Fig. 9. The Mdy2-6His fusion shows no C-terminal cleavage. (A) Alignment of predicted Mdy2 sequences from residues 74-173 with S. cerevisiae ubiquitin and human GdX. Identical residues are shaded in dark grey; similar residues are shaded in light grey. (B) Expression of MDY2 constructs with C-terminal 6xHis tag is independent of -factor. Supersensitive sst2-1 (HGX132) cells carrying the functional GST-Mdy2-H6 construct (N-terminal GST tag and a C-terminal His6 tag) were assayed with 1 µM -factor and compared with identical untreated cells and cells carrying the GST vector as a negative control. The expression levels were measured by anti-6xHis immunoblot analysis. To control for correct expression, the blot was stripped and reprobed with anti-GST antibody. No anti-His signal could be detected in the GST-negative control (lane 1) and cells not expressing GST at all (lane 4). (C) Localization of Mdy2H6 by indirect immunofluorescence. mdy2 mutant (HZH686) cells, containing functional GST-Mdy2H6 on a CEN plasmid were prepared as described in Materials and Methods and stained with anti-His antibody (Qiagen) and affinity-purified goat-anti-mouse conjugated to fluorescein (Jackson ImmunoResearch). Micrographs show FITC fluorescence (-His), DAPI fluorescence and a differential interference contrast (DIC) image. Cells were photographed with Zeiss AxioCam and AxioVision.