Figure 3: SUMOylation of SynIa promotes SV binding. | Nature Communications

Figure 3: SUMOylation of SynIa promotes SV binding.

From: SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation

Figure 3

(a) Preparation of SUMOylated SynIaDE fragment. HA-His-SynIaDE was bacterially expressed with or without co-expression of the E1/E2/Flag-SUMO construct44. Eluates containing SUMOylated SynIaDE were purified using an anti-Flag antibody resin and eluted with Flag peptide. Representative Coomassie-stained SDS–polyacrylamide gel electrophoresis (SDS–PAGE) of the input and eluate from both purification steps are shown. (b) SVs were extracted from rat brain and mixed with purified recombinant HA-His-SynIaDE (n=6) or SUMO-HA-His-SynIaDE (n=6) with active or inactive SENP (both n=4) to de-SUMOylate SynIaDE (n=4). The SVs were isolated by ultracentrifugation, resuspended and analysed by SDS–PAGE and immunoblotting for SynIaDE using anti-HA antibody. Anti-synaptophysin blot (below) shows equal loading of the SV fraction. (c) Quantification of binding of non-modified, SUMOylated, deSUMOylated and inactive SENP-treated SUMOylated SynIaDE to SVs. Data presented as mean±s.e.m. **P<0.01, *P<0.05 one-way ANOVA, post hoc test with Bonferroni correction. n numbers as above.

Back to article page