Preparation of lysates and western blot analyses

Strains with the HA-tagged allele of chk1⁺ integrated at the genomic chk1 locus were used (Table ​(Table1).1). For cell lysate preparation, approximately 20 ml of exponentially growing cells (OD = 0.8) were collected, washed once with cold water, and frozen at –80°C in 100μl of 20%TCA (Trichloroacetic Acid, Panreac). Acid-washed glass beads were added and cell homogenates were prepared in a ‘Fast Prep’ FP120 device (Savant; Bio101). Extracts were cleared by centrifugation at 3000 rpm for 10 min, and the pellets were resuspended in 50 μl of 2× sample buffer (100 mM HCl–Tris, pH 6.8, 4% SDS, 20% glycerol, 25 mM DTT and 0.4% bromophenol blue), after which 50 μl of Tris Base 2 M [pH 7.5] was added. The solution was vortexed, boiled for 5 min, and centrifuged at 13 000 rpm for 5 min to collect the supernatant (protein extract sample). Proteins were resolved by SDS-PAGE using 10% gels with an acrylamide/bisacrylamide ratio of 99:1, transferred to nitrocellulose membranes, blocked with 5% milk in Tris-buffered saline with 0.03% Tween, and subjected to immunoblotting with the α-HA antibody (Roche).