Western Blotting of Apoptosis Regulators

Standard Western blotting was carried as previously described (7 (link)). PARP, Caspase-8, Caspase-3, TRAIL-R2 and Bid antibodies were purchased from Cell Signaling Technology, cIAP1 from Enzo, FADD from BD Biosciences, FLIP antibody from AdipoGen and β-actin from Sigma. For absolute quantification of FLIP and Caspase-8, images were captured using an Odyssey Imaging System (LICOR, Lincoln, NE) at 12-bit dynamic range. Quantification of protein expression amounts was conducted as described previously using recombinant proteins and a HeLa cell line standard (8 (link)).

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Grinkevitch V., Wappett M., Crawford N., Price S., Lees A., McCann C., McAllister K., Prehn J., Young J., Bateson J., Gallagher L., Michaut M., Iyer V., Chatzipli A., Barthorpe S., Ciznadija D., Sloma I., Wesa A., Tice D.A., Wessels L., Garnett M., Longley D.B., McDermott U, & McDade S.S. (2022). Functional Genomic Identification of Predictors of Sensitivity and Mechanisms of Resistance to Multivalent Second-Generation TRAIL-R2 Agonists. Molecular Cancer Therapeutics, 21(4), 594-606.

Publication 2022

Caspase-8 Caspase-3 TRAIL-R2 cIAP1 FLIP antibody β-actin Odyssey Imaging System

Actin Antibodies Antibody Caspase 3 Caspase 8 Cell line Ciap1 Fadd Hela Protein Recombinant proteins Trail r2

Corresponding Organization : Queen's University Belfast

Other organizations : Wellcome Sanger Institute, Royal College of Surgeons in Ireland, The Netherlands Cancer Institute, Champions Oncology (United States), AstraZeneca (United States), Delft University of Technology

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Variable analysis

independent variables

PARP, Caspase-8, Caspase-3, TRAIL-R2 and Bid antibodies
CIAP1 antibody
FADD antibody
FLIP antibody
β-actin antibody

dependent variables

Protein expression amounts

control variables

Recombinant proteins and a HeLa cell line standard

controls

Positive control: Recombinant proteins
Negative control: HeLa cell line standard

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