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Fig. 6. Intranuclear distribution of ATRX in Daxx reconstituted cells. (a) Daxx wild-type (wt) reconstituted cells labeled for ATRX and PML show colocalization of the proteins at ND10 (yellow), indicating a Daxx requirement in ATRX recruitment to ND10. (d) The same cells stained with ATRX and HA show colocalization of ATRX with Daxx at ND10-like domains. (e) DaxxC accumulation at ND10-like domains but no recruitment of ATRX to ND10 (b). In cells reconstituted with Daxx{delta}C and double stained for ATRX and PML (c) or HA and ATRX (f), ATRX does not accumulate at ND10 but has exclusive accumulation in irregular domains (c) while localizing with HA-Daxx{delta}C in a subpopulation of cells at irregular heterochromatic domains (f, lower left and right cells). (g) Western-blot analysis of interaction between ATRX and Daxx. Nuclear extracts produced from Daxx–/– cell lines reconstituted with empty vector (lane pBabe), Daxx wt (lane pBabe wt), Daxx{delta}C (lane pBabeDaxx{delta}C) and DaxxC (lane pBabeDaxxC) were incubated with anti-FLAG antibody to immunoprecipitate Daxx (FLAG IP) and probed for the presence of ATRX. No ATRX-derived signal was detected in extracts from the empty vector and DaxxC-reconstituted cells, whereas the signal was present in immunoprecipitates of Daxx wt and Daxx{delta}C cell extracts. Corresponding input (20%) for the ATRX load is shown (right).





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