Fig. 5.
Secondary validation of hits with independent siRNA pools. (A) Workflow summarizing the secondary validation screen with alternative sets of ON-TARGETplus siRNA pools consisting of four distinct siRNA sequences for each gene. (B) Summary of the secondary validation rate of positive and negative regulators tested for MDP-induced NF-κB luciferase activity. (C) Comparison of relative NF-κB luciferase activity after stimulation with MDP or TNF-α. Positive controls (RIPK2, RIPK1, and RelA) are highlighted along with two selected validated hits. (D) Epistasis analysis reveals that RNF31 is a general regulator of NF-κB signaling. HEK 293 cells were transfected with the indicated siRNA pools. After 48 hours, a second transfection with an NF-κB luciferase reporter and a Renilla reporter (for normalization) was used to monitor NF-κB pathway activation with (i) a constitutively active NOD2 construct (NOD2 CA), (ii) an inducibly active (IA) RIPK2 construct (RIPK2-IA), and (iii) an inducibly active RIPK1 construct (RIPK1-IA) relative to a pcDNA3 empty vector (EV) control. The dimerization agent AP1510 (ARIAD) was added as indicated to induce signaling. Experiments were performed in triplicate, of which one representative example is shown. *, P < 0.05 by a one way ANOVA and Tukey multiple comparison test performed using GraphPad Prism software.