Cell pellets were collected by centrifugation and snap‐frozen on dry ice. Cells were thawed on ice and lysed by shaking with acid‐washed glass beads in a FastPrep FP120 device (Savant) at speed 5 for 30 s. Lysis was performed in lysis buffer A (50 mM NaCl, 50 mM Tris pH 7.6, 0.2% Triton X‐100, 0.25% NP40) containing phosphatase inhibitor cocktail 04906837001 and protease inhibitor cocktail 04693159001 (Roche). Protein concentration was determined using the BCA assay. Equal protein concentrations of each sample were loaded and proteins were separated on a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and blotted onto nitrocellulose membranes.
HA epitope‐tagged versions of Aph1, Rad1, Hus1‐, and Rad9 as well as Chk1 were detected by mouse anti‐HA Sc7392 (Santa Cruz Biotechnology) or mouse anti‐HA 2367 S from Cell Signaling Technology (Bionordika AB, Stockholm, Sweden). Cds1 was detected using Mouse anti‐c‐myc Sc40 (Santa Cruz Biotechnology). To be able to detect the low‐abundance Aph1 protein, incubation with the primary antibody was for 72 h. The loading control was α‐tubulin detected by mouse anti‐α‐tubulin T5168 (Sigma), or staining of total protein with Ponceau S Solution (Sigma). The secondary antibody was horseradish peroxidase‐coupled α‐mouse A4416 (Sigma).
HA epitope‐tagged versions of Aph1, Rad1, Hus1‐, and Rad9 as well as Chk1 were detected by mouse anti‐HA Sc7392 (Santa Cruz Biotechnology) or mouse anti‐HA 2367 S from Cell Signaling Technology (Bionordika AB, Stockholm, Sweden). Cds1 was detected using Mouse anti‐c‐myc Sc40 (Santa Cruz Biotechnology). To be able to detect the low‐abundance Aph1 protein, incubation with the primary antibody was for 72 h. The loading control was α‐tubulin detected by mouse anti‐α‐tubulin T5168 (Sigma), or staining of total protein with Ponceau S Solution (Sigma). The secondary antibody was horseradish peroxidase‐coupled α‐mouse A4416 (Sigma).