Fig. 4.

Determine the target of CinF in the NF-κB signaling pathway by epistasis analysis. (A) A diagram depicting the canonical and noncanonical branches of the NF-κB signaling pathway. The point of action for LegK1, the L. pneumophila effector that activates NF-κB, was indicated. (B) CinF does not significantly inhibit NF-κB activation induced by LegK1. HEK293T cells transfected with the reporter plasmid together with those that direct the expression of LegK1 and CinF were measured for luciferase activity. The expression of the relevant proteins was detected by immunoblotting with the appropriate antibodies, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was probed as a loading control. Results shown were one representative done in triplicate from three independent experiments with similar results. (C–F) HEK293T cells were transfected with the indicated combinations of plasmids harboring the reporter, CinF, the internal control, and the expression of luciferase driven by NF-κB was measured. Note that CinF effectively inhibits the activation induced by TRAF2 (C), TAK1 (D), and IKKβ (E) but does not affect the activation induced by p65 (F). In each case, results shown were one representative done in triplicate from three independent experiments. Statistical analysis in each panel was performed by Student’s t test. The expression of the relevant proteins was detected by immunoblotting, and GAPDH was probed as a loading control.