α rad53

From cultures in midlog phase, cell pellets of equivalent optical densities were collected, washed with 1 mL 4°C H₂O, and frozen on dry ice. Standard TCA precipitations were preformed to extract proteins. Samples were resuspended in SDS-PAGE sample buffer, boiled for 5 min, and cell lysates were analyzed by SDS-PAGE using 4–20% gradient Tis-HCl gels (BioRad, #3450034). Proteins were transferred onto 0.2-μm nitrocellulose membrane. Western blots were performed with low-salt PBS with Tween-20 (PBS-T) (15 mM NaCl, 1.3 mM NaH₂PO₄, 5.4 mM Na₂HPO₄, 0.05% Tween-20). Primary antibody incubations were performed in 5% (wt/vol) nonfat dry milk and low-salt PBS-T. Antibodies were used as follows: α-Rad53 (Abcam ab104232); α-Flag (Sigma-Aldrich, F3165); α-Myc (BioLegend, #626802), α-PSTAIR (Sigma-Aldrich P7962), α-Pds1 (generous gifts from Adam Rudner), α-TAP (Thermo-Scientific CAB1001). Western blots were visualized by the LiCor Odyssey Imaging System. Isr1 levels were quantified using the Image Studio Lite software and intensity values were normalized to the loading control for each lane.

Proteins were separated by SDS-PAGE (10% or 4–20% Mini-PROTEAN TGX gels, BioRad, Hercules, CA, USA) followed by immunoblot analysis using mouse monoclonal α-HA (BioLegend, San Diego, CA, USA) or α-GFP (Millipore Sigma) at a dilution of 1:10,000 and polyclonal rabbit α-Rad53 (Abcam) antibodies at a dilution of 1:2000 to detect Pkc1-HA or -GFP, Hrr25-HA or -GFP and Rad53, respectively. Secondary goat anti-mouse (Jackson ImmunoResearch, Westgrove, PA, USA) and donkey anti-rabbit (GE Healthcare, Chicago, IL, USA) antibodies were used at a dilution of 1:10,000. SDS-PAGE gels used to detect Pkc1 band-shifts were 10% and those used for Pkc1 association with Hrr25 were 4–20%.