cGAS antibody (D1D3G, # 15102, RRID:AB_2732795), PBRM1 antibody (D3F7O, #91894, RRID:AB_2800173), IRF-3 antibody (D83B9, #4302, RRID:AB_1904036), phospho-Histone H2A (Ser139, 20E3, #9718, RRID:AB_2118009), phospho-ATR antibody (Ser428, #2853, RRID:AB_2290281), cleaved Caspase-3 antibody (Asp175, 5A1E, #9664, RRID:AB_2070042), CHK1 antibody (2G1D5, #2360, RRID:AB_2080320), phospho-CHK1 antibody (Ser345, 133D3, #2348, RRID:AB_331212), phospho-CHK1 (Ser296, D3O9F, #90178,RRID:AB_2800153), CD11C antibody (D1V9Y, #97585, RRID:AB_2800282), CD4 antibody (D7D2Z, 25229, RRID:AB_2798898), CD8 antibody (D4W2Z, 98941, RRID:AB_2756376), PD-1 (D7D5W, 84651, RRID:AB_2800041), PD-L1 antibody (E1L3N; 13684, RRID:AB_2687655), and PD-L1 antibody (D5V3B, 64988, RRID:AB_2799672), Rad50 antibody (3427, RRID:AB_2176936) and phospho-Rad50 antibody (Ser635, 14223, RRID:AB_2798430) were from Cell Signaling Technology. SETD2 antibody (HPA042451, RRID:AB_10806239) was purchased from Sigma-Aldrich. Phospho-IRF3 (S386, EPR2346, ab76493, RRID:AB_1523836) and phospho-ATM (S1981, EP1890Y, ab81292 , RRID:AB_1640207) were purchased from Abcam. The GAPDH antibody (6C5, sc32233, RRID:AB_627679) was purchased from Santa Cruz Biotechnology. VE822 (S7102) and Prexasertib HCl (LY2606368, S7178), were purchased from Selleck Chemicals.
Pd l1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The PD-L1 antibody is a laboratory reagent used in research applications. It is a specific antibody that binds to the programmed death-ligand 1 (PD-L1) protein, which is a key regulator of the immune response. The core function of this antibody is to detect and quantify the presence of PD-L1 in biological samples, enabling researchers to investigate its role in various cellular processes and disease states.
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Lab products found in correlation
Pbrm1 antibody, by Cell Signaling Technology (1 mentions)
Cgas antibody, by Cell Signaling Technology (1 mentions)
Collagenase, by Merck Group (1 mentions)
Red blood cell lysis buffer, by BioLegend (1 mentions)
Cd11b, by BioLegend (1 mentions)
Hrp linked anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ihc kit, by Absin (1 mentions)
Axio imager m1, by Zeiss (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
β catenin 51067 2 ap, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh hrp 60004, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Anti p erk, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti erk, by Santa Cruz Biotechnology (1 mentions)
Ly294002, by Merck Group (1 mentions)
Stattic, by Merck Group (1 mentions)
11 protocols using pd l1 antibody
1
Comprehensive Immune Checkpoint Antibody Panel
2
Immunohistochemical Analysis of PD-L1 Expression
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To identify PD-L1 expression in tumor tissue, formalin-fixed sections underwent deparaffinized, rehydration, and an antigen retrieval process and then were stained using the PD-L1 antibody (Cell Signaling, USA) at 4 °C for 24 h. Next, the secondary antibody, horseradish peroxidase anti-rabbit IgG antibody, was applied to stain the section. Finally, 3,3’-diaminobenzidine staining was performed on sections by using DAB Chromogen Concentrate (Biolegend, USA). The sections were observed through microscopy (Olympus/BX43).
3
PD-L1 Expression in RB1-Deficient Cells
4
Western Blot Analysis of PD-L1 Expression
5
Evaluation of IRF4 and PD-L1 Expression in Diffuse Large B-Cell Lymphoma
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We purchased human diffuse large B lymphoma tissue Chip array (OD-CT-LY02-001) from Shanghai Outdo Biotech Co. Ltd. Od-CT-LY02-001 Chip array was purchased from Shanghai Outdo Biotech Co., Ltd. It included 30 cases from different parts of the digestive tract and different DLBCL (stomach, small intestine, cecum, colon, etc.). The webpage link of this array is (https://www.superchip.com.cn/biology/tissue.html ). IRF4 antibody (Cat. No. 62834, Cell Signaling Technology) and PD-L1 antibody (Cat. No. 13684, Cell Signaling Technology) were stained by IHC to detect the expression of IRF4 and PD-L1 (1 : 50 dilution) [16 (link)]. Briefly, 4 μm of tissue array sections were blocked with dehydrated peroxidase. Antigen recuperation was executed at 0.01 mol/L in citrate buffer and autoclaved. The primary antibody was added and incubated overnight at 4°C. Following washes with phosphate-buffered saline (PBS) and incubation with a labeled polymer-HRP second antibody for 30 min, 3, 3-diaminobenzidine tetrachloride (DAB) was applied to initiate the colorimetric reaction. Slides were restained with hematoxylin. The stained slides were observed by microscopy to obtain images. IHC scoring was also performed separately to analyze the correlation between IRF4 and PD-L1. IHC kit was purchased from Absin (Cat. No. abs957).
6
PD-L1 Expression in Breast Cancer Cells
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MDA-MB231 or MCF-7 cells were seeded 30000 cells/well. PD-L1 protein expression was evaluated after 48 hours of treatment with vorinostat (1.5μM). Cells were stained for PD-L1 antibody (red, Cell signaling Technology cod.#86744) according to the manufacturer’s instructions. After secondary antibody incubation (Alexa Fluor 555, Invitrogen cod.A31572), slides were mounted with a DAPI (blue) mounting media (ProLong® Gold Antifade Mountant with DAPI, Life Technologies). Slides were next analyzed by microscopy (Zeiss AxioImager M1, Zeiss). Representative images show PD-L1+ cells with 20x and 40x magnification.
7
Flow Cytometric Immunophenotyping Protocol
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Flow cytometry analysis was done by staining cells with fluorescently-conjugated antibodies to HLA-A2 (Novus Biologicals, NBP1-44896AF488), HLA-B7 (Novus Biologicals, NB100-64159APCCY7), HLA-C (Novus Biologicals, NBP2-50419 PE), a PE-conjugated W6/32 antibody (Santa Cruz Biotechnology, sc-32235) to detect MHC1, a PE-conjugated antibody to detect EGFR1 (Invitrogen, MA5-28544), a PE-conjugated CD119 antibody to detect IFNGR1 (Miltenyi Biotec, 130-125-851), or a PE-conjugated extracellular domain specific PD-L1 antibody (Cell Signaling Technology, 71391). Each sample of cells was obtained from one well of a 12-well dish. The cells were washed using PBS and then stained for 30 min (in darkness at 4°C) using 100 μL of a 1:100 dilution of antibody in FACS buffer solution containing 1%BSA/PBS/0.05% sodium azide. Following staining, cells were washed and then resuspended in 400 μL of FACS buffer solution containing 1%BSA/PBS/0.05% sodium azide. Cells were then visualized using a Fortessa-SORP using gates to exclude for dead cells/debris and doublets (Supplementary Figures S2A, S2B ).
8
Investigating Sintilimab's Effects on TNBC Cells
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MDA-MB-231 and 4T1 cells were purchased from Beijing Union Medical College Hospital Cell Resource Sharing Platform, SUM159 cells were a gift from Dr. Jianjun He. MDA-MB-231 was cultured in L-15 medium, SUM159 was cultured in DMEM/F-12 medium, and 4T1 was cultured in RPMI 1640 medium. All cell lines were stored in a thermostat incubator at 37 °C, 5 % CO2.
MDA-MB-231 and SUM159 were prepared extracting proteins after adding sintilimab at a concentration gradient of 0.5, 1, 2, and 4 μg/mL following with incubating for 24 h, and then, western blotting assay were conducted. ALDH1A1 antibody was purchased from Abcam (ab52492), PD-L1 antibody was purchased from Cell Signaling Technology (#13684), β-catenin (51067-2-AP) and GAPDH (HRP- 60004) antibodies were from Proteintech. Whole cell lysates were prepared with RIPA buffer, proteins were separated by SDS/PAGE gel, transferred to PVDF membrane (Millipore), incubated with primary antibody overnight, followed by HRP-conjugated secondary antibody (Proteintech, SA00001-2) and detected chemiluminescent signals.
MDA-MB-231 and SUM159 were prepared extracting proteins after adding sintilimab at a concentration gradient of 0.5, 1, 2, and 4 μg/mL following with incubating for 24 h, and then, western blotting assay were conducted. ALDH1A1 antibody was purchased from Abcam (ab52492), PD-L1 antibody was purchased from Cell Signaling Technology (#13684), β-catenin (51067-2-AP) and GAPDH (HRP- 60004) antibodies were from Proteintech. Whole cell lysates were prepared with RIPA buffer, proteins were separated by SDS/PAGE gel, transferred to PVDF membrane (Millipore), incubated with primary antibody overnight, followed by HRP-conjugated secondary antibody (Proteintech, SA00001-2) and detected chemiluminescent signals.
9
Immunomodulation Pathway Analysis
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CXCL9/10/11 were purchased from Pepro Tech (USA). The PD-L1 antibody was purchased from Cell Signalling Technology (USA). The CXCR3 antibody was purchased from ABCAM (USA). The CXCR3 antagonist AMG487 was purchased from Tocris (USA). The PI3K/AKT inhibitor LY294002 and the STAT3 inhibitor STATTIC were obtained from Sigma (St. Louis, MO, USA). The anti-STAT3, anti-pSTAT3, anti-Akt, anti-pAkt, anti-ERK, anti-pERK, and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (USA).
10
Analyzing Cell Apoptosis and Phenotypes
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MDA-MB-231 and BT549 cells were transfected with siRNF31 or siControl. 48 hours after transfection. For apoptosis analysis, the cells were treated following the Apoptosis Detection Kit (A211–01, Vazyme). For CD44/CD24 analysis, the cells were digested and then washed with PBS (with 1% FBS) for 3 times, then resuspension in 100 μl PBS, and then stained with anti-CD44-PE (BD, 1:500) and anti-CD24-FITC (BD, 1:500). The samples were then washed by PBS 3 times and finally re-suspended in 400 μl PBS. For membrane PD-L1 analysis, the cells were digested and stained with PD-L1 antibody according to PD-L1 antibody (13,684, Cell Signaling Technology). The CD45 analysis was performed on tumor tissue samples from mice after they were washed with PBS, minced, and treated with a digestive solution containing 95% RPMI 1640, 2% FBS, 1% collagenase IV, and 1% DNase I and 1% Dispase II at 37 °C. Then, single cells were centrifuged (200 rpm for 30 min) and stained with anti-mouse CD45 eFluor 450 (B220, 48–0452-82). The data was analyzed using a Beckman FACS flow cytometer.
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