TCF/LEF reporter assay.

To assess TCF/LEF transcription activity, we used TOPflash and FOPflash luciferase reporter vectors as described (44). Transcriptional induction does not occur in FOP (TOP with a mutation in the TCF/LEF-binding site), even if the WNT pathway is activated. Therefore, if reporter expression that occurs in TOP does not occur in FOP, it indicates that what is occurring is not nonspecific transcription induction, but rather canonical WNT pathway signaling.

HEK293 cells were cultured in a 24-well plate, and Lipofectamine LTX was used to transfect LRP6 expression vector (100 ng/well), FZD8 expression vector (100 ng/well), WNT3A expression vector (200 ng/well), ACLP expression vector (200 ng/well), and either TOPflash or FOPflash luciferase reporter vector. After 36 hours of culturing, intracellular expression of luciferase was quantified using the Dual-Luciferase Reporter Assay System (Promega Corp.). Cells were lysed with a passive lysis buffer included with the kit, Luciferase Assay Reagent II was added to the cell lysate, and chemiluminescence was measured with a TD-20/20 Luminometer (Turner Biosystems).