Chemiluminescent imagequant las 4000

The cell lysate was prepared from pre-adipocyte 3T3-L1 cells cultured in differentiation medium containing 0−20 μM pinostrobin for 48 h. Briefly, the cell membrane was broken by incubation with RIPA buffer supplemented with protease inhibitor cocktail on ice for 45 min. Equal amounts of protein samples as quantified by the BCA assay were separated through 10% SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The nitrocellulose membranes were further blocked with 5% (w/v) skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h, followed by incubation with primary antibody overnight at 4 °C. The membranes were then washed with TBST (3 times × 5 min) and immersed in specific secondary antibody at room temperature for 2 h. Protein bands of interest were detected and quantified under UV light after reaction with western chemiluminescent ECL substrates using a chemiluminescence instrument (Chemiluminescent ImageQuant LAS 4000, GE Healthcare Bio-Sciences AB, Björkgatan, Uppsala, Sweden). The protein expression level relative to GAPDH (internal loading control) was calculated.

After the indicated treatment, 3T3-L1 cells were washed with phosphate-buffered saline (PBS, pH 7.4), then the cell membranes were broken using RIPA buffer supplemented with a protease inhibitor cocktail. After incubation on ice for 45 min, the cell lysates were centrifuged at 12,000 rpm at 4 °C for 15 min to collect the clear supernatant containing cellular protein, which was measured for total protein content using a BCA assay kit. The total protein (30 µg) from each sample was loaded and separated onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the separated proteins were transferred onto nitrocellulose membranes, which were blocked with 5% skim milk in TBST buffer (Tris-buffered saline with Tween 20, pH 7.2) and further immunoblotted with primary antibodies against p-Akt (Thr308), Akt, p-GSK3β (Ser9), GSK3β, p-AMPKα (Thr172), AMPKα, p-AMPKβ1 (Ser128), AMPKβ1/2, p-ACC (Ser79), ACC, PPARγ, C/EBPα, and β-actin at 4 °C overnight. Before immersion in horseradish peroxidase (HRP)-linked secondary antibody at room temperature for 2 h, the membranes were washed with TBST for 7 min, three times. The reactive protein signals exposed with chemiluminescent substrates were captured and quantified using Chemiluminescent ImageQuant LAS 4000 (GE Healthcare Bio-Sciences AB, Björkgatan, Uppsala, Sweden).

After indicated treatment, MNT1 cells (1 × 10⁵ cells/well in a 6-well plate) were incubated in iced-cold RIPA lysis buffer with 1% protease inhibitor cocktail for 30 min. The cell lysate containing 30 µg protein assessed by BCA protein assay from each was mixed with loading dye before separating onto 10% SDS-PAGE. Thereafter, the separated proteins were transferred from the gel onto nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was immersed into 5% non-fat skim milk in TBST (25 mM Tris-HCl; pH 7.5, 125 mM NaCl, 0.1% Tween20) for 1 h at room temperature. Then, the membrane was washed with TBST for 5 min prior to incubation with primary antibody at 4 °C for 12 h. Before probing with HRP-secondary antibody for 2 h at room temperature, the membrane was washed three times (5 min) with TBST. The band of interested protein was examined after adding of chemiluminescence substrate (SuperSignal West Pico, Pierce, Rockford, IL, USA) onto the membrane. The signal of interested protein band was visualized and determined using Chemiluminescent ImageQuant LAS 4000 (GE Healthcare Bio-Sciences AB, Björkgatan, Uppsala, Sweden).