Fig. 2

GLS-1027 inhibits the activation of NF-κB and p38 pathways induced by LPS, but not by Poly(I:C). (A) Flow-cytometric analysis of phospho NF-κB -p65 in monocytes after LPS and poly I:C stimulation in the presence or absence of 10 µM of GLS-1027 in vitro. After stimulation, cells were fixed and permeabilized. The cells were stained with fluorochrome-conjugated antibodies against the CD14 monocyte cell surface marker and phospho NF-κB -p65. The CD14-positive monocyte population was analyzed for intracellular levels of phosphorylated NF-κB using flow cytometry with the data displayed as histograms. (B) Inhibition by GLS-1027 of NF-κB (p65) DNA binding activity in TNF-α stimulated cells. Nuclear extracts were obtained from unstimulated and TNF-α stimulated at indicated time points in the presence or absence of GLS-1027 (1 and 10 µM) cells and DNA-binding activity was examined by ELISA based EMSA assay. To confirm the specificity of this assay competitor oligo used as control. Data shown are representative of two independent experiments. (C) Cells were stimulated with TNF-α with or without GLS-1027 (10 µM) for 3-5 h. Cells were fixed and stained with an anti-NF-κB (p65) antibody, followed by anti-rabbit secondary antibody. (D-E) Phosphorylation of TAK1/IRAK1 is inhibited with GLS-1027. Human primary macrophages were treated with TNF-α and LPS, in the presence or absence of 10 µM of GLS-1027. The activation status of TAK1/IRAK was examined in the cell lysates by immunoblot analysis using the phospho-IRAK (T209) antibody (D) and phospho TAK1 activity was determined using ELISA (E), as described in Materials and methods