Non-risk or risk sequences surrounding rs10499197 or rs58905141 were cloned from EBV B cells positive for the non-risk or risk TNFAIP3 SLE risk haplotype tagged by rs10499197 into a minimal promoter firefly luciferase plasmid, pGL4.23 (Promega) (Supplementary Table S2 ) (Wang et al., 2013 (link); Pasula et al., 2020 (link); Gopalakrishnan et al., 2022 (link)). Quick change II site directed mutagenesis kit (Agilent Technologies; #200523) was used following manufacturer’s instructions to allele swap rs9494868 (Supplementary Table S2 ). Empty vector, non-risk clone, or risk clone was transiently co-transfected with the transfection control renilla luciferase plasmid, pRL-TK, using a 4D Amaxa Nucleofector Unit (Lonza) for Jurkat cells (Nucleofector SE kit; #V4XC-1032), THP-1 cells (Nucleofector SG kit; #V4XC-3032), and EBV B cells (Nucleofector SF kit; #V4XC-2032). Twenty-four hours post transfection, cells were stimulated with P/I for 2 h. Enhancer activity was determined using the Promega Dual-Luciferase Reporter Assay kit (Promega) following manufacturer’s protocol. Relative luciferase units (RLU) were determined by normalizing the firefly luciferase activity to the Renilla luciferase activity. RLU were normalized to the vector-only control and reported as normalized RLU.
Free full text: Click here
