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. Author manuscript; available in PMC: 2013 Jun 28.
Published in final edited form as: Mol Cell. 2010 Feb 12;37(3):438–446. doi: 10.1016/j.molcel.2010.01.002

FIGURE 3.

FIGURE 3

TopBP1 and BACH1 are involved in the chromatin accumulation of RPA following replication stress. (A) Both TopBP1 and BACH1 are required for RPA loading following HU treatment. U2OS cells transfected with control, TopBP1 or BACH1 siRNAs were mock treated or treated with HU (10 mM). Cells were harvested at the indicated timepoints. The soluble and chromatin fractions were prepared and immunoblotted with antibodies as indicated. Also see Figure S3. (B) The interaction between TopBP1 and BACH1 is required for RPA1 loading on chromatin at stalled replication stress. U2OS cells were infected with retroviruses expressing siRNA-resistant wild-type TopBP1 and then transfected with control or TopBP1 siRNAs. In the other case, U2OS cells stably expressing siRNA resistant wild-type, K52R mutant or T1133A mutant of BACH1 were transfected with control siRNA or BACH1 specific siRNA. 72 hours after initial siRNA transfection, cells were treated with 10mM HU and collected one hour later. Cell lysates were immunoblotted with indicated antibodies.