capillary western blot analyses  (Protein Simple Inc)

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