Western Blot Analysis of Inflammasome Proteins

Cells were lysed in RIPA lysis buffer with 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and protease inhibitor cocktail (Santa Cruz Biotech, Dallas, TX, sc-24948) for 30 min at 4 °C and then centrifuged at 16,000 g for 15 min. Samples (25–50 μg of protein/lane) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, 1704271). Blots were probed with rabbit monoclonal anti-caspase 1 (1:1000; Abcam, Cambridge, UK, ab179515), rabbit polyclonal anti-IL-1beta/IL-1F2 (1:1000; Novus Biologicals, Abingdon, UK, NB600-633), rabbit polyclonal anti-NLRP1/NALP1 (1:1000; Cell Signaling, Danvers, MA, 4990S), rabbit polyclonal anti-NLRP3/NALP3 (1:2500; Novus Biologicals, NBP2-12446), and mouse monoclonal anti-ACTB/actin (1:30,000; Sigma-Aldrich, A3853). After overnight incubation at 4 °C, bound antibodies were visualized using horseradish peroxidase-coupled secondary antibodies and the Clarity™ Western ECL Substrate (Bio-Rad, 1705061) or Clarity™ Max ECL western blotting substrate (Bio-Rad, 1705062) for low protein concentrations.

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de Dios C., Abadin X., Roca-Agujetas V., Jimenez-Martinez M., Morales A., Trullas R., Mari M, & Colell A. (2023). Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis. Translational Neurodegeneration, 12, 10.

Publication 2023

protease inhibitor cocktail nitrocellulose membranes anti-IL-1beta/IL-1F2 anti-NLRP1/NALP1 NBP2-12446 Clarity™ Western ECL Substrate Clarity™ Max ECL western blotting substrate

Actin Antibodies Biologicals Buffer Caspase 1 Cells Horseradish peroxidase Il 1beta Low protein Membranes Mouse Nitrocellulose Novus Orthovanadate Phenylmethylsulfonyl fluoride Protease inhibitor Protein Rabbit Ripa Sodium Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Corresponding Organization :

Other organizations : Institut d'Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas, Consorci Institut D'Investigacions Biomediques August Pi I Sunyer, Biomedical Research Networking Center on Neurodegenerative Diseases

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Variable analysis

independent variables

Lysis buffer composition (RIPA lysis buffer with 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and protease inhibitor cocktail)
Incubation time (30 min)
Incubation temperature (4 °C)

dependent variables

Protein expression levels of caspase 1, IL-1beta/IL-1F2, NLRP1/NALP1, and NLRP3/NALP3

control variables

Protein loading amount (25–50 μg of protein/lane)
Gel electrophoresis and membrane transfer conditions
Primary and secondary antibody dilutions

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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