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. 2017 Apr 3;216(4):1143–1161. doi: 10.1083/jcb.201606086

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© 2017 Kawabe et al.

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Figure 1. — Differential expression and subcellular localization of Munc13-2 variants in mouse hippocampal neurons. (A) bMunc13-2 and ubMunc13-2 expression levels in mouse hippocampal neurons. (Left) EYFP-fused Munc13-2 isoforms were extracted from cultured hippocampal neurons of Munc13-2^EYFP KI mice. (Right) EYFP-fused Munc13-2 isoforms were immunoprecipitated from detergent extracts of hippocampal synaptosomes from Munc13-2^EYFP KI mice. Extracted proteins or immunoprecipitates (IP) were separated by SDS-PAGE along with recombinant bMunc13-2–EGFP and ubMunc13-2–EGFP (right two lanes) and analyzed by Western blotting (WB) with anti-GFP (top), anti–bMunc13-2 (middle), and anti–ubMunc13-2 (bottom) antibodies. Two GFP-positive bands appeared in samples from Munc13-2^EYFP KI hippocampal neurons (left) and from hippocampal tissue (right), whose molecular weights corresponded to those of recombinant bMunc13-2–EGFP (arrowheads) and ubMunc13-2–EGFP (arrows), respectively. The band with larger molecular weight cross-reacted with the anti–bMunc13-2 antibody, whereas the one with lower molecular weight cross-reacted with the anti–ubMunc13-2 antibody. By densitometric quantification of GFP signals on films using ImageJ, ratios of over 4.5:1 and 6:1 were determined between expression levels of bMunc13-2–EYFP and ubMunc13-2–EYFP in Munc13-2^EYFP KI neurons and in hippocampal tissues, respectively. (B and C) Subcellular localization of Munc13-2 in mouse hippocampal neurons. Cultured autaptic hippocampal neurons from Munc13-2^EYFP KI mice were immunostained for Munc13-1, EYFP, and Bassoon. (B) The overview image of an autaptic neuron shows that AZs containing Munc13-1 only (red) are much more abundant than AZs containing Munc13-2–EYFP (green). Bar, 50 µm. (C) A high-magnification image of the boxed image section in B shows some AZs containing Munc13-1 and Munc13-2–EYFP (arrowheads) among numerous AZs containing only Munc13-1 (arrows). Only 23% of puncta double positive for Munc13-1 and Bassoon were also enriched for Munc13-2–EYFP (n = 1,942 synapses, n = 12 images). Bars, 10 µm.