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).
Pd l2
Manufactured by ProSci
The PD-L2 is a lab equipment product offered by ProSci. It is a protein that functions as a ligand for the PD-1 receptor, which is involved in the regulation of the immune system.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Acetyl alpha tubulin, by Cell Signaling Technology (1 mentions)
Lf pvdf, by Bio-Rad (1 mentions)
Galectin 9, by Abcam (1 mentions)
Icosl, by Abcam (1 mentions)
C600 imaging system, by Azure Biosystems (1 mentions)
Pd l1, by ProSci (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Bioruptor, by Diagenode (1 mentions)
2 protocols using pd l2
1
Western Blot Immunodetection of Immune Checkpoint Proteins
2
Exosome-mediated T-cell modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Purified exosomes from OSP2 cells (without or with inhibitor treatment, Manumycin A (BioViotica, 250 nM in DMSO) were mixed (1:1 (∼20 ug)) with positively selected (StemCell) CD3+ T cells activated with PMA/Ionomycin activation cocktail (1x, BioLegend) from healthy donors (HumanCellsBio) for 24 h. Where indicated, exosomes were pre‐incubated with ICP blockade at 1:100 dilution (BTLA (Cat. 7473, ProSci); PD‐L2 (Cat. 4063, ProSci); LAG‐3 (Cat. 13485, Raybiotech). Exosomes from Jurkat cells were also tested as a control. Thereafter, cells were stained with a T‐cell functional marker cocktail (Table S2 ) and underwent multi‐colour flow cytometric analyses, as previously published (Manuel, Sehgal, Connolly et al., 2013 (link); Manuel, Sehgal, Khan et al., 2013 (link)). Data were analysed by the FlowJo as described above.
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