weeks, the hatched knockdown flies were collected and cultured for an additional 14 days at 28uC to enhance the effects of dsRNAs. More than 100 heads per line were isolated and homogenized in lysis buffer consisting of 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, 0.5% Triton X-100, and complete protease inhibitor (Roche). The lysate was incubated with anti-Chp (24B10) covalently coupled with protein G- Sepharose (GE Healthcare) for 2 h. The Chp-bound Sepharose was subsequently washed with lysis buffer, and Chp was eluted with 50 mM triethylamine-acetic acid (pH 3.5). Purified Chp was spotted onto Hybond ECL nitrocellulose membranes (GE Healthcare) using a Bio Dot slot format (Bio- Rad). After the membrane was blocked with 2% bovine serum albumin, immuno- or lectin blot analysis was performed with anti- Chp (N8A), anti-HRP, or HRP-conjugated ConA, LCA, DSA, and WGA. HRP-conjugated anti-mouse or HRP-conjugated anti- rabbit IgG was used as the secondary antibody. Detection was performed using Supersignal West Pico Chemiluminescent Substrate (Thermo), and quantification performed using a LAS- 1000 Luminescent Image Analysis System (FujiFilm). An example of the dot blot is shown in Figure S2. Lectin affinity values per Chp values were converted to log values for normalization. On the basis of standard deviations from the values of GMR.GFPRNAi (as a control), genes with a z-score .3 were considered primary candidates. In the primary screen, we performed a single assay round. For the primary candidates, the lectin analysis was repeated (n = 3). Two-sided Student’s t-tests were applied to the reproducibility test data between lectin values of knockdown and control flies. Genes with p,0.005 were considered to be reproducible findings. Validation Collection of the second transgenic fly stock lines was designed in such a way that there was no overlap between the primary construct in the original library and the new dsRNA sequence, and that the new dsRNA sequence had a low off-target probability score calculated by dsCheck [22]. Using the second lines, lectin analysis was independently repeated (n = 2), and the values between the knockdown and control (GMR.GFPRNAi) were tested by two-sided Student’s t-tests. Genes with p,0.05 were considered glycosylation-related genes (Rank 1). When no change in lectin values was observed solely using GMR-Gal4, the examination was repeated using GMR-Gal4; UAS-dicer2 (VDRC). RNAi flies from VDRC whose target sequence had no overlap with the primary construct were also used as second lines. For candidate genes that were not evaluated with the second lines, first the number of 19mers with a perfect match to each Drosophila transcript was calculated using dsCheck. The highest number of 19mers with a perfect match to the off-target gene was defined as the ‘‘off-target probability score’’. The genes with a score below 3 were tested to determine whether knockdown of their suspected off-target genes led to glycosylation defects. When the knockdown of the suspected off-target genes did not lead to any evidence of glycosylation defects, the corresponding genes were added to the list of genes required for glycosylation (Rank 2). GO analysis Our final glycosylation hit list was annotated to include information on protein domain, molecular function, and protein family by cross-referencing InterPro (version 20.0 and 21.0) [47], Flybase, and Panther [48]. On the basis of the resulting annotation, genes were manually classified into 9 categories. These annotations are shown in Table S4. Large-Scale Screen for Glycosylation-Related Genes Mass spectrometry (MS) analysis of glycopeptides and glycan structure MS analysis of glycopeptides was performed as previously described [16]. Purified Chp was digested with modified trypsin (Promega). Glycopeptides and peptide mixtures were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using an Inertsil C18 column (GL Science). The matrices for peptide analysis by matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF-MS) spectra were acquired using a Voyager mass spectrometer (Applied Biosystems). MS/MS measurements were performed using an Ultraflex TOF/TOF mass spectrometer with the LIFT-MS/MS facility (Bruker Daltonics GmbH). Immunostaining To investigate the amount of a1,3-fucosylation in vivo, frozen sections of adult eye demonstrating swm or gfp knockdown were prepared. Immunostaining was performed as previously described [49]. Fluorescent images were acquired with a laser scanning confocal microscope, LSM700 (Zeiss). Cell culture, plasmid construction, transfection, and siRNA treatment BG2-c6 cells were cultured in M3 medium (Sigma) containing 10% FBS and 10 mg/mL insulin (Invitrogen) at 25uC. Transfec- tion was performed using a calcium phosphate transfection kit (Invitrogen). To construct an expression plasmid for C-terminal Flag-tagged Swm (Swm-Flag), cDNA containing the swm coding region was obtained from Canton-S total RNA by RT-PCR. A DNA fragment encoding the 3Flag sequence was ligated to the 39end of the swm coding region and inserted into the expression vector pRmHa. For the transient expression of Swm-Flag, 1.5 mg plasmid was added to each well of a 24-well plate, in which 56105 BG2-c6 cells had been seeded. Plasmid expression was induced by incubating cells in the presence of 0.1–0.5 mM CuSO4 for periods from 3 h to overnight. For knockdown experiments, dsRNA for swm or gfp was produced using in vitro transcription T7 kit (Takara). A 3-mg aliquot of dsRNA was added to each well of a 24-well plate in which 56105 cells had been seeded. After 5 days, cells were harvested for the following assays. Immunoblot For protein analysis, equal amounts of protein extracted from fly eyes were subjected to SDS-PAGE. After transfer to PVDF membranes (Millipore), membranes were blocked in PBS containing 0.05% Tween 20 and 5% skim milk and incubated overnight with the primary antibody, followed by the secondary antibody. A monoclonal anti-a-tubulin antibody was used for normalization. Detection was performed using the Supersignal West Pico Chemiluminescent Substrate. RNP-IP assay Immunoprecipitation of protein–RNA complexes was per- formed according to a protocol by Niranjanakumari et al. [50]. BG2-c6 cells transfected with swm-Flag were harvested and cross- linked in PBS containing 0.2% PFA. Following sonication in RIPA buffer (50 mM Tris-HCl pH 7.5, 1% NP-40, 0.05% SDS, 1 mM EDTA, 150 mM NaCl) with protease inhibitor (Nakarai), each supernatant was mixed with a monoclonal anti-Flag M2 antibody or normal mouse IgG and then incubated with protein-G beads (GE Healthcare) at 4uC for 6 h. Sepharose beads were washed 5 times with RIPA buffer and incubated in 50 mM Tris (pH 7.0), PLoS Genetics | www.plosgenetics.org 9 December 2010 | Volume 6 | Issue 12 | e1001254