The nano-LC was equipped with homemade precolumn (150 × 5 mm) and analytical column (75 × 150 mm) packed with Jupiter Proteo C12 resin (particle size 4 mm, Phenomenex, Torrance, CA, USA). The dried peptides were resuspended in 1% formic acid (FA) solution. Six microliters of sample solution was loaded to the precolumn for each LC-MS/MS

run. The precolumn was washed with the loading solvent (0.1% FA) for 4 min before the sample was injected onto the LC column. The eluents used for the LC were 0.1% FA (solvent A) and 95% ACN containing 0.1% FA (solvent B). The flow rate was 200 nl/min, and the following gradient was used: 3% B to 35% B in 72 min, 35% B to 80% B in 18 min, which was maintained at 80% B for 9 min. The column was finally equilibrated with 3% B for 15 min prior to the next run. Electrospray ionization was performed using a

30 mm (i.d.) nanobore stainless see more LY294002 solubility dmso steel online emitter (Proxeon, Odense, Denmark) and a voltage set at 1900 V. Sequences were searched against Swiss-Prot mouse and mammalian genomes using MASCOT software versions 2.1.0 and 2.1.04 (Matrix Science, London, UK). Peptides were required to have a rank = 1 and a score >18. We constructed a weighted correlation network as previously described using the R software package (Langfelder and Horvath, 2008). We used proteins with unique tryptic peptide counts coming from at least three IP conditions as an input (n = 411). See Supplemental Experimental Procedures for complete WGCNA procedure. Using a population of 15 age-matched (± 4 hr) virgin female flies, we estimated the percentage of animals able to climb past a line set at 9 cm in during 15 s. These tests were repeated ten consecutive times for each replicate per experimental day. The experiment was carried out in duplicate (two populations of 15 animals) in flies that were 8, 10, 12, 14, and 16 days old. Tests were always performed in the same time of the day and in the same place to avoid circadian rhythm and environmental

variability. The average number of flies climbing per day is averaged and plotted independently for each replicate. All values are presented as the mean ± SEM, and p < 0.05 was considered statistically significant. X.W.Y. is supported by the National Institute of Neurological Disorders and Stroke/National Institutes of Health (NINDS NIH) (grants R01NS049501 and R01NS074312), CHDI Foundation, the Hereditary Disease Foundation (HDF), David Weil Fund to the Semel Institute at University of California, Los Angeles, and Neuroscience of Brain Disorders Award from The McKnight Endowment Fund for Neuroscience. D.S. was supported by the NIH Chemistry-Biology Interface Research Training Program at UCLA (T32GM008496). D.S. and E.G. were supported by the UCLA Dissertation Year Fellowship Program. J.A.L. is supported by an NIH grant (R01RR20004) and by the W. M. Keck Foundation for the establishment of the UCLA Functional Proteomics Center. S.H.