Xhoi and hindiii

A modified cDNAs for mVenus, with an in-frame deletion eliminating 11 amino acids from the C-termius, and NLuc (Promega) with a deletion of 3 amino acids from the N-terminus (with permission from Promega), were amplified by PCR, respectively. The PCR products were ligated into the XhoI-ClaI and EcoRI-HindIII sites in pRSET-AT1.038 (link), respectively, to generate pPRSET-BTeam for expression of BTeam in Escherichia coli. The cDNA of BTeam was excised from pRSET-BTeam with XhoI and HindIII (Thermo Scientific). The restriction fragment was then ligated into the XhoI-HindIII sites of pcDNA3.1 (−) (Invitrogen) for mammalian expression. The constructs used in the present study are illustrated in Supplementary Fig. S1.

The 5’ flanking region of PADI3 gene was amplified by PCR using the Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, USA), the human genomic DNA from HFFs as a template, and the PAD3 primers containing Xho I and Hind III restriction enzymes sites (see primers sequences listed in S1 Table). The resulting amplification products were digested with Xho I and Hind III (Thermo Fisher Scientific, Waltham, USA) and cloned into the pGL4.20[luc2/Puro] vector (Promega, Madison, USA), which encodes the luciferase reporter gene luc2 (Photinus pyralis) with no other regulatory elements. The resulting pGL4.20-PADI3prom construct was purified using the PureYield Plasmid Miniprep System (Promega, Madison, USA) and verified by restriction mapping and complete sequencing. The resulting chromatograms were analyzed using Chromas software 2.6.6 (Technolysium Ltd.).