Figure 3

Diurnal changes of the interaction between Synaptophysin (Syp) and Synaptobrevin (Syb). (A) Western blots of proteins from synaptic vesicles prepared from either synaptophysin knock-out animals (Syp ^-/-), wild-type (wt) littermates or Per2 mutant mice. Antibodies against synaptophysin (α-Syp, left panel) and synaptobrevin (α-Syb, right panel) reveal the presence of synaptophysin (Syp) or synaptbrevin (Syb) with (+) or without (-) chemical cross-linking using disuccimidyl suberate (DSS). Cross-linked samples show in addition to the respective monomers the Syp/Syb complex at 56 kDa and the Syp or Syb dimers. In synaptic vesicles of Syp ^-/- animals, only Syb and its dimer can be observed. (B) Western blots after cross-linking of proteins show the Syp/Syb interaction over a 12 hour light/12 hour dark cycle. Synaptic vesicles were prepared at the indicated Zeitgeber times (ZT). Antibodies against Syp (left panel) and Syb (right panels) show changes in the Syp/Syb complex over time. SNAP25 served as internal control. (C) Quantification of Syp/Syb complexes using α-Syb antibodies. Data from three independent sets of animals are shown with each sample analyzed at least twice. SNAP25 served as reference for quantification. For each set, values were normalized to the value at ZT0 which was set to 1. While the Syp and Syb monomers and dimers do not change over time, the formation of the Syp/Syb complex appears to be time dependent with maximal values observed during the light period corresponding to the resting phase of mice (ZT6 and ZT12). Values represent the mean of three animals ± S.D. Significance was determined by student’s t-test between ZT6 and ZT18 ( p = 0.06) and ZT12 and ZT18 (p = 0.03).