Comprehensive Protein Profiling in Cell Lysates

Whole cell lysates (WCL) were prepared in lysis buffer containing 0.1% Triton, dithiothreitol, and protease inhibitor cocktail tablets (CPIC) (Roche Diagnostics, Indianapolis, IN) for 30min on ice and then spun at 16,000xg for 5min at 4°C. WCL samples were probed for caspase-3 (Santa Cruz Biotechnology, Dallas, TX), caspase-9 (Santa Cruz Biotechnology, Dallas, TX), caspase-8 (Santa Cruz Biotechnology, Dallas, TX), caspase-1 (Cell Signaling Technology, Danvers, MA), ICAM-1 (Santa Cruz Biotechnology, Dallas, TX), and VCAM-1 (Santa Cruz Biotechnology, Dallas, TX). Nuclear (Nuc) and cytoplasmic (Cyto) protein fractions were prepared by using the CelLytic NuCLEAR extraction kit (Millipore Sigma, Merck KGaM, Darmstadt, Germany) and probed for cyclinB1 (Santa Cruz Biotechnology, Dallas, TX), γH2AX (Cell Signaling Technology, Danvers, MA), and NF-κB-p65 (Abcam, Cambridge, UK). Protein extraction from serum-free supernatant media using an adopted methanol-mediated extraction protocol (Shi et al. 2012 (link)) was used to probe in conjunction with WCL or Nuc/Cyto fractions for pro-inflammatory molecules Gasdermin D (Santa Cruz Biotechnology, Dallas, TX), HGMB-1 (Cell Signaling Technology, Danvers, MA), IL-1α (Santa Cruz Biotechnology, Dallas, TX), IFNβ (Santa Cruz Biotechnology, Dallas, TX), and IL-1β (Cell Signaling Technology, Danvers, MA).

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Fountain M.D., McLellan L.A., Smith N.L., Loughery B.F., Rakowski J.T., Tse H.Y, & Hillman G.G. (2019). Isoflavone-Mediated Radioprotection Involves Regulation of Early Endothelial Cell Death and Inflammatory Signaling in Radiation-Induced Lung Injury.. International journal of radiation biology, 96(2), 245-256.

Publication 2019

caspase-3 caspase-9 caspase-8 caspase-1 ICAM-1 VCAM-1 CelLytic NuCLEAR extraction kit cyclinB1 γH2AX NF-κB-p65 Gasdermin D IL-1α IL-1β

Buffer Caspase 1 Caspase 3 Caspase 8 Caspase 9 Cell Cytoplasmic Diagnostics Dithiothreitol Icam 1 Il 1β Inflammatory Methanol Nf κb p65 Protease inhibitor Protein Serum Serum free media Vcam 1

Corresponding Organization :

Other organizations : Wayne State University, The Barbara Ann Karmanos Cancer Institute

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Variable analysis

independent variables

Triton concentration (0.1%) in the lysis buffer
Dithiothreitol in the lysis buffer
Protease inhibitor cocktail tablets (CPIC) in the lysis buffer
Methanol-mediated protein extraction from serum-free supernatant media

dependent variables

Levels of caspase-3, caspase-9, caspase-8, caspase-1, ICAM-1, VCAM-1, cyclin B1, γH2AX, NF-κB-p65, Gasdermin D, HGMB-1, IL-1α, IFNβ, IL-1β

control variables

Incubation time (30 min) on ice
Centrifugation speed (16,000xg) and duration (5 min) at 4°C
Serum-free culture conditions

positive controls

Not specified

negative controls

Not specified

Annotations

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