Fig. 2. HCF-1 interacts with complexes that contain splicing snRNPs and SMN. (A) Immunoprecipitation using anti-HCF-1 antibody (HC2). Lane 1 (marked CTRL1) is the positive control and contained HeLa nuclear extract. CTRL2 (lane 2) is a control immunoprecipitation using pre-immune IgG. Lanes 3–6 contained immunoprecipitates using HC2 antibodies, except that the immunoprecipitate in lane 3 was washed with PBS containing 0.01% Triton X-100 whereas the immunoprecipitates in lanes 4–6 were washed with PBS/Triton X-100 as above but with increasing concentrations of NaCl (i.e. 200, 300 and 400 mM), respectively. The beads containing immunoprecipitated proteins were loaded onto a 4–12% SDS–polyacrylamide gradient gel (Novex) and probed by western blotting using anti-HCF-1 antibodies. HCF-1 bands on the blot were revealed by ECL (Amersham-Pharmacia) and are shown by the bracket on the right of (A). (B) The experiment and the lane markings of the panel are identical to (A) except that the immunoblot was probed with the anti-Sm antibody Y12 instead of anti-HCF-1 antibodies. (C) A 10 µg aliquot of HC1 (lane 3) or HC2 (lane 4) was used to immunoprecipitate HCF-1 from ∼0.2 mg of HeLa nuclear extract. CTRL1 is a positive control lane containing ∼25 µg of HeLa nuclear extract. CTRL2 (lane 2) is a negative control immunoprecipitation reaction using 10 µg of pre-immune IgG. The immunoblot was probed with anti-SMN mAbs (MANSMA1), and the SMN bands revealed by ECL.