Figure 6. Inferring the likely targets of 5ZO based on experiments in SF and current understanding of the drug.

(a) Phosphorylation of TAK1 on activating residues (Thr184/187) 10 min after addition of 100 ng/mL TNFα or IL–1α to SF samples RA2159 in the presence of 50 nM calyculin, a serine/threonine protein phosphatase inhibitor. Error bars are SEM for detection of p–TAK1 in biological duplicate using two p–TAK1 antibodies, each at four different dilutions across three separate days (48 replicates for each stimulus; see Supplementary Fig. 17). (b) Schematic of TAK1 and MAPK signaling showing potential on–target (solid red lines) and off–target (dotted red lines) drug effects, as well as potency in inhibiting SF activation (encoded by the intensity of the yellow boxes). Analysis of DS2 and DS3 shows that secondary targets such as MEK and components of the p38 cascade have a small effects on cytokine secretion by SF relative to 5ZO. (c) Activity of signaling cascades in cells stimulated for 30 min with 100 ng/ml TNFα, IL–1α or EGF in the presence and absence of 5ZO. Activation of NFκB was scored by nuclear translocation of NFκB p65 and activation of MEK/ERK, JNK and p38 kinases by the readouts shown in panel b.