Quantifying PCNA Protein Levels

Western blotting was performed as described previously [88 (link)]. 1×10⁸ cells from log-phase cultures in YE media were collected. Cells were suspended in 150 μl of 10% trichloroacetic acid (TCA) and disrupted by a bead neater (TOMY, MS-100) in the presence of acid-washed glass beads. After adding 250 μl of 5% TCA, cell extracts were kept on ice for 30 min. The pellet recovered after centrifugation at 5,000 rpm for 10 min at 4°C (TOMY, Kitman) was suspended in 200 μl of SDS-elution buffer (0.5 M Tris base, 28.125 mM Tris-HCl (pH6.8), 11.25% glycerol, 0.9% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.0045% bromophenol blue) and incubated at 95°C for 2 min. The supernatant was recovered after centrifugation at 12,000 rpm for 10 min at room temperature. The cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (acrylamide to bisacrylamide ratio, 37.5:1) and transferred onto PolyScreen PVDF Transfer Membrane (Perkin Elmer, NEF1002001PK). Anti-PCNA antibodies (1:2,000) and Peroxidase AffiniPure HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 111-035-003; 1:10,000) were used as the primary and secondary antibodies, respectively. The blots were developed using Supersignal West Femto substrate (ThermoScientific, 34095). Images were acquired using ImageQuant LAS 500 (GE Healthcare).