polyclonal anti gnly  (Atlas Antibodies)

Journal: Cells

Article Title: Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines

doi: 10.3390/cells13161310

Figure Lengend Snippet: Cytotoxic effector proteins in LGLL cells. Analysis of internal GrzB and Gnly in NK-92 ( A ), NKL ( B ), KHYG-1 ( C ) and MOTN-1 ( D ). Cells were mildly homogenized with a balch homogenizer and subjected to differential and density centrifugation as described. Whole cell lysate (WCL), enriched organelles (EO), crude lysosomal fraction (CLF), and the cytosol (CYT) were separated together with fractions 1–6 of the density gradient on Bis-Tris NuPAGE gels, transferred to nitrocellulose, and tested for the presence of LAMP-1 and GrzB with respective mab from BD Bioscience and BioLegend and HRP-conjugated anti-mouse IgG antibodies from Cytiva. Gnly was stained with a polyclonal goat anti-Gnly antibody from R&D followed by HRP-conjugated anti-goat IgG antibodies from Abcam.

Article Snippet: To this end, NP-40 lysates or supernatants were precleared with protein G-sepharose beads (Sigma-Aldrich) for 2 h and incubated overnight with 0.5 μg of the polyclonal anti-Gnly antibody (R&D Systems Inc., Minneapolis, MN, USA) and protein G-sepharose beads at 4 °C.

Techniques: Centrifugation, Staining

Journal: Cells

Article Title: Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines

doi: 10.3390/cells13161310

Figure Lengend Snippet: Colocalization of intracellular LAMP-1 with GrzB and the 15 kDa (RF10) or 9 kDa (pc) form of Gnly in NK-92 cells. Following fixation and permeabilization, cells were stained with FITC-conjugated anti-LAMP-1 mab. After washing, cells were additionally stained with PE-conjugated anti-LAMP-1 mab (as a positive control for colocalization), PE-conjugated anti-GrzB mab, or with anti-Gnly mab RF10 or a polyclonal anti-Gnly pab (pc) and appropriate Alexa Fluor 555-conjugated secondary antibodies. A total of 10,000 cells were acquired with an ImageStream Mark II imaging flow cytometer. Only focused, single cells were considered for further analyses. ( A ) Histograms display the geometric mean value of the BDS score of respective stainings and the percentage of cells displaying a BDS score >2. ( B ) Representative images of stained cells. Scale bars represent 7 µm.

Article Snippet: To this end, NP-40 lysates or supernatants were precleared with protein G-sepharose beads (Sigma-Aldrich) for 2 h and incubated overnight with 0.5 μg of the polyclonal anti-Gnly antibody (R&D Systems Inc., Minneapolis, MN, USA) and protein G-sepharose beads at 4 °C.

Techniques: Staining, Positive Control, Imaging, Flow Cytometry

Journal: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Establishment and Validation of the Ascore Prognostic Signature. A Multivariate Cox coefficients for four ARGs (CERCAM, EMP1, GNLY, PTPRR) in the prognostic signature. B Ascore distribution among BLCA patients, sorted from lowest to highest. C Survival status categorized by Ascore for each BLCA patient. D Heatmap displaying expression levels of four genes in different Ascore groups. E Sankey diagram correlating clusters, Ascore groups, and BLCA survival status. F Kaplan–Meier analysis comparing overall survival between high and low Ascore groups in BLCA ( P < 0.0001). G Receiver Operating Characteristic (ROC) curves depicting Ascore signature’s predictive performance for 1, 3, and 5-year overall survival in BLCA, with the Area Under the Curve (AUC) values of 0.709, 0.724, and 0.745, respectively. (H–K) Kaplan–Meier analysis and time-dependent ROC curves in two external validation sets: GSE32548 and GSE32894

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: Biomarker Discovery, Expressing

Journal: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Single-Cell RNA Sequencing Analysis of Ascore Distribution and Biological Significance in Bladder Cancer. A t-SNE plot showing seven main cell types distribution in the integrated dataset, with doublets manually annotated. B Dot plot of marker genes' expression levels in each cell type. C Ascore and four genes (CERCAM, EMP1, GNLY, PTPRR) expression and distribution across cell types. D t-SNE plot showing Ascore expression levels and patterns in each cell type. E Left plot: Six main epithelial cell subgroups visualized by t-SNE dimensionality reduction. Right plot: Ascore distribution and expression in epithelial cells, highlighting Subgroup0 and Subgroup2. F GO analysis of biological function differences between Subgroup0 and Subgroup2

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: RNA Sequencing, Marker, Expressing

Journal: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Ascore Predictive Capability for Anti-PD-1 Immunotherapy Response in Gulou-Cohort2. A Representative immunohistochemistry (IHC) images illustrating the expression of four key genes (CERCAM, EMP1, GNLY, PTPRR) in two patients from Gulou-Cohort2 (Scale bars = 100 μm). Patient 4, who responded to anti-PD-1 therapy, had a low Ascore, in contrast to non-responder Patient 7, who had a high Ascore. B Distribution of Ascores among different response groups (CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease; *** P < 0.001). C ROC curves comparing the predictive accuracy of Ascore (AUC = 0.913) versus PD-L1 expression in tumor-infiltrating immune cells (ICs) (AUC = 0.662). D Decision curve analysis (DCA) indicating the net benefit of using Ascore compared to evaluating ICs' PD-L1 expression. E Kaplan–Meier curves c showing a correlation between higher Ascore values in tissue samples and reduced survival rates ( P = 0.0194)

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: Immunohistochemistry, Expressing