Figure 6.

Cyclin E is up-regulated in K5 E2F1 transgenic keratinocytes. Primary keratinocytes were isolated and cultured from the epidermis of nontransgenic (wt) and K5 E2F1 transgenic (line 1.0 and line 1.1) newborn mice. Total RNA and protein was extracted after 48 to 72 hr. (A) Twenty micrograms of total RNA was subjected to Northern analysis by using a random primed-labeled fragment of the mouse cyclin E cDNA as probe. The blot then was striped and reprobed for 7S RNA. (B) Fifteen micrograms of protein from nontransgenic (wt) and transgenic (line 1.0 and 1.1) keratinocytes was subjected to Western analysis by using polyclonal antisera specific for cyclin E. (C) One hundred and forty micrograms of whole-cell extract from K5 E2F1 transgenic keratinocytes (1.1) or keratinocytes derived from nontransgenic littermates (wt) were immunoprecipitated with 0.5 μg of antibody specific for cyclin E. A kinase assay then was performed with histone H1 as the substrate.