CSN2 and CSN5 knockdowns result in Cul-1 hyperneddylation, loss of Skp-2 and accumulation of p27. K562 cells were transiently transfected in the absence of plasmid DNA (mock), with pMACS Kk.II plasmid alone (HKK) or transiently co-transfected with HKK plasmid together with either vector control (shVC), CSN2 knockdown (shCSN2) or CSN5 knockdown (shCSN5) plasmid. HKK positive cells were sorted 24 hours post-transfection, re-cultured and harvested for analysis. (A) CSN2 (left) and CSN5 (right) protein knockdown was determined by western blot in three independent transfectants. (B) mRNA levels in vector control and knockdown cells were determined by QRT-PCR (CSN2, left, P = 0.0012; CSN5, right, P = 0.000031). Data represents 3 independent sets of triplicate transfections. (C) CSN5 and CSN2 protein levels were determined by western blot in n = 3 CSN2 (top) and CSN5 (bottom) knockdowns, respectively. (D) CSN5 mRNA levels in n = 3 vector control and CSN2 knockdown cells were determined by QRT-PCR (P = 0.011). (E) The level of Cul1 neddylation (top panels, † indicates neddylated Cul-1), Skp2 protein (second panels) and p27 protein (third panels) was determined in n = 3 shCSN2 (left) and shCSN5 (right) samples by western blot. Even gel loading was determined by β-actin signal. Graphical data indicates the mean ± s.e.m. * indicates significance with a p value of less than 0.05.
Pearce et al. BMC Cell Biology 2009 10:31 doi:10.1186/1471-2121-10-31