For α-synuclein dot blot assays, 1,000 heads were removed from CO₂ anesthetized 14-day-old w ¹¹¹⁸ and transgenic α-synuclein (UAS) flies on glass plates at 4°C. The heads were transferred into Eppendorf tubes on ice, homogenized in 10 mM PMSF and then centrifuged at 13,000 rpm for 15 min at 4°C. The supernatants were then blotted onto nitrocellulose paper, which was subsequently immersed in blocking solution for 16 h. This solution was discarded and the membrane was washed in TTBS for 15 min. Primary antibody (mouse anti-synuclein; Zymed) diluted in buffer was incubated with the membrane for 90 min followed by secondary anti-mouse antibody. The secondary antibody was decanted and the membrane was washed with TTBS for 15 min. The coloring agent, BCIP/TNBT, was added for 10 min and the membranes were then washed in water for 5 min to stop the reaction.

Transgenic α-synuclein (UAS) and w ¹¹¹⁸ flies were anesthetized in CO₂, placed in plastic vials, and quick frozen in a dry ice-ethanol bath. The frozen flies were placed on tiles that had been kept in a freezer and heads were collected from 400 flies of each genotype. Heads were then separately homogenized in 40 μL of PMSF and 100 μL of lysis buffer (Cell Signaling) containing protease inhibitor. It is important that the grinding of heads and the homogenizing process take place in an ice bath to prevent denaturation of proteins. The grinding process must be vigorous because of the very small size of the brain cells. The heads were homogenized, freeze–thawed, and sonicated three times creating three extracts which were numbered in consecutive order (see Fig. 1, top). After centrifugation, 32 μL of each extract supernatant was added to 8 μL of sample (loading) buffer (Sigma) containing beta-mercaptoethanol and Laemmli buffer. Into another Eppendorf tube 1 μg/μL of synthetic human α-synuclein (Sigma) was placed in 9 μL of sample buffer. The samples were heated for 10 min at 95°C and then subjected to gel electrophoresis at 145 V for 90 min. The gels were then transferred onto a nitrocellulose membrane at 25 V for 30 min and then incubated in 5% condensed milk solution for 60 min at 5°C. After washing for 30 min, the membrane was incubated with primary mouse antibody diluted in TBST (1:1,000) at 5°C for 16 h. After washing for 30 min, the membrane was incubated with secondary antibody diluted with TBST (1:1,000) at 5°C. The membrane was washed again and stained with BCIP/NBT overnight, washed, and blow dried. A protein ladder (Bio-Rad) was also run on each membrane.