Figure 4: Fbw7 modulates p100/p52 levels and activity.

(a) Fbw7 knockdown increases p52 protein levels. HEK293 cells were transfected with control (scr), Fbw7 or βTrCP siRNA oligos followed by transient transfection of p100 with or without NIK for 24 h. Whole-cell extracts were analysed. (b) Fbw7 alters p100 and p52 levels. HEK293 cells were co-transfected with p100 and Fbw7 or Fbw7ΔF, with or without NIK and analysed by WB. Lower panel shows quantified protein levels, for p100 (dark bars) and p52 (light bars). Error bars indicate s.d. (n=3, Fbw7+NIK n=5). (c) Fbw7 reduces p52 levels on CD40 stimulation. MDA-231 cells were transfected with control (EV) or Fbw7 plasmid. 24 h post-transfections cells were stimulated by soluble recombinant CD40 overnight and analysed by WB. (d) HEK293 cells were transfected with control (scr), Fbw7 or βTrCP siRNAs followed by transient transfection of p100 and NIK for 24 h. p100 levels in the cytosolic and nuclear extracts were analysed. (e) HEK293 cells were transfected with βTrCP siRNA oligos followed by transient transfection of control (EV) or Fbw7 for 24 h. p100 levels in the cytosolic and nuclear extracts were analysed. (f) p52:RelB complexes are more abundant in FBW7 KO cells. HCT116 FBW7 WT and KO cells were treated with 10 ng ml⁻¹ TNF-α for 24 h before lysis. 200 μg of protein was used to immunoprecipitate RelB. (g) Increased mRNA expression of NF-κB2 targets in FBW7-deficient cells. FBW7 WT and KO cells were treated or not with 10 ng ml⁻¹ TNF-α for 24 h before total RNA extraction. qPCR was performed for the indicated genes. Actin was used as a normalizing gene and all levels were compared with untreated WT. Graph depicts the mean and s.d. of a minimum of three independent experiments. Statistical significance was tested using ANOVA. *P<0.05; **P<0.01 and ***P<0.001.