The cells were lysed in RIPA buffer (Pierce, 89900) with 1X protease and phosphatase inhibitor (Pierce, A32961). Lysates were sonicated in a Bioruptor™ (Diagenode, Denville, NJ, USA) on ice for 8 minutes (8 cycles of 30 s on, 30 s off). Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) according to the manufacturer’s protocol. Samples were mixed with NuPAGE LDS 4x loading gel (NP0007) and NuPAGE 10x reducing agent (NP0009), and boiled at 95 °C. Next, samples were loaded onto 4–20% (BioRad, 4561093) or 10% gels (BioRad, 4561033) and transferred to LF PVDF (BioRad, 170–4274). Membranes were blocked with LI-COR Biosciences (Lincoln, Nebraska, USA) Odyssey Blocking Buffer (927–40100). Bands were detected using Azure Biosystems (Dublin, California, USA) Imaging System c600. The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).
Galectin 9
Manufactured by Abcam
Galectin-9 is a carbohydrate-binding protein that belongs to the galectin family. It plays a role in various biological processes, including immune regulation, cell-cell adhesion, and apoptosis. Galectin-9 can be used in research applications to study its functions and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Bioruptor, by Diagenode (1 mentions)
Pd l1, by ProSci (1 mentions)
Acetyl alpha tubulin, by Cell Signaling Technology (1 mentions)
Lf pvdf, by Bio-Rad (1 mentions)
Pd l2, by ProSci (1 mentions)
Icosl, by Abcam (1 mentions)
C600 imaging system, by Azure Biosystems (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Alpha tubulin, by Cell Signaling Technology (1 mentions)
Pacific blue anti mouse cd11b, by BioLegend (1 mentions)
Opteia, by BD (1 mentions)
Lactose, by Merck Group (1 mentions)
Apc anti mouse ly6g clone 1a8, by BioLegend (1 mentions)
Phospho irf3 ser396, by Cell Signaling Technology (1 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phospho p65 ser536, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
3 protocols using galectin 9
1
Western Blot Immunodetection of Immune Checkpoint Proteins
2
Galectin-9 Immunodetection and Neutrophil Analysis
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All reagents were purchased from Sigma-Aldrich unless otherwise indicated. For detection of galectin-9 by immunofluorescence (IF) staining, a purified rat galectin-9 antibody (Abcam, San Diego, CA) followed by Alexa-546 conjugated chicken anti-rat antibody (Molecular Probes, OR) was used. A rat anti-mouse CD11b antibody conjugated to PE (BD Pharmingen) and a purified rat anti-mouse Ly-6G (Clone Accurate Chemical, Westbury, NY, USA) followed by chicken anti-rat Alexa488 (Molecular Probes, OR) were used for detection of activated neutrophils by IF staining. For flow cytometry Pacific Blue anti-mouse CD11b and APC anti-mouse Ly6G (Clone 1A8) antibodies (Biolegend, San Diego, CA) were used. The terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining kit was purchased from Chemicon International, CA. Purified recombinant galectin-9 was purchased from R&D Systems, MN. The endotoxin level was <1.0 EU per μg of protein. Ultrapure E. coli endotoxin was purchased from Invivogen and lactose was purchased from Sigma. For detection of reactive oxygen species, Fc OxyBURST assay reagent was purchased from Molecular Probes, Eugene, OR. Mouse IL-6 ELISA kits (BD OptEIA) were from BD Biosciences, San Diego, CA.
3
Western Blot Analysis of Tumor Tissue
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Whole-cell lysates in each well were collected on ice in RIPA buffer (50 mM Tris-HCl pH 8.0, 1% Triton X-100, 0.05% SDS, 0.25% deoxycholate, 150 mM NaCl, and 50 mM NaF), supplemented with a protease inhibitor cocktail (11836170001, Roche) and Halt Phosphatase inhibitor cocktail (78420, Thermo Fisher Scientific). A total of 10 mg tumor tissue was collected from each tumor-bearing mouse and resuspended in 350 µl RIPA buffer. The mixtures were homogenized and subjected to SDS-PAGE analysis. Antibodies used were: Cd44 (Cell Signaling Technology; #37259; 1:1000 dilution), Bmi-1 (Cell Signaling Technology; #5856; 1:1000 dilution), phospho-Akt (Ser473) (Cell Signaling Technology; #4060; 1:1000 dilution), phospho-P65 (Ser536) (Cell Signaling Technology; #3033; 1:1000 dilution), P65 (Cell Signaling Technology; #8242; 1:1000 dilution), phospho-Irf3 (Ser396) (Cell Signaling Technology; #29047; 1:1000 dilution), Irf3 (Cell Signaling Technology; #4302; 1:1000 dilution), Pd-l1 (Cell Signaling Technology; #64988; 1:1000 dilution), Galectin-9 (Abcam; ab49900; 1:20,000 dilution), β-Actin (Abcam; ab49900; 1:20,000 dilution). The primary antibody was incubated at 4°C overnight. After washing the membranes four times, the secondary antibody was incubated for 1 h at room tempetature. Bands were detected by using an automatic X-ray filmprocessor (Model JP-33, JPI).
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