PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab (Saheki et al., 2016 (link)). KDEL-HRP-myc was obtained from Francesca Giordano (Galmes et al., 2016 (link)). eGFP-E-Syt1-ΔCDE plasmid was generated from mCherry-E-Syt1-ΔCDE, E-Syt1-ΔCDE was excised using SalI and SacII restriction enzymes and cloned into p-eGFP-C3 empty vector (5′-E-Syt1-ΔCDE -SalI-FW cloning primer: 5′-TAT​ATA​GTC​GAC​ATG​GAG​CGA​TCT​CCA​GGA​GA-3′; 5′ E-Syt1- ΔCDE -SacII-Rev cloning primer: 5′-TAT​ATA​CCG​CGG​TCG​AGG​TGG​GGC​ATC​CAC​A-3′). mtAEQ WT was a kind gift from Paolo Pinton (University of Ferrara, Ferrara, Italy; Robert et al., 2000 (link)). pcDNA 3.1 mCherry-STIM1 was a gift from Peter Vangheluwe. Human PERK-myc WT cDNA (µHsPERK-myc-pUC57 SC1010) was generated by GenScript and cloned into a lentiviral transfer plasmid containing ires-hygromycin (pCHMWs-HsPERK-myc-Ires-Hygro SC1017) to select for transduced cells with hygromycin. The K622A mutant was introduced using a gBlock gene fragment (IDT) by replacing a part from the human PERK-myc WT by a fragment that contains the mutation. This construct also allows selection of transduced cells with hygromycin.
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