Figure 2: FBXW7α interacts with C/EBPδ and targets it for degradation.

(a) RAW 264.7 macrophages were transfected with control or Fbxw7α siRNA oligos for 48 h, then pulse-labelled with ³⁵S-methionine/cysteine, followed by chased with excess unlabelled aminoacids for the indicated times. Quantitation of the phosphorimage of C/EBPδ signal is depicted in the graph (mean±s.e.m., n=2–3) and representative primary data are shown. (b) Western analysis of RAW 264.7 cells transfected with HA-ubiquitin expression constructs and control or Fbxw7α siRNA oligos and treated ± MG132 (20 μM, 6 h), followed by immunoprecipitation under denatured conditions with anti-C/EBPδ or IgG (with equal aliquots of the indicated extracts). Input (2.5% of lysate) was analysed as indicated. (c) Alignment of phosphodegron motifs present in known FBXW7 substrates with C/EBPδ and its TTS/AAA mutant. (d) Western analysis of RAW 264.7 cells transfected with WT- or TTS/AAA-CEBPδ expression plasmids and/or HA-FBXW7α. (e) Western analysis of RAW 264.7 cells transfected with W-T or TTS/AAA-C/EBPδ expression plasmids and immunoprecipitated with anti-C/EBPδ or IgG and input (2.5% of lysate) as indicated. (f) RAW 264.7 cells were transfected with WT- or TTS/AAA-C/EBPδ expression constructs. C/EBPδ was immunoprecipitated and the beads were incubated with FBXW7α and HA-ubiquitin as indicated (see Methods for details) and analysed by western with anti-HA and C/EBPδ antibodies. (g) Western analysis of RAW 264.7 cells transfected with WT- or TTS/AAA-C/EBPδ, treated ± MG132 (20 μM, 6 h) followed by immunoprecipitation under denatured conditions using anti-C/EBPδ or IgG and input (2.5% of lysate) as indicated. (h) Western analysis of RAW 264.7 cells transfected with WT- and TTS/DDD-C/EBPδ for 48 h followed by immunoprecipitation with anti-C/EBPδ or IgG and input (2.5% of lysate) as indicated. (i) Western analysis (right panel) of RAW 264.7 cells transfected with WT-, TTS/AAA- or TTS/DDD-C/EBPδ expression constructs and treated with CHX as indicated. Quantification (left panel) of the C/EBPδ signal was normalized to actin (n=3, *P<0.05; ***P<0.0001; NS, not significant). (j) Western analysis of RAW 264.7 cells transfected with WT- or TTS/DDD-C/EBPδ expression constructs and treated ±MG132 (20 μM, 6 h). Where applicable, data are mean±s.e.m., evaluated by two-tailed unequal variance t-test.