Mouse GLI-2 Antibody

GLI‑2 in Mouse Embryo. GLI-2 was detected in immersion fixed frozen sections of mouse embryo (15 d.p.c.) using Goat Anti-Mouse GLI-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3635) 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). Lower panel shows a lack of labeling when primary antibodies are omitted and tissue is stained only with secondary antibody followed by incubation with detection reagents. Specific staining was localized to developing muscle. View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Detection of GLI-2 by Western Blot The PY-type nuclear localization signal (PY-NLS) is required for Gli activation.(A) Western blot of cell lysates from the indicated cell lines indicated that Myc-Gli2WT and Myc-Gli2mPY were expressed at comparable levels that were slightly higher than that of endogenous Gli2. (B–C) Normalized mRNA levels of endogenous Gli1 (B) or Patch1 (C) measured by quantitative reverse transcription PCR (RT-qPCR) in control (green fluorescent protein [GFP] short hairpin RNA [shRNA]) or Gli2-depeleted NIH3T3 cells with or without lentiviral infection of the indicated Gli2 constructs. (D) Fractionation of Myc-Gli2WT and Myc-Gli2mPY from the indicated cell lines treated with or without SAG. Quantification of protein level is shown in the bottom panel. (E–F) Immunostaining (E) and quantification (F) of ciliary-localized Myc-Gli2WT or Myc-Gli2mPY in NIH3T3mGli2-shRNA cells treated with or without sonic hedgehog (Shh). N = 50 cells were examined for each Gli construct. Data are means ± SD from 2 independent experiments. **P < 0.01. (G) Gli-luciferase assay was performed in NIH3T3 cells transfected with the indicated constructs. Data are means ± SD from 2 independent experiments. *P < 0.05, NS: not significant. The underlying data for this figure can be found in S1 Data. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28777795), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of GLI-2 by Western Blot The PY-type nuclear localization signal (PY-NLS) is required for Gli activation.(A) Western blot of cell lysates from the indicated cell lines indicated that Myc-Gli2WT and Myc-Gli2mPY were expressed at comparable levels that were slightly higher than that of endogenous Gli2. (B–C) Normalized mRNA levels of endogenous Gli1 (B) or Patch1 (C) measured by quantitative reverse transcription PCR (RT-qPCR) in control (green fluorescent protein [GFP] short hairpin RNA [shRNA]) or Gli2-depeleted NIH3T3 cells with or without lentiviral infection of the indicated Gli2 constructs. (D) Fractionation of Myc-Gli2WT and Myc-Gli2mPY from the indicated cell lines treated with or without SAG. Quantification of protein level is shown in the bottom panel. (E–F) Immunostaining (E) and quantification (F) of ciliary-localized Myc-Gli2WT or Myc-Gli2mPY in NIH3T3mGli2-shRNA cells treated with or without sonic hedgehog (Shh). N = 50 cells were examined for each Gli construct. Data are means ± SD from 2 independent experiments. **P < 0.01. (G) Gli-luciferase assay was performed in NIH3T3 cells transfected with the indicated constructs. Data are means ± SD from 2 independent experiments. *P < 0.05, NS: not significant. The underlying data for this figure can be found in S1 Data. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28777795), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse GLI-2 by Western Blot HPP-9 is a long-acting Hedgehog pathway inhibitor.a SHH-GFP cells were treated for 25 h with DMSO, 1 μM HPI-1, or 1 μM HPP-9, before the medium was changed to various dilutions of ShhN-conditioned medium for 27 h. Nuclear GFP levels were quantified using fluorescence microscopy. Representative curves of three independent experiments are shown, with 9–16 images analyzed per condition. b GLI1 and GLI2 levels of cells pre-incubated with DMSO or 1 μM HPP-9 were determined. Representative blots of three independent experiments are shown. c, d The effect of 1 μM HPP-9 or HPI-1 (pre-)incubation on GLI2 ciliary trafficking was assessed through fluorescence microscopy. Representative images for GLI2 trafficking are shown in (c), and all data is quantified in (d). N independent experiments as indicated in the bars, n = 300–500 cilia analyzed per condition. Mean ± SD is plotted, two-way ANOVA, p as indicated. Scalebar 2 μm. e SHH-GFP cells were incubated with the indicated compounds for 25 h, before the medium was changed to ShhN-conditioned medium with or without competitors for 27 h. Nuclear BRD protein levels were determined using high-content fluorescence microscopy. Data shown is from N independent experiments as indicated, with n = 9–16 images analyzed per condition in each experiment. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37393376), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse GLI-2 by Western Blot Biological evaluation of HPP-9 and its inactive analog.a Chemical structures of HPP-9 and the methylated analog inact-HPP-9. b Representative micrographs showing the dose-dependent inhibition of an Shh-driven GFP reporter by HPP-9, resulting in the dose-response curves shown in (c). Scalebar 30 μm. b, c Curves and images are representative of N independent experiments (as indicated in the table below the graph), with n = 9 or 18 images analyzed per experiment. d, e NIH-3T3 cells were incubated with increasing concentrations of HPP-9 or HPI-1 in the presence of ShhN and lysates were probed for GLI1, GLI2, and GLI3. d shows a representative immunoblot illustrating that both compounds inhibit GLI1 and GLI2 but have no effect on GLI3 processing. e Quantification (mean ± SEM) of GLI1 (N = 3) and GLI2 full-length levels (N = 2) of N independent experiments. f–h qPCR for Ptch1 (f), Gli1 (g), and Gli2 (h) shows that HPP-9 reduces the expression of Hh pathway target genes, while also decreasing basal Gli2 transcript levels without affecting the fold-induction upon pathway stimulation (−: no ShhN, +: with ShhN). Data shown is the mean (f) or mean ± SD (g, h) for N independent experiments as indicated in the bar. f, g One-way ANOVA with Dunnett’s test, p as indicated in the graph, compared to DMSO + ShhN. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37393376), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse GLI-2 by Western Blot Biological evaluation of HPP-9 and its inactive analog.a Chemical structures of HPP-9 and the methylated analog inact-HPP-9. b Representative micrographs showing the dose-dependent inhibition of an Shh-driven GFP reporter by HPP-9, resulting in the dose-response curves shown in (c). Scalebar 30 μm. b, c Curves and images are representative of N independent experiments (as indicated in the table below the graph), with n = 9 or 18 images analyzed per experiment. d, e NIH-3T3 cells were incubated with increasing concentrations of HPP-9 or HPI-1 in the presence of ShhN and lysates were probed for GLI1, GLI2, and GLI3. d shows a representative immunoblot illustrating that both compounds inhibit GLI1 and GLI2 but have no effect on GLI3 processing. e Quantification (mean ± SEM) of GLI1 (N = 3) and GLI2 full-length levels (N = 2) of N independent experiments. f–h qPCR for Ptch1 (f), Gli1 (g), and Gli2 (h) shows that HPP-9 reduces the expression of Hh pathway target genes, while also decreasing basal Gli2 transcript levels without affecting the fold-induction upon pathway stimulation (−: no ShhN, +: with ShhN). Data shown is the mean (f) or mean ± SD (g, h) for N independent experiments as indicated in the bar. f, g One-way ANOVA with Dunnett’s test, p as indicated in the graph, compared to DMSO + ShhN. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37393376), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse GLI-2 by Western Blot HPP-9 is a long-acting Hedgehog pathway inhibitor.a SHH-GFP cells were treated for 25 h with DMSO, 1 μM HPI-1, or 1 μM HPP-9, before the medium was changed to various dilutions of ShhN-conditioned medium for 27 h. Nuclear GFP levels were quantified using fluorescence microscopy. Representative curves of three independent experiments are shown, with 9–16 images analyzed per condition. b GLI1 and GLI2 levels of cells pre-incubated with DMSO or 1 μM HPP-9 were determined. Representative blots of three independent experiments are shown. c, d The effect of 1 μM HPP-9 or HPI-1 (pre-)incubation on GLI2 ciliary trafficking was assessed through fluorescence microscopy. Representative images for GLI2 trafficking are shown in (c), and all data is quantified in (d). N independent experiments as indicated in the bars, n = 300–500 cilia analyzed per condition. Mean ± SD is plotted, two-way ANOVA, p as indicated. Scalebar 2 μm. e SHH-GFP cells were incubated with the indicated compounds for 25 h, before the medium was changed to ShhN-conditioned medium with or without competitors for 27 h. Nuclear BRD protein levels were determined using high-content fluorescence microscopy. Data shown is from N independent experiments as indicated, with n = 9–16 images analyzed per condition in each experiment. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37393376), licensed under a CC-BY license. Not internally tested by R&D Systems.