FIG. 7.

Symplekin-I exists in a complex containing both CstF and CPSF. (A) Immunoaffinity purification of a polyadenylation complex under mild conditions. The CSF-containing fraction obtained by Superose 6 column chromatography was subjected to immunoaffinity purification using an anti-CstF-64 MAb in the presence of 50 mM (NH₄)₂SO₄ but no NP-40. Total starting material (T) (lanes 1 and 4) and flowthrough (F) (lanes 2 and 5) and bound (B) (lanes 3 and 6) fractions were loaded on an SDS–10% polyacrylamide gel and stained with silver (lanes 1 to 3) or probed with anti-CstF-64, anti-CPSF-160, or anti-symplekin antibody (lanes 4 to 6). (B) Immunoaffinity purification of a polyadenylation complex under stringent conditions. The CSF-containing fraction was subjected to immunoaffinity purification using an anti-polyomavirus large T (αPy-lgT; lanes 1 to 3 and 7 to 9) or anti-CstF-64 (αCstF-64; lanes 4 to 6 and 10 to 12) MAb in the presence of 150 mM (NH₄)₂SO₄ and 0.05% NP-40. Total starting material (T), (lanes 1, 4, 7, and 10) and flowthrough (F) (lanes 2, 5, 8, and 11) and bound (B) (lanes 3, 6, 9, and 12) fractions were loaded on an SDS–7.5% polyacrylamide gel and stained with silver (lanes 1 to 6) or probed with anti-CstF-64, anti-CPSF-160, anti-CPSF-100/73, or anti-symplekin antibody (lanes 7 to 12).