Western Blotting for Apoptosis Signaling Proteins

Western blotting followed by enhanced chemiluminescence detection was performed as previously described.^{24 (link)} In some experiments, cell culture supernatants were also collected for the detection of HMGB1 or GAPDH. The following antibodies were used: Anti-TAK1 (4505), anti-phospho-TAK1 (Thr187; 4536), anti-α tubulin (3873), anti-HMGB1 (3935), anti-PARP (9532), anti-caspase 8 (4790), anti-cleaved caspase 8 (9429), anti-caspase 3 (9662), anti-RIP1 (3493), anti-CYLD (8462), anti-TRADD (3694), anti-cIAP1 (4952), and anti-FLIP (8510) were from Cell Signaling Biotechnology (Beverly, MA, USA); anti-RIP3 (sc-135171), anti-FADD (sc-6036), anti-CYLD (sc-74435), anti-IκBα (sc-847), anti-p65 (sc-372), anti-cIAP2 (sc-7944), and anti- GAPDH antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-RIP1 (610459) was from BD Biosciences; anti-FLIP (XA-1008) was from ProSci (Poway, CA, USA); anti-FADD (AD-I-AAM-212-E) was from Enzo Life Sciences (Farmingdale, NY, USA).

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Guo X., Yin H., Chen Y., Li L., Li J, & Liu Q. (2016). TAK1 regulates caspase 8 activation and necroptotic signaling via multiple cell death checkpoints. Cell Death & Disease, 7(9), e2381-.

Publication 2016

anti-FLIP anti- GAPDH antibodies anti-RIP1 (610459)

Anti antibodies Antibodies Caspase 3 Caspase 8 Cell culture Chemiluminescence Ciap1 Ciap2 Cyld Fadd Gapdh Hmgb1 Iκbα Rip3 Sc 372 α tubulin

Corresponding Organization :

Other organizations : University of Washington

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of TAK1, phospho-TAK1, α-tubulin, HMGB1, PARP, caspase 8, cleaved caspase 8, caspase 3, RIP1, CYLD, TRADD, cIAP1, FLIP, RIP3, FADD, IκBα, p65, cIAP2, GAPDH

control variables

None explicitly mentioned

controls

Positive controls: none mentioned
Negative controls: none mentioned

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