Co-immunoprecipitation of E-Syt1 and PERK

48 h after transfection with the selected plasmids, cells were collected and lysed at 4°C for 30 min in CHAPS buffer (1% CHAPS, 100 mM KCl, 50 mM Tris HCl, 150 mM NaCl, 1x protease inhibitor [Pierce Protease Inhibitor Tablets, Thermo Fisher Scientific Inc.]) in MQ water. Cells were centrifuged to remove debris and 500 μg of proteins from the supernatant was combined with primary antibody/conjugated GFP antibody beads overnight (ON) at 4°C. Proteins were eluted with sample buffer (62.5 μM Tris-HCl, 10% glycerol, 2% SDS, 1x protease inhibitor, 1x phosphatase inhibitor (Pierce phosphatase Inhibitor Tablets, Thermo Fisher Scientific Inc.) in MQ water and loaded on gel for Western blot analysis. For developing IPs, ChemiDoc MP Imaging system and Rabbit DyLight 680/Mouse DyLight 800 secondary antibodies were used in cells transiently co-transfected with eGFP-empty vector or eGFP-tagged E-Syt1 and myc-tagged PERK-FL or myc-tagged PERK^k618A; for the rest of the experiments, HRP-based detection using as secondary antibody the Veriblot antibody was used in order to avoid the detection of the long and short chains of the IgG.