Figure 3

TIPI-gTOW experiments of the cell cycle regulators. Copy numbers of the plasmids in the strain; (A) P_CDC19 _-500 -GFP- ^TdegF CDC15 (YSM002), (B) P _CDC19 _-500 -GFP- ^TdegF CDC20 (YSM003), and (C) P_CDC19 _-600 -GFP- ^TdegF CDC28 (YSM005) in SC medium without the indicated amino acids. Four independent measurements were performed, and the average is shown. The error bar indicates the standard deviation. *: p < 0.01 for Student’s t-test. (D) Western blotting for GFP-^TdegFCdc28 protein in strains YSM005 (P_CDC19 _-600 ) and YSM004 (P_CDC19 _-500 ) expressed in SC medium without the indicated amino acids, as detected with anti-GFP antibody. (E) Western blotting for GFP-^TdegFCdc28 protein and pTEV⁺ in strains BY4741 (wild type) and YSM005 (P_CDC19 _-600 -GFP- ^TdegF CDC28) expressed in SC medium without the indicated amino acids, as detected with anti-Cdc28 antibody (upper blot) and anti-TEV protease antibody (middle blot). In D and E; Coomassie® G-250 staining of 50 kDa bands (corresponding to the size of EF-1α) are shown as loading controls. “–U” and “–UL” indicate –Ura and –Ura–Leu, respectively.