Gal 1

Manufactured by Abcam

Sourced in United States

Gal-1 is a protein that functions as a lectin, binding to beta-galactoside carbohydrates. It is involved in various biological processes, including cell-cell interactions, cell adhesion, and immune response regulation.

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8 protocols using gal 1

Immunoblotting Analysis of Cellular Proteins

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Immunoblotting was done as described previously(23 (link)). Antibodies used in immunoblotting were Gal-1 (Abcam), AR (Abcam), E-cadherin, phospho AKT, AKT, beta-actin (Cell Signaling) and Ras (chemicon). Immunoblotting bands were quantified by Image J software.

Shih T.C., Liu R., Wu C.T., Li X., Xiao W., Deng X., Kiss S., Wang T., Chen X.J., Carney R., Kung H.J., Duan Y., Ghosh P.M, & Lam K.S. (2018). Targeting Galectin-1 impairs castration-resistant prostate cancer progression and invasion. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(17), 4319-4331.

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Immunohistochemical Analysis of Protein Expression

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Tissue microarray (TMA) sections were de-waxed using xylene twice and rehydrated with 100% ethanol for 5 minutes, 95% and 80% ethanol for 5 minute each. Then rinse in PBS. Antigen retrival was performed in 10 mM, pH 6.0 sodium citrate buffer at 95-100°C for 20 mins. After cooling down to room temperature, tissue sections were rinsed with PBS once followed by blocking endogenous peroxidase with 1% H₂O₂ and blocking non-specific binding site with Power Block (BioGenex) for 5 min at room temperature each. The tissue sections were then incubated with the specific antibody against Gal-1 (Abcam) or AR-V7 (GeneTex) or E-Cad (Cell signaling) or ki-67 (Cell signaling) overnight, followed by rinsing with PBS and incubation by an biotin-conjugated goat anti-rabbit IgG (BioGenex), as the second antibody. TMA sections were then incubated with strepavidin conjugated-HRP (BioGenex) for 20 mins at room temperature. HRP activity was detected using diaminobenzidine tetrahydrochloride (DAB) as substrate (BioGenex). Nuclei were counterstained with hematoxylin (Cell signaling). The ki-67 positive cells were counted in 3 random chosen areas.

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Western Blot Analysis of Metastatic RCC

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Dysregulated protein expression in metastatic RCC samples was verified by western blot (WB) analysis. Briefly, 30 μg of total protein were electrophoretically separated on a SDS–PAGE gel. Proteins were transferred to a nitrocellulose membrane and probed with primary antibodies overnight at 4 °C. Primary antibodies were used for Gal-1 (Abcam, Cambridge, MA, USA), HIF-1α (Novus Biologicals, Littleton, CO, USA), phospho-mTOR S2448 (serine 2448), total mTOR, phospho-Akt S473 (serine 473), total Akt and α-tubulin (Cell Signaling Technology, Danvers, MA, USA) was used as a loading control. The following day, membranes were washed with tris-buffered saline and Tween-20 and incubated with a secondary anti-rabbit antibody conjugated with horseradish peroxidase. Finally, membranes were incubated with enhanced chemoluminescence reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and protein expression was visualised after exposure to X-ray film. Each experiment was performed in triplicate and representative blots are shown. Densitometry analysis was performed using Image J software from the National Institutes of Health (http://rsbweb.nih.gov/ij/) and differences were evaluated using the Student's t-test.

White N.M., Masui O., Newsted D., Scorilas A., Romaschin A.D., Bjarnason G.A., Siu K.W, & Yousef G.M. (2014). Galectin-1 has potential prognostic significance and is implicated in clear cell renal cell carcinoma progression through the HIF/mTOR signaling axis. British Journal of Cancer, 110(5), 1250-1259.

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Histological Analysis of Mouse Tumors

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Tissues were fixed in 10% formalin for 16 h and kept in 70% ethanol for 72 h, followed by embedding in paraffin and cutting into 5-μm sections. Hematoxylin and eosin (H&E) staining was performed²² (link). Mouse tumor score was quantified by pathologists based on H&E stained slides using five criteria (i.e., the level of centrilobular vacuolar degeneration, the number of proliferation foci, scirrhous type foci of proliferation mitotic index, and inflammatory cells)²³ (link)^,²⁴ (link). Details are described in Supporting Information Table S1.
Immunostaining was performed with specific antibodies against Gal1 (Abcam, Cambridge, MA, USA) and CD8 (eBioscience, Waltham, MA). The number of positive-staining cells was counted in at least five random microscopic fields (100 × magnification) for each section.

Setayesh T., Hu Y., Vaziri F., Chen X., Lai J., Wei D, & Yvonne Wan Y.J. (2023). Targeting stroma and tumor, silencing galectin 1 treats orthotopic mouse hepatocellular carcinoma. Acta Pharmaceutica Sinica. B, 14(1), 292-303.

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Immunohistochemical Staining of Gal-1, VEGF, and CD34

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The primary antibodies used in this study were ready-to-use monoclonal mouse Gal-1 (Abcam, Cambridge, UK), anti-VEGF (Diagnostic Biosystems, Pleasanton, CA, USA), and anti-CD34 (Diagnostic Biosystems). An UnoVue HRP/DAB Detection System (Diagnostic Biosystems) was used for primary antibody visualization.
The method employed in this study has been described in our previous work [10 (link),11 (link)]. Briefly, sections of tissue (4 to 5 µm thick) were deparaffinized and washed with distilled water. Antigen retrieval was performed using a microwave. The sections were then allowed to cool and endogenous peroxidase blocking was performed in 5% hydrogen peroxide. Blocking of nonspecific antibody binding was carried out in 5% casein. Sections were washed and incubated in a moist chamber at room temperature with corresponding primary antibodies followed by incubation with anti-mouse horseradish peroxidase secondary antibodies. The reaction product was visualized after 3,3-diaminobenzidine (DAB) staining for 5 minutes followed by Mayer’s hematoxylin counterstaining [10 (link),11 (link)].

Zinovkin D.A., Achinovich S.L., Zubritskiy M.G., Whatmore J.L, & Pranjol M.Z. (2019). High Expression of Galectin-1, VEGF and Increased Microvessel Density Are Associated with MELF Pattern in Stage I-III Endometrioid Endometrial Adenocarcinoma. Journal of Pathology and Translational Medicine, 53(5), 280-288.

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Western Blot Analysis of Protein Markers

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Protein was collected and diluted with a loading buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate protein and then transferring was conducted. α-SMA, phosphor-AKT (p-AKT), AKT, phosphor-Erk1/2 (p-Erk1/2), Erk1/2, collagen I, fibronectin and Gal-1 antibodies were purchased from Abcam (Cambridge, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) antibody was obtained from Proteintech (Rosemont, IL, USA). The expression of each protein was visualized using an enhanced chemiluminescence system (ECL; Tanon, China).

Xue J, & Li S. (2023). Inhibition of Galectin-1 attenuates lung fibroblast activation and proliferation in lung fibrosis. Cellular and molecular biology (Noisy-le-Grand, France), 69(11).

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Protein Analysis Protocol for STING Pathway

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For protein analysis, cells were lysed in RIPA lysis buffer and processed for protein loading as previously published[9 (link)]. The following primary antibodies were used to detect specific proteins: Gal1 (Cat: ab138513, Abcam), phospho-STING (Cat: 72971), phospho-TBK1 (ser172, Cat: 5483), STING (Cat:13647), TBK1 (Cat: 3504), IRF-3 (Cat: 4302, RRID: AB_1904036), phospho-IRF-3 (Cat: 29047, RRID: AB_2773013), RelA/p65 (NF-κB (Cat: 8242), phospho-NF-κB (RelA/p65, Cat:3033), NF-κB1 p105/p50 (Cat: 3035), NF-κB2 p100/p52 (Cat: 4882) from Cell Signaling Technologies, and β-Actin (1:5000; Cat: sc-47778 HRP, Santa Cruz Biotechnologies). Secondary antibodies used in this study were HRP-conjugated Donkey anti-Goat IgG (H+L) (1:5000-1:10,000; Cat: A15999 Invitrogen), HRP-conjugated goat anti-rabbit (1:5000; Cat: 7074 Cell Signaling Technologies). Immunoblots were developed with Pierce West Pico (Cat: 35060; Thermo Fisher Scientific) and visualized with ChemiDoc XRS imaging system equipped with Image Lab Software (RRID: SCR_008426; Bio-Rad Laboratories). In each case, experiments were carried out in triplicate and a representative blot is shown unless otherwise stated. Blot densitometry quantification was done using ImageJ (RRID: SCR_003070)

Rørvik L.M., Aase B., Alvestad T, & Caugant D.A. (2000). Molecular Epidemiological Survey of Listeria monocytogenes in Seafoods and Seafood-Processing Plants. Applied and Environmental Microbiology, 66(11), 4779-4784.

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Differentiation of NC-like cells from ES cells

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The maintenance of ST2 cells and ES cells and differentiation of NC-like cells from ES cells were described previously (Motohashi et al., 2007) . The day when ES cells were seeded onto ST2 monolayers was defined as day 0. GAL-1 (Abcam) or rhGAL-1/Ox and CSGAL-1 (gifted from Dr. Kadoya) was also present in each experiment. Regarding GAL-1 inhibition, anti-GAL-1 (gifted from Dr. Kadoya) or control IgG (Biolegend) was used for each culture.

Motohashi T., Nishioka M., Kitagawa D., Kawamura N., Watanabe N., Wakaoka T., Kadoya T, & Kunisada T. (2017). Galectin-1 enhances the generation of neural crest cells. The International journal of developmental biology, 61(6-7).

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