previously [58]. Nuclear material was sheared using a syringe (22– 24G needle) and the homogenate boiled in LLB for 5 min. At least 3 independent protein extractions were performed for each experiment. Extracted proteins were size separated by SDS- PAGE, transferred to a nitrocellulose membrane (Protran, What- man) and probed with the antibody of interest in 2.5% milk TBS- T overnight at 4uC. The antibodies were diluted as follows: anti- VIG (CSH1801) 1:5,000; anti-Vig2 (CSH2542) 1:20,000; anti- VIG (CSH1803) 1:10,000; anti-H2B 1:10,000; anti-H3K9me2 1:20,000; anti-H4 1:2,000; anti-HP1 1:100; anti-HP1 (WA191) 1: 2,000; anti-HP2 1:10,000; anti-Actin 1:500; anti-Vasa 1:5,000; anti-Dynein 1:2,000. Secondary goat anti-rabbit (or mouse) HRP- conjugated antibodies were diluted as 1:100,000–500,000. Super- Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used for detection. Quantitative data was obtained as follows: gradually increasing amounts of the protein extract were loaded in successive lanes for all genotypes and probed with the corresponding antibodies as described above. Densitometry measurements of this dilution series showed that the antibodies applied were sensitive to the dosage of the proteins and that the optical signal response was linear in this range. ‘Standard curves’ showing the optical value dependence on the amount of loaded protein for the WT sample were generated for the protein of interest and for the loading control protein. Using these standard curves we determined the relative amount of proteins in the mutant lines. The bar graphs represent the ratio between the protein of interest and the loading control. (Note that because the two antibodies (one specific for the protein of interest and the other for the loading control) demonstrate different sensitivities and different response curves, it is not appropriate to use un-calibrated optical data for quantitation.) Purification of VIG and Vig2 from nuclear and cytoplasmic S2 cell extracts S2 cells were harvested, washed twice in PBS, resuspended in buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mMKCl, 1 mM DTT), and protease inhibitors (Roche) and lysed in ice for 309. Cells were then disrupted in a Dounce homogenizer with the B pestle for 40 strokes and spun at 800 g for 209. The supernatant was collected and clarified at 20,000 g for 309; this is the cytoplasmic fraction. The pellet from the 800 g spin was resuspended in a small amount of buffer A plus 2 mM CaCl2 and treated with Turbo DNAseI (Ambion) for 29 in a 37uC water bath with shaking. The nuclei were then spun down briefly and resuspended in buffer B (20 mM Hepes pH 7.9, 40 mM NaCl, 1.5 mM MgCl2, 20 v/v glycerol, 0.2 mM EDTA), and agitated for 309 at 4uC. The nuclear extract was then homogenized and clarified by spinning at 20,000 g for 309. The supernatant is the nuclear fraction. The pellet was then sonicated in SDS loading buffer to obtain a chromatin fraction. Both cytoplasmic and nuclear extracts were filtered through 0.4 micron and 0.8 micron filters, respectively. AKTA FPLC was used for Superose- 6 column chromatogra- phy [12,61]. Two mls of nuclear and cytoplasmic extracts (20– 35 mg/ml) were filtered through a 0.4/0.8 micron filter, loaded onto a Sepharose 6 column (Amersham Biosciences) and eluted with 1.2 column volumes of buffer A9 (100 mM NaCl instead of 10 mM KCl). 10 ul from each fraction were boiled with SDS sample buffer for 5 minutes and loaded onto a 10% SDS-PAGE. References VIG and Vig2 Impact Chromatin Immunoprecipitation experiments Whole flies or ovaries were ground in Lysis Buffer (20 mM Tris pH 7.5, 100 mM NaCl, 0.5 mM EDTA pH 8, 0.5% NP40, ‘Complete’ Protease Inhibitor Cocktail (Roche)). Fly homogenates were filtered through 1 layer of Miracloth (Calbiochem). Preclearing step: 50 ul of Protein A/G beads (Thermo Scientific) previously washed in Lysis Buffer were added to 500 ul of fly homogentate and the mixture was incubated on a rocking platform for 30 min at 4uC; the beads were collected by centrifugation (2 min at 500 g) and the liquid phase was recovered. The aliquot taken at this step served as the ‘input’ sample. 5 ug of the antibodies or purified IgG (Jackson ImmunoResearch) was added to the homogenates and the mixtures were incubated on a rocking platform for 2 h at 4uC; then 50 ul of Protein A/G beads were added and incubation continued for 1 h at 4uC. The beads were spun down and washed 3 times with Lysis Buffer. The antibodies and immunoprecipitated material were released from the beads by boiling in 50 ul of LLB for 5 min; the proteins were analyzed by Western blot. RNase treatment of extracts was conducted as outlined [62]. RNase A (100 ug/ml final concentration) was added to the precleared extracts before immunoprecipitation and the rest of procedure was performed as described above. Supporting Information Figure S1 Found at: doi:10.1371/journal.pone.0006182.s001 (0.06 PDF) Figure S2 Found at: doi:10.1371/journal.pone.0006182.s002 (0.13 PDF) Figure S3 Found at: doi:10.1371/journal.pone.0006182.s003 (0.05 PDF) Figure S4 Found at: doi:10.1371/journal.pone.0006182.s004 (0.38 PDF) Figure S5 Found at: doi:10.1371/journal.pone.0006182.s005 (0.06 DOC) Figure S6 Found at: doi:10.1371/journal.pone.0006182.s006 (0.04 PDF) MB MB MB MB MB MB Acknowledgments We thank Greg Hannon for valuable discussion. We are grateful to Paul Lasko for providing the vasaAS mutant line and to Gary Karpen and colleagues for the PEV reporter lines. We thank Paula Kiefel and Brent Brower-Toland for technical advice and assistance. We also thank members of the Elgin lab for stimulating discussions. Author Contributions Conceived and designed the experiments: EG MD SE. Performed the experiments: EG MD. Analyzed the data: EG MD SE. Contributed reagents/materials/analysis tools: MD. Wrote the paper: EG MD SE. 1. Berger SL (2007) The complex language of chromatin regulation during transcription. Nature 447: 407–412. 2. Richards EJ, Elgin SC (2002) Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 108: 489–500. PLoS ONE | www.plosone.org 10 July 2009 | Volume 4 | Issue 7 | e6182