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. 2009 Aug 24;284(42):28845–28855. doi: 10.1074/jbc.M109.042150

FIGURE 2.

FIGURE 2.

PAR1 regulates MUC18 expression via CREB. A, dual luciferase assay demonstrates that PAR1 silencing inhibits activity of the MUC18 promoter reporter (673 bp, pGL4-MUC18) in A375SM and C8161-c9 cells as compared with control cells transduced with the nontargeting shRNA. B, a schematic of the 5′-flanking sequence of the MUC18 gene depicts possible binding sites for CREB and Sp1 transcription factors, as indicated by the position of the 3′-end of each site relative to the transcription start site. A schematic also demonstrates position of primers used in the ChIP assay. C, ChIP analysis demonstrates that PAR1 silencing (shPAR1) inhibits CREB binding to MUC18 promoter as compared with control cells transduced with the nontargeting shRNA (shNT). D, Western blot shows decrease in CREB and ATF-1 phosphorylation and expression following PAR1 silencing. E, as measured by dual luciferase assay, PAR1 silencing inhibits activity of the CRE-driven luciferase reporter. F, CREB silencing (shCREB) using lentiviral shRNA results in down-regulation of CREB expression in A375SM and C8161-c9 cells as compared with control cells transduced with the nontargeting shRNA (shNT). Western blot analysis further demonstrates that CREB silencing results in down-regulation of MUC18 expression.