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. 2011 Apr 28;12(6):574–580. doi: 10.1038/embor.2011.74

Figure 4.

Figure 4

Chimeric proteins containing RBM can restore HARP function in vivo. (A) U2OS-derivative cells stably expressing HA-Flag-tagged fusion proteins or the SNF2 domain alone (BRG1 and HELLS), chimeras lacking RBM (chBRG1 and chHELLS), and chimeras with RBM (RBM-chBRG1 and RBM-chHELLS) were generated. The hydroxyurea-induced foci-forming abilities of these fusion proteins were determined by immunostaining using the indicated antibodies. (B) The endogenous and exogenous expression levels of HARP and the chimeric proteins were confirmed by immunoblotting using the indicated antibodies. The extracts were prepared from cells transfected with indicated siRNAs. (C) U2OS-derivative cells were transfected with the indicated siRNAs; 72 h later, cells were subjected to immunostaining using RPA2 and γH2AX antibodies. Foci-positive cells were quantified by counting a total of 200 cells per sample. Data are presented as mean±s.d. from three independent experiments. Scale bars, 10 μm. BRG1, Brahma-related gene 1; DAPI, 4′,6-diamidino-2-phenylindole; HA, haemagglutinin; HARP, HepA-related protein; HELLS, helicase, lymphoid specific; RBM, RPA-binding motif; RPA, replication protein A; siRNA, short interfering RNA.