2.17. Puromycin incorporation assay (SUnSET assay)

SUnSET assay was performed as per manufacturer's recommendations (Kerafast, Boston, MA, USA). Cells were incubated with puromycin (2 μg/mL) for 15 min. Post incubation, cells were washed with ice cold PBS and lysed using RIPA lysis buffer. Equal quantity of protein lysates was separated on SDS-PAGE and probed with anti-puromycin antibody. Signals were normalized with probing beta-actin (loading control).