Regulation of Cdkn1b by Hedgehog Signaling

NIH-3T3 cells were co-transfected with plasmids containing Cdkn1b promoter constructs of various lengths upstream of firefly luciferase (provided by Dr D. Everly, Rosalind Franklin University of Medicine and Science, North Chicago, IL)59 (link) and Renilla luciferase (10:1 dilution), followed by transfection of siRNA against 14-3-3ζ or the scrambled control. To determine hedgehog-dependent transcriptional activity, Shh-light2 cells were treated with the synthetic Smoothened agonist (SAG; Cayman Chemicals, Ann Arbor, MI) or transfected with siRNA against 14-3-3ζ or the scrambled control. Luciferase activity was measured after 24 or 48 h with the Dual-Luciferase Reporter Assay system (Promega, Madison, WI). Transcription factor binding sites were analysed with MotifMap34 (link). ChIP analysis of Gli3 binding to the Cdkn1b promoter was performed with antibodies against Gli3 and the Pierce Magnetic ChIP kit, as per the manufacturer's protocol (Thermo Scientific, Rockford, IL).

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Lim G.E., Albrecht T., Piske M., Sarai K., Lee J.T., Ramshaw H.S., Sinha S., Guthridge M.A., Acker-Palmer A., Lopez A.F., Clee S.M., Nislow C, & Johnson J.D. (2015). 14-3-3ζ coordinates adipogenesis of visceral fat. Nature Communications, 6, 7671.

Publication 2015

Smoothened agonist (SAG; Dual-Luciferase Reporter Assay system Pierce Magnetic ChIP kit

Antibodies Assay Binding sites Cayman Cdkn1b Cells Chip Dilution Firefly luciferase Hedgehog Luciferase Medicine Nih 3t3 cells Plasmids Promega Renilla luciferase Sirna Transcription factor Transcriptional Transfection

Corresponding Organization :

Other organizations : University of British Columbia, University of South Australia, Australian Centre for HIV and Hepatitis Virology Research, Monash University, Goethe University Frankfurt

Top 5 similar protocols

Variable analysis

independent variables

Cdkn1b promoter constructs of various lengths
SiRNA against 14-3-3ζ
Scrambled control siRNA
Smoothened agonist (SAG)
SiRNA against 14-3-3ζ
Scrambled control siRNA

dependent variables

Firefly luciferase activity
Hedgehog-dependent transcriptional activity

control variables

Renilla luciferase (10:1 dilution)

controls

Positive control: Shh-light2 cells treated with Smoothened agonist (SAG)
Negative control: Scrambled control siRNA

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