Full-length recombinant human WT α-syn, α-syn S129A, and S129D were expressed in Escherichia coli BL21 (DE3) (New England Biolabs, Cat # C2530H) using the bacterial expression vector pGEX-KGmyc. Following transformation protein expression was induced with 0.05 mM IPTG (isopropyl-β-d-thiogalactopyranoside),and either incubated at 37 °C for 2 hours or at room temperature for 6 hours,

with shaking.

The cells grown on Terrific Broth (Thermo Scientific Cat#

BP9728-2) were harvested by centrifugation at 4500× g g at 4 °C for 20 min, and pellets were stored

at -80 °C until use.

For protein purification, protein pellets were resuspended

in 30 ml Lysis Buffer containing 1X PBS, 0.5 mg/ml lysozyme, 1 mM PMSF, DNase,

and EDTA-free protease cocktail inhibitor (Roche Cat# 11836170001) for 15 min

on ice, briefly sonicated (3 sets with 33 strikes and 30-second breaks on ice

between sets), and removed the insoluble material by centrifugation at 15,000 g

at 4 °C for 30 min.

The clarified lysate was incubated with 500 ml of glutathione-Sepharose

4B (Sigma Cat# 17-0756-01), preequilibrated with 1X PBS containing 0.1% Tween

20 and 5% glycerol (binding buffer), on a tumbler at 4 °C overnight.

The GST-bound proteins were washed four times with 30 ml binding buffer and

maintained at 4 °C for pull-down assays in vitro o phosphorylation, an in vitro ro dephosphorylation experiments.

GST-bound proteins were occasionally eluted by TEV cleavage. In brief, 15 ug of the fusion protein was mixed with 5 ul of TEV protease reaction buffer (10X) and 1ul of TEV protease

(New England Biolabs, Cat#P8112), followed by overnight incubation at 4 °C.