Measuring Puromycin-Labeled Proteins in ER Stress

The effects of endoplasmic reticulum stress on puromycinylated protein levels were determined as previously described (Halliday et al., 2015 (link)). In brief, 10⁶ HEK293 cells were plated in 6-well plates. Two days later, culture media was changed to fresh media, and cells were treated with vehicle (DMSO) or thapsigargin in the presence or absence of the indicated concentration of inhibitors for 2.5 h. For puromycin labelling, 10 μg/ml puromycin was added during the last 10 min before harvest. Cells were lysed with passive lysis buffer (Sigma) supplemented with protease inhibitor cocktail (Roche). After centrifugation at 13 000 rpm for 20 min, supernatants were mixed with SDS-PAGE sample buffer. To detect puromycinylated protein 20 μg of total protein, respectively, was subjected to 12% SDS-PAGE and transferred onto PVDF membrane. Immunoblot detection was conducted using primary antibodies for puromycinylated protein (1:5000; Proteintech). Scanned images were quantified using ImageJ software.