p.Ser36Tyr (S36Y) BRCA1 variant demonstrates reduced ability to interact with BARD1 in S-phase synchronized cells.

<p>A. Exogenously expressed BRCA1 co-precipitates with BARD1 in cells that were co-transfected with BRCA1 and BARD1, S-phase synchronized and treated with HU. Immunoblot analysis of the pull-downs performed with the anti-DYKDDDDK tag antibody which recognizes only the epitope-tagged exogenous BRCA1 protein, demonstrated that BARD1 levels co-precipitated by wild type BRCA1 are higher than those co-precipitated by each of the p.Ser36Tyr and p.Cys61Gly (C61G) variants in both resting or HU treated cells. B. Immunoblot analysis of whole cell extracts detected decreased BARD1 and exogenous BRCA1 expression in p.Ser36Tyr and p.Cys61Gly BRCA1 variant transfected cells compared to the wild type transfected cells. β-actin served as loading control. C. Bar charts comparing the levels of BARD1 co-precipitated by p.Ser36Tyr and p.Cys61Gly variant with wild type BRCA1 in untreated cells or cells treated with HU, demonstrate that both variants exhibit reduced ability to interact with BARD1 compared to wild type BRCA1. D. Bar charts showing the expression levels of wild type BRCA1 and p.Ser36Tyr and p.Cys61Gly variants following normalization with β-actin in whole cell extracts, demonstrate that both variants are expressed at lower levels compared to wild type BRCA1. The results are representative of 3 experiments.