Protein was extracted by pelleting cells at OD600 of 0.8, with the pellet being resuspended in 2× sample buffer (one-third volume of 6× sample buffer containing 0.35 M Tris-HCl, 10% (w/w) SDS, 36% glycerol, 5% β-mercaptoethanol, and 0.012% bromophenol blue). The samples were boiled for 5 min at 95 °C. The cell debris was pelleted and the supernatant was separated on a 6% SDS-PAGE gel to observe changes in Cas5 mobility. Separated proteins were electrotransferred to PVDF membrane (Bio-Rad Laboratories) and blocked with 5% skim milk in phosphate-buffered saline (PBS) with 0.2% Tween-20 at room temperature for 1 h. Blots were hybridized with antibody against the HA epitope (1:5000 dilution; Roche Diagnostics), p44/42 (1:2000, Cell Signaling), PSTAIRE (1:5000, Santa Cruz Biotechnology), TAP (1:5000; Open Biosystems), Hsp90 (1:10,000; generously provided by Brian Larsen, Des Moines University) or α-β-actin (1:5000; Santa Cruz, sc-47778) overnight at 4 °C. Blots were washed with PBS with 0.1% Tween-20 and subsequently hybridized with FITC-conjugated secondary antibody diluted 1:5000 in the block solution for 45 min at room temperature. Signals were detected using an ECL western blotting kit as per the manufacturer’s instructions (Pierce).