Pcdna4 to plasmid

CXCL12 was purchased from Cedarlane. Forskolin, isobutylmethyl xanthine (IBMX), AVP, and met-enkephalin were purchased from Sigma. The following plasmids were already described: HA-CXCR4^{47 (link)}, β-arrestin-2-LucII^{48 (link)}, β-arrestin-2-YFP^{49 (link)}, Gα_i1−91RLucII^{47 (link)}, Gα_s−117RLucII^{50 (link)}, Gα_oA−91RLucII^{51 (link)}, GFP10-G_γ1^{52 (link)}, GFP10-G_γ2^{53 (link)}, GFP10-EPAC-RLucII^{54 (link)} and rGFP-CAAX^{33 (link)}. The cloning of CXCR4-RLuc and CXCR4-YFP in pcDNA3.1 was previously described^{11 (link)}. In the present study, CXCR4-RLuc and CXCR4-YFP were amplified and modified by PCR at the N-terminal end to add a myc epitope (EQKLISEEDL) or a HA epitope (YPYDVPDYA), respectively. Myc-CXCR4-RLuc and HA-CXCR4-YFP segments were then subcloned into pIREShyg3 (BsrG1/AflII) and pIRESpuro3 (Nhe1/AflII) respectively. The human μOR and V2R were amplified with a SNAP tag at their N-terminal (NEB) and subcloned in the pcDNA4/TO plasmid (Invitrogen). All the mutants were obtained by site-directed mutagenesis using the extension of overlapping gene segments by PCR technique and validated by sequencing.

The human APP695 (hAPP) gene was purchased from GeneChem co. Ltd. (Shanghai, China) and cloned into a pcDNA3 vector as described previously17 (link). The Nicastrin gene was purchased from Sangon Biotech (Shanghai, China). The cDNA encoding full length human Nicastrin was cloned into the pFastBac1 bacmids with a C-terminal 6×His-FLAG tag. The pcDNA4/TO plasmid (Invitrogen) containing C99-Gal4-VP16 was constructed according to procedures described previously17 (link)30 (link). The human Notch1-NEXT (Notch 1 extracellular truncation, amino acid residues 1721–2555 in the human sequence) gene was synthesized by GenScript Ltd. (Nanjing, China). The pGL4.31[luc2P/Gal4UAS/Hygro] plasmid was purchased from Promega.

For luciferase-based reporter gene assays, pLucNeo was constructed by inserting a firefly luciferase gene derived from pGL3-control (Promega) into the pEYFP-1 vector (Clontech) at the BglII and AflII sites19 (link). KD short hairpin RNA (shRNA) vectors for human nSMase2 (the two different target sequences: 1, 5′-CAACAAGTGTAACGACGAT-3′ and 2, 5′-GGAGATTTCAACTTTGATA-3′) and MMP1 were purchased from TaKaRa Bio. For MMP1 overexpression assays, pcDNA4-MMP1 was constructed as follow: full-length MMP1 was PCR-amplified from ES-2 EV-isolated total RNA. Three prime A-overhangs were added to the PCR products after 15 min of regular Taq polymerase treatment at 72 °C. The PCR products were cloned into pGEM-T Easy Vector (Promega). The pGEM-T Easy Vector carrying MMP1 was digested with the restriction enzymes KpnI and EcoRI. The fragments containing MMP1 were amplified and the products were ligated into a pcDNA4/TO plasmid (ThermoFisher), generating pcDNA4-MMP1. The plasmids were verified by DNA sequencing.

Plasmid pNL1.1[Nluc] carrying Nanoluciferase cDNA was a gift from Promega (Cat# N1001). The Nluc gene was amplified using PCR with an upstream HA-tag along with appropriate cloning sites for expression as either a soluble cytoplasmic protein (HANL) or as fusion at the N-terminal end of CD63 gene (HANLCD63) in pcDNA4/TO plasmid (Thermo Fisher, Cat# V102020) backbone.