Sarkosyl-insoluble proteins isolation

For the in vivo sarkosyl-insoluble fractions, 6 mated flies per genotype were fresh-frozen in liquid nitrogen and decapitated. The frozen heads were homogenized in 6.7 µl/head ice-cold Buffer A (50 mM Tris–HCl pH 7.5, 2 mM Na₃VO₄, 50 mM NaF, 50 mM β-Glycerophosphate, 150 mM NaCl, 2 mM MgCl₂, 1 × cOmplete EDTA-Free protease inhibitors mix, 1 × PhosSTOP phosphatase inhibitors mix) for 45 s on maximum speed using a Minilys homogenizer (Bertin Instruments). The homogenate was centrifuged at 6000g for 5 min at 4 °C and 34 µl of clarified homogenate was isolated. The homogenization process was repeated using an equal amount of Buffer A and 34 µl of clarified homogenate were combined with the first round, while the pellet was discarded. Subsequently N-lauroylsarcosine, Triton X-100, and SDS were added to final concentrations of 1% w/v, 1% w/v, and 0.1% w/v, respectively (Sarkosyl-Lysis Buffer). Upon the final addition of β-MeOH to 0.1 M final concentration (1% v/v), the samples were shortly vortexed and incubated at 37 °C for 1 h under 700 rpm orbital shaking. Then, the samples were centrifuged at 4 °C for 30 min at 21,000g. The supernatant corresponding to the soluble proteins was isolated and mixed with Lämmli, while the pellet was resuspended with an equal volume of Buffer A and the centrifugation step was repeated. This wash-supernatant was discarded and the pellet was resuspended in 50 µl Lämmli. Both fractions were boiled for 10 min at 95 °C and analysed in SDS-PAGE and western blotting.

The Drosophila neuronal BG2-c6 cells were cultured in 24-well plates for 48 or 72 h upon Cu²⁺ induction and were initially rinsed with PBS and before gently detached by resuspension in PBS. The cells were shortly pelleted (5000g, 5 min, 4 °C) and then lysed for 30 min on ice with Sarkosyl-Lysis Buffer. The lysate was centrifuged at 21,000g for 30 min at 4 °C and the supernatant (soluble fraction) was separated and mixed with Lämmli. The pellet was washed with 50 µl Sarkosyl-Lysis Buffer, centrifuged again and resuspended in Lämmli. Both soluble and insoluble fractions were boiled at 95 °C for 10 min before SDS-PAGE and western blotting analysis.