FIG 1.

Construction of irreversibly sensitized (IS) kinase strains. (A) Sequence alignments of IS mutant positions (2–4) in human RSK2, Kin28, Bur1, and Ctk1 kinases. The residues mutated to create the “hole” or reactive cysteine are marked by asterisks. (B) Protein expression levels of wild-type and IS mutant kinases, before (0 min) or after 90 min of treatment with 50 μM CMK. Anti-HA blots show epitope-tagged kinases at the expected sizes of Kin28 (35 kDa), Bur1 (74 kDa), and Ctk1 (61 kDa). Rpb1 and Rpb3 are two RNA polymerase II subunits used as loading control bands. Strains used: YSB3216 (Kin28 WT), YSB3221 (Kin28 V21C, L83G), YSB3229 (Bur1 WT), YSB3232 (Bur1 V74C, L149G), YSB3235 (Ctk1 WT), and YSB3237 (Ctk1 V197C, F206G).