Investigating Cell Death Mechanisms

Dimethyl sulfoxide, actinomycin D (ActD), and phosphonoformate (PFA) were purchased from Sigma. Murine TNF-alpha and caspase inhibitor zVAD-fmk were purchased from Pepro Tech and Enzo Life Sciences, respectively. GSK’840^{43 (link)} from GlaxoSmithKline has been described before. MLKL inhibitor necrosulfonamide (NSA) was purchased from Calbiochem. Immunoblotting and immunoprecipitation (IP) were performed as previously described^{13 (link)}. For non-reducing gel analysis, lysates were prepared in the absence of dithiothreitol. The following antibodies were used in cell death assays, immunoblot (IB), and IP analyses: mouse anti-mouse IFNα/β receptor 1 antibody (clone MAR1-5A3, BD Pharmingen); mouse anti-ZBP1 (clone Zippy-1, AG-20B-0010); rabbit anti-MLKL (phospho S345) (ab196436, Abcam); rat anti-MLKL (MABC604, Millipore); mouse anti-ICP0 (sc-53070, Santa Cruz); mouse anti-FLAG (clone M2, Sigma); anti-myc (clone 9E10, Santa Cruz Biotech); mouse anti-β-actin (clone AC-74, Sigma-Aldrich); rabbit anti-RIPK3 (2283, ProSci); and goat anti-RIPK3 (sc-47364, Santa Cruz).

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Guo H., Gilley R.P., Fisher A., Lane R., Landsteiner V.J., Ragan K.B., Dovey C.M., Carette J.E., Upton J.W., Mocarski E.S, & Kaiser W.J. (2018). Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1. Cell Death & Disease, 9(8), 816.

Publication 2018

Dimethyl sulfoxide zVAD-fmk ab196436 mouse anti-FLAG (clone M2, anti-myc (clone 9E10, mouse anti-β-actin (clone AC-74, rabbit anti-RIPK3

Actin Actinomycin d Antibodies Antibody Assays Caspase inhibitor Cell death Clone Dimethyl sulfoxide Dithiothreitol Goat Ifnα β receptor 1 Immunoblot Immunoprecipitation Mouse Necrosulfonamide Phosphonoformate Rabbit Ripk3 Tnf alpha Zvad fmk

Corresponding Organization :

Other organizations : The University of Texas at San Antonio, Stanford University, Emory University

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Variable analysis

independent variables

Dimethyl sulfoxide (DMSO)
Actinomycin D (ActD)
Phosphonoformate (PFA)
Murine TNF-alpha
Caspase inhibitor zVAD-fmk
GSK'840
MLKL inhibitor necrosulfonamide (NSA)

dependent variables

Cell death assays
Immunoblot (IB) and immunoprecipitation (IP) analyses

control variables

Non-reducing gel analysis (lysates prepared in the absence of dithiothreitol)

controls

Positive controls: Not explicitly mentioned.
Negative controls: Not explicitly mentioned.

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