O-propargyl-puromycin assay

Cells were treated as indicated. For the last 30 min of treatment, 20 μM O-propargyl-puromycin (OPP, {"type":"entrez-nucleotide","attrs":{"text":"C10459","term_id":"1535530"}}C10459, Thermo Scientific) was added to wells. For cycloheximide control samples, 10 µg/mL cycloheximide was added to wells 10 min prior to the addition of OPP. Cells were harvested by trypsinization and fixed with methanol at −20°C, followed by permeabilization with 0.5% Triton-X in PBS. Fixed and permeabilized cells were stained using Click-iT Plus Alexa Fluor 647 Picolyl Azide Toolkit from Thermo Scientific ({"type":"entrez-nucleotide","attrs":{"text":"C10643","term_id":"1535714"}}C10643) according to the manufacturer’s instructions and analyzed by flow cytometry.