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. 2012 Mar 23;13(6):547–553. doi: 10.1038/embor.2012.40

Figure 5.

Figure 5

Phenotype associated with Neurl4 depletion is dependent on CP110 upregulation. (A) Quantifications of CP110 level on the centrosome in U2OS cells transfected with control or Neurl4 siRNA. CP110 fluorescence intensity at centrosomes was measured using Metamorph. CP110 was stained with anti-CP110 antibody (red). DNA was stained with DAPI (blue). (B) Western blot analysis of CP110 levels after Neurl4 depletion. Samples in the right-most two lanes were treated with 10 μM cycloheximide (CHX) for 5 h before collection. (C) U2OS cells were transfected with control siRNA or siRNAs targeting Neurl4, CP110 or both Neurl4 and CP110. Representative images of centrin staining and quantification of cells with >4 centrin foci are shown. Scale bar, 2 μm; **P<0.01. (D) 293T cells were transfected with haemagglutinin–CP110, His–ubiquitin and Flag-only vector control or Flag–Neurl4. Flag–cyclin F and Flag–Fbxo32 served as positive and negative controls, respectively. Cells were incubated with 10 μM MG132 for 4 h before collection. Ubiquitylated (Ub) CP110 protein was enriched with nickel agarose and analysed by western blots. DAPI, 4,6-diamidino-2-phenylindole; Neurl4, Neuralized homologue 4; siRNA, small interfering RNA.