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over expression of wild-type MEK1 increased NTHi induced CXCL2 up-regulation. GlcNAcstatin clinical trial We sought to determine the participation of ERK12 in NTHi induced CXCL2 up-regulation, since ERKs are downstream elements of MEK1. Not surprisingly, pretreatment with AG126 and FR180204 somewhat inhibited NTHi activated CXCL2 up-regulation. Next, we performed phosphorylation assays to find out NTHi stimulated ERK activation. Interestingly, just ERK2, not ERK1, was phosphorylated upon contact with NTHi, peaking 10 min after. In consistence using the finding of the phosphorylation assays, NTHi activated CXCL2 up regulation was observed to become inhibited only by dominant negative construct of ERK2, however, not by ERK1. Constantly, ELISA analysis showed that dominant negative inhibition of ERK2 markedly inhibits NTHi caused CXCL2 up-regulation. Next, we wanted to find out if NTHi stimulated c Jun activation requires the MEK dependent signaling pathway. As shown in Fig. To determine NTHi open elements in the 5 flanking region of CXCL2, the luciferase Plastid expressing constructs containing the nested deletions of the 5 flanking region of the rat CXCL2 were made. Luciferase assays revealed that the 134 bp sized build has the least promoter activity set alongside the 3475 bp and 563 bp sized constructs, indicating that the NTHi receptive factors occur between 563 bp and 134 bp of the 5 flanking region of the rat CXCL2. The pattern analysis of the region predicted two AP 1 motifs, which agreed with the previous reports demonstrating that two AP 1 motifs exist within the 5 flanking region of the mouse CXCL2. To determine the necessity of these AP 1 motifs for NTHi stimulated CXCL2 upregulation within the SLFs, we conducted site directed mutagenesis. As shown in Fig. 4B, NTHi induced CXCL2 upregulation was inhibited by the mutation of each AP 1 design, and CXCL2 induction was completely inhibited by the mutation of both sites. Apparently, the proximal AP 1 motif were more associated PF-543 concentration with NTHi caused CXCL2 up regulation than the distal one. While in The mouse CXCL2, both AP 1 motifs were also found to be involved in NTHi induced up-regulation of CXCL2 term. Next, we wanted to find out if NTHi activated c Jun binds the AP 1 motifs of the rat CXCL2. We performed ChIP PCR assays using an anti d Jun antibody and the primers comprising either distal or proximal AP 1 pattern of CXCL2.