Figure 1: SynIa is SUMOylated at K687R. | Nature Communications

Figure 1: SynIa is SUMOylated at K687R.

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

Figure 1

All experiments were performed at least three times with consistent results. (a) Representative anti-HA immunoblot showing that SynIa is robustly SUMOylated. HA-SynIa, Flag-Ubc9 and YFP-SUMO-1 were expressed in N2A cells and the lysate then subjected to GFP-trap pull down to purify YFP-SUMOylated proteins. (b) GST-pull-down assays showing that endogenous SynI from rat cortical neurons binds Ubc9. (c) Immunoprecipitation of endogenous SUMOylated SynIa from rat brain. Whole rat brain was lysed under denaturing conditions and subjected to IP with anti-SUMO-1 antibody, then immunoblotted with anti-SynI antibody. IgG Control: mouse IgG was used instead of anti-SUMO-1; α-SUMO-1 control: RIPA buffer was used instead of brain lysate. SENP-treated pull down: before the pull down, the lysate was supplemented with 25 nM SENP (catalytic domain fragment) and incubated for 1 h at room temperature. (d) Full-length HA-SynIa and truncation constructs lacking the E domain (ΔE) or both the D and E domains (ΔDE) were expressed in HEK293T cells and the lysates subjected to GST-Ubc9 pull down. (e) Point mutations of residues in the SUMO consensus site in SynIa prevent pull down by GST-Ubc9 in HEK293T cells. (f) K687 is the only SUMOylation site in SynIa. HA-SynIa WT or HA-SynIa K687R were expressed with Flag-Ubc9 and YFP-SUMO-1 in N2A cells and lysates were immunoblotted with anti-HA antibody.

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