Quantitative Puromycin Incorporation Assay

For a puromycilation assay^{28 (link),29 (link)}, cultures were incubated for 15 min with 5.4 µM puromycin (Sigma-Aldrich, Dorset, UK), immediately followed by washing steps with PBS and fixation for 20 min with 10% formalin (VWR). Wells were washed with PBS-T and treated for 10 min with 0.1% Triton X-100. Wells were blocked for 1.5 h with 1% (m/v) skimmed milk powder (ELK, Campina, Zaltbommel, the Netherlands) in PBS-T, followed by overnight incubation at 4 °C with the anti-puromycin antibody 12D10 (Sigma-Aldrich, Dorset, UK). After washing with PBS-T, wells were incubated for 1 h at room temperature with goat anti-mouse Alexa488 (ThermoFisher Scientific). Following a final wash step, the fluorescence signal intensity was determined using a Tristar LB942 (Berthold, Bad Wildbad, Germany) equipped with excitation filter F485 and emission filter F353. Fluorescence data were normalized to DNA-content^{30 (link)}. For that, the same wells were subsequently washed with HEPES-Buffered Saline (HBS), followed by 1 h incubation with 5 µg/mL DAPI (Invitrogen) plus 5 µg/mL HOECHST 33342 (Invitrogen) in HBS. After washing steps with HBS, fluorescence signal intensity was determined using a Tristar LB942 (Berthold), using the excitation filter F355 and emission filter F460.