Figure 14.

Target validation by Q-PCR of Chromatin Immunoprecipitation (CHIP)-Based Assays. Female C57BL/6J mice were anesthetized and instilled with 200 μl of control peptide or PAR2-AP on days 1 and 2, as described in methods. Mice were euthanized 24 hours after the last instillation; the urinary bladders were removed rapidly, frozen, and shipped to Genpathway [40] for querying the chromatin for transcription of selected genes. After isolation, the chromatin was incubated with TFEB antibody to precipitate the CHIP DNA. The final CHIP DNAs were then used as templates in Q-PCR reactions, performed in triplicate, using specific primer pairs (Table 1). Results are presented as average and standard error of Transcription Binding Events Detected Per 1000 Cells. Asterisks indicate a statistical significant difference (p < 0.05) between a specific gene and the un-transcribed region used as control and a plus sign indicates a statistical significant difference (p < 0.05) between CHIPs isolated from PAR2-AP- and control peptide-treated bladders.