Fig. 3

hSSB1 is required for recruitment of BLM to chromatin in response to DNA damage. a, b, c U2OS cells were transfected with control or hSSB1 mRNA-targeting siRNA. 72 h later, cells were then either left untreated or exposed to 6 Gy ionising radiation (IR), 4 h prior to harvesting and subcellular fractionation. Proteins from the indicated fractions were separated by electrophoresis and immunoblotted with antibodies against BLM, hSSB1, H3 (for chromatin fractions) and actin (for the soluble nuclear fraction). BLM levels were determined by densitometry, normalized to the levels of H3 or actin and expressed relative to the untreated lane. d, e, f U2OS cells were depleted of hSSB1 as per above and treated with 1 μM camptothecin (CPT) for 3 h or 2 mM hydroxyurea (HU) for 20, 72 h post siRNA transfection. Cells were then harvested, subcellular fractionation performed and the indicated fractions immunoblotted with antibodies against BLM, hSSB1, Pol II CTD, RPA70, pS33 RPA32, RPA32, SP1 and H3 as indicated. BLM levels were determined as per above. g HeLa cells were transfected with hSSB1 transcript-targetting siRNA and harvested after 48 or 96 h. Whole cell lysates were prepared and immunoblotted with antibodies against BLM, hSSB1 and actin