Las 4000 imaging device

The MCF-7 and T47D cell lines were treated with nobiletin for 24 h. Whole cells were lysed on ice with radio immunoprecipitation lysis buffer containing phosphatase and protease inhibitors. Cells were disrupted and centrifuged at 15,000 rpm for 10 min at 4 °C to remove cellular debris. Protein concentrations were measured using the Bradford method. Equal amounts of proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane. The blots were blocked for 1 h with 5% BSA. Membranes were probed overnight at 4 °C with a primary antibody followed by HRP-conjugated secondary antibodies. Detection was performed using the ECL Plus detection kit and an LAS-4000 imaging device (Fujifilm, Japan).

Whole cell lysates were prepared from untreated or TA-treated NCCIT cells by incubating them on ice with radio immunoprecipitation lysis buffer (20-188; EMD Millipore) containing phosphatase and protease inhibitors to isolate protein. The protein concentrations were measured via the Bradford method (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts of protein (100 μg/well) were resolved with 10% SDS-PAGE. Then, the separated proteins were transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skim milk (BD Biosciences, San Jose, CA, USA; 90002-594) in TBS-T buffer (20 mM Tris-HCl (Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA; 10708976001), pH 7.6, 137 mM NaCl (Formedium, Norfolk, UK; NAC03), 0.1X Tween 20 (Scientific Sales, Inc. Oak Ridge, TN, USA; 0777)). The membranes were then incubated overnight at 4 °C in a shaker with primary antibodies diluted in 5% bovine serum albumin (EMD Millipore). The membranes were then washed with TBS-T and incubated for 1 h at room temperature with Horseradish peroxidase (HRP)-conjugated secondary antibodies. Detection was performed with a Femto Clean Enhanced Chemiluminescence Solution Kit (GenDEPOT; 77449; Katy TX) and a LAS-4000 imaging device (Fujifilm, Tokyo, Japan).

The MCF-7 and MDA-MB-231 cell lines were treated with nobiletin, recombinant CD36 protein, SSO or palmitic acid for 24 h. Whole cells were lysed on ice with radio immunoprecipitation lysis buffer containing protease and phosphatase inhibitors. Cells were scrapped out and centrifuged at 15,000 rpm for 10 min at 4 °C to remove cellular debris. Protein concentrations were measured using the Bradford method. Equal amounts of proteins were resolved on sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane. The blots were blocked for 1 h with 5% BSA (Bovine serum albumin). Membranes were probed over night at 4 °C with a primary antibody followed by HRP-conjugated secondary antibodies. Detection was performed using the ECL Plus detection kit and an LAS-4000 imaging device (Fujifilm, Japan).

Protein samples were isolated from untreated (control) or silibinin-treated H292, A549, and H460 cells using radioimmunoprecipitation lysis buffer (20-188; EMD Millipore) containing phosphatase and protease inhibitors. The concentration was measured using Bradford’s method (Thermo Fisher Scientific). Equal amounts of protein (100 μg/well) were dissolved with 10% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Separated proteins were then transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skim milk (BD Biosciences, San Jose, CA, USA) in TBS-T buffer (20 mM Tris-HCl (Sigma-Aldrich; Merck KGaA), pH 7.6, 137 mM NaCl (Formedium, Norfolk, UK; NAC03), and 0.1× Tween 20 (Scientific Sales, Inc., Oak Ridge, TN, USA)). The membranes were then incubated overnight at 4 °C in a shaker with primary antibodies diluted in 5% bovine serum albumin (EMD Millipore). The membranes were then washed with TBS-T and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. Detection was performed using a Femto Clean Enhanced Chemiluminescence Solution Kit (77449; GenDEPOT, Katy, TX, USA) and a LAS-4000 imaging device (Fujifilm, Tokyo, Japan).

Whole-cell lysates were prepared by incubating the untreated or UA-treated A549 and H460 cells on ice with the RIPA lysis buffer (20–188; EMD Millipore) containing protease and phosphatase inhibitors. Protein concentrations were measured using the Bradford assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts of protein at 100 µg/well were resolved with 10–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skim milk (BD Biosciences, San Jose, CA, USA) in TBS-T buffer (20 mM Tris–HCl) (Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA), pH 7.6, 137 mM NaCl (Formedium, Norfolk, UK; NaCO₃), and 0.1 × Tween 20 (Scientific Sales, Inc., Oak Ridge, TN, USA). Next, the membranes were incubated with primary antibodies (0.5–1 µg/mL) diluted in 5% bovine serum albumin (EMD Millipore) overnight at 4 °C on a shaker, washed with TBS-T, incubated with HRP-conjugated secondary antibodies (1–2 µg/mL) for 1 h at room temperature, and detected using a Femto clean enhanced chemiluminescence solution kit (GenDEPOT; 77449; Katy, TX, USA) and an LAS-4000 imaging device (Fujifilm, Tokyo, Japan).

Protein samples were isolated from untreated (control) or 6-gingerol-treated NCCIT or NTERA-2 cells using radioimmunoprecipitation (RIPA) lysis buffer (20–188; EMD Millipore), which contained protease and phosphatase inhibitors. First, the concentration of proteins was measured using Bradford’s method (Thermo Fisher Scientific). Next, 100 μg of protein from each sample were resolved with sodium dodecyl sulfate-polyacrylamide gel (10%–15%) electrophoresis. Then, the separated proteins were transferred onto nitrocellulose membranes, followed by blocking with 5% skim milk (BD Biosciences, San Jose, CA, USA) in TBS-T buffer [20-μM Tris-HCl (Sigma-Aldrich; Merck KGa A), pH 7.6, 137-μM NaCl (Formedium, Norfolk, UK; NAC03), and 0.1× Tween20 (Scientific Sales, Inc. OakRidge, TN, USA)] for 1 h. Next, the membranes were incubated overnight at 4°C in a shaker with specific primary antibodies diluted in 5% bovine serum albumin (EMDMillipore). Then, the membranes were washed with TBS-T and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the detection was performed using a Femto Clean Enhanced Chemiluminescence Solution Kit (77449; GenDEPOT, Katy, TX, USA) in a LAS-4000 imaging device (Fujifilm, Tokyo, Japan). Quantifications were conducted using ImageJ software (v.1.8.0_172; National Institutes of Health).

Whole-cell lysates were prepared by incubating untreated or LPS, mannitol, 5.5 mM glucose, or 25 mM glucose-treated THP-1 cells on ice with the radioimmunoprecipitation lysis buffer (20–188; EMD Millipore) containing protease and phosphatase inhibitors. Protein concentrations were measured using the Bradford method (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The same amounts of protein (100 μg/well) were resolved with 10–15% SDS-PAGE. Separated proteins were then transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skim milk (BD Biosciences, CA, USA) in a TBS-T buffer (20 mM Tris-HCl (Sigma-Aldrich; Merck KGaA, St. Louis, Missouri), pH 7.6, 137 mM NaCl (Formedium, Norfolk, UK; NAC0₃), 0.1× Tween 20 (Scientific Sales, Inc., Oak Ridge, TN, USA)). The membranes were then incubated overnight at 4 °C in a shaker with primary antibodies diluted in 5% skim milk. The membranes were then washed with TBS-T and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies. Detection was performed using a Femto Clean Enhanced Chemiluminescence Solution Kit (GenDEPOT; 77449; Katy, TX, USA) and a LAS-4000 imaging device (Fujifilm, Tokyo, Japan).