Te70x semidry blotter system

Protein extraction was done by lysing the cells in a FastPrep FP120 (BIO101) machine with 20% TCA and glass beads. Three repetitions of a 20 s cycle, power setting 5.5. Proteins were precipitated with TCA 7.5% and centrifuging at 15,000 r.p.m. for 10 min at 4°C. Then, the pellet was resuspended in Laemmli buffer ×1.5. Western blots were resolved in 7.5% SDS-PAGE gels. Proteins were transferred to polyvinylidene fluoride membranes using the TE70X semidry blotter system (Hoefer). The antibodies used were anti-HA (Roche, 3F10), anti-Myc (Roche, 9E10), anti-PGK1 (Thermo Fisher Scientific, 459250) anti-V5 (Abcam, ab9116), and anti-FLAG (Invitrogen MA1-142). Blots were incubated with the ECL Prime Western blotting detection reagent (GE Healthcare). Blots were developed by exposure to high-performance chemiluminescence films (Amersham Hyperfilm ECL, GE Healthcare) or in an ImageQuant LAS 4000 mini machine (GE Healthcare).

All proteins were resolved in 7.5% SDS-PAGE gels, except Sgs1 (6% SDS-PAGE gel), Rmi1 (10% SDS-PAGE gel), and histone H3 (15% SDS-PAGE gel). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes using the TE70X semidry blotter system (Hoefer). The antibodies used were anti-HA (Roche, 3F10), anti-H3 (Abcam, ab1791), anti-myc (Roche, 9E10), anti-PGK1 (Thermo Scientific, 459250), anti-RFA (Agrisera, AS07 214), and anti-SMT3 (Abcam, ab14405). Blots were incubated with the ECL Prime Western blotting detection reagent (GE Healthcare) followed by exposure to high-performance chemiluminescence films (Amersham Hyperfilm ECL, GE Healthcare) to detect the signal.