Goat anti gli3

Manufactured by R&D Systems

Goat anti-Gli3 is a primary antibody that recognizes the Gli3 protein. Gli3 is a transcription factor that plays a role in the Hedgehog signaling pathway, which is involved in embryonic development and cell differentiation.

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Lab products found in correlation

Irdye antibodies, by LI COR (2 mentions) Complete mini, by Roche (2 mentions) Odyssey system, by LI COR (2 mentions) Radioimmunoprecipitation assay (ripa), by Merck Group (2 mentions) Phosstop, by Roche (2 mentions) α tubulin, by Abcam (2 mentions) Image studio software version 4, by LI COR (2 mentions) Polyvinylidene difluoride pvdf membrane, by Bio-Rad (2 mentions) Odyssey infrared imaging system, by LI COR (2 mentions) Goat anti gli2, by R&D Systems (2 mentions) Rabbit anti phospho nf κb p65, by Cell Signaling Technology (1 mentions) Rabbit anti β actin, by Affinity Biosciences (1 mentions) Rabbit anti mmp3, by Proteintech (1 mentions) Rabbit anti gli1, by Cell Signaling Technology (1 mentions) Rabbit anti nf κb p65, by Cell Signaling Technology (1 mentions) Mouse anti e cadherin, by Cell Signaling Technology (1 mentions) Mouse anti iκbα, by Cell Signaling Technology (1 mentions) Rabbit anti phospho ampkα, by Cell Signaling Technology (1 mentions) Rabbit anti ampkα, by Cell Signaling Technology (1 mentions) Anti lamin a, by Abcam (1 mentions)

Spelling variants of this product

Anti-GLI3 (3 citations)

9 protocols using goat anti gli3

Western Blot Protocol for Protein Analysis

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Cortical tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma) supplemented with protease (Complete Mini, Roche, Basel, Switzerland) and phosphatase (PhosStop, Roche) inhibitors according to standard protocols. Western blot analyses were conducted according to standard protocols. Briefly, soluble extracts were loaded onto Criterion, 4%–15% Tris-HCI 4 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Berkeley, CA, USA), separated at 120 V, and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) at 30 V for 2 h or overnight at 4 °C. Membranes were blocked with 5% BSA/1X TBS-T (Tris-buffered saline with 0.1% Tween 20) for 1 h at room temperature, and incubated with primary antibodies diluted in blocking buffer overnight at 4 °C, and secondary antibodies (1:5000 dilution; IR-Dye antibodies, LI-COR, Lincoln, NE, USA) for 1 h at RT. Membranes were washed in 1X TBS-T and scanned using the Odyssey Infrared Imaging System (LI-COR). Primary antibodies were used as follows: goat anti-Gli3 (1:500; R&D Systems) and α-Tubulin (1:5000, Abcam). Quantification and analysis were conducted using the Odyssey System and Image Studio Software version 4.0.21 (LI-COR, Lincoln, NE, USA).

Yabut O.R., Ng H.X., Fernandez G., Yoon K., Kuhn J, & Pleasure S.J. (2016). Loss of Suppressor of Fused in Mid-Corticogenesis Leads to the Expansion of Intermediate Progenitors. Journal of Developmental Biology, 4(4), 29.

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Protein Expression Analysis in Transfected Cells

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Transfected cells or grinded tumor tissues were lysed in modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% vol/vol NP-40, 1% n-Dodecyl β-D-maltoside, 0.25% wt/vol sodium deoxycholate, 1 mM DTT, and 1 × Roche complete Protease Inhibitor Cocktail) for 1 h at 4 ℃. The lysate was clarified by centrifugation for 20 min at 14,000 × g. The protein concentration was determined using a bicinchoninic acid assay and equal amounts of total protein from each of the samples was supplemented with 5 × SDS loading buffer, incubated at 95 ℃ for 5 min, subjected to SDS-PAGE, followed by western blot analysis. The following antibodies were used: rabbit anti-β-actin (Affinity; 1:5000), rabbit anti- AMPKα (Cell Signaling Technology; 1:1000), rabbit anti-Phospho-AMPKα (Cell Signaling Technology; 1:1000), mouse anti-E-cadherin (Cell Signaling Technology; 1:1000), rabbit anti-N-cadherin (Elabscience; 1:1000), rabbit anti-MMP3 (Proteintech; 1:1000), rabbit anti-GLI1 (Cell Signaling Technology; 1:1000), rabbit anti-GLI2 (NOVAS; 1:1000), goat anti-GLI3 (R&D; 1:1000), mouse anti-IκBα (Cell Signaling Technology; 1:1000), rabbit anti-NF-κB p65 (Cell Signaling Technology; 1:1000) and rabbit anti-Phospho-NF-κB p65 (Cell Signaling Technology; 1:1000).

Cai J., Wang Y., Wang X., Ai Z., Li T., Pu X., Yang X., Yao Y., He J., Cheng S.Y., Yu T., Liu C, & Yue S. (2023). AMPK attenuates SHH subgroup medulloblastoma growth and metastasis by inhibiting NF-κB activation. Cell & Bioscience, 13, 15.

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Subcellular Fractionation and Immunoblotting

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Subcellular fractionation was performed as reported (Chen et al. 2009 (link)). The purity of the cytoplasmic and nuclear fractions was assessed by cytoplasmic- and nuclear-specific markers, including anti-tubulin (1:5000; Sigma) and anti-Lamin A (1:3000; Abcam). The following antibodies were used for Western blotting of cytoplasmic and nuclear fractions: rabbit anti-Sufu (1:3000; Santa Cruz Biotechnology), goat anti-Gli2 (1:500; R&D Systems), and goat anti-Gli3 (1:500; R&D Systems).

Lin C., Yao E., Wang K., Nozawa Y., Shimizu H., Johnson J.R., Chen J.N., Krogan N.J, & Chuang P.T. (2014). Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling. Genes & Development, 28(22), 2547-2563.

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FLAG-tagged Protein Immunoprecipitation

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HEK293T cells were lysed using NP-40 buffer (50 mM Tris-Cl pH8.0, 0.1 M NaCl, 10 mM Sodium fluoride, 1 mM Sodium vanadate, 1% NP-40, 10% Glycerol, 1.5 mM EDTA, Protease Inhibitor Cocktail). Cell lysates were rotated for 30 min at 4°C and cleared by centrifuge at full speed. Lysates were then incubated with M2 beads (Sigma-Aldrich, St Louis, MO) for 24 hr at 4°C. After 4-time wash (15 min/time) with NP-40 buffer, the protein samples were collected by boiling in 1×SDS loading buffer and subjected to standard SDS-PAGE and Western Blot. Primary antibodies used in this study: Mouse anti-flag (Sigma-Aldrich), Mouse anti-Set7 (Cell signaling, Danvers, MA), Goat anti-Gli3 (R&D), Rabbit anti-GAPDH (Sigma-Aldrich), Rabbit anti-monomethylated K436 (Me-K436, Abcolony, China), Rabbit anti-monomethylated K595 (Me-K595, Abcolony, China). Please also refer the figure legend of Figure 1—figure supplement 3 for more detailed information. Secondary antibodies used in this study were purchased from Millipore Company.

Fu L., Wu H., Cheng S.Y., Gao D., Zhang L, & Zhao Y. (2016). Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals. eLife, 5, e15690.

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Protein Extraction and Immunoblotting Protocol

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Cells were lysed using RIPA buffer (150nm NaCl, 50mM Tris, pH 7.6, 0.1% SDS, 0.1% NP-40 and 0.5% sodium deoxycholate) supplemented with protease inhibitors (Roche). Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Zebrafish embryos were deyolked before lysis in RIPA buffer supplemented with protease inhibitors, as previously described (Link et al., 2006 (link)). All lysates were boiled for 5 minutes in 4x SDS-PAGE loading buffer, except prior to immunoblotting for DREADD expression, for which lysates were incubated at room temperature for 45 minutes. Protein samples were separated on 4–15% gradient TGX precast gels (Bio-Rad) and transferred to PVDF membrane (Bio-Rad). 5% non-fat dried milk in TBS with 0.1% Tween was used to block membranes and to dilute antibodies. HRP signal was detected using Clarity Western ECL substrate (Bio-Rad). Primary antibodies used were goat anti-GFP (1:1,000, Rockland, RRID:AB_218182), mouse anti-GAPDH (1:100,000, Proteintech, RRID:AB_2107436), goat anti-GLI3 (1:200, R&D Systems, Cat #AF3690), goat anti-c-Myc (1:5,000, Novus, RRID:AB_10004121) and mouse anti-β-actin (1:100,000, Proteintech, RRID:AB_2687938). We used HRP-conjugated secondary antibodies at 1:5,000 dilution (Jackson ImmunoResearch Laboratories, Inc).

Truong M.E., Bilekova S., Choksi S.P., Li W., Bugaj L.J., Xu K, & Reiter J.F. (2021). Vertebrate cells differentially interpret ciliary and extraciliary cAMP. Cell, 184(11), 2911-2926.e18.

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Immunostaining of Embryonic and Tissue Samples

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For immunostaining, embryos and tissue were processed as previously described²⁴ and staining was performed on 100 µm thick vibratome sections in PBS with 0.3% Triton (PBST) and 3% BSA (Sigma A3608). Antisera were as follows: chicken anti-GFP 1/1000 (Abcam ab13970), rabbit anti-RFP 1/500 (Abcam ab62341), goat anti-Sox2 1/500 (R&D AF2018), rabbit anti-Sox9 (Millipore AB5535), rabbit anti-Prdm16 1/200 (gift from P. Seale⁶⁷), rabbit anti-Pax6 1/500 (Covance PRB-278P), rabbit anti-Hes1 1/100 (Cell Signalling D6P2U), goat anti-Gli3 1/500 (R&D AF3690). DNA was stained with DAPI. Fluorescent images were acquired using a Leica SP8 confocal microscope and analysed using ImageJ.

van den Ameele J., Krautz R., Cheetham S.W., Donovan A.P., Llorà-Batlle O., Yakob R, & Brand A.H. (2022). Reduced chromatin accessibility correlates with resistance to Notch activation. Nature Communications, 13, 2210.

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Western Blot Protocol for Protein Analysis

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Immunoprecipitation and Western Blot Analysis

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HEK293T cells were lysed in lysis buffer (25 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5 mM phenylmethyl sulphonyl fluoride (PMSF, ThermoFisher) and protease inhibitor cocktail (Roche). The cell lysates were sonicated and cleared by centrifugation at 13,000 g for 10 min at 4 °C. IPs were performed at 4 °C for 6–8 h with the following agarose beads: Anti-HA-agarose (Sigma-Aldrich), Anti-flag-agarose (Sigma-Aldrich), Anti-myc-agarose (Sigma-Aldrich), GFP-Trap affinity resin (Chromotek). Western blot analysis was performed using anti-Flag-HRP-conjugated (1:5,000, Sigma-Aldrich), anti-HA-HRP-conjugated (1:5,000, Sigma-Aldrich), anti-myc-HRP-conjugated (1:5,000, Sigma-Aldrich), anti-GFP-HRP-conjugated (1:5,000, Rockland Immunochemicals), goat anti-Gli2 (R&D Systems), goat anti-Gli3 (R&D Systems), rabbit anti-Gli3 (Novus biologicals), goat anti-Zic5 (Novus biologicals) and mouse anti-alpha-tubulin-HRP-conjugated (Proteintech). Secondary antibodies used were goat anti-rabbit-HRP-conjugated (Cell Signaling Technology) and mouse anti-goat-HRP-conjugated (Santa Cruz Biotechnology).

Sun J., Yoon J., Lee M., Lee H.K., Hwang Y.S, & Daar I.O. (2022). Zic5 stabilizes Gli3 via a non-transcriptional mechanism during retinal development. Cell reports, 38(5), 110312.

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Co-Immunoprecipitation of AKT-Regulated Proteins

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HEK293T cells (ATCC, #CRL-3216) were cultured in DMEM media supplemented with 10% FBS. For co-immunoprecipitation, HEK293T cells were seeded on 10 cm tissue culture dishes, cultured in DMEM media supplemented with 10% FBS, and transfected with the expression vectors tagged with Flag, and HA using Superfect (Qiagen) or calcium phosphate. Cells were treated with AKT1/2 kinase inhibitor, iAKT1/2 (Sigma Aldrich, A6730) at 10 μM for 20 hr and then cells were harvested and lysed in IP buffer (20 mM Tris-HCl, pH 8.0, 0.5 % NP-40, 1 mM EDTA, 150 mM NaCl, 2 mM PMSF, 10% Glycerol, 4 mM Na3VO4, 200 mM NaF, 20 mM Na-pyroPO4, and protease inhibitor cocktail). In these studies, immunoprecipitations were performed with mouse anti-HA antibody (Covance). Immunoblotting assays were performed using goat anti-Gli3 (R and D Systems, AF3690, 1:250), rabbit anti-ARHGAP36 (Sigma, HPA-002064, 1:2000), mouse anti-HA (Covance, 1:5000), rabbit anti-β-tubulin (Santa Cruz, sc-9104, 1:2000), rabbit anti-pSER (Cell Signaling, #9651, 1:5000), mouse anti-TuJ1 (Covance, 1:5000), rabbit anti-FoxP1 (abcam, ab16645, 1:1000) and anti-pCREB (Cell Signaling, 9198S, 1:1000) antibodies.

Nam H., Jeon S., An H., Yoo J., Lee H.J., Lee S.K, & Lee S. (2019). Critical roles of ARHGAP36 as a signal transduction mediator of Shh pathway in lateral motor columnar specification. eLife, 8, e46683.

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