NF-κB luciferase reporter assay

Transient transfection of HEK293 T cells was performed using Lipofectamine 2000 according to the manufacturer's instructions. Luciferase reporter assays were performed with pκB-Luc plasmid which encodes a firefly luciferase reporter gene under the control of a NF-κB-responsive promoter⁷¹ and Renilla luciferase reporter vector pRL-TK (Promega). Briefly, cells were co-transfected with 200 ng pκB-Luc plasmid, 200 ng constitutive expression plasmid Renilla luciferase reporter vector pRL-TK (Promega), and 5 ng pCMV6-hCD137 (GeneCopoeia), 500 ng pcDNA 3.1+HA-ubiquitin K63R (a kind gift from Gijs Versteeg, Mount Sinai School of Medicine), 500 ng pCMV6-mCYLD (Origene) and/or 500 ng pCMV6-mA20 (Origen) as indicated. Five hours after transfection cells were stimulated for 18 hours with 10 μg/ml anti-CD137 mAb (clone 6B4) or mAb control (mIgG1, Biolegend) and lysed in dual reporter lysis buffer (Promega). Firefly luciferase signal were normalized using Renilla luciferase values.

For luciferase reporter experiments in lymphoid cell lines, 40 × 10⁶ CEM or MOLT4 cells per condition were electroporated using the Gene Pulser MX System (300 V, 950 μF Bio-Rad, Hercules, CA) with plasmids encoding human CD137 (40 μg), K63R (80 μg), A20 (80 µg), CYLD (80 µg), NF-κB-firefly luciferase reporter (40 μg), pRL-TK (40 μg), or empty vector (20–30 μg) as indicated. After 18 h, live cells were isolated using Ficoll density gradient separation, washed, and stimulated during 24 h with 10 μg/ml anti-CD137 mAb (clone 6B4) or control Ab (Mouse IgG1; Biolegend).