Red from Act.lqfRa-gfp and Act.lqfRENTH-gfp embryos: GFP-positive embryos were

Red from Act.lqfRa-gfp and Act.lqfRENTH-gfp embryos: GFP-positive embryos were homogenized in 100 ml lysis buffer (1 NP40, 0.5 deoxycholate, 1 mM DTT, 150 mM NaCl, 50 mM Tris pH 8.0 with protease PHCCC site inhibitor cocktail [Roche, complete-mini, EDTA-free] and 2 mM PMSF). Lysis buffer (300 ml) was added followed by centrifugation at 12,000 rpm at 4uC. A 300 ml aliquot was removed and mixed with 20 ml of a 50 slurry of GFP-trapA (Chromotek) and a 10 ml aliquot was mixed with 26 SDS loading buffer as a loading control. After incubating 2 hrs. with mild shaking at 4uC, the 300 ml aliquot was spun down, the pellet collected and washed for 5 min. with shaking in 1 ml lysis buffer, and then washed again for 10 min. with shaking in 1 ml of 500 mM NaCl. The pellet was washed 4 times more in 1 ml of 500 mM NaCl and then mixed with 20 ml of 26 Laemmli Buffer. Each sample was boiled for 5 min, microfuged, and the supernatant subjected to SDS-PAGE in a 7.5 gel. Western blotting was performed as described (Chen et al., 2002). Primary antibodies were: rat anti-E-cadherin (DSHB:DCAD2, used 1:1000), mouse anti-Armadillo (DSHB:N27A1, used 1:500), rat anti-a-catenin (DSHB:Homatropine methobromide site DCAT-1, used 1:100), rat anti-GFP (Chromotek:3H9, used 1:1000). Secondary antibodies were from Santa Cruz Biotechnology and used at 1:5000: goat anti-rat HRP , goat anti-mouse HRP, goat anti-rat HRP.Protein blot in FigureProtein extracts of 2 adult flies containing one copy each of the transgene indicated and the ey-gal4 driver were made byFigure 9. The effect of Tel2 on Wingless signaling. A model for how Wingless signaling is compromised in the absence of Tel2 is illustrated. We speculate that in the absence of Tel2, increased Ecadherin at the plasma membrane sequesters Armadillo (Arm) so that little remains free in the cytoplasm to enter the nucleus in response to Wingless signaling. doi:10.1371/journal.pone.0046357.gSupporting InformationFigure S1 Amino acid sequence alignment of human and yeast Tel2 and Drosophila LqfR-exon 6. The amino acid sequences of H. sapiens Tel2, D. melanogaster LqfR exon 6, andOnly Tel2 Portion of Fly EpsinR/Tel2 Is EssentialS. cerevisiae Tel2 were aligned using MacVector and the results are shown. H. sapiens vs. S. cerevisiae: aligned length = 850, gaps = 23, identities = 116 (13 ), similarities = 102 (12 ). H. sapiens vs. D. melanogaster: aligned length = 929, gaps = 15, identities = 181 (19 ), similarities ?158 (17 ). D. melanogaster vs. S. cerevisiae: aligned length = 924, gaps = 18, identities = 110 (11 ), similarities = 121 (13 ). (TIF)Figure S2 Rescue of E-cadherin accumulation abnormality in lqfR- clones by transgene expression. Confocal microscope images of three third instar larval eye discs immunostained with antibodies to E-cadherin (red). lqfR- clones are marked by the absence of GFP (green). The images at bottom are identical to the ones at the top except only the red layer is shown and the clone is outlined. (A 9) The discs express the transgenes indicated. The genotype is ey-flp; FRT82B lqfRD117/FRT82B ubi-gfp in all panels, with the addition of Act5C-gal4, UASlqfRa/ + (B,B9) and Act5C-gal4, UAS-lqfRaexon6/ + (C,C9) on chromosome 2. scale bar: ,10 mm in A 9; ,25 mm in C,C9 (TIF)AcknowledgmentsWe are grateful to Konrad Basler, Xinhua Lin, and the Bloomington Drosophila Stock Center for flies. We acknowledge the DNA sequencing and confocal microscope facilities of the ICMB at UT Austin, and we thank Paul Macdonald for the use of his confocal micr.Red from Act.lqfRa-gfp and Act.lqfRENTH-gfp embryos: GFP-positive embryos were homogenized in 100 ml lysis buffer (1 NP40, 0.5 deoxycholate, 1 mM DTT, 150 mM NaCl, 50 mM Tris pH 8.0 with protease inhibitor cocktail [Roche, complete-mini, EDTA-free] and 2 mM PMSF). Lysis buffer (300 ml) was added followed by centrifugation at 12,000 rpm at 4uC. A 300 ml aliquot was removed and mixed with 20 ml of a 50 slurry of GFP-trapA (Chromotek) and a 10 ml aliquot was mixed with 26 SDS loading buffer as a loading control. After incubating 2 hrs. with mild shaking at 4uC, the 300 ml aliquot was spun down, the pellet collected and washed for 5 min. with shaking in 1 ml lysis buffer, and then washed again for 10 min. with shaking in 1 ml of 500 mM NaCl. The pellet was washed 4 times more in 1 ml of 500 mM NaCl and then mixed with 20 ml of 26 Laemmli Buffer. Each sample was boiled for 5 min, microfuged, and the supernatant subjected to SDS-PAGE in a 7.5 gel. Western blotting was performed as described (Chen et al., 2002). Primary antibodies were: rat anti-E-cadherin (DSHB:DCAD2, used 1:1000), mouse anti-Armadillo (DSHB:N27A1, used 1:500), rat anti-a-catenin (DSHB:DCAT-1, used 1:100), rat anti-GFP (Chromotek:3H9, used 1:1000). Secondary antibodies were from Santa Cruz Biotechnology and used at 1:5000: goat anti-rat HRP , goat anti-mouse HRP, goat anti-rat HRP.Protein blot in FigureProtein extracts of 2 adult flies containing one copy each of the transgene indicated and the ey-gal4 driver were made byFigure 9. The effect of Tel2 on Wingless signaling. A model for how Wingless signaling is compromised in the absence of Tel2 is illustrated. We speculate that in the absence of Tel2, increased Ecadherin at the plasma membrane sequesters Armadillo (Arm) so that little remains free in the cytoplasm to enter the nucleus in response to Wingless signaling. doi:10.1371/journal.pone.0046357.gSupporting InformationFigure S1 Amino acid sequence alignment of human and yeast Tel2 and Drosophila LqfR-exon 6. The amino acid sequences of H. sapiens Tel2, D. melanogaster LqfR exon 6, andOnly Tel2 Portion of Fly EpsinR/Tel2 Is EssentialS. cerevisiae Tel2 were aligned using MacVector and the results are shown. H. sapiens vs. S. cerevisiae: aligned length = 850, gaps = 23, identities = 116 (13 ), similarities = 102 (12 ). H. sapiens vs. D. melanogaster: aligned length = 929, gaps = 15, identities = 181 (19 ), similarities ?158 (17 ). D. melanogaster vs. S. cerevisiae: aligned length = 924, gaps = 18, identities = 110 (11 ), similarities = 121 (13 ). (TIF)Figure S2 Rescue of E-cadherin accumulation abnormality in lqfR- clones by transgene expression. Confocal microscope images of three third instar larval eye discs immunostained with antibodies to E-cadherin (red). lqfR- clones are marked by the absence of GFP (green). The images at bottom are identical to the ones at the top except only the red layer is shown and the clone is outlined. (A 9) The discs express the transgenes indicated. The genotype is ey-flp; FRT82B lqfRD117/FRT82B ubi-gfp in all panels, with the addition of Act5C-gal4, UASlqfRa/ + (B,B9) and Act5C-gal4, UAS-lqfRaexon6/ + (C,C9) on chromosome 2. scale bar: ,10 mm in A 9; ,25 mm in C,C9 (TIF)AcknowledgmentsWe are grateful to Konrad Basler, Xinhua Lin, and the Bloomington Drosophila Stock Center for flies. We acknowledge the DNA sequencing and confocal microscope facilities of the ICMB at UT Austin, and we thank Paul Macdonald for the use of his confocal micr.