5 mM EDTA, 10 mM DTT, and 1% SDS at 70uC for 45 min to reverse cross-linking. RNA was extracted from the immunopre- cipitates using Sepasol reagent (Nakarai) followed by DNase I treatment. The extracted RNA was used as a template to synthesize cDNA using Superscript III (Invitrogen) according to the manufacturer’s protocol. Real-time PCR Total RNA was extracted using the RNeasy mini kit (Qiagen) or Sepasol reagent (Nakarai). Complementary DNAs were synthe- sized using Superscript III according to the manufacturer’s protocol. Real-time PCR was carried out using a 7900HT Fast Real Time PCR system (Applied Biosystems) with Power Syber Green. Samples were normalized with Drosophila rpl32. RNA degradation analysis BG2-c6 cells transfected with gfp or swm dsRNA were grown for 5 days, and then actinomycin D (Nakarai) was added to a final concentration of 5 mg/mL to arrest de novo RNA synthesis. At 30, 60, and 90 min after actinomycin D treatment, the cells were harvested, and fucTA and rpl32 (control) mRNA was quantified by real-time PCR as described above. PAT assay PolyA measurement of fucTA mRNA was carried out as described [51]. PCR fragments from the PAT assay were separated on 2% agarose gels and visualized by Southern blot using DIG DNA Labeling and Detection kit (Roche). Subcellular fractionation Fractionation and RNA isolation were performed as described [52]. The cytoplasmic fraction was extracted by hypotonic buffer (10 mM Tris (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonident P-40, 40 units/mL RNase inhibitor, 1 mM DTT) and then extracted by EDTA buffer (10 mM Tris (pH 7.5), 10 mM NaCl, 40 mM EDTA, 0.5% (v/v) Nonident P-40, 40 units/mL RNase inhibitor, 1 mM DTT). The remaining pellet was used as the nuclear fraction. Sepasol solution was used to purify mRNA from each fraction as described above. Human homologs The InParanoid database (version 7.0) was searched for mammalian homologs [53]. Supporting Information Figure S1 Schemes of Chp glycan attachment sites and glycan structures. (upper) The glycosylated sites in Chp are indicated as ‘Y’ with amino acid positions. The colors represent the glycan types. Open boxes in Chp protein indicate leucine-rich repeats. (lower) Schemes for three types of glycan structures are represented. The number of sugars is indicated in parentheses following the sugar name. The moieties recognized by lectins are encircled by lines colored to indicate the lectin species. Found at: doi:10.1371/journal.pgen.1001254.s001 (9.83 MB TIF) Figure S2 An example of dot blot analysis for the screen. Chp purified from adult heads with the knockdown of each gene (1A,11H) and the control gfp (12A,12H) was blotted and detected with anti-Chp (left) and anti-HRP (right). The control Chp was sequentially diluted and blotted at the points from 12A to 12H. Dots in red squares represent anti-Chp and anti-HRP signals against Chp purified from swm knockdown flies. Found at: doi:10.1371/journal.pgen.1001254.s002 (4.28 MB TIF) Large-Scale Screen for Glycosylation-Related Genes Figure S3 Glycan structures attached to N1012 in Chp. Six glycan structures that were identified by mass spectrometry analysis are schematically represented. Found at: doi:10.1371/journal.pgen.1001254.s003 (5.23 MB TIF) Figure S4 Knock-in of the fucTA gene by ends-out recombination. (A) The donor DNA was generated by FLP and I-SceI action from the X chromosome (top). Homologous recombination was used to insert the 3Myc sequence into the endogenous fucTA gene and the white gene into the region between fucTA and Msh6 genes (middle). The expected structure (bottom) was verified by PCR with two sets of primers (colored thick arrows). (B) Expected bands were amplified with two sets of primers (red, fucTA locus; blue, Msh6 locus) using chromosomal DNA from a fucTA knock-in fly (fucTA-MycKI) but not in white flies (w). (C) Myc-tagged FucTA protein (arrow) was detected by immunoblot using anti-Myc antibody in the adult eye extracts of fucTA knock-in flies (fucTA-MycKI) but not in white flies (w). The amount of Myc-tagged FucTA protein was reduced in swm knockdown eyes (swm KD) compared to the control eyes (gfp KD). Found at: doi:10.1371/journal.pgen.1001254.s004 (8.80 MB TIF) Figure S5 The amount of Mgat1 and fdl mRNA in swm knockdown BG2-c6 cells. The amount of mRNA encoding Mgat1 was slightly but significantly increased, whereas that of fdl mRNA was not changed in swm knockdown cells compared with control gfp knockdown cells, n = 3. *p , 0.05. Found at: doi:10.1371/journal.pgen.1001254.s005 (1.77 MB TIF) Table S1 Specificity of the lectins used in this study. Found at: doi:10.1371/journal.pgen.1001254.s006 (0.02 XLS) MB Table S2 Glycosylation Screening Results using NIG RNAi fly strains. Found at: doi:10.1371/journal.pgen.1001254.s007 (1.39 MB XLS) Table S3 Glycosylation Screening Results using VDRC RNAi fly strains. Found at: doi:10.1371/journal.pgen.1001254.s008 (0.06 MB XLS) Table S4 Glycosylation-related genes identified in this study. Found at: doi:10.1371/journal.pgen.1001254.s009 (0.08 MB XLS) Table S5 Manmalian homolog. Found at: doi:10.1371/journal.pgen.1001254.s010 (0.03 XLS) Table S6 Primer Sequences for transgenic fly. Found at: doi:10.1371/journal.pgen.1001254.s011 (0.03 XLS) MB MB Table S7 Primer Sequences used in this study in cultured cells. Found at: doi:10.1371/journal.pgen.1001254.s012 (0.02 MB XLS) Acknowledgments Specific antibodies, plasmids, and fly stocks were provided by the Develop- mental Studies Hybridoma Bank, Bloomington Drosophila Stock Center, the Drosophila Genetic Resource Center, and the Vienna Drosophila RNAi Center. We thank members of S. Goto’s laboratory and Y. Sado for technical support. Author Contributions Conceived and designed the experiments: MYH YK KFAK OK SG. Performed the experiments: MYH YK WA HY SG. Analyzed the data: MYH YK WA KFAK OK SG. Contributed reagents/materials/analysis tools: MYH YK WA KFAK SN HO RU OK SG. Wrote the paper: MYH SG. PLoS Genetics | www.plosgenetics.org 10 December 2010 | Volume 6 | Issue 12 | e1001254