Fig. 5

Cyclin K counteracts cyclin E1 to promote pre-RC assembly. a Expression profiling of cyclin proteins with or without cyclin K knockdown for 48 and 72 h in HCT116 cells. Ab1 denoted a commonly used commercial anti-cyclin E1 polyclonal antibody. Asterisk denoted the non-specific signal detected by several batches of Ab1. b Verification by a monoclonal anti-cyclin E1 antibody (Ab2). Note that Ab2 only detected the faster mobility band of cyclin E1. c Demarcation of epitope regions of Ab1 and Ab2 by a series of cyclin E1 truncation mutants. 1–410 denoted full-length cyclin E1. All proteins were FLAG-tagged at the C-terminus, and expression constructs were transfected into HEK293 cells. d Alkaline phosphatase (AP) treatment of HCT116 cell lysates derived from cells with or without cyclin K knockdown, followed by protein blotting analyses. e Differential recognition of phosphorylation mimics by Ab2 and Ab3. S366A, alanine substitution of serine to mimic unphosphorylated state. S366D, aspartate substitution of serine to mimic phosphorylated state. Expression constructs were transfected into HEK293 cells. Right panel, summary of the specificity of antibodies used in Fig. 5. f Endogenous cyclin E1 protein detected by Ab2 and Ab3 with or without cyclin K knockdown in HCT116 cells. g Similar experiment as in d except that CDK12 (12) was knocked down in HCT116 cells. h Detection of endogenous chromatin-bound cyclin E1 by Ab2 with or without cyclin K knockdown in HCT116 cells. i Distribution of wild-type and mutant cyclin E1 proteins in soluble and chromatin-bound fractions derived from HCT116 cells. j Endogenous CDK2 in HEK293 cells was immunoprecipitated by wild-type or mutant cyclin E1 proteins, followed by protein blot analyses. k Endogenous CDK2 in HCT116 cells was immunoprecipitated by anti-CDK2 antibody. Immunoprecipitates were then treated with or without alkaline phosphatase (AP), and associated endogenous cyclin E1 was analyzed. A/G, protein A/G beads without anti-CDK2 antibody. l Small-molecule inhibitor of CDK2 (dinaciclib, 500 nM) partially rescued CDT1 loading defect caused by cyclin K knockdown in HCT116 cells. m S366A and wild-type but not S366D cyclin E1 prevented pre-RC assembly (indicated by MCM4 loading onto chromatin) in HEK293 cells. All experiments were repeated at least three times and representative results are shown