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Vpr and VPRBP associate on chromatin.

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posted on 02.09.2010, 00:52 by Jean-Philippe Belzile, Levon G. Abrahamyan, Francine C. A. Gérard, Nicole Rougeau, Éric A. Cohen

A) HeLa cells were transfected with a plasmid expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Soluble and chromatin-bound fractions were isolated and were subjected to anti-HA immunoprecipitation as described in Materials and Methods. B) HeLa cells were transfected with a plasmid expressing HA-tagged Vpr (WT) or Vpr (1–78). Soluble and chromatin-bound fractions were isolated and subjected to anti-HA immunoprecipitation as described in Materials and Methods. Half of the immunoprecipitations (lanes 8, 10, 12, and 14) were conducted in presence of 25 µg/ml ethidium bromide (EtBr) to displace proteins from DNA. For both panels, input controls and immunoprecipitates were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody.

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