FIGURE 4.

A, endotoxin LPS induces phosphorylated NFκBp65 Ser(P)⁵²⁹ (pNFκB) in BMC-2 macrophage cells. Cells were cultured on circular glass slides in 24-well tissue culture plates in medium containing 10% fetal calf serum for 24 h at 37 °C in a humidified incubator. Cells were pretreated with the indicated concentrations of MMP9i, in combination with 10 μg/ml LPS for 15 min or left untreated (no ligand). Cells were fixed, permeabilized, and immunostained with phospho-specific polyclonal rabbit antibody against the human NFκBp65 Ser(P)²⁷⁶ (pNFκB), followed by Alexa Fluor594 goat anti-rabbit IgG. Stained cells were visualized by epifluorescence microscopy using a ×40 objective. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean corrected density of staining ± S.E. (error bars) for all cells within the respective images. The data are a representation of one of five independent experiments showing similar results. B, Western blot analyses of phosphorylated NFκB (Ser(P)³¹¹) in nuclear lysates. BMA macrophage cells were pretreated with 100 μg/ml MMP9i or 200 μm Tamiflu for 30 min followed by 5 μg/ml LPS. Nuclear lysates from the cells were separated by SDS-PAGE, and the blot was probed with phospho-specific polyclonal rabbit antibody against NFκBp65 Ser(P)³¹¹ with minimal reactivity with non-phosphorylated p65. Specific NFκBp65 Ser(P)³¹¹ blocking peptide was added to the anti-NFκBp65 Ser(P)³¹¹ antibody in probing the blot. MCM2 (highly conserved minichromosome maintenance complex protein-2) was used as an internal control protein for loading of the nuclear lysate. Cytoplasmic cell lysates from the same samples were separated by SDS-PAGE, and the blot was probed with anti-IκB antibody. Specific IκB blocking peptide was added to the anti-IκB antibody in probing the blot. β-actin was used as an internal control protein for loading of the cytoplasmic cell lysate. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio of NFκBp65 Ser(P)³¹¹ to MCM2 or mean ratio of IκB to β-actin of band density ± S.E. (error bars) for 5–10 replicate measurements. The data are a representation of one of three independent experiments showing similar results. C, Western blot analyses of IκB in cell lysates. BMC-2 macrophage cells were pretreated with 200 μm Tamiflu for 30 min followed by 5 μg/ml LPS. Cell lysates from the cells were separated by SDS-PAGE and the blot probed with anti-IκB antibody. β-Actin was used as an internal control protein for loading of the cytoplasmic cell lysate. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio of IκB to β-actin of band density ± S.E. (error bars) for 5–10 replicate measurements. The data are a representation of one of three independent experiments showing similar results.