Journal: Development (Cambridge, England)

Article Title: Ptch2 mediates the Shh response in Ptch1 −/− cells

doi: 10.1242/dev.110056

Figure Lengend Snippet: The Shh-binding loop 2 of Ptch1 can mediate the Shh response in Ptch1–/– fibroblasts independently of the proton-driven antiporter activity. (A) After Ptch1–/– MEFs were grown to confluence, cells were cultured overnight in low-serum medium and treated with ShhN-conditioned medium, 200 µM SAG or 1 µM cyclopamine. Cells were lysed and lacZ activity was assessed by determining β-galactosidase levels. Data show mean±s.e.m. from three experiments performed in triplicate. (B,C) Ptch1–/– MEFs were co-transfected with Ptch1, Ptch1 mutants or Disp1 as control vector, and a Gli-luciferase reporter and CMV-Renilla. When transfected cells reached confluence, cells were cultured overnight in low serum and treated with control conditioned medium (mock), ShhN-conditioned medium from HEK293T cells or 5E1-conditioned medium. Cells were lysed the next day and luciferase activity was measured. Data are shown relative to control (cells transfected with Disp1) and treated with control conditioned medium (mock); data are mean±s.e.m. from three experiments performed in duplicate. In B and C, levels were normalized to the induction level measured in the Disp1-transfected cells (100). Statistical significance was tested by ANOVA for all forms of Ptch1 versus Disp1. (B) One-way ANOVA, P=0.0015. (C) Two-way ANOVA, P<0.0001. Relevant pair-wise Student's t-tests are indicated: *P<0.05; **P<0.01; ***P<0.005. (D) Schematic diagram of Ptch1 mutants. The aspartic acid residue in red denotes the antiporter mutation in Ptch1 that is located in the sterol-sensing domain labeled in blue. The Shh-binding domain located in loop 2 is the second large extracellular loop between TM domains 7 and 8.

Article Snippet: NEBs treated with antibody were cultured in the presence of α-Shh (5E1) or α-Myc (9E10) (Developmental Studies Hybridoma Bank) conditioned in DFNB medium at 1:5 for the duration of the experiment.

Techniques: Binding Assay, Activity Assay, Cell Culture, Transfection, Control, Plasmid Preparation, Luciferase, Residue, Mutagenesis, Labeling

Journal: Development (Cambridge, England)

Article Title: Ptch2 mediates the Shh response in Ptch1 −/− cells

doi: 10.1242/dev.110056

Figure Lengend Snippet: Activation of the Shh response in Ptch1–/– mESCs is induced by Shh. (A-D) Embryoid bodies (NEBs) derived from Ptch1–/– mESCs were neuralized with 1 µM retinoic acid (RA) in the presence of 1:5 α-Shh 5E1 supernatant (B,D) or control α-Myc 9E10 supernatant (A,C). (C,D) SAG (200 nM) was added. Nkx2.2 and Isl1/2 expression was assessed by immunofluorescence after 6 days. (E) RT-PCR analysis for indicated transcripts was performed on RNA isolated from Ptch1–/– NEBs. (F) Shh expression was assessed by immunofluorescence using 5E1. (G-I) Numbers of Isl1/2+ (G), Nkx2.2+ (H) or Pax7+ (I) cells per NEB were quantified. Data are mean±s.e.m.; n≥20; ***P<0.005; **P<0.01. (J-M) Ptch1–/– mESCs were mixed with Smo–/– mESCs at indicated ratios. Derived NEBs were neuralized with 1 µM retinoic acid and after 7 days, and Nkx2.2, Isl1/2 and Shh expression was assessed. (N) Number of Isl1/2+ or (O) Nkx2.2+ cells per NEB was quantified. (P) Ptch1–/– mESCs were mixed with Smo–/– mESCs, neuralized and treated with 1:5 α-Shh 5E1 supernatant. Isl1/2 expression was assessed. (Q) Wild-type (AB1) mESCs were mixed with Smo–/– mESCs, neuralized and Isl1/2 expression was assessed. Data are mean±s.e.m. n≥20; **P<0.01; Student's t-test.

Article Snippet: NEBs treated with antibody were cultured in the presence of α-Shh (5E1) or α-Myc (9E10) (Developmental Studies Hybridoma Bank) conditioned in DFNB medium at 1:5 for the duration of the experiment.

Techniques: Activation Assay, Derivative Assay, Control, Expressing, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Development (Cambridge, England)

Article Title: Ptch2 mediates the Shh response in Ptch1 −/− cells

doi: 10.1242/dev.110056

Figure Lengend Snippet: Fibroblast chemotaxis to Shh does not require Ptch1, but is sensitive to Ptch1-mediated inhibition. (A) Ptch1+/+ and Ptch1–/– MEFs were transfected with vector or Ptch1Δloop2, and net migration to 5 nM recombinant ShhN was assessed in the absence or presence of 5E1. Vector transfected Smo–/– MEFs were included as a control. Data are net migration from six experiments±s.e.m.; ***P<0.005. (B) As for A, using 2 µM purmorphamine rather than SAG, because it is a more-consistent Smo agonist in chemotaxis experiments. (C) Ptch1–/– MEFs were stably transduced with shRNA constructs against indicated genes or non-silencing controls (ctrl). RT-PCR was performed to assess knockdown efficiency, and net migration to ShhN was assessed. Average net migration from three experiments±s.e.m. is shown; *P<0.05; ***P<0.005. Statistical significance was assessed using Student's t-test. (D) Ptch1–/– MEFs were stably transduced with indicated constructs and western blot analysis was performed to assess expression levels. Subsequently, net migration of transduced MEFs to ShhN was measured. Average net migration from three experiments±s.e.m. is shown; **P<0.01.

Article Snippet: NEBs treated with antibody were cultured in the presence of α-Shh (5E1) or α-Myc (9E10) (Developmental Studies Hybridoma Bank) conditioned in DFNB medium at 1:5 for the duration of the experiment.

Techniques: Chemotaxis Assay, Inhibition, Transfection, Plasmid Preparation, Migration, Recombinant, Control, Stable Transfection, Transduction, shRNA, Construct, Reverse Transcription Polymerase Chain Reaction, Knockdown, Western Blot, Expressing

Journal: Developmental biology

Article Title: SHH E176/E177-Zn 2+ conformation is required for signaling at endogenous sites

doi: 10.1016/j.ydbio.2017.02.006

Figure Lengend Snippet: Factors affecting the formation of SHH non-reducible cross-linked dimers (CLdimer) A. uSHHN (lanes 1–9), lanes 1–2, pH 6.5, lanes 3–4, pH 7.0, lanes 5–9, pH 7.5, lanes 2, 4, 6, 9, 1 mM Zn2+, lane 7, 1 mM Mg2+, lanes 8 and 9, 1 mM EDTA. Western analysis was performed using anti-SHH antibody H160. B. uSHHN (lanes 1–5), lane 1, Zn2+ addition 30 minutes after cross-linking reaction starts, lane 2, minus Zn2+, lane 3, Zn2+ addition from the beginning of the cross-linking reaction, lanes 4–5, 100ng heparin, lane 5, 1 mM Zn2+. Western analysis was performed using anti-SHH antibody H160. Solid arrows indicate CLdimer and monomer, and dashed arrow indicates CLintra. C. SHHN proteins affinity purified from secreted cell culture supernatants, affinity purified on a 5E1 column (5E1, monoclonal anti-SHH, Developmental Hybridoma Studies Bank), and assayed for cross-linking. SHHN (lanes 1–2, 1′–2′), SHHN-E176A (lanes 3–4, 3′–4′), wtSHH (lanes 5–6, 5′–6′), and SHH-E176A (lanes 7–8, 7′–8′) were incubated in the presence (lanes 2, 4, 6, 8) or absence (lanes 1, 3, 5, 7) of 1mM Zn2+. Western analysis was performed using α-SHHCL/P (lanes 1′–8′), stripped and reprobed with H160 (lanes 1–8). Arrows indicate CLdimer and monomer. D. Description of recombinant proteins used in A–D: *Full-length construct, produces N-and C-lipid modified SHH proteins. SHH proteins are affinity purified from culture supernatants of transfected C17 neural cells, as previously described for wtSHH (Feng et al., 2004), **N-terminal construct produces N-lipid containing, C-lipid lacking SHH proteins. SHH proteins are affinity purified from supernatants of transfected C17 neural cells, previously described for (Feng et al., 2004), §N-terminal construct lacks both N-and C-lipid modifications. uSHHN is produced in E.coli, Ni-Agarose affinity purified, histidine tag cleaved, as previously described in (Williams et al., 1999). + (antibody recognizes), − (antibody does not recognize), weak (antibody weakly recognizes), ND (not determined), +ç (recognizes wtSHH crosslinked form after storage at −80°C for 6 months). Note: Recombinant proteins (1–5) do not contain tags at the N-or C-termini. Proteins 1–4 were purified by affinity chromatography on a 5E1 anti-SHH-antibody column, while the 6Xhis-tag on uSHHN (protein 5) was removed prior to crosslinking assays.

Article Snippet: 5E1 purified antibodies were obtained from Developmental Studies Hybridoma Bank (DSHB, University of Iowa); 5E1 is a well-characterized mouse monoclonal antibody that recognizes SHH ( Ericson et al., 1996 ). α-SHH CL;M/P (rabbit sera raised against SHH aa24–197, gift from Jessell lab) was made by affinity purification on uSHHN column (aa24–197), and specificity verified by western and immunohistochemistry on Shh −/− spinal cord sections.

Techniques: Western Blot, Affinity Purification, Cell Culture, Incubation, Recombinant, Construct, Modification, Transfection, Produced, Purification, Affinity Chromatography

Journal: Cilia

Article Title: Mammalian Clusterin associated protein 1 is an evolutionarily conserved protein required for ciliogenesis

doi: 10.1186/2046-2530-1-20

Figure Lengend Snippet: Cluap1 localizes to primary cilia in vitro. Antibody against acetylated α-tubulin (red) and Cluap1 (green) label primary cilia (arrows) in ( A-C ) NIH3T3 cells (scale bars are 14 μm). ( D-F ) 176-6C collecting duct epithelium (scale bars are 21 μm) and ( G-I ) IMCD3 cells (scale bars are 20 μm). Arrows indicate primary cilium. Nuclei are stained blue with Hoechst.

Article Snippet: Cells and embryos were labeled with the following antibodies: anti-acetylated α-tubulin, 1:1,000 (T-6793; Sigma-Aldrich, St. Louis, MO); anti-Arl13b, 1:1,000 (a gift from Dr. Tamara Caspary, Emory University); anti-Cluap1, 1:1,000 (generated as described above); and anti_ShhN, 1:1,000 (5E1, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA).

Techniques: In Vitro, Staining

Journal: Cilia

Article Title: Mammalian Clusterin associated protein 1 is an evolutionarily conserved protein required for ciliogenesis

doi: 10.1186/2046-2530-1-20

Figure Lengend Snippet: Cluap1 KO embryos fail to form primary cilia. ( A,C,E ) Cluap1 WT E9.5 embryos were immunolabeled for the cilia marker acetylated α-tubulin (red) and Cluap1 (green) in the lateral plate mesenchyme of Cluap1 WT embryos. ( B,D,F ) Cluap1 KO embryos show a total loss of cilia in the same region. Hoechst nuclear stain in blue. Scale bar is 31.5 μm.

Article Snippet: Cells and embryos were labeled with the following antibodies: anti-acetylated α-tubulin, 1:1,000 (T-6793; Sigma-Aldrich, St. Louis, MO); anti-Arl13b, 1:1,000 (a gift from Dr. Tamara Caspary, Emory University); anti-Cluap1, 1:1,000 (generated as described above); and anti_ShhN, 1:1,000 (5E1, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA).

Techniques: Immunolabeling, Marker, Staining