Other alleles were the following: w; endoAΔ4 ( Verstreken et al., 2002), w chc1 ( Bazinet et al., 1993), w; dap160Δ2 ( Koh et al., 2004), lap1 ( Zhang et al., 1998), yw; eps15e75 ( Koh et al., 2007), and w; α-ada1 ( González-Gaitán and Jäckle, 1997). endoA+ in Figure 2 is endo+(21B) ( Venken Dactolisib research buy et al., 2008). Genomic

EndoA phosphomutants were generated by chimeric PCR using endo+(21B) in P(acman) and primers in Table S1. Wild-type endoA, endoA[S75A], endoA[S75D], and endoA[S75E] were cloned into pStinger using AscI and PacI. Transgenes were inserted into VK37 on the second chromosome at Genetivision. Flies expressing human LRRK2 (LRRK2WT), LRRK2KD (LRRKD2017A), and LRRK2G2019S were generated by site-directed mutagenesis using pCDNA3.1 LRRK2V5 and primers in Table S1. These constructs were cloned into

pUAST-attB using XhoI and NotI. Transgenes were inserted into VK22 on the second chromosome. FM1-43, electrophysiology, and electron microscopy were performed and the data quantified as described (Uytterhoeven et al., 2011; Miśkiewicz et al., 2011) and as outlined in the Supplemental Experimental Procedures. Antibodies used were the following: anti-HRP rabbit pAb (1:1,000; Jackson ImmunoResearch), anti-Dlg4F3 mouse mAb (1:50), anti-GluRIIA8B4D2 mouse mAb (1:50, DSHB), anti-endophilin Ab-EndoAGP69 guinea pig (1:1,000), and Alexa-conjugated secondary antibodies (Invitrogen). Cold in vitro LRRK2-phosphorylated human EndoA1 was separated by SDS-PAGE and Coomassie-stained bands were excised and in-gel digested with trypsin. secondly One part CH5424802 concentration was analyzed by LC-MS/MS using an Orbitrap XL MS (Ghesquière et al., 2009); a second part was used to isolate phosphopeptides by titanium dioxide beads (GE Healthcare, TiO2 Mag Sepharose). MASCOT generic files from the LC-MS/MS data were used to search the human Swiss-Prot database with trypsin as the protease, allowing for one missed cleavage. Mass tolerance of the precursor ions was set to 10 ppm (with MASCOT’s C13 option set to 1) and of fragment

ions to 0.5 Da. Variable modifications were as follows: N-acetylation, N-pyroglutamate, oxidation of methionine, phosphorylation of serine, threonine, and tyrosine and propionamide formation of cysteine. Only peptides ranked first, and those of which the MASCOT ion score exceeded the corresponding identity threshold score set at the 99% confidence level were withheld. A total of four in vitro human EndoA preparations with LRRK2 and four with LRRK2KD were analyzed by LC-MS/MS, and, for one preparation, an in-gel N-propionylation step was done (Arnesen et al., 2010). Rabbit EndoA S75 phosphoantibodies were generated at Pickcell against VKGIphSKLSGQA and affinity purified using the same peptide and the nonphosphopeptide. For lambda phosphatase, fly heads were collected on ice and crushed in lysis buffer (10 mM HEPES, 150 mM NaCl, 1% triton) (pH 7.4) with complete protease inhibitor (Roche).