Trans it 293 reagent

The mouse Tgfbr2 promoter (702 bp, from −650 to +52 relative to the transcription start site) was amplified from genomic DNA of E0771GFP cells by PCR using Q5 Hot Start High-Fidelity 2X Master Mix (NEB). The Tgfbr2 promoter fragment was cloned into the Kpn I and Hind III restriction sites of the pGL3-Basic firefly luciferase reporter vector (Promega). A mutant Tgfbr2 promoter where putative ZBTB18 binding sites (5′-[AC]ACATCTG[GT][AC]-3′) are substituted with two Sma I binding sites (5′-CCCGGGCCCGGG-3′) was generated by cloning a gBlock into the Kpn I and Hind III restriction sites of the pGL3-Basic firefly luciferase reporter vector. 293FT cells were plated in 96-well plates at 10⁴ cells per well. After 24 hours, cells were cotransfected with 75 ng of luciferase reporter plasmid (pGL3-Basic, pGL3-Tgfbr2 promoter, or pGL3-mutant Tgfbr2 promoter); 75 ng of expression plasmid ZBTB18OE, ZBTB18-Nucl, ZBTB18-Cyto, or empty expression vector (EV); and 50 ng of pRL-TK Renilla luciferase vector (to normalize for transfection efficiency; Promega) using TransIT-293 reagent in Opti-MEM I. After 24 hours, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase values were normalized to Renilla luciferase values and protein content and reported relative to the EV condition.