Fig. 4

MMP excretion in female and male rats post-CCI. a MMP activity in urine of female rats. The urine samples collected at day 0 (prior to injury) and days 1 and 28 post-CCI (n = 4/group) were equilibrated in MMP buffer, pH 7.5, and then the protein concentrations were determined using the Bradford assay and made even by sample dilution in MMP buffer, pH 7.5. Dialyzed urine (50 μl each) was co-incubated with the fluorescent Mca-PLGL-Dpa-AR-NH₂ MMP substrate. Where indicated, GM6001 (10 μM) was added to the samples to inhibit MMP activity. The numbers indicate the MMP-specific cleavage activity. Data are means ± SD from multiple individual measurements performed at least in duplicate. b Gelatin zymography of female rat urine (5 μl of each normalized sample) collected at day 0 and days 1 and 28 post-CCI (representative of n = 4/group). Gels were incubated in the absence and the presence of 20 mM EDTA (− EDTA and + EDTA, respectively). c Specific urinary MMP activity in female and male rats. The urine samples collected at day 0 and 28 post-CCI (n = 4–5/group) were equilibrated in MMP buffer, pH 7.5, and then the protein concentrations were determined using the Bradford assay. Dialyzed urine samples (50 μl, each) were co-incubated with the fluorescent Mca-PLGL-Dpa-AR-NH₂ MMP substrate in the presence and the absence of GM6001 (10 μM). The specific MMP activity (RFU without GM6001–RFU with GM6001) was normalized to the protein concentrations. Data are means ± SD from multiple individual measurements performed at least in duplicate. RFU relative fluorescence unit, MMP-9 the latent MMP-9 control from HT1080 cells, NS non-MMP activity band