Quantitative Protein Analysis in Flies

Western blotting was performed as described previously [59 (link)]. Ten fly heads for each genotype were homogenized in Tris-Glycine SDS sample buffer, and the same amount of the lysate was loaded to each lane of 10%, 12.5% or 15% Tris-Glycine gels and transferred to nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk, blotted with the antibodies described below, incubated with appropriate secondary antibody and developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences). The membranes were also probed with Anti-tubulin, and used as the loading control for other blots in each experiment. Anti-tubulin (Sigma-Aldrich), anti-tau (Merck Millipore), anti-non phospho tau (Merck Millipore), anti-pSer202 tau (Thermo Fisher Scientific), anti-pThr231 tau (Thermo Fisher Scientific), anti-pSer404 tau (Abcam), anti-pSer422 tau (Abcam), anti-Nervana (Developmental Studies Hybridoma Bank), anti-VDAC1 (Abcam), anti-NDUFS3 (Abcam), anti-MnSOD (Enzo Life Sciences), anti-Atg8 (Merck Millipore) and anti-ref(2)P (Abcam) antibodies were purchased. Anti-MANF is a kind gift from Dr. T. I. Heino (University of Helsinki). Imaging was performed with ImageQuant LAS 4000 (GE Healthcare Life Sciences), and the signal intensity was quantified using Image J (NIH).