Figure 3. MMP-9 interacts with CD44 in GBM xenograft cells.

(A) Expression of CD44 and MMP-9 in astrocytes and GBM xenograft cells (4910 and 5310) were determined by immunoblot analysis using specific antibodies. The immunoblot was stripped and re-probed with GAPDH antibody as a loading control. The experiments were repeated three times and a representative blot is shown. (B) 4910 and 5310 cells were cultured for 24 hrs, collected, and lysed in radioimmunoprecipitation (RIPA) buffer. Equal amounts of proteins were co-immunoprecipitated (IP) with anti-CD44, anti-MMP-9, or non-specific antibodies (for negative control). The immunocomplexes were subjected to SDS-PAGE for immunoblotting with specific antibodies. A band of IgG is shown as a control. For gelatin zymography, immunocomplexes were incubated in Laemmli sample buffer for 30 min at room temperature and supernatants were analyzed over gelatin containing SDS-PAGE. The experiments were repeated three times, and a representative blot is shown. (C) Cells were cultured in 8-well chamber slides for 24 hrs. Immunocytochemistry was performed for co-localization (yellow) of CD44 (red) and MMP-9 (green) using specific antibodies. Nucleus was counter stained with DAPI. Also shown is the negative control where the primary antibody was replaced by non-immune serum (Inset). The experiments were repeated three times, and representative pictures are shown. (Micrographs 60X)