Mouse anti gli1

Manufactured by Cell Signaling Technology

Sourced in United States

Mouse anti-GLI1 is a primary antibody that specifically recognizes the GLI1 protein, a key transcription factor in the Hedgehog signaling pathway. This antibody can be used to detect and quantify GLI1 expression in various experimental systems.

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5 protocols using mouse anti gli1

Western Blotting and Cell Fractionation

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Western blotting was performed as already described^{55 (link)}. The following antibodies were used: mouse anti-GLI1 (#2643), mouse anti-cyclin A2 (#4656), rabbit anti-BCL2 (#2876), rabbit anti-BAX (#2772), rabbit anti-cyclin B1 (#12231), rabbit anti-PARP-1 (#9532), rabbit anti-phospho-ATR (Ser428) (#2853), rabbit anti-phospho-CHK1 (Ser345) (#2348), rabbit anti-phospho-CDC2 (Tyr15) (#4539), rabbit anti-phospho-H2A.X (Ser139) (#9718), rabbit anti-phospho-Histone H3 (Ser10) (#3377), rabbit anti-phospho-WEE1 (Ser642) (#4910) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-CDC2 (sc-954), mouse anti-Myc (sc-40), mouse anti-HSP90 (sc-13119) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-SMO (ST1718) (Merck Millipore, Burlington, MA, USA). Chemiluminescent detection was used. Cell fractionation was performed as previously described^{56 (link)}. The following antibodies were used: mouse anti-GLI1 (#2643) (Cell Signaling Technology), goat anti-fibrillarin (D-14), and goat anti-GAPDH (V-18) (Santa Cruz Biotechnology).

Pietrobono S., Santini R., Gagliardi S., Dapporto F., Colecchia D., Chiariello M., Leone C., Valoti M., Manetti F., Petricci E., Taddei M, & Stecca B. (2018). Targeted inhibition of Hedgehog-GLI signaling by novel acylguanidine derivatives inhibits melanoma cell growth by inducing replication stress and mitotic catastrophe. Cell Death & Disease, 9(2), 142.

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Western Blot Analysis of Gli1 Protein

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Western blots were done as described in Ringuette et al., 2016 (link) with the following modifications. For E15.5 samples, 10 retinas (5 embryos) per genotype were pooled and 6 retinas (3 pups) were pooled for P0 samples. A total of 50 μg protein was loaded per lane. Primary antibodies used were mouse anti-Gli1 (1:3000, Cell Signaling Technologies) and mouse anti-γ-Tubulin (1:1000, Sigma, Cat#T6557), the latter serving as the loading control. Detection was done with donkey anti-mouse IgG horseradish peroxidase at 1:5000 (1:5000, Millipore Cat#AP308P) and Luminata Crescendo Western HRP substrate (Millipore Cat#WBLUR0100). Blots were probed for Gli1 first, stripped, and reprobed for γ-Tubulin.

Li X., Gordon P.J., Gaynes J.A., Fuller A.W., Ringuette R., Santiago C.P., Wallace V., Blackshaw S., Li P, & Levine E.M. (2022). Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis. eLife, 11, e78342.

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Immunocytochemical Detection of Gli1 and Numb

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To detect Gli1 and Numb, neurospheres were blandly disaggregated and plated on poly-lysine-coated Lab-Tek chamber slides (cover slips) for 2 h. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, incubated in blocking solution (5% normal goat serum, 1% BSA, 0.1% Triton X-100) and stained overnight with primary antibodies diluted in blocking solution and for 2 h with secondary antibodies. Primary antibodies were mouse anti-Gli1 and rabbit anti-Numb (Cell Signaling Technology Inc). 594- or 488-conjugated anti-mouse and anti- rabbit secondary antibodies were purchased from Molecular Probes (Invitrogen, Eugene, OR). Nuclei were counterstained with Hoechst reagent. Cover slips were mounted with fluorescence mounting medium (Dako, Carpinteria, CA). Images were acquired with Carl Zeiss microscope (Axio Observer Z1) and AxioVision Digital Image Processing Software.

Abballe L., Mastronuzzi A., Miele E., Carai A., Besharat Z.M., Moretti M., De Smaele E., Giangaspero F., Locatelli F., Ferretti E, & Po A. (2018). Numb Isoforms Deregulation in Medulloblastoma and Role of p66 Isoform in Cancer and Neural Stem Cells. Frontiers in Pediatrics, 6, 315.

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Western Blot Analysis of Hedgehog Pathway

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Cells were lysed in cold RIPA buffer (50mM Tris-HCl pH 7.5, 1% NP-40, 150mM NaCl, 5mM EDTA, 0.25% NaDOC, and 0.1% SDS) supplemented with protease and phosphatase inhibitors and centrifuged at 20,000× g for 20 min at 4°C [26 (link)]. Supernatant was collected as whole cell extract (WCE). Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and incubated for 1 h in blocking buffer at room temperature. The following primary antibodies were used: mouse anti-GLI1 (#2643, Cell Signaling Technologies, Danver, MA, USA), mouse anti-SMO (sc-166685, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse anti-HSP90α/β (sc-13119, Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with the corresponding horseradish peroxidase (HRP)-coupled secondary antibody, membranes were developed by using SuperSignal West Femto (Thermo Fisher Scientific, Waltham, MA, USA) and imaged with ChemiDoc Imaging Systems (Bio-Rad, Hercules, CA, USA).

Pietrobono S., Franci L., Imperatore F., Zanini C., Stecca B, & Chiariello M. (2021). MAPK15 Controls Hedgehog Signaling in Medulloblastoma Cells by Regulating Primary Ciliogenesis. Cancers, 13(19), 4903.

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Western Blot Analysis of Gli1 in Retina

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Western blots were done as described in Ringuette et.al. (2016) with the following modifications. For E15.5 samples, 10 retinas (5 embryos) per genotype were pooled and 6 retinas (3 pups) were pooled for P0 samples. 50 ug protein was loaded per lane. Primary antibodies used were mouse anti-Gli1 (1:3000, Cell Signaling Technologies) and mouse anti-g-Tubulin (1:1000, Sigma, Cat#T6557), the latter serving as the loading control. Detection was done with donkey anti-mouse IgG horseradish peroxidase at 1:5000 (1:5000, Millipore Cat#AP308P) and Luminata Crescendo Western HRP substrate (Millipore Cat#WBLUR0100). Blots were probed for Gli1 first, stripped, and reprobed for g-Tubulin.

Li X., Gordon P.J., Gaynes J.A., Fuller A., Ringuette R., Santiago C.P., Wallace V.A., Blackshaw S., Li P., & Levine E.M. (2021). Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis.

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