Geminin

After treatment, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then washed twice with PBS. Cells were then permeabilized with 0.1% Triton-X100 at 4 °C for 15 min and subsequently blocked with 3% BSA for 1 h at room temperature. Primary antibody staining was performed overnight at 4 °C in the presence of 3% BSA. Primary antibodies used in this study were: Geminin (1:500, #52508, Cell Signaling Technology), Phospho-Rb (1:1000, (Ser807/811), #8516, Cell Signaling Technology), Recombinant Anti-RAB7 (1:1000, (ab137029), abcam), and Phospho-S6 (1:250, (Ser240/244), #2215, Cell Signaling Technology) and visualized by incubation with secondary antibody conjugation (1:500–1:1000). ​​Secondary antibodies conjugated to Alexa Fluor 488 (#A32731, Thermo Fisher), Alexa Fluor 546 (#A11010, Thermo Fisher) or Alexa Fluor 647 (#A21245, Thermo Fisher) were used in this study and incubated for 1 −2 h at room temperature followed by three washes with PBS. Hoechst staining was added at 1:5000 dilution for 5 min. Immunofluorescence images were taken on a Nikon Ti-E microscope using either a 10×0.45 NA objective or a 20×0.75 NA objective. Images were processed and analyzed using custom MATLAB scripts and ImageJ analysis tools.