Immunoprecipitation with a MMP-2 antibody had no effect (Fig

Immunoprecipitation with a MMP-2 antibody had no effect (Fig. property. These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the Mangiferin CNS in vivo. and FCS was purchased from PAA. All other chemicals were purchased from at 4C, the pellet was collected and resuspended in 2 vol of 2.25 M sucrose in PBS. The plasma membrane fraction was isolated by centrifugation at 150,000 for 1 h p85-ALPHA at 4C on a discontinuous sucrose density gradient at the 1.52C0.8 M sucrose interphase, resuspended in PBS, and then stored in 500-l fractions at ?70C for further use. Plasma membranes were pelleted, resuspended in Mangiferin 1 vol PBS containing 2 M NaCl, homogenized, and then centrifuged for 1 h at 4C at 100,000 to remove associated proteins. This salt-washed plasma membrane fraction (PM) was resuspended in PBS and 100-l fractions were stored at ?70C for further use. The same procedure, on a smaller scale, was used for the preparation of the fibroblast membranes. Preparation of the bNI-220Cenriched CNS Substrate A CNS-derived inhibitory protein fraction was prepared as described by Spillmann et al. (1997, 1998) with some modifications. In brief, bovine spinal cord (obtained from Schlacthaus Der Stadt Zrich) was cleaned from the meninges, Mangiferin minced, and subsequently homogenized on ice in 1 vol of extraction buffer (100 mM Tris-HCl, pH 8.0, 60 mM CHAPS, 10 mM EDTA, 2.5 mM iodacetamide, 1 mM PMSF, 0.1 g/ml aprotonin, 1 g/ml leupeptin, and 1 g/ml pepstatin A) and extracted for 10 min on a rotary shaker. After pelleting the insoluble material at 100,000 for 1 h at 4C, the clear extract was enriched for inhibitory activity Mangiferin on a Q-Sepharose anion exchange column. bNI-220 is a main inhibitory protein constituent of this fraction. Western Blotting For the evaluation of bNI-220 degradation Mangiferin properties, 10 g of the samples were incubated for 1 h at 37C with 30 g bNI-220Cenriched CNS substrate (see Cell Spreading Assay). Cell homogenates and plasma membrane fractions were prepared as described above and analyzed by 10 or 5% (MT1-MMP blot, NI-220 blot, respectively) PAGE according to Laemmli et al. (1970). The separated proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with 3% gelatin in TBS containing 1% Tween 20 (TBST) for 16 h at 4C and probed with 5 g/ml antiCMT1-MMP antibody or a rabbit anti-NI-220 polyclonal antibody 472 (1:5,000) (Huber et al., 1997). After extensive washing with TBST, the membrane was incubated with goat antiCmouse Ig coupled to HRP or goat antiCrabbit Ig coupled to HRP for 1 h at room temperature. Finally, the blot was developed with the ECL-Western blot detection kit. Zymography Zymography was performed as described by Sawaya et al. (1996). In brief, 15 ng of proMMP2 were preincubated with 10 g plasma membrane for 2 h at 37C and electrophoresed on 10% SDS-PAGE containing gelatin. After electrophoresis, the gels were rinsed twice in 2.5% Triton X-100 and incubated at 37C for 20 h in 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij35. For the evaluation of the different protease inhibitors, the inhibitors were added to the development buffer; for the evaluation of the effect of the inhibitors on the MT1-MMPCmediated activation of proMMP-2, the plasma membranes were.