Extended Data Fig. 1. Short duration exposure to AZD5582 activates the ncNF-κB pathway.

(a-d) Isolated total human CD4⁺ T cells treated with 100 nM of AZD5582 and then either not washed (a) or washed three times with PBS at (b) 8 h, (c) 4 h, or (d) 1 h post-treatment. Whole cell lysates were then analyzed by immunoblot for components of the ncNF-κB pathway. (e) Densitometry analysis of the ratio of p52 to p100 from the pulse-wash assay immunoblots. Points represent values for the densitometric ratio from one western blot, representative of several independent experiments. (a-e) Entire set representative of one experiment with 5 replicates of the 1 h wash condition conducted. (f) Fold induction of ncNF-κB target gene expression in isolated CD4⁺ T cells from an uninfected donor treated with the indicated concentration of AZD5582, and either washed after 1 h or not washed, then cultured for 24 h as measured by quantitative RT-PCR. Points represent two technical replicates and lines represent the average. The data presented are representative of three independent experiments. (g) DMSO-normalized induction of luciferase activity from the Jurkat reporter model after exposure to AZD5582 (10, 100, or 1000 nM) for either 30 min (blue), 1 h (red), 2 h (green), or continued exposure (purple). Points represent three replicates in one assay run, representative of two independent experiments. Lines represent the average of the three replicates. For gel source data, see Supplementary Figure 1.