Phospho iκbα

Curcuma Longa Linn (powdered form) and lecithin (L-α-phosphatidylcholine) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The organic solvents such as toluene and dichloromethane were purchased from Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from GE Healthcare (Logan, UT, USA). The following antibodies were obtained: c-Src, phospho-c-Src, PKC, phospho-PKC, JNK, phospho-JNK, p38, phospho-p38, ERK, phospho-ERK, IκBα, phospho-IκBα, NF-κBp65, phospho-NF-κBp65, Bcl-2, Bax, cleaved caspase-3, and β-actin antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA); The following reagents were obtained: N-acetylcysteine (NAC) (Tocris, KOMA Biotech, Seoul, Korea) and 5-(and-6)-chloromethyl-2′,7′- dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) (Invitrogen, Carlsbad, CA, USA). PP2, SP600125, Bisindolylmaleimide I, and Bay11-7082 were obtained from MedChemExpress (Monmouth Junction, NJ, USA). All other reagents did not show any critical cytotoxic effects by themselves.

Fisetin (>98% pure), PD98059 and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO). Caffeic acid phenethyl ester (CAPE) was purchased from MP Biologicals (Solon, OH). Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA). The monoclonal and polyclonal antibodies for MEK1/2, phospho-MEK1/2 (Ser²¹⁷/Ser²²¹), ERK1/2 (phospho-p44/42, Thr²⁰²/Tyr²⁰⁴), E-cadherin, N-cadherin, vimentin and snail were obtained from Cell Signaling Technology (Beverly, MA). Monoclonal antibodies for NFκB p50, NFκB p65, IKKα, IκBα, phospho-IκBα, fibronectin and desmoglein were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-mouse, anti-goat and anti-rabbit secondary antibodies horseradish peroxidase conjugate were obtained from Millipore Corporation (Billerica, MA). Alexa Flour 488 or 594 labeled anti-mouse, anti-goat and anti-rabbit secondary antibodies were obtained from Life Technologies Corporation (Grand Island, NY).

Carnosic acid was purchased from Cayman Chemical (Ann Arbor, MI, USA). Fetal bovine serum (FBS) was obtained from Biowest (Riverside, MO, USA). Dulbecco`s modified Eagle`s medium (DMEM), bovine calf serum (BCS), Fetal bovine serum (FBS), trypsin-EDTA, phosphate-buffered saline (PBS, pH 7.4), and Hank`s balanced salt solution (HBSS) were purchased from Welgene Inc. (Gyeongsan, Korea). Dimethylsulfoxide (DMSO), antibiotic-antimycotic solution 100X, insulin, 3-isobutyl-1-methyl-xanthine (IBMX), dexamethasone, isopropanol, formaldehyde, DCFH-DA, DHE, protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), Triton X-100, 1,4-dithiothreitol (DTT), skim milk, and DPAI (4′,6-diamidino-2-phenylindole) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies of Nox4, p47^phox, p22^phox, NF-κB (p65), phospho-NF-κB (p65), IκBα, phospho-IκBα, C/EBPα, C/EBPβ, C/EBPγ, HO-1, γ-GCSc, GST, and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-adiponectin was obtained from Cell Signaling Technology (Beverly, MA, USA). 3T3-L1 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Livers were homogenized in RIPA lysis buffer (EMD Millipore Corp, Temecula, CA, USA), containing 10 µL/mL of phosphatase I and II inhibitor and protease inhibitor (Sigma-Aldrich, St. Quentin Fallavier, France). Tissue homogenates were centrifuged for 15 min at 10,000 g, and supernatants were stored at −80 °C. Equal amount of proteins (40 µg) were run on a 4–15% gradient Mini-protean TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred onto Trans-Blot Turbo nitrocellulose membranes (Bio-Rad, Marnes-la-Coquette, France) and blotted with the following primary antibodies: phospho-IκBα (1/500), total-IκBα (1/500), Nuclear Factor kappa-B (NFκB)p65 (1/200) (all Santa Cruz Biotechnology, Heidelberg, Germany) and β-actin (1/5000, Sigma-Aldrich, St. Quentin Fallavier, France). Anti-mouse, anti-goat, or anti-rabbit IgG labeled with Dylight 800 or Dylight 680 was used as secondary antibodies. Proteins were determined by Infrared fluorescent detection (Odyssey, LI-COR Biosciences, Boulogne-Billancourt, France) and quantified using Image Studio Lab v5.2 software (LI-COR Biosciences, Boulogne-Billancourt, France).

Dried BV was purchased from You-Miel Bee Venom Ltd. (Hwasoon, Jeonnam, Korea). The composition of the BV was as previously described [26 (link)]. Caspase-3, caspase-8, caspase-9 antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA). DR3, DR4, DR5, Fas, TRAIL, IκBα, phospho-IκBα, p50, p65, p21, p53, Bcl-2, Bax, Histone-H1 and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The cell culture materials were obtained from Invitrogen (Carlsbad, CA), and other chemical reagents were from Sigma Chemical Co.

Western blotting was performed as previously described.^{43 (link), 44 (link)} Briefly, the cells were collected and lysed using lysis buffer containing protease inhibitor (Sigma-Aldrich) on ice for 30 min followed by centrifugation. Protein concentrations in the supernatants were determined using a protein assay kit (Bio-Rad), and samples were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking for nonspecific binding, the blots were incubated with specific antibodies against BST2 (Abcam, Cambridge, MA, USA), cleaved PARP (c-PARP), Bcl-X_L, livin (Cell Signaling Technology, Beverly, MA, USA), IκBα, phospho-IκBα, actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GAPDH (Proteintech Group, Chicago, IL, USA), followed by reaction with an HRP-conjugated secondary antibody (Cell Signaling Technology). Signals were enhanced with the ECL detection system (Amersham Biosciences, Piscataway, NJ, USA) and captured using X-ray film.