Figure 1: FHL1 interacts with CHK2/CDC25C/14-3-3É›.
(a) Yeast CG1945 cells were transformed with the indicated plasmids (bait and prey for the two-hybrid assay) and grown on selective media. Positive interaction is indicative of colonies that grow on selective media and have β-galactosidase activity. (b) HeLa cells were treated with or without IR (8 Gy). Six hours later, cells were fractionated into cytoplasmic and nuclear fractions, and IP with the indicated antibodies or preimmune control serum (IgG). Precipitates were analysed by immunoblot using the indicated antibodies. Lamin A/C and α-tubulin were used as the nuclear and cytoplasmic marker, respectively. MW, molecular weight. (c,d) Glutathione-sepharose beads bound with GST-FHL1 (c), GST-CDC25C (d) or GST were incubated with purified His-tagged CHK2 (c) or FHL1 (d). After washing the beads, the bound proteins were subjected to SDS–PAGE and immunoblot with anti-His antibody. (e) Glutathione-sepharose beads bound with GST-14-4-3ɛ or GST were incubated with FLAG-FHL1-expressing HeLa cell lysates or purified His-tagged-FHL1 treated with or without the protein phosphatase λPPase. The bound proteins were analysed by immunoblot with anti-FLAG or anti-His. As a positive control for λPPase, HeLa whole cell lysates were treated with λPPase, and the treated lysates showed decreased ERK1/2 phosphorylation (pERK1/2) with immunoblot. (f) HeLa cell lysates were IP with antibodies specific for CDC25A or CDC25B, followed by immunoblotting with the indicated antibodies. (g) HeLa cell extracts were fractionated on Superose 6 size exclusion columns. FPLC chromatographic elution profiles are shown. The elution positions of calibration proteins with known molecular masses (kDa) are indicated by arrows, and an equal volume from each chromatographic fraction was analysed by immunoblot with the indicated antibodies. Red frame indicates a potential complex with FHL1, CDC25C and CHK2, and blue frame shows a potential complex with FHL1, CDC25C and 14-3-3ɛ.
