Figure 4.

MERIT40 mediates the stability of the BRCA1–RAP80 complex at DSBs. (A) MERIT40 depletion results in decreased Rap80 and BRCC36 protein levels. IB was performed on HeLa cell lysates at 72 h following siRNA transfection as indicated. (B) MERIT40 is required for endogenous Abraxas stability (left) and ectopic Abraxas in an eAbraxas cell line (right). IB was performed for Abraxas in nontransduced (mock) or eAbraxas-expressing HeLa cells at 72 h after Ct or MERIT40 siRNA. Note that eAbraxas expression causes reduced levels of endogenous Abraxas protein. (C) MERIT40 depletion reduces Rap80 and BRCC36 protein levels and half-life. HeLa cells were transfected with Ct or MERIT40 siRNAs. Untreated (0) or treated cells were lysed at the indicated time following the addition of CHX (60 μg/mL). IB was performed as indicated. (D,E) MERIT40 knockdown reduces Rap80 DSB localization. IF was performed for Rap80 following Ct or MERIT40 siRNA treatment at IR-induced (D) or laser-induced (E) DSBs as indicated. Images are representative of >200 cells for each condition in three independent experiments. (E) Quantification of Rap80 colocalization with γH2AX at stripes is shown in Supplemental Figure S8A. (F) HeLa S3 cells containing stably expressed eRap80 were transfected with Ct- or MERIT40-targeted siRNA and subsequently examined for IRIF with α-HA and α-53BP1 antibodies. eRap80 is still able to localize to IRIF following MERIT40 depletion.