Protein was extracted by T-PER tissue protein extraction reagent (Thermo, Rockford, IL) with protease inhibitor cocktail set III and phosphatase inhibitor cocktail set II (Millipore, Germany) according to the manufacturer’s protocol. Primary antibodies included GAPDH as loading controls (Proteintech, #60004-1-Ig), RAB17 (Proteintech, #17501-1-AP), RAB34 (SANTA, #SC376898), HA-tag (CST, #3724), CDK2 (CST, #2546), CyclinB1 (Proteintech, 55004-1-AP), PD-L1 (CST, #13684), PD-L2 (Abcam, #ab187662).
Pd l2
Manufactured by Abcam
Sourced in United States
PD-L2 is a protein that serves as a ligand for the programmed cell death 1 (PD-1) receptor. It plays a role in the regulation of the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Pd l1, by Abcam (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail set 3, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail set 2, by Merck Group (1 mentions)
Rab17, by Proteintech (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Cyclin b1, by Proteintech (1 mentions)
Tgf β, by Abcam (1 mentions)
Human pd l1 elisa kit, by Abcam (1 mentions)
Vimentin, by Signalway Antibody (1 mentions)
N cadherin, by Signalway Antibody (1 mentions)
Mouse antibody against β actin, by Proteintech (1 mentions)
Anti rabbit or anti mouse secondary antibody, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Histone h3, by Proteintech (1 mentions)
Anti β actin antibody, by Thermo Fisher Scientific (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Il 17, by BioLegend (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
6 protocols using pd l2
1
Protein Extraction and Western Blot
2
Comprehensive Tumor Immunomodulatory Protein Analysis
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E-cadherin (Signalway Antibody 48801), N-cadherin (Signalway Antibody 48779), Vimentin (Signalway Antibody 38104), Tubulin (Signalway Antibody 48885), Galectine 9 (Abcam ab153673), CEACAM1 (CD66) (Abcam ab108397), PD-L2 (Abcam ab256386), Vista (Abcam ab230950), IκBα (pS32) (Cell Signaling #2859), IκBα (Cell Signaling #9242), RelA(pS536) (Cell Signaling #3033), TGFβ (Abcam ab92486), TGFβ inhibitor (LY-364947, MCE, USA), NFκB inhibitor (BAY11, MCE, USA). PD-L1 was measured using the Human PD-L1 ELISA kit (Abcam ab214565).
3
Western Blot Analysis of Immune Markers
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Proteins were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer and boiled for denaturation. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed on samples containing 30 µg of protein using a 10% polyacrylamide gel. After electrotransfer and blocking with 3% bovine serum albumin, the membranes were incubated overnight at 4 °C with primary antibodies. Rabbit antibodies against TFEB, HLA-A, GAPDH, Histone-H3 (1:2000; Proteintech, China), PD-L1, PD-L2 (1:5000; Abcam, USA), and mouse antibody against β-actin (1:5000; Proteintech) were used as primary antibodies. After incubating with anti-rabbit or anti-mouse secondary antibody (1:5000; Proteintech) for 60 min at room temperature, enhanced chemiluminescence (ECL, NJ, USA) was used to visualize the bands.
4
Multiparameter Immunophenotypic Analysis
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Anti-CD34, CD44, CD45, CD73, Sca-1, CD90, CD105, and 7AAD antibodies were purchased from BD Biosciences. Anti- CD3, CD8, CD4, CD25, FOXP3, IL17, Annexin V, IFN- γ, and IL2 antibodies were purchased from BioLegend. Anti-Runx2, GAPDH, MCP-1, AKT, p-AKT, Erk, p-Erk, NF-κB p65, p-NF-κB p65, p-IKKα/β, and TNF-α antibodies were purchased from Cell Signaling Technology. Anti-ALP, CD3, PD-L1, PD-L2, and NEMO antibodies, were purchased from Abcam. CFSE cell division tracker Kit, anti-β-actin antibody, Lipofectamine™ RNAiMAX transfection reagent, and trypan blue were purchased from Invitrogen. Anti-PPAR-γ, OCN and Fas-L antibodies were purchased from Santa Cruz. Hoechst 33342 was purchased from Sigma. The EDU cell proliferation assay kit was purchased from KeyGEN Biotech. CCK8 Kit was purchased from DOJINDO.
5
Flow Cytometry Analysis of Immune Markers
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After cordycepin treatment, 4NAOC-1 cells were washed with PBS, fixed, permeabilized with 0.1% triton x-100, blocked with anti-CD16/32 monoclonal antibody (eBioscience, CA, USA), and stained with IL-17RA (eBioscience, CA, USA), PD-L1 (Biolegend, CA, USA), and PD-L2 (Abcam, Cambridge, UK) monoclonal antibodies for 2 hours at room temperature. Subsequently, cells were analyzed with flow cytometry (Beckman Coulter FC500, CA, USA), and results were plotted with Flowing Software 2.5.1 (Cell Imaging Core, Turku Centre for Biotechnology).
6
Quantitative Immunoblotting Analysis of Immune Checkpoint Proteins and Metabolic Regulators
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Whole-cell lysates of A498, 786-O or CTLs isolated from co-culture experiments were prepared using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Roche). The protein concentration was quantified using a BCA protein assay kit (ThermoScientific) and twenty microgram of total protein per lane was used for immunoblotting. The blots were probed with primary antibodies against PD-L1 (Abcam), PD-L2 (Abcam) and phospho-MTOR, MTOR, phospho-p70S6K, p70S6k, phosphor-fructokinase, hexokinase, pyruvate kinase, phospho-LDHA, LDHA, pyruvate dehydrogenase and beta-actin (all from Cell Signaling). Immunoreactivity was revealed with an ECL-Plus kit (ThermoScientific) using the ChemiDoc MP system (Bio-Rad).
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