Figure 2

Loss of CSN2 and CSN5 results in sequential loss of F-box proteins. K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and cells re-cultured. (A) shVC and shCSN2 cells were harvested day 6 and 9 post transfection and the level of Skp2, Cdc4, β-TrCP and β-actin protein determined by western blot. (B) The level of Skp2, Cdc4 and β-TrCP proteins (normalised for loading using β-actin) in shCSN2 at each time point was normalised to expression in shVC cells and the data plotted as the mean ± s.e.m. (C) shVC, shCSN2 and shCSN5 cells were treated with DMSO (control) or the proteasome inhibitor MG132 (10 μM) for the final 18 hours of culturing and the level of Skp-2, Cdc4, β-TrCP and β-actin protein determined by western blot. (D, E) The level of Skp2, Cdc4 and β-TrCP mRNA in shCSN2 (D) and shCSN5 (E) cells was determined at each time point post transfection relative to expression in vector control scramble cells by QRT-PCR. Data shown is the mean ± s.e.m of n = 3 transfections. * indicates a significant difference to vector controls with a p value of less than 0.05.