A-USU cells. HeV-G only and HeV-F only transfections were carried out in parallel as controls. At 48 hrs post Title Loaded From File transfection, cells were lysed in buffer containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 Triton X-100 and protease inhibitors at 4uC for 30 min, clarifiedHendra Virus Entry Mechanism Implied by Structureby centrifugation, and divided into two equal portions. One half of each cell lysate was used to precipitate the HeV-F glycoprotein and the other half for precipitation of HeV-G. For total HeV-G present for precipitation, 1 ml of rabbit polyclonal anti-G antiserum was added followed by addition of 50 ml of a 20 slurry of Protein G-Sepharose 4B beads. For HeV-F precipitation, 30 ml of a 50 slurry S-protein agarose beads (EMD Biosciences Inc., Madison, WI) was added to a separate and equal amount of lysate. In both cases, the complexed products and beads were washed twice in lysis buffer, and then boiled in sample loading buffer and the precipitated proteins were analyzed by SDS-PAGE and Western blotting under reducing Fexinidazole chemical information conditions with either F or G specific mouse mAbs. The images of the precipitated F and G glycoproteins were quantified by densitometry using ImageQuantTL Software (GE Healthcare, Piscataway, NJ).conformations of residue W122 (labeled in sticks). The different packing modes in the crystal prevent the “up” and “down” W122 rotamers from switching their conformations. (TIF)Table S(DOC)AcknowledgmentsWe thank Nikolov lab members, including Alexander Antipenko, Juha Himanen, David Grandy and Dorothea Robev for general assistance, Haitao Li and Yehuda Goldgur (Structural Biology Department of MSKCC) for assistance with data collection, Professor Min Lu (Weill Medical College of Cornell University) for analytical ultracentrifugation assay. The views expressed in the manuscript are solely those of the authors, and they do not represent official views or opinions of the Department of Defense or The Uniformed Services University.Supporting InformationFigure S1 Related to Figure 6; Crystal packing helps to trap the “up” rotamer of W122. (A) Ephrin-B2 with the “down” W122 conformation contacts the adjacent HeV-G in crystal packing. (B) Ephrin-B2 with the “up” W122 conformation contacts the adjacent HeV-G in crystal packing. (C) Superimposition of the two HeV-G/ephrin-B2 complexes with differentAuthor ContributionsConceived and designed the experiments: KX DK YC CCB DBN. Performed the experiments: KX DK YC LY. Analyzed the data: KX DK YC KRR CCB DBN. Contributed reagents/materials/analysis tools: KRR YC MVK. Wrote the paper: KX DK YC CCB DBN.
Studies of variation in ovulation rate and litter size in populations of prolific sheep led to the conclusion that gene(s) with a large effect on prolificacy segregate in several breeds, including the Booroola 1527786 [1,2], Romney [3], Cambridge [4], Belclare [5], Icelandic [6], Javanese [7], Olkuska [8], Lacaune [9] and Woodlands sheep [10]. Subsequent studies led 11967625 to the identification of the genes responsible in most of these cases: the FecBB mutation in BMPR1B (bone morphogenetic protein receptor type 1B) in Booroola Merino [11] and Javanese [12] sheep; mutations in BMP15 (bone morphogenetic protein 15) in Romney (FecXI, FecXH) [13], Cambridge and Belclare (FecXG, FecXB) [14], Lacaune (FecXL) [15], and Rasa Aragonesa sheep (FecXR) [16,17]; and mutations in GDF9 (growth differentiation factor 9) in Belclare, Cambridge (FecGH) [14], Icelandic [FecGT] [18] and Santa Ines sheep (FecG.A-USU cells. HeV-G only and HeV-F only transfections were carried out in parallel as controls. At 48 hrs post transfection, cells were lysed in buffer containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 Triton X-100 and protease inhibitors at 4uC for 30 min, clarifiedHendra Virus Entry Mechanism Implied by Structureby centrifugation, and divided into two equal portions. One half of each cell lysate was used to precipitate the HeV-F glycoprotein and the other half for precipitation of HeV-G. For total HeV-G present for precipitation, 1 ml of rabbit polyclonal anti-G antiserum was added followed by addition of 50 ml of a 20 slurry of Protein G-Sepharose 4B beads. For HeV-F precipitation, 30 ml of a 50 slurry S-protein agarose beads (EMD Biosciences Inc., Madison, WI) was added to a separate and equal amount of lysate. In both cases, the complexed products and beads were washed twice in lysis buffer, and then boiled in sample loading buffer and the precipitated proteins were analyzed by SDS-PAGE and Western blotting under reducing conditions with either F or G specific mouse mAbs. The images of the precipitated F and G glycoproteins were quantified by densitometry using ImageQuantTL Software (GE Healthcare, Piscataway, NJ).conformations of residue W122 (labeled in sticks). The different packing modes in the crystal prevent the “up” and “down” W122 rotamers from switching their conformations. (TIF)Table S(DOC)AcknowledgmentsWe thank Nikolov lab members, including Alexander Antipenko, Juha Himanen, David Grandy and Dorothea Robev for general assistance, Haitao Li and Yehuda Goldgur (Structural Biology Department of MSKCC) for assistance with data collection, Professor Min Lu (Weill Medical College of Cornell University) for analytical ultracentrifugation assay. The views expressed in the manuscript are solely those of the authors, and they do not represent official views or opinions of the Department of Defense or The Uniformed Services University.Supporting InformationFigure S1 Related to Figure 6; Crystal packing helps to trap the “up” rotamer of W122. (A) Ephrin-B2 with the “down” W122 conformation contacts the adjacent HeV-G in crystal packing. (B) Ephrin-B2 with the “up” W122 conformation contacts the adjacent HeV-G in crystal packing. (C) Superimposition of the two HeV-G/ephrin-B2 complexes with differentAuthor ContributionsConceived and designed the experiments: KX DK YC CCB DBN. Performed the experiments: KX DK YC LY. Analyzed the data: KX DK YC KRR CCB DBN. Contributed reagents/materials/analysis tools: KRR YC MVK. Wrote the paper: KX DK YC CCB DBN.
Studies of variation in ovulation rate and litter size in populations of prolific sheep led to the conclusion that gene(s) with a large effect on prolificacy segregate in several breeds, including the Booroola 1527786 [1,2], Romney [3], Cambridge [4], Belclare [5], Icelandic [6], Javanese [7], Olkuska [8], Lacaune [9] and Woodlands sheep [10]. Subsequent studies led 11967625 to the identification of the genes responsible in most of these cases: the FecBB mutation in BMPR1B (bone morphogenetic protein receptor type 1B) in Booroola Merino [11] and Javanese [12] sheep; mutations in BMP15 (bone morphogenetic protein 15) in Romney (FecXI, FecXH) [13], Cambridge and Belclare (FecXG, FecXB) [14], Lacaune (FecXL) [15], and Rasa Aragonesa sheep (FecXR) [16,17]; and mutations in GDF9 (growth differentiation factor 9) in Belclare, Cambridge (FecGH) [14], Icelandic [FecGT] [18] and Santa Ines sheep (FecG.
