E spots with a greater than Ligand Blocking of Neogenin Receptor Leads to Apoptosis in EBased on previous work that suggests Neogenin may function as a dependence receptor

was incubated in 10 mM HEPES, 10 mM PIPES, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT at pH 7.0, 0.5 mM GTP in a final volume of 50 mL at 37uC for 9349566 15 minutes. Mixtures were then spun at 16,000 r.p.m. for 20 minutes in a microfuge refrigerated at 4uC to obtain supernatant and pellet fractions. The pellet fraction was resuspended in 50 mL of the same buffer to obtain equal volumes of supernatant and pellet fractions. Samples were heated at 95uC for 5 minutes after addition of 10 mL of 6 X SDSAGE buffer, resolved on a 8% polyacrylamide gel and visualized by Coomassie staining. 9 January 2011 | Volume 6 | Issue 1 | e14583 Preparation of heavy and light membrane fractions by velocity sucrose gradient centrifugation The preparation was performed according to reference. Briefly, 26107 cultured HeLa cells transfected with or without Ndel1 Regulates Dyn2 Activity Supporting Information trans-Golgi network and endoplasmic reticulum. Confocal pictures of HeLa cells transfected with a control siRNA or Ndel1 siRNA and stained for Ndel1 and p230 trans-Golgi, a protein associated with the trans-Golgi network. The depletion of Ndel1 does not alter the structure and intracellular localization of the TGN. Scale bar, 5 mm. Confocal pictures of HeLa cells transfected with a control siRNA or Ndel1 siRNA and cotransfected with a construct encoding an endoplasmic reticulum -targeted fluorophore. The three upper panels represent the three ER phenotypes observed in both treatments: perinuclear, partially dispersed and fully dispersed ER. The bar graph shows the distribution of ER phenotypes among cells. Note that the distribution of the ER, labelled with the ER-mCherry remains largely unchanged in Ndel1 siRNA-transfected cells when compared to control siRNA-transfected cells. The bar graph reports the results of one experiment and is representative of the data found in 3 independent experiments. Chi-square analysis. ns, not significant. Scale bar, 10 mm. Analysis by Western blots of the content of the KDEL ER marker and TGN marker p230 trans-Golgi in the light and heavy membrane fractions isolated from cells overexpressing GluR1 together with either Dyn2, a mutant inactive form of Dyn2 ), Ndel1, or depleted of Ndel1 by siRNA. Note that Ndel1 does not affect the distribution of the KDEL ER marker and TGN marker among the fractions: p230 is for instance mostly found in the HM fraction of cells overexpressing or lacking Ndel1 in a similar way to untransfected control cells. Dyn2, which is important for TGN biology affects p230 trans-Golgi fractionation but not KDEL ER marker distribution. On the contrary, the inactive Dyn2 mutant does not affect p230 trans-Golgi distribution. Found at: doi:10.1371/journal.pone.0014583.s001 Acknowledgments We are grateful to Dr. S Wasiak for critical reading of the manuscript and Dr. M MedChemExpress Odanacatib McNiven for the Dyn2 cDNA templates. Author Contributions Conceived and designed the experiments: MC JW MDN. Performed the experiments: MC JW HCT GN SYS YUP HTL. Analyzed the data: MC JW HCT GN YUP CB SKP MDN. Contributed reagents/materials/ analysis tools: SKP MDN. Wrote the paper: MC MDN. 10 January 2011 | Volume 6 | Issue 1 | e14583 Ndel1 Regulates Dyn2 Activity 37. McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, et al. Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape. J Cell Biol 151: 18798. 38. Orth JD, Krueger EW, Cao H, McNiven MA The large GTPase dynamin regulates actin comet formation and movement in liv

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