Pro prep lysis buffer

Total protein was extracted using PRO-PREP lysis buffer (iNtRON Biotechnology, Seongnam, Korea), which contains phosphatase inhibitors and a protease inhibitor cocktail. The lysates were centrifuged at 13,000 rpm for 15 min. The protein samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes, blocked using 5% non-fat skim milk, and incubated overnight at 4 °C with antibodies against PPARγ, LPL, aP2, adiponectin (Abcam, Cambridge, UK), C/EBPα (Cell Signaling Technology Beverly, MA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Total proteins were extracted from intestine using Pro-Prep™ lysis buffer (Intron Biotechnology, Seoul, Korea) and quantitated with Bradford assay reagent (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol. Immunoblots were probed using the following antibodies: VEGF (1:1000, sc-1836, Santa Cruz Biotechnology, CA, USA), eNOS (1:1000, sc-634, Santa Cruz Biotechnology) and β-actin (1:3000, Sigma, MO, USA). The blots were incubated with these antibodies overnight at 4 °C and detected by the luminescence method using ECL solution (NEL104001EA, PerkinElmer, Waltham, MA, USA).

B16F10 melanoma cells were cultured at a density of 1 × 10⁴ cells/mL in 6 well plate overnight. Then, the cells were treated with the indicated concentrations of fisetin for 96 h and lysed with PRO-PREP lysis buffer (iNtRON Biotechnology). In a parallel experiment, the cells were washed with ice-cold PBS, and the cytosolic and nuclear protein was extracted using NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA). After cleaning lysates by centrifugation, protein was quantified by the Bio-Rad protein assay reagents (Bio-Rad, Hercules, CA, USA). An equal amount of protein was separated by SDS-polyacrylamide gel, transferred onto nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA) and then immunoblotted with the indicated antibodies. The expressional value was normalized to the intensity of β-actin or nucleolin.

B16F10 cells were seeded at 1 × 10⁴ cell/mL in 6 well plates for 18 h at 37 °C and then pretreated with α-MSH (500 ng/mL) for 24 h prior to treatment with different concentrations of PS and PTS (0-400 μg/mL). The cells were lysed with PRO-PREP lysis buffer (iNtRON Biotechnology). The lysate was incubated for 20 min on ice and centrifuged at 16,000 rpm at 4 °C for 15 min. The supernatant was collected and protein concentrations were measured using Bio-Rad protein assay reagents (Bio-Rad, Hercules, CA, USA). Equal amount of protein was separated by electrophoresis on 10% SDS-polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA) and immunoblotted with specific antibodies overnight at 4 °C. The membrane was washed three times and reincubated with peroxidase-labeled secondary antibody for 2 h. Bound antibodies were detected using an enhanced chemiluminescence plus kit (Thermo Scientific, Rockford, IL, USA). The images were visualized by a Chemi-Smart 2000 (Vilber Lourmat, Marne-la-Vallee, France). Images were captured using Chemi-Capt (Vilber Lourmat) and transported into Adobe Photoshop.

Liver and eWAT tissues were homogenized and lysed in ice-cold PRO-PREP lysis buffer (iNtRON Biotechnology, Seongnam, Korea), containing phosphatase inhibitors and a protease inhibitor cocktail for 30 min at 4 °C. Then, the lysates were centrifuged at 12,000 rpm for 20 min at 4 °C and the supernatants of these tissue lysates were separated. The tissue protein quantification was measured using Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich, St. Louis, MO, USA). Each 20 μg of protein samples were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked using 5% bovine serum albumin (BSA) in tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and incubated with primary antibodies against PPAR-α, PPAR-γ, C/EBP-α and β-actin overnight at 4 °C. Afterward, the membranes were washed two times with TBST, incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature and rewashed four times with TBST and one time with TBS. The proteins were visualized using an enhanced chemiluminescence (ECL) detection kit (BIO-RAD, Hercules, CA, USA) and a Davinch-In vivo & western imaging system (Davinch-K, Seoul, South Korea).