Fig 8. Evidence that GST-Yih1 preferentially binds to the Cdc28 active complex.

yih1Δ cells (MSY-Y2) expressing Yih1 fused to GST from a galactose-inducible promoter were grown to log-phase in S medium containing galactose as carbon source (SGal). Cells were synchronized with α-factor, released into fresh SD media and samples were collected at the indicated times. (A) Representative histograms of DNA content (PI staining) of arrested (G1 – time 0) and released cells measured by flow cytometry. The distribution of cells in G1 (1C), S and G2/M (2C), analyzed with the Flowjo software, 9.3.3 version is shown. (B) Representative immunoblot of in vivo GST-pull-down assays. The collected cells were promptly harvested and equal amounts of proteins (1 mg) were subjected to glutathione-mediated pull-down assays. All the precipitated material (upper-panels) and 2% of the input (lower-panels) were subjected to immunoblots to detect GST proteins and Cdc28. As a negative control, GST alone was expressed in asynchronous yih1Δ cultures, pulled-down and analyzed as above. (C) The relative amount of Cdc28 bound to GST-Yih1 was determined with data from B using the NIH image J software. The amount of precipitated Cdc28 was normalized to the levels of precipitated GST-Yih1. Blue, yellow and pink shaded boxes indicate the estimated G1, S and G2/M cell cycle stages, respectively. Data represent mean ±S.E. of three independent experiments.