Human/Mouse Sonic Hedgehog/Shh N‑Terminus Antibody

Detection of Human Sonic Hedgehog/Shh by Western Blot. Western Blot shows lysates of HEK293 human embryonic kidney cell line either mock transfected or transfected with mouse Sonic Hedgehog/SHH. PVDF membrane was probed with 1 µg/ml of Goat Anti-Human/Mouse Sonic Hedgehog/Shh N-Terminus Antigen Affinity-purified Polyclonal Antibody (Catalog # AF464) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Sonic Hedgehog/Shh at approximately 23 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

Sonic Hedgehog/Shh in Mouse Embryo. Sonic Hedgehog/Shh was detected in immersion fixed frozen sections of mouse embryo (13 d.p.c.) using Goat Anti-Human/Mouse Sonic Hedgehog/Shh N-Terminus Antigen Affinity-purified Polyclonal Antibody (Catalog # AF464) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to developing brain. View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Detection of Sonic Hedgehog/Shh N-Terminus by Western Blot Impaired Shh release from Disp−/− cells. (A) Alignment of targeted disp gene sequences from Disp−/− cells and from non-targeted (nt Ctrl) cells. (A′) Schematic representation of the Disp protein structure. An asterisk indicates the CRISPR/Cas9-generated stop codon introduced at position 323, deleting 11 of 12 TM domains that together represent ∼80% of the protein sequence. L1 and L2 indicate extracellular loops. TM2–TM6 (colored red) constitute the SSD. (B,C) Immunoblots of cellular (c) and released (into the medium, m) Shh (B) and unlipidated control C25SShhN (C) in nt Ctrl and Disp−/− cells in the presence of Scube2. Arrows indicate solubilized Shh and the arrowhead indicates accumulated cellular material in Disp−/− cells. (D) In the absence of Scube2, Shh processing into serum-free medium was abolished in nt Ctrl and Disp−/− cells. Instead, both cell types released similar amounts of unprocessed protein. In B,C,D, anti-beta -actin blots ( alpha beta -actin) and Ponceau S staining of residual serum albumin (PonS) serve as loading controls. (B′,C′,D′) Quantifications of relative Shh (B′,D′) and C25SShhN (C′) release from nt Ctrl and Disp−/− cells. Ratios of solubilized versus cellular Shh were determined and expressed relative to Shh release from nt Ctrl cells (black bars). Data are mean±s.d. n=21 in B′, n=8 in C′ and n=5 in D′. ****P<0.0001; ns, not significant (two-tailed unpaired t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34308968), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Sonic Hedgehog/Shh N-Terminus by Western Blot Overexpressed Disptg locates to the cell surface and restores Shh release from Disp−/− cells. (A,A′) Representative confocal planes of Disp−/− cells expressing Shh (A, red) or Disptg (A′, red). Both transgenes were secreted to the cell surface. Nuclei were counterstained with DAPI (blue). Dashed lines indicate the border of cytoplasmic eGFP signals (green). Images are representative of three experiments. Scale bars: 2 µm. (B) Co-expressed transgenic Disptg enhanced processed Shh release from Disp−/− cells (c) into the medium (m). Transgenic Disp delta L2, which lacks most of the second extracellular loop, did not release significantly increased amounts of truncated Shh. Empty-vector (EV)-transfected Disp−/− cells served as negative controls. (B′) Quantified relative processed Shh release, as shown in B. Data are mean±s.d. n=10. **P<0.01; ns, not significant; P=0.377 for Disp delta L2 (one-way ANOVA with Dunnett's multiple comparison post hoc test). (C) Co-expressed transgenic Disptg or Disp delta L2 did not significantly increase Shh release from nt Ctrl cells. (C′) Quantified relative processed Shh release as shown in C. Data are mean±s.d. n=14. ns, not significant (one-way ANOVA with Dunnett's multiple comparison post hoc test). In B and C, arrows indicate solubilized truncated Shh from Disptg- or Disp delta L2-expressing cells and the arrowhead indicates solubilized Shh from EV-transfected cells. Anti-beta -actin blots ( alpha beta -actin) and Ponceau S staining of residual serum albumin (PonS) serve as loading controls. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34308968), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Sonic Hedgehog/Shh N-Terminus by Western Blot Overexpressed Disptg locates to the cell surface and restores Shh release from Disp−/− cells. (A,A′) Representative confocal planes of Disp−/− cells expressing Shh (A, red) or Disptg (A′, red). Both transgenes were secreted to the cell surface. Nuclei were counterstained with DAPI (blue). Dashed lines indicate the border of cytoplasmic eGFP signals (green). Images are representative of three experiments. Scale bars: 2 µm. (B) Co-expressed transgenic Disptg enhanced processed Shh release from Disp−/− cells (c) into the medium (m). Transgenic Disp delta L2, which lacks most of the second extracellular loop, did not release significantly increased amounts of truncated Shh. Empty-vector (EV)-transfected Disp−/− cells served as negative controls. (B′) Quantified relative processed Shh release, as shown in B. Data are mean±s.d. n=10. **P<0.01; ns, not significant; P=0.377 for Disp delta L2 (one-way ANOVA with Dunnett's multiple comparison post hoc test). (C) Co-expressed transgenic Disptg or Disp delta L2 did not significantly increase Shh release from nt Ctrl cells. (C′) Quantified relative processed Shh release as shown in C. Data are mean±s.d. n=14. ns, not significant (one-way ANOVA with Dunnett's multiple comparison post hoc test). In B and C, arrows indicate solubilized truncated Shh from Disptg- or Disp delta L2-expressing cells and the arrowhead indicates solubilized Shh from EV-transfected cells. Anti-beta -actin blots ( alpha beta -actin) and Ponceau S staining of residual serum albumin (PonS) serve as loading controls. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34308968), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Sonic Hedgehog/Shh N-Terminus by Western Blot Overexpressed Ptctg restores Shh release from Disp−/− cells. (A,A′) Schematic representations of Disp (blue) and Ptc (green). Twelve transmembrane domains (TM1–TM12), two extracellular loops (L1 and L2), and the N- and C-termini are labeled. Conserved SSDs (TM2–TM6) are highlighted in red. Disp delta L2 and Ptc delta L2 lack most of the second extracellular loops (L2). (B,C) Co-expression of transgenic Ptctg or Ptc delta L2 increases Shh shedding from Disp−/− (B) and nt Ctrl (C) cells (c, cellular Shh; m, Shh released into the medium). Arrows indicate solubilized processed Shh from Ptctg- or Ptc delta L2-expressing cells, and arrowheads indicate reduced amounts of solubilized Shh from empty vector (EV)-transfected cells. Anti-beta -actin blots ( alpha beta -actin) and Ponceau S staining of residual serum albumin (PonS) serve as loading controls. (B′,C′) Quantification of relative processed Shh release as shown in B and C. Data are mean±s.d. n=6 in B′, n=9 in C′. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Dunnett's multiple comparison post hoc test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34308968), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Sonic Hedgehog/Shh N-Terminus by Western Blot Impaired Shh release from Disp−/− cells. (A) Alignment of targeted disp gene sequences from Disp−/− cells and from non-targeted (nt Ctrl) cells. (A′) Schematic representation of the Disp protein structure. An asterisk indicates the CRISPR/Cas9-generated stop codon introduced at position 323, deleting 11 of 12 TM domains that together represent ∼80% of the protein sequence. L1 and L2 indicate extracellular loops. TM2–TM6 (colored red) constitute the SSD. (B,C) Immunoblots of cellular (c) and released (into the medium, m) Shh (B) and unlipidated control C25SShhN (C) in nt Ctrl and Disp−/− cells in the presence of Scube2. Arrows indicate solubilized Shh and the arrowhead indicates accumulated cellular material in Disp−/− cells. (D) In the absence of Scube2, Shh processing into serum-free medium was abolished in nt Ctrl and Disp−/− cells. Instead, both cell types released similar amounts of unprocessed protein. In B,C,D, anti-beta -actin blots ( alpha beta -actin) and Ponceau S staining of residual serum albumin (PonS) serve as loading controls. (B′,C′,D′) Quantifications of relative Shh (B′,D′) and C25SShhN (C′) release from nt Ctrl and Disp−/− cells. Ratios of solubilized versus cellular Shh were determined and expressed relative to Shh release from nt Ctrl cells (black bars). Data are mean±s.d. n=21 in B′, n=8 in C′ and n=5 in D′. ****P<0.0001; ns, not significant (two-tailed unpaired t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34308968), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Sonic Hedgehog/Shh N-Terminus by Western Blot Impaired Shh release from Disp−/− cells. (A) Alignment of targeted disp gene sequences from Disp−/− cells and from non-targeted (nt Ctrl) cells. (A′) Schematic representation of the Disp protein structure. An asterisk indicates the CRISPR/Cas9-generated stop codon introduced at position 323, deleting 11 of 12 TM domains that together represent ∼80% of the protein sequence. L1 and L2 indicate extracellular loops. TM2–TM6 (colored red) constitute the SSD. (B,C) Immunoblots of cellular (c) and released (into the medium, m) Shh (B) and unlipidated control C25SShhN (C) in nt Ctrl and Disp−/− cells in the presence of Scube2. Arrows indicate solubilized Shh and the arrowhead indicates accumulated cellular material in Disp−/− cells. (D) In the absence of Scube2, Shh processing into serum-free medium was abolished in nt Ctrl and Disp−/− cells. Instead, both cell types released similar amounts of unprocessed protein. In B,C,D, anti-beta -actin blots ( alpha beta -actin) and Ponceau S staining of residual serum albumin (PonS) serve as loading controls. (B′,C′,D′) Quantifications of relative Shh (B′,D′) and C25SShhN (C′) release from nt Ctrl and Disp−/− cells. Ratios of solubilized versus cellular Shh were determined and expressed relative to Shh release from nt Ctrl cells (black bars). Data are mean±s.d. n=21 in B′, n=8 in C′ and n=5 in D′. ****P<0.0001; ns, not significant (two-tailed unpaired t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34308968), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Sonic Hedgehog/Shh N-Terminus by Western Blot Overexpressed Ptctg restores Shh release from Disp−/− cells. (A,A′) Schematic representations of Disp (blue) and Ptc (green). Twelve transmembrane domains (TM1–TM12), two extracellular loops (L1 and L2), and the N- and C-termini are labeled. Conserved SSDs (TM2–TM6) are highlighted in red. Disp delta L2 and Ptc delta L2 lack most of the second extracellular loops (L2). (B,C) Co-expression of transgenic Ptctg or Ptc delta L2 increases Shh shedding from Disp−/− (B) and nt Ctrl (C) cells (c, cellular Shh; m, Shh released into the medium). Arrows indicate solubilized processed Shh from Ptctg- or Ptc delta L2-expressing cells, and arrowheads indicate reduced amounts of solubilized Shh from empty vector (EV)-transfected cells. Anti-beta -actin blots ( alpha beta -actin) and Ponceau S staining of residual serum albumin (PonS) serve as loading controls. (B′,C′) Quantification of relative processed Shh release as shown in B and C. Data are mean±s.d. n=6 in B′, n=9 in C′. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Dunnett's multiple comparison post hoc test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34308968), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Sonic Hedgehog/Shh N-Terminus by Western Blot Loading controls.(A–D) Actin, PonceauS, and FLAG-tagged Scube2 loading controls for blots shown in Figure 3. Arrowheads indicate Shh specifically solubilized by Disp and Scube2.Figure 3—figure supplement 1—source data 1.Uncropped western blots for Figure 3—figure supplement 1.Uncropped western blots for Figure 3—figure supplement 1. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/39297609), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Sonic Hedgehog/Shh N-Terminus by Western Blot Loading controls.(A–D) Actin, PonceauS, and FLAG-tagged Scube2 loading controls for the blots shown in Figure 6A and B. (E, F) Reverse-phase high-performance liquid chromatography (RP-HPLC) profiles of samples [1] and [2] shown in (D). Note that proteins released from non-targeting control (nt ctrl) cells and Disp-/- cells are both cholesterylated. This suggests that overexpressed monolipidated Shh is only loosely associated with the plasma membrane and can ‘leak out’, or desorb, and thereby associate with high-density lipoprotein (HDL) in a non-enzymatic manner. (G) Purified commercial 8908-SH Shh and HDL were mixed, incubated for 10 min, and analyzed by size-exclusion chromatography (SEC). This revealed rapid spontaneous association and explains the background desorption of dual-lipidated Shh in our cellular assays. Note the excellent 8908-SH Shh co-elution with the ApoE4-bearing HDL fraction, suggesting that this mobile apolipoprotein may have facilitated spontaneous 8908-SH Shh association, or that other properties of ApoE-bearing ‘late’ HDL (Sacks and Jensen, 2018) somehow facilitate Shh association. (H) Dual-lipidated mCherry is efficiently secreted to the outer plasma membrane leaflet of producing Bosc23 cells. Intracellular mCherry fluorescence is shown in white, alpha -mCherry antibody binding to the surface of non-permeabilized cells is shown in magenta, and DAPI staining of the nucleus is shown in blue. Scale bar: 10 μm.Figure 7—figure supplement 1—source data 1.Uncropped western blots of Figure 7—figure supplement 1.Uncropped western blots of Figure 7—figure supplement 1. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/39297609), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Sonic Hedgehog/Shh N-Terminus by Western Blot Loading controls.(A, B) Actin, PonceauS, and FLAG-tagged Scube2 loading controls for (the same stripped) blots shown in Figure 2.Figure 2—figure supplement 1—source data 1.Uncropped western blots of Figure 2—figure supplement 1.Uncropped western blots of Figure 2—figure supplement 1. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/39297609), licensed under a CC-BY license. Not internally tested by R&D Systems.