letters to nature Methods Cell lines All cell lines were obtained from the American Tissue Culture Collection or the Coriell Institute apart from ATLD1 and ATLD3 cells (a gift from J. Petrini), ATM cells (a gift from Y. Shiloh and J. Engelhardt), and cells with mutant ligase IV (180BRM; a gift from H. Wang and G. Iliakis). To complement cells, the complementary DNAs for Mre11 and NBS1 were amplified by PCR and cloned under control of the CMV promoter in a modified version of the pCLNC retrovirus vector27. Retroviruses were generated by transfecting gag/pol packaging cell lines, together with a plasmid expressing the VSV-G envelope protein. ATLD or NBS cells were infected with retrovirus supernatants and selected in 900 mg ml21 G418. We assessed expression of Mre11 and NBS1 by immunoblotting and used pools of resistant clones for complementation experiments. The mre11-3 mutant contains the HD129/130LV mutation generated by site-directed mutagenesis using QuickChange (Stratagene).
Antibodies We visualized viral replication centres by staining for DBP with either a mouse monoclonal antibody (B6-8) or rabbit polyclonal antisera. Antibodies were purchased from Novus (NBS1, ATM), Genetex (Mre11-12D7, Rad50-13B3), Santa Cruz (haemagglutinin A (HA), Ku86, DNA-PKcs, NBS1-S343) and Roche (BrdU). Antisera to E4orf6, pTP, PML, RPA70 and DBP were gifts from P. Branton, J. Schaack, T. Sternsdorf, T. Melendy, A. Levine and P. van der Vliet, respectively. All secondary antibodies were from Jackson Laboratories.
Viruses, infections, PFGE and western blotting The E4 mutant viruses have been described8,11 and were propagated and titred by plaque assays on W162, a Vero-derived E4-complementing cell line28. Wild-type Ad type 5 (Ad5) and dl110 were propagated in human 293 cells. We purified all viruses by two sequential rounds of ultracentrifugation in CsCl gradients and stored them in 40% glycerol at 220 8C. HeLa cells were infected with a multiplicity of infection (MOI) of 25 plaqueforming units (p.f.u.) per cell and collected at the indicated hour post-infection (h.p.i.). Other cell lines were infected with MOIs of 25–100 and collected between 48 and 72 h.p.i. We carried out PFGE essentially as described5,6 and included l DNA size markers. Western blotting was done as described20.
Immunofluorescence Cells were grown on glass coverslips in 24-well dishes and infected with wild-type or mutant viruses at an MOI of 25 p.f.u. per cell. After 8–18 h, the cells were washed with PBS and fixed at 220 8C for 10 min with ice-cold methanol:acetone (1:1). We carried out immunofluorescence essentially as described29. In all cases, control staining experiments showed no crossreaction between the fluorophores, and images obtained by staining with individual antibodies were the same as those shown for double-labelling. We stained nuclear DNA with 4 0 ,6-diamidino-2-phenylindol (DAPI) and mounted coverslips using Fluoromount-G (Southern Biotechnology Associates) or Vectashield (Vector Labs). Immunoreactivity was visualized by using a Nikon microscope in conjunction with a CCD camera (Cooke Sensicam), or by deconvolution using a DeltaVision microscope system with software from the supplier (Applied Precision). Images were obtained in double or triple excitation mode and processed using SlideBook and Adobe Photoshop.
Acknowledgements We thank G. Ketner for the E4 mutant viruses and W162 cells, I. Verma for retrovirus constructs and cell lines, T. Paull for cDNAs; B. Gilbert for technical assistance; S. Malpel for complemented NBS cells; J. Bailis for advice on PFGE; M. Blower and the Center for Cytometry and Molecular Imaging at the Salk Institute for deconvolution assistance; J. Simon for help with figures; J. Weitzman, M. Grifman, T. Cathomen, C. Barlow, R. Bushman and S. Forsburg for critically reading the manuscript; R. Evans, T. Hunter, G. Wahl, B. Sefton, D. Spector, T. de Lange and J. Petrini for discussions; S. Stampfer for encouragement; and the James B. Pendleton Charitable Trust for providing the Pendelton Microscopy Facility. M.D.W. is supported in part by a grant from NIH, and by gifts from the Joe W. & Dorothy Dorsett Brown Foundation and the Lebensfeld Foundation. T.S. was supported by an NIH Graduate Training Grant to UCSD, and by fellowships from the Chapman Foundation, the Legler Benbough Foundation and the Salk Institute Association.
Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to M.D.W. (e-mail: [email protected]
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The mammalian sodium channel BNC1 is required for normal touch sensation Margaret P. Price, Gary R. Lewin, Sabrina L. Mcllwrath, Chun Cheng, Jinghul Xie, Paul A. Heppenstall, Cheryl L. Stucky, Anne G. Mannsfeldt, Timothy J. Brennan, Heather A. Drummond, Jing Qiao, Christopher J. Benson, Deirdre E. Tarr, Ron F. Hrstka, Baoll Yang, Roger A. Williamson & Michael J. Walsh
Nature 407, 1007–1011 (2000). .............................................................................................................................................................................
In this Letter, the x axes for the stimulus response functions shown in Fig. 2a–f were incorrectly labelled 5, 10, 20, 40 (and 80) mm. The axes should read 50, 100, 200, 400 (and 800) mm. This error does not alter our conclusions. A
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