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Reporter assay

CF C-W. Fan
NY N. Yarravarapu
HS H. Shi
OK O. Kulak
JK J. Kim
CC C. Chen
LL L. Lum
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The HH-responsive firefly luciferase reporter (GLI-BS) and a control Renilla luciferase reporter (SV40-RL) were transfected into indicated cell lines using Effectene (Qiagen) either alone or with indicated DNAs. 24 hrs after transfection, cells were switched to low serum media (3% calf serum), and grown for another 48 hrs. in 5% CO2 in the presence/absence of compounds. 3T3-ShhFL cell line that stably expressing SHH and the two reporters were cultured in a similar fashion. FL and RL activities in lysate generated using Passive Lysis Buffer (Promega) were then assessed using the Dual-Luciferase kit (Promega) and a 96-well plate reading luminometer (BMG). The ratio of FL/RL was calculated and the averaged ratios from three replicates were reported.

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