Quantity one image analyzer

Total protein concentration of each VLP preparation and ammonium sulfate precipitated samples was determined using a Nanodrop ND-8000 (Thermo Scientific, USA) at 280 nm and the Bio-Rad Bradford protein assay reagent (Bio-Rad Laboratories, USA) with bovine serum albumin (BSA; Pierce, USA) as a standard. The yield of L1 protein was quantified using SDS-PAGE analysis. Serially diluted HPV16 L1 VLPs from leaves and insect cells were loaded on an SDS-PAGE gel, and their band densities were measured by Quantity one image analyzer (version 4.5, Bio-Rad).

Homogenized liver tissue specimens were boiled with 2 × SDS-PAGE loading buffer at 95 °C for 10 min. Then, proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred onto a PVDF membrane. After blocking with 5% nonfat dried milk in TBS-T (10 mM Tris-HCl, pH7.8, 150 mM NaCl and 0.05% [v/v] Tween-20) for 2 h at room temperature, the membrane was incubated with anti-TXNIP antibody (Cell Signaling Technology) or anti-β-actin antibody, diluted 2,000 to 5,000 fold, for at least 3 h at room temperature. The membrane was then washed and incubated with anti-rabbit secondary antibodies (5,000 to 10,000 dilution) for 1 h at room temperature. Protein bands were detected using the ChemiDoc XRS⁺ detection system (ECL, Bio-Rad); densitometric analysis was performed using Quantity One^® Image Analyzer (Bio-Rad).

RIPA buffer supplemented with a cocktail of protease and phosphatase inhibitors was used to lyse the liver. Liver protein concentrations of the extracts were measured by a BCA kit according to the manufacturer’s protocol. The proteins were loaded onto 10–15% SDS-PAGE, transferred to polyvinylidene difluoride membranes (PVDF) and blocked with 5% nonfat milk for 1 h. The membranes were incubated overnight with primary antibodies at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, then visualized with a chemiluminescence reagent. The protein levels were quantified by the Quantity One Image Analyzer software program (Bio-Rad) and β-actin content was used as an internal control.