Ru ski 43

SHH release assays were performed essentially as described (Tukachinsky et al., 2012 (link)). For blotting-based assays, confluent HEK293T cultures were washed three times with serum-free DMEM, and were then incubated for 24 h in DMEM. Conditioned media and cell lysates were analyzed by immunoblotting for SCUBE2, SHH, and other relevant species. For NanoLuc-based assays using purified release factors, HEK293T cells stably expressing SHH(NL5) were used (Petrov et al., 2020 (link)). After washing with DMEM without serum, the cells were treated with cycloheximide (100μg/mL) (Sigma) for 30 min prior to the addition of purified proteins. Aliquots of conditioned medium were collected at the indicated times, centrifuged to remove cellular debris, and NanoLuc luciferase activity was measured using Nano-Glo Luciferase Assay Substrate (Promega), according to the manufacturer’s instructions. In experiments testing the role of palmitoylation on SHH release, cells were first treated overnight with HHAT inhibitor RU-SKI 43 (Petrova et al., 2013 (link)) (Tocris Bioscience).