MS and pull-down controls to show Ist1 is ubiquitinated. (A) Amino acid sequence of MALDI identified proteins Car2 (top), Hsp4 (middle), and Met17 (bottom) with matched peptide sequences annotated in bold red. (B) Table showing previously identified contaminants from yeast lacking His6 tagged proteins purified on a Ni2+-NTA column. Table also includes which contaminants were identified by MS from purification of Ist1-HA-His6. (C) Histogram depicting average amino acid percentage distribution in yeast proteome (gray) and Ist1 (red). (D) WT and His6-ubiquitin expressing cells were grown to log phase before ubiquitinated proteins were isolated from a denatured lysate on Ni2+-NTA beads. Original lysates left and purified samples (right) were analyzed by SDS-PAGE followed by immunoblot with the indicated antibodies. (E) Ist1 amino acid sequence (from residue 1–154 from S. cerevisiae yeast) alignment across species indicated with the highly conserved lysine residue at position 135 (blue arrow).
