Fig. 6.

CGRP prevents NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 and LPS-induced IκBα degradation in HMEC-1 cells. (A) CGRP inhibits NF-κB DNA binding to promoters. Nuclear extracts were prepared from HMEC-1 cells stimulated for 4 h with LPS (1 μg/ml) in the presence or absence of CGRP with CGRP added to cultures 1 hr before LPS. NF-κB binding was assayed by EMSA. Nuclear extracts were incubated with antisera against p50, p65 or IgG control for 15 min before adding the radiolabeled probe. Similar results were observed in 3 independent experiments. (B) CGRP prevents LPS-induced IκBα degradation. HMEC-1 cells were stimulated with LPS in the presence or absence of CGRP as in (A). Cytosolic amounts of IκBα at different time points were determined by Western blot. One representative experiment of 3 is shown. (C) Bay11-7085 blocked LPS-induced CXCL8, CCL2 and CXCL1 production by HMEC-1 cells. HMEC-1 cells were stimulated with LPS (1 μg/ml) in the presence or absence of graded concentrations of Bay11-7085. Supernatants were collected 24 h after LPS stimulation and chemokine content assayed. Each result is the mean ± SD of three separate replicates (each with duplicate wells) performed at the same time. This result is representative of 2 such experiments. (***p<0.001 vs LPS, no Bay11-7085; ^*p<0.05, ^**p<0.01, ^***p<0.001 vs no LPS, no Bay11-7085).