Computational protocol: Essential Roles of Epithelial Bone Morphogenetic Protein Signaling During Prostatic Development

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Protocol publication

[…] Genomic sequences of the 3ā€²-region of the Nkx3.1 locus, which includes the prostatic regulatory region (), were obtained from NCBI (http://www.ncbi.nlm.nih.gov/nuccore/) and were submitted for analysis by rVISTA (http://genome.lbl.gov/vista) and MultiPipmaker (http://pipmaker.bx.psu.edu/pipmaker/). For promoter analysis, the genomic DNA fragments from C57BL were obtained by a standard PCR procedure and were inserted into the pGL4.24 vector (Promega Corp) using the Infusion system (Takara). Mouse Smad1 and Smad4 were amplified with RT-PCR and inserted into the pFLAG-cytomegalovirus vector (Sigma).The expression and reporter plasmids were transfected into PC3 cells with Lipofectamine LTX plus (Life Technologies) according to the manufacturer's instruction. Twenty-four hours after transfection, luciferase activity was measured by chemiluminescence by employing the Dual-Luciferase Reporter Assay System (Promega Corp). The values were normalized against Renilla luciferase activity under the control of the cytomegalovirus promoter vector pGL4.74 (Promega). Further addition of Bmp7 (50 ng/mL) (R&D system) was treated after 16ā€“20 hours after transfection of Smad1/4 expression vector, and samples were collected and their luciferase activities measured after 24 hours. More than 3 independent experiments were performed. Statistical analysis was performed using Student's t test or Welch's t test followed by an F test (P < .05 was considered as significant). […]

Pipeline specifications

Software tools rVISTA, MultiPipMaker
Application Miscellaneous
Organisms Mus musculus
Diseases Prostatic Neoplasms