Fig. 3

Nucleocytoplasmic distribution of Irr1 depends on growth conditions. Cells expressing Irr1-GFP were grown in YPD medium to exponential phase (YPD) or subjected to nitrogen starvation for 22 h (SD-N) or treated with α-factor for 3 h (α-factor) and whole-cell lysates (WCL) were prepared and fractionated into crude nuclear (3000×g) and cytoplasmic (cpl) fractions as detailed in “Methods” section. The unfractionated lysate and the fractions were separated by SDS-PAGE (85 μg of protein per lane), transferred to Hybond-C extra membrane, probed with anti-GFP antibodies (a and top panel in b) or anti-histone H3 antibodies (bottom panel in b), developed as detailed in “Methods” section and relative signal intensity was plotted taking WCL from YPD-grown cells as 1. All bands below that corresponding to intact Irr1-GFP are represented as “proteolysis” (c). All values are shown as mean ± SD of four independent experiments. The two panels in b represent the same membrane. Lane M contains molecular mass markers and ctrl is a negative control—whole cell lysate from cells expressing untagged Irr1. Western blots as in a were quantified using Fiji Image J software