Image studio lite version

Cells were lysed in ice-cold RIPA buffer containing protease (cOmplete tablets mini) and phosphate (PhosSTOP EASYpack) inhibitors (both Roche Diagnostics, Mannheim, Germany) and centrifuged, the supernatant was collected. Denatured protein samples (25μg) were separated by gel electrophoresis for 50 min at 200 V and transferred onto polyvinylidene difluoride membranes for 105 min at 30 V. Blots were blocked in 5% dry milk and incubated at 4 °C overnight with rabbit polyclonal antibody against NHP2L1 (1:500; #15802–1-AP), RABAC (PRAF1; 1:250; #10542–1-AP, both Proteintech, Rosemount, IL), BRIX1 (1:1000, NBP1–91708, Novus Biologicals, Centennial, CO) and rabbit monoclonal antibody against Sec61B (1:500; #14648, Cell Signaling, Danvers, MA) and PKR (EIF2AK2; 1:2000; ab184257,abcam). The signal was visualized using Amersham ECL anti-rabbit horseradish peroxidase-linked (1:1000; Cytiva, Marlborough, MA) supersignal chemiluminescent reagents (Thermo Fisher Scientific) and a Chemidoc Touch Imaging System (BIO-RAD, Hercules, CA). Blots were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) and reprobed using mouse monoclonal antibody against β actin (1:8000; NB600–501, Novus Biologicals) and reimaged. Densitometry was quantified using Image Studio Lite, version 5.2.5 (LI-COR Biosciences, Lincoln, NE); each protein was normalized relative to β actin.