Fig. 5. Wild-type HA-RPA32-containing heterotrimers are hyperphosphorylated following siRNA downregulation of endogenous RPA32 and HU treatment. (A) SCC38 cells were transfected with HA-wt-RPA32 (lanes 2,3) or HA-A4A8-RPA32 (lanes 3,4). Cells were treated with siRNA directed towards the 3' non-coding sequence of RPA32 for 48 hours. Following siRNA downregulation of endogenous RPA32, cells were subjected to HU (5 mM) treatment for 3 hours. Lysates from either control or HU-treated cells were analyzed for the presence of RPA32 by western blot analysis using RPA32 antibodies that recognize both endogenous and transfected RPA32. The membrane was reblotted without stripping with antibodies that recognize phosphorylated S4S8 forms of RPA32 (bottom panel). (B) HA-tagged RPA32 formed a complex with endogenous RPA70 and RPA14, as demonstrated by co-immunoprecipitating HA-tagged RPA32. Lysates prepared from UM-SCC38 cells transfected with an empty vector or HA-wt-RPA32 were subjected to immunoprecipitation using HA antibodies. Immunoprecipitates were then analyzed for RPA70, RPA32 and RPA14 by western blot analysis (lanes 1-2).