Fig. 2. Impaired formation of RPA32-S(P)4-S(P)8 foci in NBS-ILB1 (vector only) and NBS-ILB1 (S343A) cells. (A-R) The indicated isogenic cell lines were treated with 5 mM HU for 3 hours prior to detergent-extraction, fixation and immunofluorescence using RPA32-S(P)4-S(P)8 (green) or RPA32 (red) antibodies. (D-F) HU treatment induced RPA recruitment to sites of DNA damage and normal RPA hyperphosphorylation occurred in NBS-ILB1 (NBS1) cells. (J-L,P-R) NBS-ILB1 (vector only) and NBS-ILB1 (S343A) cells recruited RPA to sites of DNA damage; however, RPA32 was not phosphorylated at these sites. HU toxicities were comparable for all cell lines tested (supplementary material Fig. S3).