Figure 2.

Overexpression of A20 and/or CYLD downregulates NF-κB activation elicited by CD137 ligation. (A) 293 T cells transiently transfected with the indicated expression plasmids for 18 hours were stimulated for 5 hours with anti-CD137 (6B4) or control mIgG1. Experiments were read out by means of cotransfection with the NF-κB luciferase reporter construction and subsequent incubation with luciferin. Results are shown as relative luminescence units between firefly luciferase activity and renilla luciferase as an internal control (mean ± SEM) given as fold change with respect to the empty vector (left panel). Immunoblots in lysates of the indicated transfectants confirm expression of the corresponding proteins (right panel). (B) Immunoblots for IκBα and anti-GAPDH (loading control) in lysates from 293 T cells transfected as in A with the indicated constructs. (C and D) Experiments performed as in A in MOLT4 and CEM T cells electroporated with the plasmids encoding the indicated expression cassettes. A representative set of experiments out of at least 6 performed is shown. In this case, cells were electroporated 18 hours before the experiment and cells were exposed to antibodies for 24 hours before NF-κB activity measurement in lysates. Quantitative RT-PCRs confirming mRNA expression of the plasmid were performed and CD137 surface expression was checked by flow cytometry (not shown). Shown experiments were consistent with at least 5 similarly performed.