p.Ser36Tyr (S36Y) BRCA1:BARD1 complex is withheld within the cytoplasm following HU treatment.

<p>Lysates of transfected cells were fractionated into nuclear and cytosolic extracts following S-phase synchronization and treatment with HU. BARD1 was co-precipitated with the ectopically expressed BRCA1 in the A. Nuclear and B. Cytosolic extracts with the anti-DYKDDDDK tag antibody which recognizes only the exogenous BRCA1. A. Immunoblot analysis of the pull-downs demonstrated that unlike wild type BRCA1, both the p.Ser36Tyr and p.Cys61Gly (C61G) variants exhibit reduced ability to co-precipitate BARD1 in the nuclear extracts of stressed cells. B. However, in the cytosolic extracts the p.Ser36Tyr variant, but not p.Cys61Gly, co-precipitated a higher proportion of BARD1 in treated cells compared to wild type transfected cells. In untreated cells, both variants, including wild type BRCA1, co-precipitated larger amounts of BARD1 compared to treated cells. Bar charts showing that C. in the nuclei of treated cells, both the p.Ser36Tyr and p.Cys61Gly variants exhibit reduced ability to co-precipitate with BARD1, whereas D. in the cytosol the p.Ser36Tyr variant co-precipitates similar levels of BARD1 compared to wild type BRCA1, but it is not statistically significant. Immunoblot analysis of nuclear and cytoplasmic extracts demonstrating their purity is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093400#pone.0093400.s002" target="_blank">Fig. S2</a>. The results are representative of 2 experiments.