Protein Isolation and Western Blotting

Proteins were isolated from the yeast cells by TCA (tri-chloroacetic acid) extraction, as described in [40 (link),51 (link)]. Briefly, about 4 × 10⁸ cells in the exponentially growing phase were collected and resuspended in 200 μL of a 20% TCA solution. These cells in the TCA solution were lysed using the glass bead method. Proteins were then precipitated and resuspended in 200 μL of a Laemmli buffer and were neutralized with the addition of 100 μL of 1 M Tris, pH 8.8. The protein sample was boiled for 3 min, and 10 μg of the proteins was fractionated using SDS-PAGE in 4–12% gradient gels followed by Western blotting. The blot was probed in an Odyssey blocking buffer with the goat polyclonal anti-Rad53 (yC-19: sc6749, Santa Cruz Biotechnology, Dallas, TX, USA); the rabbit polyclonal anti-H2A phospho S129 antibody (ab15083, Abcam, Cambridge, UK); the rabbit polyclonal anti-HA (H6908, Sigma-Aldrich, St. Louis, MO, USA); or the rabbit polyclonal anti-G6PD (A9521, Sigma-Aldrich), followed by the secondary antibody LI-COR IRDye 800CW of the goat polyclonal anti-rabbit IgG (H+L) 926-32211 or the LI-COR IRDye 800CW donkey anti-goat IgG (H+L) 926-32214. Imaging was performed using the LI-COR Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).