gli1  (Novus Biologicals)

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Gli1 mRNA fold changes show the responsiveness of SMO mutants to SHH. Untreated Gli1 levels indicate low SMO activity, while SHH-treated values correspond to the level of SMO activation induced by SHH ligand. A t-test with Welch’s correction was used to compute statistical significance. (p values: untreated vs treated: WT: 1.327 × 10 -3 , G 2.57 f V: 9.212 × 10 -3 , I ECL2 A: 4.2 × 10 -5 , A 2.60 f M: 7.1 × 100 -5 , R 5.64 f A: 2.062 × 10 -3 , R 5.64 f Q: 1.192 × 10 -3 , F 6.36 f I: 2.163 × 10 -3 , L 5.62 f A: 1.948 × 10 -3 , treated WT vs treated mutant: G 2.57 f V: 9.1 × 10 -3 , I ECL2 A: 0.02734, A 2.60 f M: 0.7477, R 5.64 f A: 0.08858, R 5.64 f Q: 0.02766, F 6.36 f I: 1.923 × 10 -3 , L 5.62 f A: 2.306 × 10 key: Not significant (ns) p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001, All experimental data represent biological replicates, N=4.) ( b ) ΔGli1 mRNA fold change (SHH vs untreated) and Δ \begin{document}$\Delta$\end{document} PMF (difference of peak PMF, calculated as P M F W T \begin{document}$PMF_{WT}$\end{document} - P M F m u t a n t \begin{document}$PMF_{mutant}$\end{document} ) plotted for the mutants in Pathway 1. ( c ) Example mutant A 2.60 f M shows that cholesterol is able to enter SMO through Pathway 1 even on a bulky mutation. ( d ) Same as ( b ) but for Pathway 2 ( e ) Example mutant L 5.62 f A shows that cholesterol can enter SMO through Pathway 2 due to lesser steric hindrance. All snapshots presented are frames taken from MD simulations.

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Activity Assay, Activation Assay, Mutagenesis

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure abundance of mSMO and GLI1 proteins in SMO -/- cells stably expressing either mSMO-WT or Pathway 1 mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 3—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure abundance of mSMO and GLI1 proteins in SMO -/- cells stably expressing either mSMO-WT or Pathway 2 mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 3—figure supplement 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—figure supplement 4—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Gli1 mRNA fold changes show the responsiveness of SMO mutants to SHH. Untreated Gli1 levels indicate low SMO activity, while SHH-treated values correspond to the level of SMO activation induced by SHH ligand. A t-test with Welch’s correction was used to compute statistical significance. (p values: untreated vs treated: WT: 3 × 10 -6 , Y LD A: 2.46 × 10 -4 , F 6.65 f A: 1.08 × 10 -3 , I ECL 3 A: 1.12 × 10 -4 , treated WT vs treated mutant: F 6.65 f A: 1.6 × 10 -5 , I ECL 3 A: 1.6 × 10 -5 , Y LD A: 1.4 × 10 -5 , key: Not significant (ns) p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001, All experimental data represent biological replicates, N=4.) ( b ) Δ \begin{document}$\Delta$\end{document} Gli1 mRNA fold change (SHH vs untreated) and Δ \begin{document}$\Delta$\end{document} PMF (difference of peak PMF, calculated as P M F W T \begin{document}$PMF_{WT}$\end{document} - P M F m u t a n t \begin{document}$PMF_{mutant}$\end{document} ) are plotted for mutants along the TMD-CRD pathway. ( c, d ) Example mutants Y LD A and F 6.65 f A show that cholesterol is unable to translocate through this pathway because of the loss of crucial hydrophobic contacts provided by Y207 and F484 and along the solvent-exposed pathway.

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Activity Assay, Activation Assay, Mutagenesis, Solvent

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure the abundance of mSMO and GLI1 proteins in SMO −⁄− cells stably expressing either mSMO-WT or Common Pathway mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 6—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Gli1 mRNA fold changes show the responsiveness of SMO mutants to SHH. Untreated Gli1 levels indicate low SMO activity, while SHH-treated values correspond to the level of SMO activation induced by SHH ligand. A t-test with Welch’s correction was used to compute statistical significance. (p values: untreated vs treated: WT: 1.327 × 10 -3 , G 2.57 f V: 9.212 × 10 -3 , I ECL2 A: 4.2 × 10 -5 , A 2.60 f M: 7.1 × 100 -5 , R 5.64 f A: 2.062 × 10 -3 , R 5.64 f Q: 1.192 × 10 -3 , F 6.36 f I: 2.163 × 10 -3 , L 5.62 f A: 1.948 × 10 -3 , treated WT vs treated mutant: G 2.57 f V: 9.1 × 10 -3 , I ECL2 A: 0.02734, A 2.60 f M: 0.7477, R 5.64 f A: 0.08858, R 5.64 f Q: 0.02766, F 6.36 f I: 1.923 × 10 -3 , L 5.62 f A: 2.306 × 10 key: Not significant (ns) p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001, All experimental data represent biological replicates, N=4.) ( b ) ΔGli1 mRNA fold change (SHH vs untreated) and Δ \begin{document}$\Delta$\end{document} PMF (difference of peak PMF, calculated as P M F W T \begin{document}$PMF_{WT}$\end{document} - P M F m u t a n t \begin{document}$PMF_{mutant}$\end{document} ) plotted for the mutants in Pathway 1. ( c ) Example mutant A 2.60 f M shows that cholesterol is able to enter SMO through Pathway 1 even on a bulky mutation. ( d ) Same as ( b ) but for Pathway 2 ( e ) Example mutant L 5.62 f A shows that cholesterol can enter SMO through Pathway 2 due to lesser steric hindrance. All snapshots presented are frames taken from MD simulations.

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Activity Assay, Activation Assay, Mutagenesis

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure abundance of mSMO and GLI1 proteins in SMO -/- cells stably expressing either mSMO-WT or Pathway 1 mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 3—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure abundance of mSMO and GLI1 proteins in SMO -/- cells stably expressing either mSMO-WT or Pathway 2 mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 3—figure supplement 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—figure supplement 4—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Gli1 mRNA fold changes show the responsiveness of SMO mutants to SHH. Untreated Gli1 levels indicate low SMO activity, while SHH-treated values correspond to the level of SMO activation induced by SHH ligand. A t-test with Welch’s correction was used to compute statistical significance. (p values: untreated vs treated: WT: 3 × 10 -6 , Y LD A: 2.46 × 10 -4 , F 6.65 f A: 1.08 × 10 -3 , I ECL 3 A: 1.12 × 10 -4 , treated WT vs treated mutant: F 6.65 f A: 1.6 × 10 -5 , I ECL 3 A: 1.6 × 10 -5 , Y LD A: 1.4 × 10 -5 , key: Not significant (ns) p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001, All experimental data represent biological replicates, N=4.) ( b ) Δ \begin{document}$\Delta$\end{document} Gli1 mRNA fold change (SHH vs untreated) and Δ \begin{document}$\Delta$\end{document} PMF (difference of peak PMF, calculated as P M F W T \begin{document}$PMF_{WT}$\end{document} - P M F m u t a n t \begin{document}$PMF_{mutant}$\end{document} ) are plotted for mutants along the TMD-CRD pathway. ( c, d ) Example mutants Y LD A and F 6.65 f A show that cholesterol is unable to translocate through this pathway because of the loss of crucial hydrophobic contacts provided by Y207 and F484 and along the solvent-exposed pathway.

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Activity Assay, Activation Assay, Mutagenesis, Solvent

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure the abundance of mSMO and GLI1 proteins in SMO −⁄− cells stably expressing either mSMO-WT or Common Pathway mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 6—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay

Journal: JCI Insight

Article Title: Stem cell–associated osteogenic deficiency causes craniofacial deformities with progeroid accumulation of prelamin A

doi: 10.1172/jci.insight.196932

Figure Lengend Snippet: ( A ) Limiting dilution analysis of stem cell–mediated bone formation with renal capsule transplantation. Representative images of whole-mount von Kossa staining detecting ectopic bone formation in the mouse recipients transplanted by the indicated number of suture cells into the renal capsule. Arrowheads indicate the ectopic bones. ( B ) Representative images showing the analysis of Axin2-expressing cells using the Axin2 mGFP allele in the indicated 1-month-old (1M) suture. ( C ) Representative images examining the BMPR1A + and GLI1 + cell population within the indicated 1-month-old (1M) suture. ( D ) Graphs indicate the quantitation of the average percentage of BMPR1A + and GLI1 + cells in 3 independent experiments ( P < 0.005 or 0.05, n = 3, mean ± SEM, 2-tailed Student’s t test). SAG, sagittal; COR, coronal; AF, anterior frontal. Scale bars: 1 mm ( A ) and 50 μm ( B and C ).

Article Snippet: Mouse monoclonal antibodies against Bmpr1a (NBP2-37421, 1:75, Novus Biologicals), fascin1 (SC-21743, 1:100, Santa Cruz), and Alexa Fluor 568 phalloidin (A12380, 1:400, Invitrogen); rabbit polyclonal antibodies Osx (ab22552, 1:800, Abcam), OCN (23418-1-AP, 1:50, Proteintech), Gli1 (NBP1-78259, 1:100, Novus Biologicals), Myl2 (3671, 1:100, Cell Signaling Technology), and GM130 (2296, 1:200, Cell Signaling Technology); rabbit monoclonal antibodies lamin A/C (MA5-35284, 1:200, Invitrogen) and SUN2(EPR6557, 1:100, Abcam); anti-rabbit secondary antibody (BA-1000, 1:200, Vector laboratories); and anti-mouse IgG secondary antibodies (PK-2200, 1:250, Vector laboratories), were used in the immunostaining studies.

Techniques: Transplantation Assay, Staining, Expressing, Quantitation Assay