Skip to main content
. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Mol Psychiatry. 2015 Apr 14;20(8):986–994. doi: 10.1038/mp.2015.45

Figure 2.

Figure 2

Distinct NF populations in synapses and axons. Ultrastructural colocalization of NFL and NFH (a, b, c) or NFL with NFM (d, e, f) on the same NF or on a filamentous web within the postsynaptic terminal by pre-embedding (a) or post-embedding immunoelectron microscopy (b–g). Paraformaldehyde-fixed samples were incubated with mouse anti-NFL or rabbit anti-NFH or NFM antibodies and probed with goat anti-mouse IgG and goat anti-rabbit IgG conjugated to 15 and 10 nm gold beads. a shows an enlarged spine containing NF decorated by both NFL (arrowheads) and NFH (arrows). b shows a filament decorated by only NFH (arrows). d shows a filament decorated by both NFL (arrowheads) and NFM (arrows). f shows two linear structures decorated by NFM (arrows) and one of them is in close contact with a vesicle (arrow). d shows a NF decorated by both NFL (arrowhead) and NFM (arrows) in an axon. Scale bars, 200nm in a; 100nm in b, c, d, e, g; 50nm in f. Pre, presynaptic terminal; Post, postsynaptic terminal. Western blot profiles of fractions 1–18 from 0–30% Optiprep gradient separation of hippocampal post-nuclear supernatant showing NF proteins are associated with synaptosomes (j). j Ponceau S stain and immunoblots stained with corresponding antibodies. Total NF (mainly axonal) and synaptosomal NF were isolated from striatum (i, j, k) or hippocampus (l, m, n) and quantified after immunoblotting with corresponding antibodies. (i) relative ratios of α-internexin to NFL in fractions of total NF and synaptosomal NF. j relative ratio of phosphorylated NFM (RMO55) to total NFM(RMO44) in total NF and synaptosomal NF. (k) relative ratio of phosphorylated NFH (SMI31) to non-phosphorylated NFH (SMI33) in total NF and synaptosomal NF. (l) relative ratios of α-internexin to NFL in fractions of total NF and synaptosomal NF. (m) relative ratio of phosphorylated NFM (RMO55) to total NFM(RMO44) in total NF and synaptosomal NF. (n) relative ratio of phosphorylated NFH (SMI31) to non-phosphorylated NFH (SMI33) in total NF and synaptosomal NF. (o) proportion of NFH relative to NFL increased in the synaptic pool compared to the total pool in striatum of MKO mice. (p) proportion of NFH relative to α-internexin increased in the synaptic pool compared to the total pool in striatum of MKO mice. (q) relative ratio of phosphorylated NFH (SMI31) to non-phosphorylated NFH (SMI33) in total NF and synaptosomal NF from striatum of MKO mice. ANT: α-internexin; PSD95: postsynaptic density protein 95; WT, wild-type; TKO, α-internexin, NFH and NFL triple knockout mice. IHL-TKO, α-internexin, NFH and NFL triple knockout mice. t-NF, total NF; s-NF, synaptic NF; MKO, NFM knockout mice.