Figure 6

The effect of proteasome inhibitors on intracellular levels of P-IκB, protein in LPS-stimulated RAW 264.7 cells (Western blots analyses). RAW 264.7 cells (1 × 10⁶ cells/500 μL/well) for 2 h, after 2 h, the cells were treated with 100 μL dissolved in 0.5% DMSO of dexamethasone (10 μM), mevinolin (20 μM), α,-tocopherol (100 μM), δ-tocotrienol (10 μM), riboflavin (40 μM), or quercetin-HCL (40 μM) for 1 h. Then all the wells were challenged with LPS (10 ng/well; 400 μL) and incubated at 37°C 5% CO₂ for 36 h. Cells were washed with phosphate buffer saline, and cytoplasmic extracts were prepared using cell extraction buffer supplemented with phenylmethylsulfonyl fluoride and phosphatase inhibitors, according to the directions of manufacture [20]. Protein concentrations were measured with BCA protein assay kits, and P-IκB-α, levels were measured by Western blots analyses [7, 20]. Data are presented as the percent of P-IκB compared to controls. The treatments 1-7 correspond to: 1. Control (media + cells + LPS [10 ng/mL] + 0.5% DMSO); 2. dexamethasone; 3. mevinolin; 4. α-tocopherol; 5. δ-tocotrienol; 6. riboflavin; 7. quercetin-HCL.