Gas7 localizes at the presynaptic region of the NMJ and is associated with axon terminal sprouting.

<p>(A) Gas7 aggregates at the axon terminal region of NMJ in wild-type (WT) soleus. Only weak Gas7 expression was detected in the NMJs of the soleus from the Gas7-deficient (MT) mice. (B) Gas7 co-localizes with synaptophysin partially. Images show separate and merged signals of Gas7, synaptophysin, nAChR cluster, and nuclei. FITC-conjugated Gas7 antibody was used to visualize Gas7 localization (green); Cy5-conjugated NFM antibody indicates neurites of axon terminals (red) or Cy5-conjugated synaptophysin indicates the presynaptic vesicles (red); Cy3-conjugated α-BTX was used to identify nAChR cluster (blue), and nuclei were visualized using Hoechst stain. White arrows mark co-localization areas. Scale bar, 10 µm. (C) The 3-D construction was obtained from 13 slides. It shows that Gas7 co-localizes with synaptophysin in the NMJ (black arrows). The resolution in the z-direction in this image is 1 µm. (D) and (F) The shape of the NMJ and the number of branches of the axon terminal were determined by immunofluorescence. The presence of small nAChR clusters containing nerve termini can be observed at the end of a sprout (red arrow). NFM staining was used to determine nerve axons (green). nAChRs were stained with α-BTX (red). (E) The number of major nerve terminal branches (numbered 1–6 in D) per NMJ did not differ significantly in old wild-type (WT) and Gas7-deficient (MT) mice. (G) Compared to wild-type (WT) mice, the percentage of NMJs exhibiting terminal sprouts was significantly lower in Gas7-deficient (MT) mice (n = 3 mice in each group, 50 NMJs). Mean ± SEM, **<i>P</i><0.01.