MBP-mediated pull-down assay

For pull-down assays, the Ptch1 and MBP-ShhN, WT or indicated variants, were incubated with amylose resin resuspended in 200 μl of buffer containing 25 mM Tris pH 8.0, 150 mM NaCl, and 0.06% digitonin or the indicated detergents at concentration of their respective 2× CMC at 4°C for ~1 hour. Then the mixture was spun down at 500g for 3 min. After removing the supernatant, the resin was extensively rinsed with the assay buffer to remove unbound proteins. Finally the resin was resuspended in 200 μl of buffer, out of which 20 μl was applied to SDS-PAGE analysis followed by Coomassie blue staining.

In the assay reported in Fig. 3C, all the indicated Ptch1 and MBP-ShhN variants were applied at concentrations of ~0.15 mg/ml. In the titration pull-down assay reported in Fig. 5B, ShhN was used at ~0.15 mg/ml, while Ptch1 was serially diluted fourfold at concentrations of ~0.60, ~0.15, ~0.04, and ~0.01 mg/ml, respectively. In the assay to test the effects of different detergents on the complex formation, the molar ratio of the two proteins was approximately 1:1 with Ptch1 and MBP-ShhN at concentrations of ~0.15 and 0.08 mg/ml, respectively.