Quantifying Protein Synthesis in Single Cells

To measure protein synthesis in single living cells, we adapted a protocol to detect the incorporation of puromycin by fluorescent staining (51 (link)). Forty-eight hours post-transient transfection of wild-type or mutant RACK1-HaloTag in HEK293 cells, we replated the cells. We treated the cells with either the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (Sigma-Aldrich) at 10 μM or dimethyl sulfoxide (DMSO) for 8 h. Then, 30 min prior to fixation, we added the HaloTag TMR ligand (Promega) to the medium, followed by supplementation with 9.2 μM puromycin (Gibco) 20 min prior to fixation. We changed the medium 10 min prior to fixation in order to remove any excess HaloTag TMR dye. We incubated fixed samples with an antipuromycin antibody (clone 12D10; Millipore) for 1 h at room temperature and then with an Alexa Fluor 488-labeled goat anti-mouse secondary antibody (Invitrogen) according to the manufacturer's instructions.
We quantified fluorescence in the cells with a FACSAria III system (BD Biosciences) and selected and analyzed populations with FlowJo, v10.3 (Tree Star).