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. 2011 Sep 28;31(39):13972–13980. doi: 10.1523/JNEUROSCI.1289-11.2011

Figure 2.

Figure 2.

An EHD1/3 ortholog with conserved properties is enriched at synaptic release sites. A, Liposome sedimentation assay with l-EHD analyzed by Western blot. B, Binding of Alexa 488-labeled EHD (0.5 μm) to unlabeled fluid-supported bilayers with excess membrane (SUPER templates). Scale bar, 5 μm. C, Tubulation of liposomes by l-EHD. Electron micrographs show negatively stained liposomes with no protein added (upper panel) or incubated with 1.6 μm l-EHD (lower panel). Scale bars: 0.25 μm. D, Antibodies against the EH domain of l-EHD recognized a 60 kDa protein in lamprey brain extract (left lane). No cross-reactivity with bands corresponding to intersectin (middle lane: Western blot with intersectin antibodies) or eps15 (right lane: Western blot with eps15 antibodies) was observed. E, Immunoprecipitation of lamprey brain extract with l-EHD antibodies and control antibodies (Coomassie staining). F, Western blot of bound and unbound material from immunoprecipitation with l-EHD antibodies. Neither intersectin nor eps15 could be detected in the bound material. The l-EHD antibodies efficiently depleted l-EHD, but neither intersectin nor eps15, from the extract. G, Tissue distribution of l-EHD. Extracts from different lamprey tissues were blotted with l-EHD antibodies and with antibodies to VAMP2. H, l-EHD visualized at synaptic release sites in living reticulospinal axons following double microinjection of Alexa 488-labeled l-EHD antibodies (upper panels), and Alexa 546-labeled synaptotagmin antibodies (middle panels; lower panels show merged images). Low-magnification (left panels, scale bar, 30 μm) and high-magnification (right panels, scale bar, 2 μm) images are shown. Each spot corresponds to a synaptic vesicle cluster. The diffuse labeling in the axon may reflect unbound antibodies or antibodies bound to a cytoplasmic pool of EHD. The labeling pattern observed with the microinjected l-EHD antibodies is similar to that seen with antibodies to other endocytic proteins (Evergren et al., 2004a; Andersson et al., 2008; Jakobsson et al., 2008).