Transient Transfection of PERK and E-Syt1 Constructs in HEK293T and HeLa Cells

HEK293T cells were transiently transfected with PERK full length-myc, PERK-K622A -myc, myc-empty vector, eGFP-empty vector, eGFP-tagged E-Syt1, eGFP-E-Syt1-ΔCDE, eGFP-E-Syt1-ΔSMP or eGFP-E-Syt1-ΔDE using Trans-IT X2 transfection reagent accordingly to the manufacturer’s instructions. HeLa cells were transiently transfected with human HRP-KDEL-myc, Mitochondrial Aequorin WT, mCherry-empty vector, mCherry-tagged E-Syt1, mCherry-E-Syt1-ΔCDE, eGFP-empty vector, eGFP-E-Syt1, eGFP-E-Syt1-ΔCDE, eGFP-Syt1-ΔSMP or eGFP-E-Syt1-ΔDE using Lipofectamin 2000 (Thermo Fisher Scientific) or electroporated with 4D-Nucleofector (Lonza Bioscience) using SE Cell Line kit (Lonza Bioscience). 24 h after transfections, cells were replated to (microscopy) culture dishes (Mattek corporation) or collected for lysate after 48 h. For Oxygen Consumption Rate (OCR) analysis, cells were plated after nucleofection, and OCR analysis was performed using a Seahorse XF24 (Agilent) Extracellular Flux Analyzer 24 h later.