ELISA kits for human TGFβ1, PD-L1 (R&D Systems, UK) and NmU (Peninsula Laboratories, CA) were used according to the manufacturer's instructions. For analysis of conditioned media, HCC1954, SKBR3 and EFM192A cell variants were seeded at 1 × 105 cells/well in a 24-well plate and left to attach overnight. The following day, cells were washed and fed with serum-free media and incubated for a further 72hr. Supernatant was then collected and analyzed immediately or stored at −80°C until analysis. Results were normalized by total cell protein contained in the well. 10µg of protein from lysed or intact EVs, as stated, were tested per well.
Pd l1
Manufactured by R&D Systems
Sourced in United States, United Kingdom
PD-L1 is a protein that plays a role in the regulation of the immune system. It is expressed on the surface of various cell types, including tumor cells, and can interact with the PD-1 receptor on T cells, leading to the suppression of T cell activity. PD-L1 is an important target in cancer immunotherapy.
Automatically generated - may contain errors
Lab products found in correlation
Pd l2, by R&D Systems (4 mentions)
P erk, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Granzyme b, by Abcam (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
P38 mapk, by Cell Signaling Technology (2 mentions)
S6 ribosomal protein, by Cell Signaling Technology (2 mentions)
Desmin, by Thermo Fisher Scientific (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
β tubulin, by Merck Group (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Ripk3, by Novus Biologicals (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
P nf κb, by Abcam (1 mentions)
P mapk, by Cell Signaling Technology (1 mentions)
P mtor, by Merck Group (1 mentions)
P stat5, by Abcam (1 mentions)
25 protocols using pd l1
1
Quantification of secreted factors
2
Western Blot Analysis of Signaling Pathways
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Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF). Proteins (20-40 μg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking procedure, membranes were incubated with primary antibodies (1:1000), HRP-conjugated secondary antibodies (1:5000), and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, 34095). Antibodies used were; PD-L1 (R&D, MAB1086), p-JAK1 (pY1022, Assay Biotech, A7125), p-JAK2 (pY1007 + 1008, Abbomax, 601-670), p-MAPK (cell Signaling, 9101S), p-Erk (Cell Signaling, 4695), p-MEK (Ser 217/221, Cell Signaling, 9121), p-Akt (S473, Cell Signaling, 9271), and p-Stat3 (pY705, Abcam, ab76315), p-Stat1 (pY701, Abbomax, 620-160), p-Stat5 (Abcam, pY694, ab32364), p-NFκB (S536, Abcam ab86299), p-mTOR (Millipore, 09-345), JAK1 (Abgent, AP20699a), JAK2 (Abgent, AP20700c), Stat1 (Abgent, AP19835b), MEK (Ameritech, ATB-T2715), MAPK (Signalway, 21245), ERK (Ameritech, ATB-T5371), Akt (Santa Cruz, sc5298) and GAPDH (Cell Signaling, 2118S).
3
Immunoblotting for PD-L1, PD-L2, and Signaling Molecules
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Cells were treated as described and then lysed in Strawn buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.2% Triton × 100 and 10% glycerol) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany), sonicated and boiled for 5 min, and separated on SDS-polyacrylamide gels. Consecutively, proteins were immunoblotted on a PVDF membrane. The membrane was blocked in blocking buffer (TBS [pH 7.6], 0.1% Tween-20 and 5% nonfat dry milk) for at least 3 h at 4 °C, followed by incubation with the primary antibody (PD-L1 and PD-L2 both from R&D Systems, Wiesbaden, Germany) in TBS (pH 7.6), 0.05% Tween-20 and 5% BSA. Bound primary antibodies were detected using rabbit anti-goat IgG-horseradish peroxidase conjugates (Dako, Frankfurt, Germany) and visualized with the LumiGlo detection system (CST, Frankfurt, Germany). In the case of PD-L2 a tertiary antibody (anti-rabbit IgG-HRP, CST) was used to enhance the signal strength. Equal loading was controlled by anti-GAPDH or anti-actin (Santa Cruz, Biotechnology, Heidelberg, Germany). In order to detect signaling molecules, the following antibodies were used: p-STAT-1 (Tyr701), STAT-1, p-STAT-2 (Tyr 690), STAT-2, p-STAT-3 (Tyr705), STAT-3, p-JAK-1 (Tyr1022/1023), JAK-1, p-JAK-2 (Tyr1008), JAK-2 and IRF-1, all from CST.
4
Cultivation and Antibody Analysis of Cancer Cell Lines
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The cancer cell lines B16F10, DBT, 4 T1, LLC and LM8 were obtained from American Type Culture Collection (Rockville, MD, USA). B16F10, DBT, 4 T1, LLC and LM8 cells were cultured in Dulbecco modified Eagle medium (DMEM)/F12 (Sigma) supplemented with 10% fetal bovine serum and 10 U/ml penicillin and streptomycin (Life Technologies, Grand Island, NY) at 37°C in 5% CO2. Cells were detached using 1 mM EDTA in phosphate-buffered saline solution (PBS) and used for further experiment. Antibodies used for western blotting included PD-L1 (1:1000, R&D Systems, Minneapolis, MN), p-p38 and β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX). Antibodies for flow cytometry, including pp38, pERK, pJNK pAKT, pmTOR, pp70-S6K, pSTAT1, pSTAT3, pSTAT4 and pSTAT5, were purchased from Cell Signaling Technology.
5
Immunohistochemical Analysis of Immune Markers
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After fixing the tissues in 10% formalin followed by paraffin embedding, the FFPE tissue sections were washed with xylene (3 times) and rehydrated with a gradient of ethanol (twice in 100%, 90%, 80%, and 70%). The tissue sections were heated in antigen retrieval buffer (AR9) in a boiling water bath for 12 min for antigen retrieval and then incubated in blocking buffer (5% BSA in PBS with 0.4% Triton-X (PBST)) for 30 min. Slides were incubated in primary antibodies: 1:30 Cep55 (Santa Cruz, Santa Cruz, CA, USA), 1:200 CD8 (Abcam, Cambridge, UK), 1:100 CD4 (Novus Biologicals, Centennial CO, USA), 1:50 FOXP3 (Biotechne), 1:50 TCF1/7 (Cell Signaling, Danvers, MA, USA), Granzyme B (Abcam, Cambridge, UK), 1:100 PD-L1(R&D Systems, Minneapolis, MN, USA), and 1:200 CD3 (Abcam, Cambridge, UK) overnight at 4 °C. The following day, the slides were washed with PBST and incubated in secondary antibodies conjugated with fluorescence for 1 h at room temperature. The slides were washed, mounted with Prolong gold antifade mountant with DAPI, and imaged.
6
Comprehensive Antibody Panel for EGFR Signaling
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The following antibodies were used: Flag (F3165; Sigma-Aldrich, St Louis, MO, USA; 1:5,000), Myc (11667203001; Roche Diagnostics, Indianapolis, IN, USA; 1:5,000), HA (11666606001; Roche Diagnostics; 1:5,000), PD-L1 (13684; Cell Signaling Technology, Danvers, MA, USA; 1:1,000), PD-L1 (329702; BioLegend; 1:1,000), PD-L1 (GTX117446; GeneTex, Irvine, CA, USA; 1:1,000), PD-L1 (AF156; R&D Systems, Minneapolis, MN, USA; 1:1,000), PD-1 (ab52587; Abcam, Cambridge, MA, USA; 1:2,000), Granzyme B (ab4059; Abcam; 1:1,000), EGFR (4267; Cell Signaling Technology; 1:1,000), GSK3β (9315; Cell Signaling Technology; 1:1,000), phospho-GSK3β Ser 9 (9336; Cell Signaling Technology; 1:1,000), β-TrCP (4394; Cell Signaling Technology; 1:1,000), α-Tubulin (B-5-1-2; Sigma-Aldrich; 1:5,000), β-Actin (A2228; Sigma-Aldrich; 1:10,000). EGF, cycloheximide and TM were purchased from Sigma-Aldrich. Gefitinib, erlotinib, lapatinib, cetuximab and AG1478 were obtained from Calbiochem Corp (Billerica, MA, USA).
7
Western Blot Analysis of Cellular Proteins
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Cells were lysed in a modified lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 10 nM calyculin A, 1 mM Na3VO4, and protease inhibitors), followed by SDS-PAGE and western blotting analysis as previously described55 (link). The primary antibodies against STAG2 (Santa Cruz, SC-81852), IRF9 (Cell Signaling, #76684), IRF7 (Cell Signaling, #4920), USP18 (Cell Signaling, #4813), ISG15 (Santa Cruz, SC-166755), IRF1 (Cell Signaling, #8478), IRF3 (Cell Signaling, #11904), GAPDH (Cell Signaling, #2118), GAPDH (Cell Signaling, #51332), STAG1 (Novus Biologicals, NB100-298), PD-L1 (R&D Systems, AF156), CTCF (Cell Signaling, #2899), and Flag M2 (Sigma, F3165) were used. All primary antibodies were used at a 1:1,000 dilution, with the exception of GAPDH (1:3000) and PD-L1 (1:200).
8
Immunohistochemical Profiling of Immune Markers
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IHC was performed on formalin-fixed, paraffin-embedded tissue sections. Serial sections (5-μm thick) were dewaxed and rehydrated. Heat-induced antigen retrieval was followed by endogenous peroxidase activity blockade with hydrogen peroxide. The sections were incubated overnight at 4°C with the following primary antibodies: CD207 (1:50; Atlas Antibodies), VE1 (1:50; Spring Bioscience), FOXP3 (1:75; Abcam), and PDL1 (1:50; R&D Systems). For antigen visualization, a peroxidase-labeled secondary antibody (EnVision/HRP system; DAKO) was used. Subsequently, the sections were rinsed in the kit buffer and immersed in diaminobenzidine stain. The expression of GATA3 (1:100; Cell Signaling Technology) and T-bet (1:50; Abcam) was performed via double IHC staining using DouMaxVision immunohistochemical double dye kits (KIT-9998; Maixin Biotech) according to the manufacturer's instructions, with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as the blue-black chromogen for alkaline phosphatase and 3-amino-9-ethylcarbazole as the red chromogen for horseradish peroxidase. The sections were counterstained with Harris' hematoxylin and then mounted. For the negative controls, phosphate-buffered saline was used instead of primary antibody.
9
Western Blot Analysis of Phospho-STAT3
10
Immunomodulatory Evaluation of PD-1 Axis
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AML-12 cells (Lot#: HTX2091) and MC38 cells (Lot#: HYC0116) as a kind of mouse hepatocytes and mouse colon cancer cells, respectively, were both bought from Shanghai Bangjing Industrial Co., Ltd (Shanghai, China). Pembrolizumab was bought from Selleck Chemicals LLC (Texas, USA). Human IL-2, IFN-γ ELISA kits, recombinant PD-1, PD-L1 and PD-L2 were purchased from R&D System (Minnesota, USA). C57BL/6 or NOD/SCID mice, human PD-1 (hPD-1) knock-in (C57BL/6 background) mice were bought from Shanghai Model Organisms Science and Technology Co., Ltd. (Shanghai, China). All the animal studies were approved by the Animal Use and Care Committee.
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