Fig. 3. PRH suppresses cyclin D1 protein production and cyclin D1 mRNA transport. (A) Induction of PRH, PRHΔ and PRHLL cells is the same in the U937 system as observed by both PRH polyclonal antibody and FLAG tag antibody. PRHLL refers to the PRHLL2324AA mutant and PRHΔ to the deletion mutant. (B) Cyclin D1 levels are suppressed by overexpression of PRH wild-type but not mutant constructs. (C) Addition of 10 µM lactacystin does not reverse the effects of PRH on cyclin D1 protein levels. (D) Northern analysis of RNA isolated from total cell lysates of PRH-overexpressing cells indicates that there are no alterations in the total levels of cyclin D1 mRNA. (E) Fractionation studies in conjunction with northern analysis reveal that PRH overexpression leads to retention of cyclin D1 mRNA in the nucleus. Cells transfected with vector or overexpressing PRH mutants do not have these effects. n indicates the nuclear, and c the cytoplasmic fraction. tRNALys is a marker for the cytoplasmic fraction, and U6snRNA for the nuclear fraction. The numbers 0, 2 and 3 indicate days post-tetracycline withdrawal. W.B., western blot; N.B., northern blot.