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western blot analyses  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc western blot analyses
    Targeting CD47 with SRF231 induces phagocytosis and cell death in lymphoid malignant cells. A. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in Jurkat cells cocultured with hMDM ( n = 2). B. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in 11 CLL patient samples cocultured with hMDMs from 3 different donors (MAC1—MAC3). Reported P values were calculated by paired Student’s t test. C. Diagram showing gating strategy – Phagocytosis is identified as the CD14 + population with uptake of CFSE + lymphocytes. SRF231-mediated cell death is identified as the non-phagocytosed CD14 − cells with CFSE and Violet Live/Dead uptake. Violet Live/Dead (Zombie Violet™) is an amine-reactive fluorescent dye that stains cells that have compromised membranes as compared to AnnV stain that binds phosphatidylserine. Representative flow cytometry <t>analyses</t> showing the effect of hIgG4 isotype (10 µg/ml, top panel) and SRF231 (10 µg/ml, bottom panel) on phagocytosis and cell death of non-phagocytosed cells, cocultured with hMDMs. D. Population of non-phagocytosed Jurkat cells following treatment with either hIgG4 isotype or SRF231 were analyzed by flow cytometry. Tumor cell death denoted by % of death of non-phagocytosed cell population ( n = 2). E . Cells from 24 CLL patient samples were subjected to Protein G-bound SRF231 (10 µg/ml) and cell viability were measured by AnnV/Hoechst cell death assay. Decrease in percent live (AnnV-/Hoechst+) cells in SRF231 vs. hIgG4 isotype treatment was reported. Reported P values were calculated by paired Student’s t test. F. Healthy B cells (HB, n = 8), healthy T cells (HT, n = 8), healthy monocytes (HM, n = 3) and PBMC cells from CLL patients (CLL, n = 24) were subjected to Protein G-bound SRF231 or hIgG4 isotype treatment (10 µg/ml). Cell death induction was measured using AnnV/Hoechst assay at 0, 2, 4, 6 and 10 h, and data presented were % live cells of SRF231 normalized to their respective hIgG4 isotype. Reported P values were calculated by Sidak’s multiple comparison test. G-H. <t>Western</t> <t>Blot</t> showing CD47 and β-actin protein expressions in primary HB and CLL cells, Ri-1, Raji and Jurkat (Wild type and CD47 Knockout) cells. I. Cell death inductions of Ri-1 ( n = 3, 6 h; n = 4, 24 h), Jurkat ( n = 4) and Raji ( n = 4) cells were measured following Protein G-bound SRF231 incubation with AnnV/Hoechst assay at indicated timepoints. Reported P values were calculated by paired Student’s t test
    Western Blot Analyses, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/western+blot+analyses/Phospho-RIP+(Ser166)+Antibody/pmc12810019-36-5-14
    Average 95 stars, based on 230 article reviews
    western blot analyses - by Bioz Stars, 2026-11
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    1) Product Images from "CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies"

    Article Title: CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-025-01774-3

    Targeting CD47 with SRF231 induces phagocytosis and cell death in lymphoid malignant cells. A. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in Jurkat cells cocultured with hMDM ( n = 2). B. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in 11 CLL patient samples cocultured with hMDMs from 3 different donors (MAC1—MAC3). Reported P values were calculated by paired Student’s t test. C. Diagram showing gating strategy – Phagocytosis is identified as the CD14 + population with uptake of CFSE + lymphocytes. SRF231-mediated cell death is identified as the non-phagocytosed CD14 − cells with CFSE and Violet Live/Dead uptake. Violet Live/Dead (Zombie Violet™) is an amine-reactive fluorescent dye that stains cells that have compromised membranes as compared to AnnV stain that binds phosphatidylserine. Representative flow cytometry analyses showing the effect of hIgG4 isotype (10 µg/ml, top panel) and SRF231 (10 µg/ml, bottom panel) on phagocytosis and cell death of non-phagocytosed cells, cocultured with hMDMs. D. Population of non-phagocytosed Jurkat cells following treatment with either hIgG4 isotype or SRF231 were analyzed by flow cytometry. Tumor cell death denoted by % of death of non-phagocytosed cell population ( n = 2). E . Cells from 24 CLL patient samples were subjected to Protein G-bound SRF231 (10 µg/ml) and cell viability were measured by AnnV/Hoechst cell death assay. Decrease in percent live (AnnV-/Hoechst+) cells in SRF231 vs. hIgG4 isotype treatment was reported. Reported P values were calculated by paired Student’s t test. F. Healthy B cells (HB, n = 8), healthy T cells (HT, n = 8), healthy monocytes (HM, n = 3) and PBMC cells from CLL patients (CLL, n = 24) were subjected to Protein G-bound SRF231 or hIgG4 isotype treatment (10 µg/ml). Cell death induction was measured using AnnV/Hoechst assay at 0, 2, 4, 6 and 10 h, and data presented were % live cells of SRF231 normalized to their respective hIgG4 isotype. Reported P values were calculated by Sidak’s multiple comparison test. G-H. Western Blot showing CD47 and β-actin protein expressions in primary HB and CLL cells, Ri-1, Raji and Jurkat (Wild type and CD47 Knockout) cells. I. Cell death inductions of Ri-1 ( n = 3, 6 h; n = 4, 24 h), Jurkat ( n = 4) and Raji ( n = 4) cells were measured following Protein G-bound SRF231 incubation with AnnV/Hoechst assay at indicated timepoints. Reported P values were calculated by paired Student’s t test
    Figure Legend Snippet: Targeting CD47 with SRF231 induces phagocytosis and cell death in lymphoid malignant cells. A. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in Jurkat cells cocultured with hMDM ( n = 2). B. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in 11 CLL patient samples cocultured with hMDMs from 3 different donors (MAC1—MAC3). Reported P values were calculated by paired Student’s t test. C. Diagram showing gating strategy – Phagocytosis is identified as the CD14 + population with uptake of CFSE + lymphocytes. SRF231-mediated cell death is identified as the non-phagocytosed CD14 − cells with CFSE and Violet Live/Dead uptake. Violet Live/Dead (Zombie Violet™) is an amine-reactive fluorescent dye that stains cells that have compromised membranes as compared to AnnV stain that binds phosphatidylserine. Representative flow cytometry analyses showing the effect of hIgG4 isotype (10 µg/ml, top panel) and SRF231 (10 µg/ml, bottom panel) on phagocytosis and cell death of non-phagocytosed cells, cocultured with hMDMs. D. Population of non-phagocytosed Jurkat cells following treatment with either hIgG4 isotype or SRF231 were analyzed by flow cytometry. Tumor cell death denoted by % of death of non-phagocytosed cell population ( n = 2). E . Cells from 24 CLL patient samples were subjected to Protein G-bound SRF231 (10 µg/ml) and cell viability were measured by AnnV/Hoechst cell death assay. Decrease in percent live (AnnV-/Hoechst+) cells in SRF231 vs. hIgG4 isotype treatment was reported. Reported P values were calculated by paired Student’s t test. F. Healthy B cells (HB, n = 8), healthy T cells (HT, n = 8), healthy monocytes (HM, n = 3) and PBMC cells from CLL patients (CLL, n = 24) were subjected to Protein G-bound SRF231 or hIgG4 isotype treatment (10 µg/ml). Cell death induction was measured using AnnV/Hoechst assay at 0, 2, 4, 6 and 10 h, and data presented were % live cells of SRF231 normalized to their respective hIgG4 isotype. Reported P values were calculated by Sidak’s multiple comparison test. G-H. Western Blot showing CD47 and β-actin protein expressions in primary HB and CLL cells, Ri-1, Raji and Jurkat (Wild type and CD47 Knockout) cells. I. Cell death inductions of Ri-1 ( n = 3, 6 h; n = 4, 24 h), Jurkat ( n = 4) and Raji ( n = 4) cells were measured following Protein G-bound SRF231 incubation with AnnV/Hoechst assay at indicated timepoints. Reported P values were calculated by paired Student’s t test

    Techniques Used: Staining, Flow Cytometry, Comparison, Western Blot, Knock-Out, Incubation

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    Article Snippet: For Western blot, endothelium was treated with IFN-β (R&D Systems, Cat 8234-MB, 10 ng/mL) for 15 minutes, and protein samples were prepared and quantified, then loaded on a SDS-PAGE gels for separation and transferred to a PVDF membrane. .. The following antibodies were used for Western blot analyses purchased from Cell Signaling Technology: STING (CST, Cat 13647S), STAT1 (CST, Cat 14994S), P-STAT1(CST, Cat 9167S), JAK1 (CST, Cat 50996S), and P-JAK1 (CST, Cat 74129S); Protein bands were visualized by chemiluminescence using an ECL detection kit. .. For coimmunoprecipitation, endothelium treated with IFN-β (R&D Systems, Cat 8234-MB, 10 ng/mL) or not were collected, extracted, and quantified; Protein lysates were incubated with anti-STING antibody (CST, Cat 13647S, 2 μL) and rotated over night; Protein A beads (Thermo Scientific, Cat 20333, 10 μL) were added and incubated for another 2 hours.

    Article Title: Neat1 promotes acute kidney injury to chronic kidney disease by facilitating tubular epithelial cells apoptosis via sequestering miR-129-5p
    Article Snippet: The proteins from cultured cells or tissues were extracted using RIPAlysis buffer (Beyotime) with protease inhibitor (Sigma) and quantified with the bicinchoninic acid BCA protein quantitation kit (Thermo Fisher Scientific). .. Western blot analyses were conducted as described previously.16 The primary antibodies used were as follows: anti-FADD (2782S, Cell Signaling Technology, Danvers, MA, USA; AV30294, Sigma), anti-caspase-8 (4790S, Cell Signaling Technology), anti-cleaved caspase-8 (8592S and 9748S, Cell Signaling Technology), anti-caspase-3 (9662S, Cell Signaling Technology), anti-cleaved caspase-3 (9661S, Cell Signaling Technology), anti-caspase-7 (9492S, Cell Signaling Technology), anti-cleaved caspase-7 (8438S, Cell Signaling Technology), anti-Bax (5023S and 14796S, www.moleculartherapy.org Cell Signaling Technology), anti-Bcl2 (3498S, Cell Signaling Technology), anti-b-actin (4970S, Cell Signaling Technology), anti-p53 (2524S, Cell Signaling Technology), anti-phospho-p53 (Ser15) (9284S, Cell Signaling Technology), anti-fibronectin (F3648, Sigma), anti-collagen I (72026S, Cell Signaling Technology), anti-aSMA (alpha smooth muscle actin; 19245S, Cell Signaling Technology), anti-Rela (SAB5701125, Sigma), anti-E-cadherin (14472S, Cell Signaling Technology), anti-vimentin (5741S, Cell Signaling Technology), and anti-FSP-1 (13018S, Cell Signaling Technology). .. Western blot images were quantitatively analyzed using the ImageJ program (NIH, Bethesda, MD, USA).

    Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling.
    Article Snippet: .. Antibodies to the following proteins were used for Western blot analyses: pAKT (S473, CST #4060S; 1:1000), AKT (CST #4685S; 1:1000), pERK1/2 (CST #4370S; 1:1000), ERK1/2 (CST #4695S; 1:1000), GAPDH (CST #2118S; 1:000), Caveolin-1 (CST #3238S; 1:1000), Flotillin-1 (Abcam #ab41927; 1:500), GFP (Invitrogen #MA1-952; 1:1000), VEGFR2 (CST #2479L; 1:1000), pY1175-VEGFR2 (CST #2478S; 1:1000), 4G10 (CST #96215S; 1:1000), GRB2 (CST #3972S; 1:1000), and VEGFR3 (Santa cruz #SC321, 1:400). ..

    Article Title: Orally Bioavailable and Site-Selective Covalent STING Inhibitor Derived from a Macrocyclic Marine Diterpenoid.
    Article Snippet: Blot images were visualized by chemiluminescent substrate (Thermo Scientific, 34580) and imaged on a ChemiDoc MP imaging system (Bio-Rad). .. The antibodies used in this study at the following dilutions for Western blot analyses: Antiphospho-STING (#72971, 1:1000), anti-STING (#13647, 1:1000), antiphospho-TBK1 (#5483, 1:1000), anti-TBK1 (#3504, 1:1000), antiphospho-IRF3 S396(#29047, 1:1000), anti-IRF3 (#4302, 1:1000), anti-α-tubulin (#2144, 1:2000) were purchased from Cell Signaling Technology. .. The antibodies used in this study at the following dilutions for Western blot analyses: Antiphospho-STING (#72971, 1:1000), anti-STING (#13647, 1:1000), antiphospho-TBK1 (#5483, 1:1000), anti-TBK1 (#3504, 1:1000), antiphospho-IRF3 S396(#29047, 1:1000), anti-IRF3 (#4302, 1:1000), anti-α-tubulin (#2144, 1:2000) were purchased from Cell Signaling Technology.

    Article Title: CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies
    Article Snippet: RosetteSep Human Monocyte Enrichment Cocktail (Stem Cell Technologies, #15068), APC mouse anti-human CD14 (BD Biosciences, #555399), APC mouse anti-human CD206 (BD Biosciences, #550889), Magrolimab (ProSci, #10–950), mouse IgG (BD Biosciences, #554721), B6H12 (BD Biosciences, #556043), Recombinant human M-CSF (Life Technologies, #PHC9501), Cell trace CFSE (Life Technologies, # C34554 ), Zombie Violet fixable viability kit (Biolegend, #423113), Z-VAD-FMK (Santa Cruz Biotechnology, #sc-3067), BAPTA/AM (Santa Cruz Biotechnology, sc-202488), GSK’872 (Selleckchem, #S8465), Necrostatin-1 (Selleckchem, #S8037), Mounting medium with DAPI (ThermoFisher, # P36931 ), Alexa Fluor-488 anti-Rabbit IgG (Invitrogen, #A11034), Hoechst (Invitrogen, #H3570), Alexa Fluor 488-Annexin V (Life Technologies, #A13201). .. The primary antibodies used for Western blot analyses were as follows: RIPK1-phospho-Ser166 rabbit antibody (Cell Signaling Technology, #65746), RIPK1 rabbit antibody (Cell Signaling Technology, #3493), MLKL-phospho-ser358 rabbit antibody (Cell Signaling Technology, #91689), anti-MLKL rabbit antibody (Cell Signaling Technology, #14993), β-Actin mouse antibody (Santa Cruz Biotechnology, #sc-47778), caspase 3 rabbit antibody (Cell Signaling Technology, #9662), anti-LC3 rabbit antibody (Cell Signaling Technology, #4108), anti-ERK rabbit antibody (Cell Signaling Technology, #4695), anti-PGAM rabbit antibody (Abcam, #ab126534), anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, #7074), anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, #7076). .. The primary antibodies used for immunohistochemistry were as follows: MLKL-phospho-Ser358 rabbit antibody (Abcam, #ab187091), MLKL-phospho-Thr357 mouse antibody (Novus Biologicals, #MAB91871) and RIPK1-phospho-ser166 rabbit antibody (Thermofisher, #PA5-104645).

    Article Title: Endothelial STING-JAK1 interaction promotes tumor vasculature normalization and antitumor immunity
    Article Snippet: .. The following antibodies were used for Western blot analyses purchased from Cell Signaling Technology: STING (CST, Cat 13647S), STAT1 (CST, Cat 14994S), P-STAT1(CST, Cat 9167S), JAK1 (CST, Cat 50996S), and P-JAK1 (CST, Cat 74129S); Protein bands were visualized by chemiluminescence using an ECL detection kit. .. For coimmunoprecipitation, endothelium treated with IFN-β (R&D Systems, Cat 8234-MB, 10 ng/mL) or not were collected, extracted, and quantified; Protein lysates were incubated with antiSTING antibody (CST, Cat 13647S, 2 μL) and rotated over night; Protein A beads (Thermo Scientific, Cat 20333, 10 μL) were added and incubated for another 2 hours.

    other:

    Article Title: Orally Bioavailable and Site-Selective Covalent STING Inhibitor Derived from a Macrocyclic Marine Diterpenoid
    Article Snippet: Blot images were visualized by chemiluminescent substrate (Thermo Scientific, 34580) and imaged on a ChemiDoc MP imaging system (Bio-Rad).



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    Targeting CD47 with SRF231 induces phagocytosis and cell death in lymphoid malignant cells. A. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in Jurkat cells cocultured with hMDM ( n = 2). B. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in 11 CLL patient samples cocultured with hMDMs from 3 different donors (MAC1—MAC3). Reported P values were calculated by paired Student’s t test. C. Diagram showing gating strategy – Phagocytosis is identified as the CD14 + population with uptake of CFSE + lymphocytes. SRF231-mediated cell death is identified as the non-phagocytosed CD14 − cells with CFSE and Violet Live/Dead uptake. Violet Live/Dead (Zombie Violet™) is an amine-reactive fluorescent dye that stains cells that have compromised membranes as compared to AnnV stain that binds phosphatidylserine. Representative flow cytometry analyses showing the effect of hIgG4 isotype (10 µg/ml, top panel) and SRF231 (10 µg/ml, bottom panel) on phagocytosis and cell death of non-phagocytosed cells, cocultured with hMDMs. D. Population of non-phagocytosed Jurkat cells following treatment with either hIgG4 isotype or SRF231 were analyzed by flow cytometry. Tumor cell death denoted by % of death of non-phagocytosed cell population ( n = 2). E . Cells from 24 CLL patient samples were subjected to Protein G-bound SRF231 (10 µg/ml) and cell viability were measured by AnnV/Hoechst cell death assay. Decrease in percent live (AnnV-/Hoechst+) cells in SRF231 vs. hIgG4 isotype treatment was reported. Reported P values were calculated by paired Student’s t test. F. Healthy B cells (HB, n = 8), healthy T cells (HT, n = 8), healthy monocytes (HM, n = 3) and PBMC cells from CLL patients (CLL, n = 24) were subjected to Protein G-bound SRF231 or hIgG4 isotype treatment (10 µg/ml). Cell death induction was measured using AnnV/Hoechst assay at 0, 2, 4, 6 and 10 h, and data presented were % live cells of SRF231 normalized to their respective hIgG4 isotype. Reported P values were calculated by Sidak’s multiple comparison test. G-H. Western Blot showing CD47 and β-actin protein expressions in primary HB and CLL cells, Ri-1, Raji and Jurkat (Wild type and CD47 Knockout) cells. I. Cell death inductions of Ri-1 ( n = 3, 6 h; n = 4, 24 h), Jurkat ( n = 4) and Raji ( n = 4) cells were measured following Protein G-bound SRF231 incubation with AnnV/Hoechst assay at indicated timepoints. Reported P values were calculated by paired Student’s t test

    Journal: Journal of Hematology & Oncology

    Article Title: CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies

    doi: 10.1186/s13045-025-01774-3

    Figure Lengend Snippet: Targeting CD47 with SRF231 induces phagocytosis and cell death in lymphoid malignant cells. A. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in Jurkat cells cocultured with hMDM ( n = 2). B. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in 11 CLL patient samples cocultured with hMDMs from 3 different donors (MAC1—MAC3). Reported P values were calculated by paired Student’s t test. C. Diagram showing gating strategy – Phagocytosis is identified as the CD14 + population with uptake of CFSE + lymphocytes. SRF231-mediated cell death is identified as the non-phagocytosed CD14 − cells with CFSE and Violet Live/Dead uptake. Violet Live/Dead (Zombie Violet™) is an amine-reactive fluorescent dye that stains cells that have compromised membranes as compared to AnnV stain that binds phosphatidylserine. Representative flow cytometry analyses showing the effect of hIgG4 isotype (10 µg/ml, top panel) and SRF231 (10 µg/ml, bottom panel) on phagocytosis and cell death of non-phagocytosed cells, cocultured with hMDMs. D. Population of non-phagocytosed Jurkat cells following treatment with either hIgG4 isotype or SRF231 were analyzed by flow cytometry. Tumor cell death denoted by % of death of non-phagocytosed cell population ( n = 2). E . Cells from 24 CLL patient samples were subjected to Protein G-bound SRF231 (10 µg/ml) and cell viability were measured by AnnV/Hoechst cell death assay. Decrease in percent live (AnnV-/Hoechst+) cells in SRF231 vs. hIgG4 isotype treatment was reported. Reported P values were calculated by paired Student’s t test. F. Healthy B cells (HB, n = 8), healthy T cells (HT, n = 8), healthy monocytes (HM, n = 3) and PBMC cells from CLL patients (CLL, n = 24) were subjected to Protein G-bound SRF231 or hIgG4 isotype treatment (10 µg/ml). Cell death induction was measured using AnnV/Hoechst assay at 0, 2, 4, 6 and 10 h, and data presented were % live cells of SRF231 normalized to their respective hIgG4 isotype. Reported P values were calculated by Sidak’s multiple comparison test. G-H. Western Blot showing CD47 and β-actin protein expressions in primary HB and CLL cells, Ri-1, Raji and Jurkat (Wild type and CD47 Knockout) cells. I. Cell death inductions of Ri-1 ( n = 3, 6 h; n = 4, 24 h), Jurkat ( n = 4) and Raji ( n = 4) cells were measured following Protein G-bound SRF231 incubation with AnnV/Hoechst assay at indicated timepoints. Reported P values were calculated by paired Student’s t test

    Article Snippet: The primary antibodies used for Western blot analyses were as follows: RIPK1-phospho-Ser166 rabbit antibody (Cell Signaling Technology, #65746), RIPK1 rabbit antibody (Cell Signaling Technology, #3493), MLKL-phospho-ser358 rabbit antibody (Cell Signaling Technology, #91689), anti-MLKL rabbit antibody (Cell Signaling Technology, #14993), β-Actin mouse antibody (Santa Cruz Biotechnology, #sc-47778), caspase 3 rabbit antibody (Cell Signaling Technology, #9662), anti-LC3 rabbit antibody (Cell Signaling Technology, #4108), anti-ERK rabbit antibody (Cell Signaling Technology, #4695), anti-PGAM rabbit antibody (Abcam, #ab126534), anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, #7074), anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, #7076).

    Techniques: Staining, Flow Cytometry, Comparison, Western Blot, Knock-Out, Incubation