Quant las 500 system

Proteins were extracted from 3T3-L1 cells and samples of epididymal adipose tissues using a protein lysis buffer (iNtRON Biotechnology, Seongnam, Korea) with phosphatase and protease inhibitors. The protein samples (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated bands were transferred to a PVDF membrane, and subsequently blocked in 5% skim milk. The membranes were incubated overnight with the primary antibodies for 2 h at room temperature (RT) or 4 °C. The membranes were subsequently incubated with horseradish peroxidase-labeled secondary antibodies for 1 h. The reactive bands of target proteins were detected using an ECL system (iNtRON Biotechnology, Seongnam, Korea), and the reactive band signals were visualized using a Quant LAS 500 system (GE Healthcare Bio-Sciences, Björkgatan, Uppsala, Sweden).

To analyze the expression of proteins such as PPARγ, C/EBPα, SREBP-1, FAS, CPT-1, AMPK, ACC, and β-actin, the cells treated with different concentrations of neferine were subjected to western blot analysis. 3T3-L1 adipocytes and primary white adipocytes were extracted with a protein lysis buffer (iNtRON Biotechnology, Seongnam, Korea) containing protease and phosphatase inhibitors (Thermo Fisher, San Jose, CA, USA). After incubation for 30 min on ice, total protein from each sample was quantified using a PRO-MEASURE protein measurement solution (iNtRON Biotechnology, Seongnam, Korea). Samples with same protein amounts were separated on SDS-PAGE gel, and the separated bands were electro-transferred to a polyvinylidene fluoride (PVDF) membrane. For 1 h, the membrane was blocked and immunoblotted with primary antibodies for 2 h, followed by probing with horseradish peroxidase-labeled secondary antibodies for 1 h. The reactive bands of target proteins were detected by the Quant LAS 500 system (GE Healthcare Bio-Sciences AB, Björkgatan, Uppsala, Sweden) using an enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK).