Fig. 2

Downregulation of SKP2 pathway by proteasome inhibition causes accumulation of ubiquitinated proteins and upregulates the expression of p27Kip1. a Bortezomib-mediated ubiquitination of various proteins in CML cells. AR230 and K562 cells were treated with different doses of bortezomib for 24 h as indicated. After cell lysis, equal amounts of proteins were separated by SDS–PAGE, transferred to PVDF membrane, and immunoblotted with antibodies of anti-ubiquitin and GAPDH as indicated b Bortezomib treatment down-regulated the expression of SKP2 and increased the level of p27Kip1. AR230 and K562 cells were treated with various doses of bortezomib for 24 h as indicated. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immuno-blotted with antibodies against SKP2, p27Kip1 and GAPDH as indicated. c Bortezomib treatment of K562 cells causes the stabilization of p27. K562 cells were treated with and without 25 nm of Bortezomib for 24 h. Cells were then treated with 10 μM Cyclohexamide for 30, 60, 120 and 240 min. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immuno-blotted with antibodies against p27Kip1 and GAPDH as indicated. Each band was quantified by densitometry and ratio of p27/GAPDH was plotted. d SKP2 siRNA expression downregulates SKP2 and accumulated p27Kip1. AR230 and K562 cells were transfected with Scrambled siRNA (100 nm) and SKP2 siRNA (50–100 nm) using Lipofectamine 2000 as described in “Methods” section. After 48 h of transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against SKP2, P27Kip1, and GAPDH as indicated