SUnSET Assay
To perform surface sensing of translation (SUnSET) assays, cells were
grown to 70-80% confluency in fully supplemented medium. Medium was
then replaced for Earle’s Balanced Salts Solution (EBSS). For one well,
medium was replaced with EBSS containing 1 µM cycloheximide (CHX) as a
positive control. After 30 min, puromycin was added to the cells to a
final concentration of 1 µM and allowed to incorporate for 30 min. Cell
lysates were then harvested for SDS-PAGE immunoblot analysis with
detection of incorporated puromycin via an anti-puromycin antibody.