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. Author manuscript; available in PMC: 2013 Aug 10.
Published in final edited form as: Mol Cell. 2012 Jun 14;47(3):410–421. doi: 10.1016/j.molcel.2012.05.025

Figure 4. A C-terminal conserved region in AH2 is required for its tethering to DNA damage sites.

Figure 4

(A) HeLa cells were transfected with the plasmid encoding SFB-tagged AH2 and dPIPB mutant. Immunostaining experiments were performed 6 hr after HU treatment using indicated antibodies. (B) Schematic representation of wild type and mutant AH2 used in this study. Four distinct conserved regions are presented: SNF2 helicase domain (residues 45–498); PIP box (residues 510–528); HARP-like (HPL) domain (residues 712–820); and HNH motif (residues 1011–1051). (C and D) Experiments were performed as in (A). (E) Foci-positive transfected cells were quantified by counting a total of 100 transfected cells with positive staining. Data are presented as mean ± s.d. from 3 different experiments. (F) Deletion of C-terminal conserved region (dC80) failed to rescue HU hypersensitivity in cells with AH2 depletion. AH2 depleted (AH2 shRNA#55) HeLa derivative cell lines stably expressing shRNA-resistant wild-type AH2 or dC80 mutant were generated. The empty vector was included as control. Survival curves are shown for indicated cell lines in response to increasing doses of HU. Data are presented as mean ± s.d. from three different experiments. (G) The endogenous and exogenous AH2 expression was confirmed by immunoblotting using indicated antibodies. See also Figure S2 and S3.