The pGL3 Firefly luciferase basic vector (Promega) was digested with KpnI and XhoI in order to insert SH2D1A promoter inserts of 1000 bp (maximum activity) versus 100bp (minimal activity) as previously published [25 (link)]. Segments of the SH2D1A promoter were amplified using these primers: SAPprom-F1: 5′ atttgcattaatacagtttagcctcaatcgaag-3, SAPprom-F2: 5′ -atttgcggtaccgttgttggggtgcttctctc-3′, SAPprom-R: 5′-ttactcaccggtggcctggtggactcttgg-3′.
Expression cassettes encoding WT FOXP3 or GFP were PCR amplified and cloned into the pmax vector (Lonza) after cutting with KpnI and XhoI. PCR primers used were: pmaxFOXP3-F 5′-atttgcggtaccgccgccaccatgcccaaccccaggcctg-3′, pmaxFOXP3-R 5′-ttactcctcgagtcaggggccaggtgtaggg-3′, The loss of function mutation in the DNA binding domain of FOXP3 (R397W) was introduced by site-directed mutagenesis using DpnI digestion and the following primers for amplification: R397W-F 5′-tgctttgtgTgggtggagagcgagaagg-3′, R397W-R 5′-gctctccacccAcacaaagcacttgtgcagac-3′. For luciferase assays, Jurkat T cells were triple transfected with 5 μg pmax-GFP or FOXP3 expression vector, 5 μg pSAP-GL3 firefly luciferase vector, 0.5 μg pRL-TK renilla luciferase vector, and the pmax vector and assayed after 24 hours. Luciferase activity was quantified using a Dual Luciferase Assay Kit (Promega) and a Synergy H1 microplate reader (Bio-Tek).
Expression cassettes encoding WT FOXP3 or GFP were PCR amplified and cloned into the pmax vector (Lonza) after cutting with KpnI and XhoI. PCR primers used were: pmaxFOXP3-F 5′-atttgcggtaccgccgccaccatgcccaaccccaggcctg-3′, pmaxFOXP3-R 5′-ttactcctcgagtcaggggccaggtgtaggg-3′, The loss of function mutation in the DNA binding domain of FOXP3 (R397W) was introduced by site-directed mutagenesis using DpnI digestion and the following primers for amplification: R397W-F 5′-tgctttgtgTgggtggagagcgagaagg-3′, R397W-R 5′-gctctccacccAcacaaagcacttgtgcagac-3′. For luciferase assays, Jurkat T cells were triple transfected with 5 μg pmax-GFP or FOXP3 expression vector, 5 μg pSAP-GL3 firefly luciferase vector, 0.5 μg pRL-TK renilla luciferase vector, and the pmax vector and assayed after 24 hours. Luciferase activity was quantified using a Dual Luciferase Assay Kit (Promega) and a Synergy H1 microplate reader (Bio-Tek).