anti ciap1 polyclonal antibody  (R&D Systems)

Journal: Pharmacology Research & Perspectives

Article Title: The preclinical pharmacokinetics of Tolinapant—A dual cIAP1/XIAP antagonist with in vivo efficacy

doi: 10.1002/prp2.70030

Figure Lengend Snippet: Measurement of relative cIAP1 levels by MSD assay in PBMC lysates prepared at the indicated time after the indicated dose of tolinapant. PBMC pellets were prepared for pharmacodynamic analysis from whole blood by density gradient centrifugation (Histopaque). PBMC lysates were prepared in Triton‐X100 lysis buffer and normalized by total protein assay (BCA assay). Equivalent amounts of total protein (100 μg) were analyzed by immunoassay (MSD platform) using biotinylated and sulfo‐tagged anti‐cIAP1 capture and detection antibodies.

Article Snippet: After washing, detection of levels of cIAP1 bound was achieved by incubating the plates with sulfo‐tagged anti‐cIAP1 polyclonal antibody (R&D Systems, cat no. AF8181) for 2 h at room temperature with shaking, followed by washing and reading the plates in the presence of MSD read buffer.

Techniques: Gradient Centrifugation, Lysis, BIA-KA

Journal: bioRxiv

Article Title: Inhibition of IAPs induces programmed cell death and inflammatory signaling in patient-derived metastatic breast cancer organoids

doi: 10.1101/2024.08.28.610103

Figure Lengend Snippet: (A) hMOs were grown for three to seven days in growth medium followed by 24 h treatment with 10 µM of BV6, Birinapant (Bi), ASTX-660 (ASTX) and LCL-161 (LCL), respectively, or DMSO as vehicle control. Subsequently, CellTiter-Glo® viability assays were performed and the values were normalized to non-treated controls. Error bars represent standard deviation. * p < 0.05, ** p< 0.01, *** p < 0.005. (B) hMOs, treated as in (A) were harvested and Western blotting was performed with antibodies recognizing cIAP1, cIAP2, XIAP as well as total and cleaved caspase-3. Vinculin served as loading control. Representative blots of three independent experiments are shown.

Article Snippet: The following antibodies were used in this study: α-Vinculin (V9131, Sigma), α-RIPK1 (610459, BD Biosciences), α-phospho-RIPK1 S166 (657465, Cell Signaling Technologies), α-RIPK3 (13526, Cell Signaling Technologies), α-phoshpo-RIPK3 S227 (ab209384, Abcam), α-MLKL (14993, Cell Signaling Technologies), α-phospho-MLKL S358 (91689, Cell Signaling Technologies), α-caspase-3 (9662S, Cell Signaling Technologies), α-cleaved-caspase-3 (9661, Cell Signaling Technologies), α-cIAP1 (AF8181, R&D Systems), α-cIAP2 (3130, Cell Signaling Technologies), α-XIAP (610716, BD Biosciences).

Techniques: Control, Standard Deviation, Western Blot

Journal: Cancer Research

Article Title: Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

doi: 10.1158/0008-5472.CAN-23-3007

Figure Lengend Snippet: Impaired death receptor signaling in malignant cells caused resistance to CAR T-cell cytotoxicity. A, Hierarchical cluster analysis of transcriptomes for OvCAR3 and G164 cells in monolayer and spheroids. B, Heatmap illustrating normalized gene expression of differentially expressed genes relating to death receptor signaling in OvCAR3 and G164 cells (adjusted P value < 0.05). C, cIAP1/2 expression on human HGSOC omental metastasis ( n = 16) and adjacent omentum ( n = 10). Statistics performed using two-way ANOVA. D and E, Representative images (left) and quantification (right) of Incucyte killing assay in which monolayers of G164 ( D ) and G33 ( E ) cells were treated with birinapant and CAR T cells. F, Western blot showing the expression of cIAP2 in OvCAR3 and G164 cells treated with birinapant. Representative images of three repeats. G, ELISA data showing TNFα concentration after coculturing CAR T cells with monolayers of OvCAR3 and G164 cells for 2 days at 1:5 T:E ratios. Data plotted as mean ± SD for three CAR T-cell donors. Statistics performed using two-way ANOVA. H, Representative images (left) and quantification (right) of Incucyte killing assay in which OvCAR3 monolayer was treated with anti-TNFα antibody and CAR T cells. D–F, Data shown for one CAR T-cell donor at 1:5 T:E ratio. Images shown are 3 days after treatment. Red, dead cells. Scale bars, 400 μm.

Article Snippet: The membrane was incubated with cIAP2 antibody (R&D Systems, Cat. AF8171) at 4°C overnight, followed by incubation with anti-goat horseradish peroxide (HRP)-conjugated antibody (Dako, Cat. P0160) for 1 hour at room temperature.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay