Pierce bca protein assay reagent

E. coli BL21 cells (Novagen, Madison, WI) containing p0809C304 were grown to late log phase at 25°C in APS^™ superbroth (Difco, Detroit, MI) containing 25 μg/mL chloramphenicol. Protein expression was induced with 0.02% arabinose for three hours. Cells were harvested by centrifugation, resuspended 1:4 (w/v) with phosphate buffered saline (PBS) containing 5 mM imidazole, and lysed by two cycles of microfluidization (model M-110Y apparatus; Microfluidic Corp., Newton, Mass.). The lysate was centrifuged at 17000 x g. Supernatant was loaded onto a XK16 column (GE Healthcare, Piscataway, NJ) containing 10 mL of Talon® Superflow resin (Clontech Laboratories, Inc., Mountain View, CA). The column was washed with 10 mM imidazole and chimera was eluted with 50 mM imidazole. The eluate was pooled, diluted tenfold with 20 mM sodium phosphate buffer pH 6.5, and applied to a 5 ml HiTrap SP column (GE Healthcare). Chimera was eluted using a 0–400 mM sodium chloride (NaCl) linear gradient over 30 column volumes (CVs). Eluates were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and western blot. Protein concentrations were determined by Pierce^™ BCA protein assay Reagent (Thermo Fisher Scientific, Waltham, MA). Fractions containing chimera were pooled, sterilized by passage through a 0.22 μM filter (Millipore, Billerica, MA) and stored at -80°C.

Human breast carcinoma cells (MDA-MB-231, MDA-MB-435, T47D, and MCF7) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, MD, USA). Mouse mammary carcinoma cells (4 T1) were kindly provided by Dr. Jeong-Seok Nam (Gwangju Institute of Science and Technology, Gwangju, Korea). Cells were grown in Dulbecco’s modified Eagle’s medium (Corning Cellgro, Manassas, VA, USA) supplemented with 10 % (v/v) heat-inactivated foetal bovine serum (Corning Cellgro). TNFα, methanol, n-hexane, chloroform, ethyl acetate, n-butanol, dimethylsulfoxide (DMSO), Tris-base, HEPES, Triton X-100, glycerol, leupeptin, phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). SDS, acrylamide, bisacrylamide, ammoniuk persulfate, nitrocellular membranes were from Bio-Rad Laboratories (Hercules, CA, USA). Pierce™ BCA Protein Assay Reagent was obtained from Thermo Scientific (Rockford, IL, USA).

Cells were collected by centrifugation at 10,000 g for 2 min and were suspended with BugBuster protein extraction reagent (Novagen) for protein extraction according to the manufacturer's instructions. The supernatant of the cell crude extract was subjected to a protein assay (Pierce BCA protein assay reagent, Thermo Fisher Scientific) according to the manufacturer’s protocol. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of cell pellets, which were suspended with phosphate-buffered saline (PBS) buffer (Sigma), was performed with a precast gel (12% Mini-PROTEAN TGX Precast Gel, Bio-Rad) and was blotted with a semidry system (Trans-Blot Turbo Transfer System and mini PVDF Transfer Pack, Bio-Rad). The proteins were detected with primary antibodies (0.1–0.5 μg/mL) derived from mice or rabbits followed by the relevant IgG-HRP conjugates (1,000- to 5,000-fold dilution). After development with a chemiluminescent substrate (Advance Western Blotting Detection Kit, GE Healthcare), the proteins were detected using a luminescent imaging analyzer (Image Quant 350, GE Healthcare). The standard curves for the quantitative evaluation of cellular GFP and S1 ranged from 1–50 ng per lane. The number of cells loaded per lane varied from 1×10⁶ to 2×10⁷ cells according to the preliminary estimated sensitivity in detection.

3T3-L1 cells were differentiated into adipocytes for 6 days in DMEM in the presence or absence of CG-1. Intracellular triglyceride levels were measured by using a WAKO LabAssay Triglyceride Kit (FUJIFILM Wako Pure Chemical, Osaka, Japan). Protein concentrations were determined by the use of a Pierce BCA Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The intracellular triglyceride concentration was normalized to the protein concentration.