Figure 5

Brca1 and Wwox interact in a complex through Brca1 central region. (a) Western blot (WB) and corresponding Coomassie blue staining of GST pulldown using GST, and GST-Wwox WW1/WW2 fragment, GST-Wwox WW1/WW2 fragment with mutation in WW1 domain, GST-Wwox WW1/WW2 fragment with mutation in WW2 domain, GST-Wwox SDR fragment and GST-Wwox (full-length) to IP HA-Brca1 following transfection in HEK293T cells. GST-Wwox with WW1 mutation and GST-Wwox SDR fragment do not IP HA-Brca1, suggesting that Wwox interacts with Brca1 through its first WW domain. (b) WB of HEK293T cells transfected with HA-Brca1 plus myc-Wwox and corresponding IP using IgG and HA (Covance) antibodies as bait. (c) Schematic of Wwox domain. (d) Schematic of Brca1 domain and deletion mutants. (e) WB and corresponding HA (SCBT) co-IP of MDA MB-231 cells following transfection with ΔN, ΔM1, ΔM2, ΔM3 and ΔC Brca1 deletion mutants as well as myc-Wwox. Brca1 deletion mutants are detected by polyclonal anti-Brca1³⁷ directed against Brca1 amino acids 400–1100, resulting in weaker detection of ΔM1 and ΔM2 mutants. (f) Graph depicting % survival of doxycycline-inducible 231 cells that express Wwox WW1 mutant with (231/WW1 mut-pos) and without (231/WW1 mut-neg) doxycycline after increasing IR doses. (g) Graph depicting % survival of doxycycline-inducible 231 cells that express Wwox SDR mutant with (231/SDR mut-pos) and without (231/SDR mut-neg) doxycycline after increasing IR doses.