Ser10-phosphorylated p27^KIP1 is predominantly cytoplasmic in PEL cells. (A) BC-3 cells were separated into cytoplasmic and nuclear extracts as described in “Materials and methods.” The resulting fractions were resolved on SDS-PAGE (12%) and analyzed by immunoblotting with anti-p-p27 (Ser10), anti-p27^KIP1, and anti-v-cyclin antibodies (top 3 panels). The distribution of the nuclear marker Sp1 and the cytoplasmic marker β-tubulin confirmed the purity of the fractions (bottom 2 panels). (B) U2OS cells were transfected with expression vector for Myc-p27^KIP1 alone or together with v-cyclin. Cells were analyzed 48 hours after transfection by indirect immunofluorescence. The cells were labeled by anti-Myc and anti-p-p27^KIP1 Ser10 antibodies as indicated and their nuclear morphology was visualized by Hoechst staining. (C) U2OS cells were transfected with expression vector for Myc-p27^KIP1 alone or together with v-cyclin or with S10A-p27^KIP1 alone. Total-cell lysates of transfected cells were resolved by SDS-PAGE (12%) and immunoblotted with anti-p-p27 (Ser10), anti-p27^KIP1, and anti-v-cyclin antibodies as indicated (WB).