. Primary antibodies (Absin, Shanghai, China) were used including anti-MMP2, anti-MMP-9, anti-p53, anti-Bcl-xL, anti-CylinD1, anti-BAX and anti-pAkt. The signal was evaluated using an enhanced chemiluminescence detection kit (Beyotime). An antibody against GAPDH (Absin) served as an endogenous reference.

Cells and liver tissues were lysed with RIPA buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, and 1 × protease inhibitor cocktail. The lysates were then centrifuged at 14,000 × g for 20 min at 4 °C. The protein concentrations of the supernatants were assessed using the BCA assay kit (Beyotime, China) according to the manufacturer’s protocol. Lysates containing equal quantities of protein were resolved by 10% SDS-PAGE and then transferred to a PVDF membrane (Millipore, USA) in transfer buffer for 90 min at 200 mA. Subsequently, the membranes were blocked with 5% nonfat milk in TBST (1.5 mM Tris-Base, 8.5 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) for 2 h, and then incubated at 4 °C overnight with the following primary antibodies: anti-CPT1 (Abcam, USA), anti-Bax, and anti-Bcl-2 (both from Proteintech, Wuhan, China). An antibody against GAPDH (Absin, China) was used as an internal control. The membranes were then washed four times with TBST for 20 min each and then incubated with anti-rabbit or anti-mouse secondary antibodies (1:10,000 in TBST; ZSGB-BIO, Beijing, China) for 1.5 h at room temperature. The immunoreactive protein bands were visualized using the ECL kit (Thermo Fisher Scientific, USA).