Tk renilla luciferase plasmid

The TCF/LEF luciferase reporter plasmid, NF-κB luciferase reporter plasmid, and TK-Renilla luciferase plasmid were purchased from Promega (Madison, WI, USA). Smo plasmid was purchased from Origene (Rockville, MD, USA). The Smo mutant plasmids were generated from wild type Smo plasmid using QuickChange Site-Directed Mutagenesis kit from Agilent (Santa Clara, CA, USA) and confirmed by sequencing. Information on Smo mutants plasmids is provided in Supplementary data 2. The Gli1 plasmid and Gli2-HA plasmid were obtained from Addgene (Cambridge, MA, USA). Transient transfections were performed by Lipofectamine 2000 reagent from Invitrogen (Grand Island, NY, USA) according to the manufacturer's instructions.

Cells were routinely cotransfected with a TK-Renilla luciferase plasmid (Promega, Madison, WI, USA) to normalize for the transfection efficacy. The Dual Luciferase Reporter Assay Kit from Promega was used following the manufacturer's protocol. Luciferase activity was measured with a Victor X3 2030 multi-label plate reader (PerkinElmer, Eden Prairie, MN, USA). The data are represented the mean values obtained from three independent experiments.

The 8 × Gli1-binding site luciferase reporter (8 × GBS-luciferase) plasmid was a kind gift from Dr. Hiroshi Sasaki. The TCF/LEF-luciferase reporter plasmid, NF-κB –luciferase reporter plasmid, and TK-Renilla luciferase plasmid were purchased from Promega (Madison, WI). The Gli2 lacking the N-termianl (Gli2ΔN) plasmid and ShhN plasmids were obtained from Addgene (Cambridge, MA). The Myc-DKK-tagged ORF clone of Homo sapiens Smo plasmid was purchased from Origene (Rockville, MD). The mutant human plasmid SmoM2 (W535L) was generated from wild type Smo plasmids using QuickChange Site-Directed Mutagenesis kit from Agilent (Santa Clara, CA) and was confirmed by sequencing. The Sufu-shRNA was purchased from Santa Cruz (Santa Cruz, CA).
Transient transfections were performed using Lipofectamine 2000 reagent from Invitrogen according to the manufacturer’s instructions. The lentiviral stocks were prepared according to previous report [19 (link)]. Briefly, the plasmid carrying the Sufu-shRNA and three packaging plasmids were co-transfected into 293T cells using Lipofectamine 2000. The viruses were harvested 24 h post transfection and 4 ml viruses were used for infected NIH-3T3 cells seeded in 10-cm dishes. Infected cells were analyzed 5–7 days post infection by western blot analyses of the expression of Sufu.