FIGURE 5.

Cleavage-resistant NgR1 is insensitive to MMPs. A, schematic of the WT-NgR1 cleavage site (indicated by a vertical line) and the mutations generated and tested for cleavage resistance. CE, cleavage enhanced. CR2–CR5, cleavage-resistant 2–5. B, Western blot analysis of conditioned medium and lysate from SH-SY5Y cells transfected with FLAG-tagged WT-NgR1 or mutant constructs. C, COS-7 cells were transfected with FLAG-NgR1 and p75 constructs. Cells were subjected to immunoprecipitation (IP) with an anti-FLAG antibody and analyzed for precipitating NgR1 and p75 by Western blotting. D, cell-surface FLAG staining of COS-7 cells transfected with FLAG-WT-NgR1 or FLAG-CR4-NgR1 and treated with 0.75 μm MT1-MMP (MT1), 1.5 μm MT3-MMP (MT3), or 1 unit/ml PI-PLC for 3 h. Scale bar = 10 μm. E, FLAG-positive cells were counted and are expressed as a percentage of total cells as identified by Hoechst counterstaining. Quantification is from three separate experiments. *, p < 0.001 by two-way ANOVA with a Bonferroni post hoc test. rec, recombinant.