Figure 4. MT3-MMP mediates synaptic NgR1 shedding from cortical neurons. a, Schematic representation of soluble Ecto-NgR1 (1–358) and CT-NgR1 (359–410) fragments generated by surface NgR1 proteolysis. b, Crude membrane extract from WT and NgR1-KO mice probed with commercially available mouse anti-NgR1 polyclonal antibody. c, Cell-surface biotinylation of mouse dissociated cortical neurons treated with 0.75 μm rec-MT1-MMP and PI-PLC, probed with commercially available mouse anti-NgR1 polyclonal antibody. d, Membrane extracts from mouse cerebral cortex (P10–P30) probed for NgR1 and PSD95. e, Densitometry analysis of full-length NgR1 and CT-NgR1 during cortical mouse development. f, Synaptosomes from mouse cerebral cortex fractionated into extrasynaptic (Extra), presynaptic (Pre) and postsynaptic (Post) compartments. PSD95, SynPhy, and Lingo-1 were used as markers for synaptic subfractions. g, Conditioned media and lysates from cortical synaptosomes treated with pan-metalloprotease inhibitors, BB-94 and GM6001, DMSO, and GM6001-inactive control (GM-I). N-Cadherin (N-Cad) expression in media and lysates was used as a control MMP-substrate. h, i, NgR1 shed fragments in the conditioned media and quantification (protein densitometry) from MT3-MMP and MT5-MMP knockdowns in cortical neurons aged for 14 DIV. N = 3–7 from independent brains, or dissociated cortical cultures. Data are mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, by Bonferroni post hoc test.