Figure 5: eLIM binds CHK2/CDC25C and promotes phosphorylation and nuclear accumulation of CDC25C.

(a) Alignment analysis of 11-aa motif in the indicated LIM proteins. Conserved amino acids are marked in red. (b) Glutathione-sepharose beads bound with GST or the indicated GST fusion proteins were incubated with purified His-tagged CHK2. After washing the beads, the bound proteins were subjected to SDS–PAGE and immunoblot with anti-His antibody. (c) Glutathione-sepharose beads bound with GST-FHL1 were incubated with HeLa cell lysates expressing Myc-tagged CHK2 plus the indicated 11-aa peptides and analysed as in b. The two amino acids after the conserved amino acids WH are marked in blue. m-FHLs represent mutant FHLs. (d) Glutathione-sepharose beads bound with the indicated GST fusion proteins were incubated with HeLa cell lysates expressing Myc-tagged CHK2, CDC25C, 14-3-3ɛ or CDC2 and analysed as in b. (e) The eLIM-1, eLIM-2 and eLIM-C peptides (c) dissolved in deionized water were added to culture media for HeLa cells at final concentrations of 5 and 50 nM, respectively. Cells were irradiated (8 Gy) and harvested after 6 h of treatment and analysed by immunoblot with the indicated antibodies. The aa sequences of the peptides are shown in c. (f) HeLa cells were treated with the indicated peptides (50 nM) as in e, irradiated (8 Gy), fractionated into cytoplasmic and nuclear fractions, and analysed by immunoblot with the indicated antibodies. (g) HeLa cells were treated with the indicated peptides (50 nM) as in e, irradiated (8 Gy) and IP with anti-CDC25C or normal IgG, followed by immunoblot with the indicated antibodies. (h) HeLa cells were treated with the indicated peptides labelled with FITC (50 nM) at the indicated times. The nuclei were stained with DAPI (blue). The ___location of the peptides (green) was visualized by a fluorescent microscope. Scale bars, 100 μm.