FIGURE 9.

A, MMP9 colocalizes with TLR4. BMA macrophage cells were treated with 5 μg/ml LPS for 5, 15, 30, and 45 min or left untreated as controls. Cells were fixed, non-permeabilized, and immunostained with rat anti-mouse TLR4 (HTS510, Santa Cruz Biotechnology, Inc.) and rabbit anti-mouse MMP9 (H-129, Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor594 goat anti-rabbit IgG or Alexa Fluor488 rabbit anti-rat IgG. Stained cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted confocal microscope) with a ×100 oil objective. Images were captured using a z-stage of 8–10 images/cell at 0.5-mm steps and were processed using ImageJ version 1.38x software. To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using ImageJ version 1.38x software. B, flow cytometry analysis of MMP9 expressed on the cell surface of live BMA macrophage cells following LPS treatment for 5 min as described in the legend to Fig. 7A. MMP9 co-immunoprecipitates with TLR4 (C), and conversely, TLR4 co-immunoprecipitates with MMP9 (D). BMA macrophage cells are left cultured in medium or in medium containing 5 μg/ml LPS. Cells (1 × 10⁷ cells) are pelleted and lysed in lysis buffer. MMP9 and TLR4 in cell lysates from BMA cells are immunoprecipitated with 1.0 μg of rabbit anti-MMP9 or 2 μg of rat anti-TLR4 antibodies for 24 h. Following immunoprecipitation, complexes are isolated using protein A or G magnetic beads and resolved by 8% gel electrophoresis (SDS-PAGE). The blots are probed for TLR4 (88 kDa) with anti-TLR4 or MMP9 (78 or 84 kDa) with anti-MMP9 antibodies followed by Clean-Blot IP Detection Reagent for immunoprecipitation/Western blots and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with x-ray film. Sample concentration for gel loading was determined by Bradford assay. Quantitative analysis was done by assessing the density of TLR4 or MMP9 bands corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio corrected density of TLR4 over the MMP9 band for 6–8 replicate measurements within each lane. The data are a representation of one of five independent experiments showing similar results. IP, immunoprecipitation.