Figure 4

Skp1 forms DNA-damage-induced foci in an Fbh1-dependent manner. (A) YFP-Skp1 complements the skp1^tsmutants. The skp1^tsmutants expressing YFP-wild type skp1 or none in the absence of thiamine were streaked onto EMM2 plates and incubated at 26°C (left) or 37°C (right) for 3 days. (B) DNA damage-induced YFP-Skp1 foci. The fbh1 Δ strains harbouring the vector pREP41, pREP42 Myc-fbh1 or pREP42 Myc-fbh1-fb were transformed with pREP42 YFP-skp1. Cells were treated with or without MMS (0.1%) for 2 h at 30°C and observed by fluorescence microscopy. The top, middle, and bottom panels show YFP, DAPI, and phase contrast images, respectively. The scale bar indicates 10 μm. (C) Quantitative analysis of YFP-Skp1 foci. Cells with Skp1 foci were counted and divided by the total number of cells. At least 100 cells were counted per strain. The result represents the average of three independent measurements. (D) Expression of Myc-tagged Fbh1. Cells were treated as in (B) and protein extracts were analysed by SDS-PAGE followed by Western blotting with anti-Myc or anti-α-tubulin antibodies.