Cyclone storage phosphor system

NIH3T3 cells were treated with 100 nM SAG for 48 h to activate the HH pathway, harvested and lysed in lysis buffer (20 mM HEPES pH 7.9, 25% glycerol, 0.5% Nonidet P-40, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT) to obtain whole-cell extracts (WCE).^{64 (link)} To analyze the binding of Gli proteins to Site1 at E2F1 promoter we used the following oligonucleotide and its complementary strand: site1F, 5′-AGCTACCCTGGAGGCGTCT-3′ site1R, 5′-TCGAAGACGCCTCCAGGGT-3′. The DNA-binding reaction was performed by incubating 20 fmol of double-stranded ³²P-labeled oligo with 40 μg of WCE in a total volume of 20 μl containing 1 μg poly-dC, 100 mM KCl in 40 mM HEPES (pH 7.9), 10 mM MgCl2, 0.4 mM EDTA, 4 mM DTT and 40% glycerol^{64 (link)} for 20 min at room temperature. In indicated competition experiments, a 100-fold excess of unlabeled oligo was used. When using antibodies, proteins were preincubated with anti-GLI1 (N-16, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-GLI2 (#AF3635, R&D Systems, Minneapolis, MN, USA) antibodies for 20 min at room temperature before adding the radiolabeled probe. The samples were separated on native polyacrylamide gel (6% polyacrylamide:bisacrylamide, 29:1) at 4 °C for 3 h and the signal was detected by Cyclone Storage Phosphor System (Perkin Elmer, Waltham, MA, USA).