C. albicans Cas5-HA cells were harvested from log phase cultures in YPD (+/−caspofungin) with lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7, 15 mM EDTA, 1% Triton 100-X, 10% glycerol) plus phosphatase inhibitors (Sigma, P5726 and P0044) and protease inhibitors (Roche complete Mini Protease Inhibitor Cocktail Tablets 04693124001) using glass beads and bead beating 10 × 20 s with 1 min ice in between. Cell extracts were precipitated with methanol–chloroform and resuspended in rehydration buffer (Bio-Rad Cat#163-2105). Approximately 30 μg protein was loaded onto 7 cm IPG strips (pH 3-10, Bio-Rad Cat#163-2000) and resolved using Bio-Rad Protean i12 IEF System. The IPG strips were then resolved using 10% SDS-PAGE gels and processed for western blotting following the procedures described above for one-dimensional western blot analysis.