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. 2018 Jun 6;38(23):5399–5414. doi: 10.1523/JNEUROSCI.3214-17.2018

Figure 2.

Figure 2.

ISP promotes GAG chain degradation in coverslip-bound aggrecan/laminin spot assays. A, CM from 4 DIV or 0.1 U/ml ChABC-treated DRGs were incubated with aggrecan spots for 2 DIV, then stained with CS-56 and analyzed. B, Representative aggrecan spot images. Scale bar, 200 μm. C, ISP degrades aggrecan GAG-CSPGs in a dose-dependent manner (n = 120, 73, 69, 92, 77, 59, 50; F(7,173) = 47.21, CON vs 2.5 μm ISP, p = 0.0001; CON vs 5 μm ISP, p = 0.0001; CON vs 2.5 μm S-ISP, p = 0.0006; CON vs ChABC, p = 0.0001; 2.5 μm ISP vs S-ISP, p = 0.0097; ANOVA). D, ISP degradation of GAG chains is time-dependent and significant at 48 h of incubation with DRGs (n = 47, 31, 32, 41, 30, 39, 78; F(6,291) = 30.77, p = 0.001, ANOVA). E, As a control, equal molar concentrations of peptide alone in media do not degrade GAG chains (n = 34, 27, 28, 22; F(3,107) = 1.684, not significant, p = 0.1748, ANOVA). F, Western blots of CM from DRGs treated with varying concentrations of ISP incubated with 20 μg/ml aggrecan confirm CS-56 spot degradation; n = 4 blots. G, DIPEN, a neo-epitope present once aggrecan is cleaved, increases with ISP dose; n = 3. H, Western blots of CM from vehicle control, 2.5 μm ISP, and 2.5 μm S-ISP incubated with 20 μg/ml aggrecan blotted with CS-56 and DIPEN; n = 2. I, Control Western blots of media incubated for varying times show intact CS-56 and no DIPEN signal; n = 3. Lines indicate median. Boxes represent quartiles. Whiskers indicate range. **p < 0.01, ***p < 0.001.