FIGURE 7.

MgSO₄ influences NF-κB activation. (A) LPS-induced IκBα gene expression is decreased in the presence of magnesium supplementation. CBMCs were stimulated with 50 pg/ml LPS, and relative IκBα gene expression, normalized to a stably expressed housekeeping gene, was assessed using real-time PCR at various time points. Gray circles/dashed line delineate magnesium-supplemented samples; black squares/solid line delineate control samples; error bars indicate the SEM of triplicate samples. (B) Magnesium exposure decreases phosphorylated NF-κB p65 (S536) levels following TLR stimulation. CBMCs were stimulated for 30 min, lysed, and proteins resolved by SDS-PAGE followed by Western blotting. Proteins were identified with specific rabbit polyclonal Abs and detected via HRP-conjugated secondary Abs and electrochemi-luminescence. Detection of β-actin demonstrates comparable protein loading; magnesium supplementation and LPS exposure are indicated at the bottom. (C) Magnesium reduces NF-κB p65 nuclear localization. Neonatal monocytes were stimulated with LPS for 30 min; nuclear extracts were prepared and analyzed via Western blotting using labeled Abs and an infrared imaging system (Odyssey; Li-Cor Biosciences). TFIID quantitation was used as a protein loading control and the p65/TFIID ratio, representing relative p65 abundance in the nucleus, is shown beneath the blot. Results are representative of three experiments.