Figure 7

CERK inhibition downmodulates TNF-α-induced activation of MAPK and NF-κB signaling pathways in THP-1 cells. THP-1 monocytic cells were pretreated with CERK inhibitor (NVP-231: 12 nM) and then incubated with TNF-α. Cell lysates were prepared as described in “Materials and methods” section. Samples were run on denaturing gels. Immuno-reactive bands were developed using an Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Chicago, IL, USA) and visualized by Molecular Imager ChemiDoc MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA). (A) Phosphorylated proteins of SPAK/JNK, (B) p38 and (C) NF-κB are shown in the upper panels with the lower panel representing respective total proteins. The phosphorylation intensity was quantified by using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and presented in bar graphs as arbitrary unit (AU) of corrected protein expression. Signaling proteins were also determined by flow cytometry. Cell were immediately fixed and permeabilize for 20 min at 4 °C, then stained to visualize the JNK, p38 and NF-κB phosphorylation. Flow cytometry data are presented as a bar graph of mean staining index (SI) as well as by representative histograms (D,E). Bar graphs depict the mean values ± SEM of staining intensity (SI). P < 0.05 was considered as statistically significant (*p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). The data in all figures are representative of three independent experiments. NF-κB/AP-1 reporter monocytic cells were pretreated with CERK inhibitor (NVP-231: 12 nM) or vehicle for 1 h and then incubated with TNF-α for 12 h. Cell culture media were assayed for SEAP reporter activity, representing NF-κB/AP-1 activation (F). Reporter cells were also tested for surface expression of CD11c (G).