NIH3T3 cells were cultured in DMEM containing 10% Bovine Calf Serum (ATCC). HEK 393T and Sufu -/- MEF cells were cultured in DMEM with 10% Fetal Bovine Serum (FBS) (Sigma Aldrich). Shh treatment was done by serum starvation for 24 hours (0.5% Bovine Calf Serum), then adding a recombinant mouse Shh N-terminal fragment (R&D Systems #464-SH) at 1 ug/ml overnight. Smoothened agonist SAG (Sigma Aldrich) treatment was done at 200 ng/ml for 8–12 hours. Cell transfections were performed using PolyJet in vitro DNA Transfection Reagent (SignaGen) following manufacturer’s instruction. Immunoprecipitation, immunostaining, and western blot analyses were carried out as described previously [47 (link)]. The antibodies used in this study are listed as follows: anti-Myc (9E10, Santa Cruz Biotechnology), anti-β-galactosidase (A11132, Life Technologies), anti-acetylated tubulin (T7451, Sigma Aldrich), anti-mGli2 (AF3635, R&D Systems), anti-mGli3 (AF3690, R&D Systems), anti-mKapβ2 (Ab10303, Abcam), anti-α-tubulin (T9026, Sigma Aldrich), anti-Histone3 (Ab1791, Abcam), and anti-BrdU (B8434, Sigma Aldrich).
Free full text: Click here
