One glo luciferase assay system reagent

The transcription factor-binding sites analysis was performed on the hypothetical promoter region of Inpp5a (from −2000 to 0) using an online transcription factor analysis tool (Matin spector). The proximal promoter of Inpp5a (from −1419 to −109), which contains 2 GC boxes, was linked to luciferase reporter pGL4.14 (Promega). The reporter assays were performed in both PC12 and HEK293 cells. The PC12 cell lines expressing TBP-13Q or -105Q were transfected with Inpp5a reporter with or without SP1 plasmids. HEK293 cells were transfected with Inpp5a reporter and SP1 with TBP plasmids (TBP-13Q or -105Q). Cells were transfected for 24 h, then collected for luciferase promoter activity assay. Independent experiments were repeated three times. ONE-Glo Luciferase Assay System reagent (Promega, E6120) was used to detect reporter activity using a microplate reader (Synergy H4, BioTek).

HEK293T ATAD5-luciferase cells were grown at 37 °C in 5% CO₂ and 50:50 DMEM:F12 medium supplemented with 10% fetal bovine serum and 1% antibiotics. 1,500 cells were plated in each well of a 384 well plate, 20 ul of fresh medium was added to each well of a 384 well plate, followed by 0.5 μl of each drug at 5 mM or DMSO and 30 μl of 5 × 10⁴ cells/ml. The plates were incubated at 37 °C for 18 hours and equilibrated at room temperature for 30 minutes. To measure the ATAD5-luciferase activity, 50 ul of ONE-Glo® luciferase assay system reagent (Promega) was dispensed into each well and the luminescence signal was measured using a Wallac plate reader.

For determining the capacity of antibodies to induce the NF‐ĸB signaling pathway in Jurkat cells overexpressing NF‐ĸB‐Luc and human GITR (Jurkat‐hGITR‐NF‐ĸB‐Luc cell), the cells were seeded at 5 × 10⁴ cells/well in 50 μL of cell assay medium (RPMI1640 + 5% FBS) in 96‐well assay plates (Corning, Corning, NY, USA; #3903). Plates were then incubated with 50 μL of a dilution series of GITR antibodies in the non‐crosslinking format, or 50 μL of a dilution series of F(ab')2 Fragment anti‐Human IgG, Fcγ fragment specific (Jackson Immunoresearch, West Grove, PA, USA; #109–006‐008) and GITR antibody at a ratio of 4 : 1 in the crosslinking format. After incubation at 37 °C in 5% CO₂ for 5 h, 100 μL of ONE‐Glo™ Luciferase Assay System Reagent (Promega, Madison, WI, USA; #E6120) was added to each well, and luminescence values were measured using a Varioskan LUX (Thermo, Carlsbad, CA, USA).