Coomassie brilliant blue solution

The IC₅₀ of TQ; 5.5 µM and 15 μM were used to treat MV4-11 and K562 cells, respectively, for 48 h. After that, the treated and untreated cells were lysed with RIPA buffer containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor solution (Nacalai Tesque, Kyoto, Japan). The protein concentrations were measured by Bradford protein assay [49 (link)], using bovine serum albumin (BSA) and Coomassie brilliant blue (CBB) solution from Nacalai tesque, Inc. (Kyoto, Japan).

Dulbecco’s modified Eagle’s medium (DMEM), LPS (E. coli O128:B12), and dexamethasone (Dex) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, antibiotics, and cloning vectors were purchased from Life Technologies (Carlsbad, CA, USA). HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The anti-mouse LCN2 antibody was purchased from Abnova (Taipei, Taiwan). Goat anti-rabbit IgG was obtained from GE Healthcare Life Sciences (Buckinghamshire, UK). Complete Mini, EDTA-free protease inhibitor cocktail was from Roche (Mannheim, Germany). The DNA ligation kit and EX Taq were purchased from Takara Bio (Shiga, Japan). PNGase F, Endo H, and Neuraminidase (NA) were obtained from New England BioLabs (Herts, UK). Coomassie brilliant blue (CBB) solution and the Rapid Stain CBB kit were procured from Nacalai Tesque (Kyoto, Japan). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

DC2.4 cells were placed into 24-well plates (Corning Inc., Corning, NY, USA) at a density of 1.0 × 10⁴ cells/well and cultivated in 500 µL culture medium. After 24 h of pre-incubation, the cells were treated with 0.25 μg/mL, 0.5 μg/mL, 1 μg/mL, and 2 μg/mL of each pDNA ternary complex for 2 h. After 22 h of incubation, the cells were washed with phosphate-buffered saline and then lysed in 100 µL lysis buffer (pH 7.8; 0.1 M Tris/HCl buffer containing 0.05% Triton X-100 and 2-mM ethylenediaminetetraacetic acid). A protein assay Coomassie brilliant blue solution (Nacalai tesque Inc., Kyoto, Japan) was used to determine the protein content of the lysate, with BSA as a standard, and a microplate reader (Infinite-200Pro M-Plex, Tecan Japan Co., Ltd., Kanagawa, Japan) at 595 nm was used to measure absorbance. A PicaGene luminescence kit and a luminometer (Lumat LB 9507; EG & G Berthold, Bad Wildbad, Germany) were used to determine luciferase activity. Luciferase activity is shown as relative light units (RLUs) per mg protein.
To assess the time-dependent transfection activity of the promoters, DC2.4 cells were treated with 2 μg/mL of each pDNA ternary complex for 2 h as described above. Luciferase activity was measured at 6, 12, 24, and 48 h after treatment.