The small Jab1 complex is required for down-regulation of p27 in K562 cells. (A) K562 cells were transfected with the siRNA expression vectors indicated at the top of the panel together with the GFP expression vector. After 60 hours of transfection, cell lysates were separated by SDS-PAGE (first to fourth rows) and native-PAGE (fifth and sixth rows), and analyzed by immunoblotting using antibodies against Jab1, p27, Cul1, and γ-tubulin. The relative amounts of Jab1 and p27 are presented as the ratio of Jab1–γ-tubulin and p27–γ-tubulin and calculated with the level of K562 cells transfected with the empty siRNA expression vector as 1.0. (B) The cell-cycle distributions of the GFP-positive cells in panel A were analyzed using a flow cytometer after staining with Hoechst 33342. The averages of 3 independent experiments are shown. (C) K562 cells transfected with the siRNA expression vectors were treated with DMSO and STI571 for 8 hours. Cell lysates were separated by SDS-PAGE (first to fourth rows) and native-PAGE (fifth and sixth rows), and analyzed by immunoblotting using antibodies to Jab1, p27, Cul1, and γ-tubulin. The relative amounts of Jab1 and p27 are presented as the ratio of Jab1–γ-tubulin and p27–γ-tubulin and calculated with the level of untreated K562 cells transfected with the mJab1 siRNA expression vector as 1.0.