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Anti gli1 h300

Manufactured by Santa Cruz Biotechnology
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Anti-Gli1 H300 is a primary antibody that targets the Gli1 protein. Gli1 is a transcription factor that plays a key role in the Hedgehog signaling pathway. This antibody can be used for various applications, such as Western blotting and immunohistochemistry, to detect and study the Gli1 protein.

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Lab products found in correlation

N cadherin 6b3, by Developmental Studies Hybridoma Bank (2 mentions) Py 489, by Developmental Studies Hybridoma Bank (2 mentions) Anti tbx5 ab, by Santa Cruz Biotechnology (2 mentions) Anti mitf ab, by Abcam (2 mentions) Fluorescein oregon green ab, by Thermo Fisher Scientific (2 mentions) Anti pax2 ab, by Fortrea (2 mentions) Anti abcg2 h 70, by Santa Cruz Biotechnology (1 mentions) Imaris 8, by Oxford Instruments (1 mentions) Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions) Phosphate buffered saline (pbs), by Merck Group (1 mentions) Fv1200 mpe laser scanning confocal microscope, by Olympus (1 mentions) Igg rabbit, by Thermo Fisher Scientific (1 mentions) Magnify chromatin immunoprecipitation system, by Thermo Fisher Scientific (1 mentions) Anti acetyl histone 3, by Merck Group (1 mentions) Anti flag m2 peroxidase hrp, by Merck Group (1 mentions) Anti gli2 h 300, by Santa Cruz Biotechnology (1 mentions) Anti nanog, by Cosmo Bio (1 mentions) Anti actin 1 19, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

Gli1 H300 Anti-GLI1

6 protocols using anti gli1 h300

1

Immunofluorescence Staining of Gli1 and ABC Transporters

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Immunofluorescence experiments were performed as previously described43 (link) using permanox Labtek chamber slides as support. Briefly, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS (Sigma-Aldrich, St. Louis, MO). Cells were then blocked with 5% BSA in PBS for 30 min at room temperature and incubated overnight with the following primary antibodies: anti-Gli1 H300 (sc-20687, Santa Cruz Biotechnology Inc.), anti-Gli1 (AF3455, R&D Systems), anti- ABCB1 (MDR1, D-11; sc-55510 Santa Cruz Biotechnology Inc.) and anti‐ABCG2 H-70 (sc‐2582; Santa Cruz Biotechnology, Inc.) diluted in blocking solution. Secondary antibodies conjugated with Alexa Fluor 488 or 594 were purchased from Molecular Probes (Invitrogen) and diluted 1:400 and 1:200, respectively, in blocking solution. Nuclei were Hoechst-counterstained and cover slips were mounted with fluorescence mounting medium (Prolong Gold, Thermo Fisher Scientific, MA, USA). Images were acquired using a FV1200 MPE laser scanning confocal microscope (Olympus) with a UPlanSAPO 20x/0.75 NA objective. Imaris 8.1 software (Oxford Instruments, https://imaris.oxinst.com/) was used for image-processing.
Po A., Citarella A., Catanzaro G., Besharat Z.M., Trocchianesi S., Gianno F., Sabato C., Moretti M., De Smaele E., Vacca A., Fiori M.E, & Ferretti E. (2020). Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells. Scientific Reports, 10, 13988.
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2

Chromatin Immunoprecipitation of Gli1 and Acetyl-H3

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ChIP was performed using the MAGnify Chromatin Immunoprecipitation System (Invitrogen). Protocol was performed as described in44 (link). For each ChIP reaction 300,000 cells were used, cell lysates were added to their respective antibody/beads for 2 h. Eluted DNA was PCR amplified with primers encompassing the Gli- responsive elements of human ABC promoter. The following antibodies were used: IgG rabbit (Invitrogen), rabbit polyclonal anti-Gli1 H300 (sc-20687, Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-acetyl-histone 3 (06599, Millipore). Eluted DNA has been analysed with Q-PCR. Primers were designed with Primer-Blast designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and Primers tool (Genomatix Genome Analyzer, GGA, v3.30126, https://www.genomatix.de/) and are reported in Supplementary Table 2.
Po A., Citarella A., Catanzaro G., Besharat Z.M., Trocchianesi S., Gianno F., Sabato C., Moretti M., De Smaele E., Vacca A., Fiori M.E, & Ferretti E. (2020). Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells. Scientific Reports, 10, 13988.
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3

Immunohistochemistry for Neural Development

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Antibodies against Pax6 (1:10), N-cadherin (6B3, 1:10), β-catenin (PY-489, 1:20) and Laminin (3H11, 1:10) were obtained from Developmental Studies Hybridoma Bank (Iowa City, Iowa, USA). Anti-Pax2 Ab (1:100) was obtained from Covance (Princeton, NJ, USA). Anti-Tbx5 Ab (1:100) and Anti-Gli1 (H-300, 1:50) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Anti-Mitf Ab (1:500) was obtained from Abcam (Cambridge, MA, USA). Anti-Chx-10 (Vsx2, 1:250) Ab was purchased from ExAlpha (Shirley, MA, USA). Fluorescein/Oregon Green Ab (1:100) was purchased from Molecular Probes (Grand Island, NY, USA). The production of Abs against C3 (targets amino acids 741–761) and C3aR as well as the blocking peptides are described in Haynes et al. (2013) (link). Preimmune IgG was isolated from the corresponding rabbit serum using Protein A affinity purification. All secondary Abs were purchased from Molecular Probes (Grand Island NY, USA) and used at 1:100 dilutions.
Grajales-Esquivel E., Luz-Madrigal A., Bierly J., Haynes T., Reis E.S., Han Z., Gutierrez C., McKinney Z., Tzekou A., Lambris J.D., Tsonis P.A, & Del Rio-Tsonis K. (2017). Complement component C3aR constitutes a novel regulator for chick eye morphogenesis. Developmental biology, 428(1), 88-100.
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4

Immunohistochemistry for Neural Development

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Antibodies against Pax6 (1:10), N-cadherin (6B3, 1:10), β-catenin (PY-489, 1:20) and Laminin (3H11, 1:10) were obtained from Developmental Studies Hybridoma Bank (Iowa City, Iowa, USA). Anti-Pax2 Ab (1:100) was obtained from Covance (Princeton, NJ, USA). Anti-Tbx5 Ab (1:100) and Anti-Gli1 (H-300, 1:50) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Anti-Mitf Ab (1:500) was obtained from Abcam (Cambridge, MA, USA). Anti-Chx-10 (Vsx2, 1:250) Ab was purchased from ExAlpha (Shirley, MA, USA). Fluorescein/Oregon Green Ab (1:100) was purchased from Molecular Probes (Grand Island, NY, USA). The production of Abs against C3 (targets amino acids 741–761) and C3aR as well as the blocking peptides are described in Haynes et al. (2013) (link). Preimmune IgG was isolated from the corresponding rabbit serum using Protein A affinity purification. All secondary Abs were purchased from Molecular Probes (Grand Island NY, USA) and used at 1:100 dilutions.
Grajales-Esquivel E., Luz-Madrigal A., Bierly J., Haynes T., Reis E.S., Han Z., Gutierrez C., McKinney Z., Tzekou A., Lambris J.D., Tsonis P.A, & Del Rio-Tsonis K. (2017). Complement component C3aR constitutes a novel regulator for chick eye morphogenesis. Developmental biology, 428(1), 88-100.
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5

Western Blotting and Immunoprecipitation Assay Protocol

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Cells were lysed using RIPA buffer (Tris-HCl pH 7.6 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, sodium pyrophosphate 2 mM and protease inhibitors). Lysates were separated on 8% acrylamide gel and immunoblotted using standard procedures. The following antibodies were used: anti-Arrb1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-Nanog (Cosmo Bio Co, Japan), anti-Actin I-19 (sc-1616; Santa Cruz Biotechnology), anti-β-III-Tubulin (MAB 1637 Millipore), anti-Gli1 H-300 (sc-20,687; Santa Cruz Biotechnology), anti-acetyl-Gli1 (Lys518) (Eurogentec) [32 (link)], anti-p300 C-20 (sc-585; Santa Cruz Biotechnology), anti-FLAG M2-Peroxidase (HRP) (A8592 Sigma), anti-HA (sc-7392 Santa Cruz), anti-Gli2 H-300 (sc-28,674; Santa Cruz Biotechnology), anti-Smo N-19 (sc-6366; Santa Cruz Biotechnology), anti-Sox2 (MAB4343 Millipore). HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) were used in combination with enhanced chemo-luminescence (ECL Amersham).
For immunoprecipitation assay antibody sources and concentrations used were: Protein G Plus-Agarose (sc-2002; Santa Cruz Biotechnology); anti-FLAG M2 Affinity Gel (Sigma A2220, IP 30⌠l), anti-FLAG M2-Peroxidase (HRP) (A8592 Sigma, western blotting 1:5000), anti-HA (sc-7392 Santa Cruz, 1:1000); anti-myc-HRP.
Miele E., Po A., Begalli F., Antonucci L., Mastronuzzi A., Marras C.E., Carai A., Cucchi D., Abballe L., Besharat Z.M., Catanzaro G., Infante P., Di Marcotullio L., Canettieri G., De Smaele E., Screpanti I., Locatelli F, & Ferretti E. (2017). β-arrestin1-mediated acetylation of Gli1 regulates Hedgehog/Gli signaling and modulates self-renewal of SHH medulloblastoma cancer stem cells. BMC Cancer, 17, 488.
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6

Investigating Gli1-Maml1 Protein Interactions

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In situ PLA was performed in NIH3T3 cells using the Duolink In situ-Fluorescence Technology, Olink Bioscience (Sigma-Aldrich). All the steps were performed according to the manufacturer’s protocol. Primary antibodies: anti-Gli1 (H300) and anti-Maml1 (N-20) (cat.#sc-18506) from Santa Cruz Biotechnology. Hybridization between the two PLA anti-rabbit PLUS and anti-goat MINUS probes leading the fluorescent red signal only occurs when the distance between the two antigens is less than 40 nm. In control experiment, cells were incubated with only one primary antibody and no significant binding was detected (only Gli1; only Maml1). Single plane confocal images were acquired using an inverted Olympus iX73 confocal microscope as described in immunofluorescence staining.
Quaranta R., Pelullo M., Zema S., Nardozza F., Checquolo S., Lauer D.M., Bufalieri F., Palermo R., Felli M.P., Vacca A., Talora C., Di Marcotullio L., Screpanti I, & Bellavia D. (2017). Maml1 acts cooperatively with Gli proteins to regulate sonic hedgehog signaling pathway. Cell Death & Disease, 8(7), e2942-.
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