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Fig. 2. Pds5A and Pds5B associate with cohesin in HeLa cell nuclear extracts. (A) Immunoblot of HeLa cell nuclear extracts with antibodies against Pds5A (lane 1), Pds5B (lane 2) and the cohesin subunit Smc3 (lane 3). (B) The same antibodies were used in immunoprecipitations carried out on HeLa cell nuclear extracts in the absence (lanes 1, 3 and 5) or presence (lanes 2 and 4) of the corresponding antigen peptide (0.5 mg/ml). The immunoprecipitates were fractionated in a 7.5% SDS-polyacrylamide gel and stained with silver. The positions of cohesin subunits, Pds5A and Pds5B are marked by arrowheads, a white circle and a black circle respectively, in lanes 1 and 3. The two polypeptides of 105 and 110 kDa present in the Pds5B precipitate (asterisks in lane 3) are likely to be non-specific because neither of them was detectable when a different antibody against Pds5B was used. (C) Immunoblot analysis of the immunoprecipitates obtained with non-immune rabbit IgG as control (lane 1), anti-Pds5A (lane 2), anti-Pds5B (lane 3) or anti-Smc3 (lane 4). (D) The immunoprecipitates obtained with anti-Smc3 were washed with a buffer containing increasing concentrations of KCl (0.2 M, 0.4 M and 0.8 M, lanes 1-3) before being analyzed by immunoblotting.
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