Generating Murine Hh Protein Variants

We used murine Shh (Dierker et al., 2009 (link)) and Hh sequences (nucleotides 1–1416, corresponding to amino acids 1–471 of D. melanogaster Hh) that were generated from cDNA by PCR using primer sequences that can be provided upon request. PCR products were inserted into pENTR for sequence confirmation and subsequently into pUAST for protein expression in S2 cells or the generation of transgenic flies. Mutations were introduced via the QuickChange Lightning site-directed mutagenesis kit (Stratagene, La Jolla, United States). S2 cells were cultured in Schneider’s medium (Invitrogen, Carlsbad, United States) supplemented with 10% fetal calf serum and 100 μg/mL penicillin/streptomycin. The cells were transfected with constructs encoding Hh and Hh variants together with a vector encoding an actin-Gal4 driver using Effectene (Qiagen, Hilden, Germany) and cultured for 36 h in Schneider’s medium before protein was harvested from the supernatant.