PMC:4925210 / 39573-40698 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4925210","sourcedb":"PMC","sourceid":"4925210","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4925210","text":"TOP/FOP Flash reporter assays\nOne million of MEFs or CHO-K1 cells were transfected with a Super TOP-Flash or FOP-Flash plasmid and Renilla luciferase pRL-TK vector together with pcDNA3 or ZNRF3 expression vector, using the Ingenio Electroporation Kit (Mirus-Bio) and the Nucleofector IIb Device (Lonza). Renilla luciferase was used as the internal control reporter to normalize transfection efficiency. All transfections were performed with 0.5 μg of Super TOP-Flash or FOP-Flash, 0.05 μg of pRL-TK, and 2 μg of pCDNA3 or ZNRF3. After 18 h following transfection, cells were split into 18 wells of a 24 well plate. After 24 hr, cells were treated with PBS, WNT3A (500 ng/ml), WNT9B (1 μg/ml), or a mixture of WNT3A and WNT9B for another 24 hr and lysed in a reporter lysis buffer (Promega, Madison, WI, USA). Luciferase assay was performed using the Dual Luciferase Assay System kit according to the manufacturer's protocols (Promega, Madison, WI, USA). Relative luciferase activity was reported as fold induction after normalization for transfection efficiency. Three independent experiments were performed in sextuplicates.","divisions":[{"label":"Title","span":{"begin":0,"end":29}}],"tracks":[]}