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. Author manuscript; available in PMC: 2018 Feb 1.
Published in final edited form as: Brain Behav Immun. 2016 Nov 7;60:282–292. doi: 10.1016/j.bbi.2016.11.003

Figure 5. MT1-MMP releases MBP69-86 peptides in vitro and in CCI nerve.

Figure 5

A, SDS-PAGE of MBP (11 μM) co-incubated for 60 min at 37°C with MT1-MMP (0.1 μM) at an enzyme-substrate ratio of 1:100 and 1:1,000. Where indicated, GM6001 was added to the reactions. B, Representative MALDI-TOF MS spectra of the resulting MBP peptide sample in A. The high and low molecular mass fragments are shown in the top and bottom panels, respectively. The intact and the digested MBP samples are in red and magenta, respectively. The buffer alone, green line. The numbers in the parentheses show the numbering of the peptide in the MBP sequence. a.u., arbitrary unit. C, MBP69-86 immunofluorescence (AB5864, Millipore, red) in nerve after i.n. hAb-DX2400 or IgG1 (1.0 mg/ml, each) injection at day 9 post-CCI alone (top panel) or with CD68 (green) in IgG1-treated CCI nerve (bottom panel); DAPI nuclear stain, blue. The algesic MBP69-86+ fragments are not phagocytosed by macrophages. Scale bar, 50 μm. D, The graph of the mean MBP69-86 immunoreactive (IR) as the percentage of total area of C. in n=3/group, 3 sections (≥90 μm apart) per n and 3 randomly selected areas per section at 400x magnification ± SEM, *, p≤0.05; **, p ≤0.0001, by one-way ANOVA with Tukey’s post-hoc test.