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. 2016 Feb 4;61(3):434–448. doi: 10.1016/j.molcel.2015.12.017

Figure 6.

Figure 6

Abraxas Promotes BRCA1 BRCT Dimerization In Vivo

(A) Abraxas-dependent BRCA1 dimerization in vivo. The differentially Myc- and FLAG-tagged BRCA1 full-length constructs were transiently transfected into parental 293T (Ctrl) or Abraxas KO cells. The lysates from cells treated with 10 Gy IR followed by 1 hr incubation at 37°C were used for FLAG-immunoprecipitation. The intensity of individual bands was quantified by densitometric analysis using NIH ImageJ software. The normalized value (IPed_mycBRCA1/Input_mycBRCA1) was shown in the bar graph.

(B) Abraxas-dependent BRCA1-BRCT domains dimerization in vivo. The differentially Myc- and HA-tagged BRCA1-BRCT domains constructs were transiently transfected into parental 293T (Ctrl) or Abraxas KO cells. The lysates from cells treated with 10 Gy IR followed by 1 hr incubation at 37°C were used for HA-immunoprecipitation. The band intensity was quantified with NIH imageJ software. The normalized value (IPed_mycBRCT/Input_mycBRCT) was shown in the bar graph.

(C and D) BRCA1 germline mutations F1662S or M1663K decrease BRCA1 dimerization in vivo. Myc-tagged BRCA1 full-length (WT-FL) and HA-tagged BRCA1 BRCT (WT-BRCT) or Myc-tagged mutant full-length (F1662S or M1663K) and HA-tagged BRCT triple mutant (TM, F1662S/M1663K/R1670E) were co-expressed in cells. The lysates from cells treated with 10 Gy IR followed by 1 hr incubation at 37°C were prepared for either Myc- immunoprecipitation (C) or reciprocal IP with HA- immunoprecipitation (D). The normalized value was shown in the bar graph.

(E) Abraxas dimerize/oligomerize in vivo independent of binding to BRCA1. The differentially tagged GFP- and HA-FLAG-tagged WT or GFP-tagged and HA-FLAG-tagged Abra1 mutants (S404A or S406A) were co-expressed in cells. The anti-GFP immunoprecipitation was carried out with lysates from 293T cells treated or not treated with 10 Gy IR followed by 1 hr incubation at 37°C. The images are from the same blot.

(F) Abraxas dimerizes/oligomerizes, in vivo, through the CC domain. The immunoprecipitations were carried out with lysates prepared from cells co-expressing HA-FLAG-tagged WT Abra1 or HA-FLAG-tagged CC domain deletion mutant (ΔCC) and GFP-tagged WT or ΔCC (see also Figure S6).