4.5. Treatment with o-propargyl Puromycin and Localization of Nascent Peptides

To evaluate DDX6 distribution after translation was inhibited with puromycin and to identify nascent peptide localization, we used Click-it Plus OPP protein synthesis assay Alexa 488 (Invitrogen, Eugene, OR, USA). The cells in culture were incubated with 20-µM o-propargyl puromycin (OPP, diluted in culture medium) for 30 min (37 °C), washed once with PBS and incubated with fixation solution (4% paraformaldehyde in PBS) for 15 min. The cells were washed, submitted to permeabilization (incubation with 0.5% Triton X-100 in PBS for 10 min) and washed again with PBS. Then, we performed the reaction for fluorescence-labeling of nascent peptides containing OPP according to the manufacturer’s instructions.

After OPP-labeling, the hASCs were subjected to immunofluorescence for the identification of DDX6 and TIAR localization. The samples were analyzed by confocal microscopy (Leica SP5 AOBS, Leica, Germany).

To evaluate nascent peptide localization under stress, the same experiment was performed with (1) hASCs treated only with OPP for 30 min and (2) hASCs treated with both OPP and sodium arsenite (0.5 mM) for 30 min.