Figure 2: NF-κB2 ubiquitylation and levels are regulated by Fbw7.

(a) p100 turnover rate is prolonged in FBW7 KO HCT116 cells. p100 protein half-life was analysed following treatment with cycloheximide. p100 band intensity was normalized to β−actin and then normalized to t=0 controls. p100 half-life in WT=6.7 h (R²=0.87) and in KO=14.7 h (R²=0.78) (b) RelB turnover rate is prolonged in FBW7 KO HCT116 cells. RelB protein half-life was measured as in (a). WT=1.9 h (R²=0.91) and in KO=4.6 h (R²=0.71) (c) Total RNA was prepared from WT and KO cells and NF-κB2 mRNA expression was analysed by qRT–PCR. The results are shown as means. Error bars indicate s.d. (n=3). (d) p100 protein levels increase on Fbw7 silencing. HCT116 FBW7 WT cells were transfected with the scrambled (scr) or FBW7 siRNA for 48 h. Endogenous p100 protein was analysed by WB. ±indicated s.d. (n=3). (e) qRT–PCR of FBW7 and NF-κB2 mRNA levels from siRNA transfected cells in (d), n=3, error bars indicate s.d. (f) Fbw7 knockdown stabilizes p100 protein. HEK293 cells were transfected with the indicated siRNAs for 48 h and NIK for 24 h and treated with cycloheximide. p100 and p52 half-lives were analysed. The graphs to the right show quantification of the protein bands in the blot. p100 half-life control=12 h (R²=0.99) and Fbw7=58 h (R²=0.72). Degradation of p52 was marginal under these conditions and time frames. (g) Fbw7 ubiquitylates endogenous p100. FBW7 WT and KO cells were transfected with HA-ubiquitin. NF-κB2 was immunoprecipitated from whole-cell extracts and NF-κB2 ubiquitylation was analysed by WB using HA antibodies (see also methods). (h) Ubiquitylation of endogenous p100 is restored by ectopic Fbw7 expression. FBW7 KO cells were transiently transfected with FBW7. Ubiquitination assay was performed as described in (g).