Anti cdt1

Cells were pulse-labeled with 10 μM EdU for 30–45 min before harvest. EdU staining was performed using the Click-iT EdU kit (ThermoFisher, C10634 or C10633) according to the manufacturer’s protocol. For immunodetections with Phospho-MCM2 (Ser139) (MCM2-pS139, Cell Signaling, 12958), and anti-CDT1 (Cell Signaling, 8064), cells were incubated on ice for 10 min in a cytosol extraction buffer (10μM HEPES, pH7.9; 10 μM KCl; 1 μM EGTA; 0.25% NP40; 1× protease inhibitor cocktail and phosphatase inhibitor cocktail), then centrifuged at 800 × g for 5 min at 4 °C. The cell pellets were washed once with a cytosol extraction buffer, then cells were fixed with 4% PFA. EdU click were followed by CDT1 (dilution 1:100) or MCM2-pS139 (dilution 1:100) at 4 °C overnight prior to flow analysis to visualize changes in protein levels during the cell cycle. For the fluorophores, EdU using Alexa 647, MCM2-pS139 (dilution 1:100), and CDT1 using Alexa 488 conjugated anti-rabbit IgG (Thermo Fisher Scientific, A11008) (dilution 1:100). DAPI was used for DNA staining. A BD LSR Fortessa cell analyzer with FACSDiva software and/or FlowJo10.6 were used for cell cycle analysis.