FIG. 3.

Interaction of newly translated cyclin E with CCT in human cells. (A) Cyclin E is a substrate of the CCT complex. Extracts from thymidine-blocked HeLa cells were aliquoted into three parts and incubated with either 15 mM EDTA, 5 mM MgCl₂, or 5 mM Mg plus ATP at room temperature for 20 min. Human CCTβ was immunoprecipitated from these samples, and the immune complexes (IP) along with whole-cell extract (Total) were separated by SDS-PAGE and processed for Western blot analysis. The upper half of the blot was probed sequentially with anti-cyclin E antibody and anti-β-tubulin antibody (Boehringer Mannheim). The lower half was probed with anti-PCNA antibody (Santa Cruz) and anti-Cdk2 antibody (Transduction Laboratories). (B) Newly synthesized cyclin E in vivo associates with the CCT complex. A549 cells transduced with either an adenovirus expressing cyclin E (E) or a control virus (V) were pulse-labeled with [³⁵S]methionine for 1.5 min (p), chased for 10 min (c), extracted and immunoprecipitated with anti-CCTβ antibody (lanes 1 to 4) or anti-cyclin E antibody (lanes 5 to 8). The CCT immune complexes were dissociated and reimmunoprecipitated with anti-cyclin E antibody (lanes 1 to 4), and cyclin E immune complexes were subjected to SDS-PAGE analysis.