Fig. 1: Purification of human Disp1 (hDisp1) in pre- and post-proteolytic cleavage states.

a Topological diagram of hDisp1. Disp homologues contain 12 transmembrane segments (TMs), two large extracellular domains (ECD1 and ECD2), as well as flexible N- and C-terminal intracellular domains. TMs 2-6 constitute a conserved sterol-sensing domain (SSD), found in various membrane proteins related to cholesterol transport and metabolism, including Ptch, NPC1, and the endoplasmic reticulum cholesterol sensor SCAP⁵⁶. The N-terminal Flag tag, C-terminal His₁₀ tag, and the predicted Furin cleavage site between Arg280 and Glu281 are indicated. b Purified hDisp1^NNN was separated by SDS-PAGE, and was detected by Coomassie staining (left) or Western blot (right). Full-length and cleavage products are indicated. Purified hDisp1^NNN consists of a mix of cleaved and uncleaved species. c The Disp1-3C construct, in which the Furin cleavage site is replaced by a 3C protease cleavage site. d Purified hDisp1^NNN-3C consists entirely of uncleaved protein (lane 2), which is quantitatively cleaved by incubation with 3C protease (lane 3). Purified, partially Furin-cleaved hDisp1^NNN is shown for comparison (lane 1). e Disp1-3C supports Shh release from cells in cleavage-dependent manner. Wild-type (WT) hDisp1 or hDisp1-3C was stably co-expressed with NanoLuc-tagged Shh (Shh-NL) in Disp1-null HEK293T cells. The cells were incubated with purified recombinant mouse Scube2 (1 μM) in serum-free media, in the presence of the protein synthesis inhibitor, cycloheximide (100 μg/mL). Time course of background-subtracted Shh-NL release was measured for six time points in a single independent biological experiment, by NanoLuc luminescence. Initial rates of Shh-NL release were normalized to Shh-NL expression levels in each cell line. Bars represent best-fit slope of a linear regression fit to the release time points, and error bars represent standard error of the regression fit. Shh release is reduced for Furin-uncleavable Disp1-3C compared to WT Disp1, which is cleaved by Furin. ANCOVA was used for testing for differences in release rate for each Disp1 variant upon 3C protease addition. Pretreating the cells with 3C protease enhances Shh-NL release in Disp1-null cells rescued with hDisp1-3C (p = 0.0185), but not in unrescued cells (p = 0.1044), or cells rescued with WT hDisp1 (p = 0.9987). f Immunoblot for the experiment in (e), showing Disp1-3C cleavage by exogenously added 3C protease. Both WT hDisp1 and hDisp1-3C are tagged with mCherry at the C-terminus, and hDisp1-3C is also tagged with HPC at the N-terminus, as indicated in the diagram to the left. Note the 3C protease-dependent appearance of an N-terminal fragment of hDisp1-3C. g Pull-down assay showing that uncleaved hDisp1-3C and hDisp1^NNN-3C exhibits greatly reduced binding to Shh compared to hDisp1 and hDisp1^NNN, which are cleaved. The WT and mutant hDisp1 constructs were co-expressed with Shh in HEK293F cells, and immunoprecipitated hDisp1 and Shh were analyzed by Western blotting. Source data for (b) and (d–g) are provided as a Source Data file.