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. 2016 Jan 13;36(2):590–606. doi: 10.1523/JNEUROSCI.2300-15.2016

Figure 4.

Figure 4.

Hasp localizes Hig to synaptic clefts of cholinergic synapses. A, Localization of Hig in synaptic regions of haspD1 brains. Hig signals disappeared throughout the synaptic regions in haspD1 (indicated by brackets) but were present in neuronal cell bodies (arrowheads). Loss of Hig in synaptic regions was restored in rescued flies (elav-Gal4/Y; haspD1; UAS-hasp-HA/+). The level of Hasp was unchanged in higdd37 brains, including synaptic regions. Anti-Brp antibody was used to label the synaptic regions. al, Antenna lobe; sog, subesophageal ganglion. B, Quantifications of Hig, Hasp, and Brp in antennal lobes (al). The number of samples examined: wild-type, N = 22; haspD1, N = 12; higdd37, N = 8; Rescued, N = 10. Each optical section was obtained from a distinct brain. Error bars indicate ± SEM. **p < 0.01, relative to wild-type (Steel–Dwass test). NS, Not significantly different. C, Western blotting of Hig and Hasp proteins. Extracts from adult heads of wild-type, higdd37, and haspD1 flies were probed with antibodies for Hig (left) or Hasp (middle and right). Right blot, One-fourth of each sample used in the middle blot was loaded to clarify the patterns of S3 and S4 bands at the cost of full-length (FL) bands. β-Actin was used as a loading control. All Hig bands (two FLs and S) in haspD1 flies and most Hasp bands (FL and S1–3) in higdd37 flies were present nearly at wild-type levels. The level of Hasp S4 was elevated in higdd37 (see right blot). The two FL bands of Hig may have been produced by splicing (Hoshino et al., 1993). Several Hasp bands are likely to be generated by either splicing or cleavage (Fig. 1A; see below). Molecular markers (kDa) are indicated on the right of each blot. D, Quantification of Hig and Hasp bands in wild-type, higdd3, and haspD1 flies. The levels of Hasp S1 and S2 were slightly changed in higdd37. The signal intensity of each band was compared with the corresponding wild-type band in each of four blots. Error bars indicate ± SEM. *p < 0.05, relative to wild-type (Steel–Dwass test). NS, Not significantly different.