Fig. 1

hSSB1 associates with BLM in cells. BLM (a) or hSSB1 (b)-associating proteins were immunoprecipitated from U2OS whole cell lysates prepared from cells that had been either left untreated or exposed to 6 Gy ionising radiation (IR) and harvested after the indicated time periods. Control immunoprecipitations with an isotype IgG were performed from combination (c) samples comprised of equal amounts of each sample. Eluted proteins and whole cell lysates were separated by electrophoresis and immunoblotted with antibodies against BLM, hSSB1 and INTS3 (b only). hSSB1 (a) or BLM (b) levels were determined by densitometry, normalised to the levels of BLM (a) or hSSB1 (b) as well as to the level of input protein and expressed relative to the untreated lane. c Line graph illustrating hSSB1: BLM association from three independent repeats of a and b. d BLM was immunoprecipitated from whole cell lysates, prepared from U2OS cells that were either untreated or had been treated with 2 mM hydroxyurea (HU) for 6 h. Eluted proteins and whole cell lysate samples (input) were immunoblotted with antibodies against BLM and hSSB1. hSSB1 levels were determined and expressed as per (a). e U2OS cells were transfected with plasmids encoding wild type (WT) or F98A 3× FLAG hSSB1, 24 h prior to cell lysis and immunoprecipitation of BLM-association proteins. Eluent was immunoblotted with antibodies against BLM, FLAG, INTS3, MRE11, RPA70 and RPA32. FLAG levels were determined by densitometry and normalised to the levels of BLM