Fig. 3. Association of Pds5A with chromatin requires functional cohesin in HeLa cells. (A) HeLa cells were transfected with siRNAs specific to the cohesin subunit Scc1, Pds5A or Pds5B or mock-transfected with buffer as control. To estimate the extent of depletion, aliquots of siRNA-treated cell extracts were analyzed by immunoblotting (lanes 5-7) alongside increasing amounts of a control cell extract (expressed as percentage, lanes 1-4). The levels of Orc2 were analyzed as a loading control. (B,C) HeLa cells transfected as in A were pre-extracted with detergent before fixation and stained with DAPI and anti-Smc3 (B) or anti-Pds5A (C). All images were acquired over the same exposure time. Bar, 50 µm.