Figure 2 - NPM1 is a substrate for K-cyclin–CDK6 and its phosphorylation is essential for cyclin-driven centrosome duplication.

From the following article

p53-Driven apoptosis limits centrosome amplification and genomic instability downstream of NPM1 phosphorylation

Maria Emanuela Cuomo, Axel Knebel, Nick Morrice, Hugh Paterson, Philip Cohen & Sibylle Mittnacht

Nature Cell Biology 10, 723 - 730 (2008) Published online: 4 May 2008


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(a) Recombinant glutathione-S-transferase fused human NPM1 (GST–NPM1) and GST–pRB (amino acids 763–928) were produced in Escherichia coli, purified and phosphorylated in the presence of [gamma-32P]ATP using the indicated cyclin–CDK complexes. Reactions were subjected to SDS–PAGE and autoradiography. (b) U2OS cells were transfected with plasmids encoding the indicated proteins. Cells lysates were prepared after 24 h, enriched for the tagged proteins by immobilized metal affinity chromatography (IMAC) and analysed by immunoblotting. (c) Two-fold dilutions of cell lysates from KSHV-positive (BLBL-1, BCP-1 and BC-3) and KSHV-negative (LCL3) cell lines were probed as indicated. (d) Lysates from U2OS cells infected with a recombinant KSHV or mock-infected control for 3 days were probed as indicated. (e) BC-3 cells were transfected with siRNA targeting two independent regions of the K-cyclin encoding transcript (1 and 2) or an irrelevant siRNA (C). After 48 h, lysates were prepared and two-fold dilutions probed as indicated. (f) Cells were transfected with the indicated plasmids together with a vector encoding EGFP. Experimental details and analysis were as for Fig. 1a, b. Data are mean plusminus s.d. (n = 3). (g) CHO cells transfected with indicated plamids and EGFP were exposed to hydroxyurea for 40 h (+HU) or left untreated (–HU). Centrosomes were analysed as in Fig. 1a, b. Graphs indicate the percentages of EGFP-positive cells with 1–2 or more than 2 centrosomes (mean plusminus s.d., n = 3). Full-length images of blots in ae are presented in Supplementary Information, Fig. S8.