Antibodies used were: Flag (Sigma-Aldrich), HA (Roche), LC3 (Sigma-Aldrich), AMPK, ULK1 p-Ser 317, and p-Ser 555 (Cell Signaling Technology), NLRP1 (Cell Signaling Technology), NLRP3 (Adipogen), Caspase-1, and ULK1 (Santa Cruz Biotechnology, Inc.), and GFP, IRF3, Myc, and Actin (Abcam). To determine autophagic activity by immunoblotting, cells were cultured in the presence of bafilomycin A1, and lysates were subjected to immunoblotting as described previously (Mizushima et al., 2010 (link)). The reagents used were Ultrapure LPS (InvivoGen), IFN-γ (PeproTech), Cytotoxic LDH assay (Promega), and TO-PRO-3 Iodide (Life Technologies). Immunoblotting and immunostaining were conducted as previously described (Kyei et al., 2009 (link)). FAM-YVAD-FMK stainings (FLICA; ImmunoChemistry Technologies) were performed according to the manufacturer’s instructions.
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Autophagy and Inflammasome Activation Assays
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Actin
Antibodies
Assay
Autophagic
Bafilomycin a1
Caspase 1
Cells
Ifn γ
Iodide
Irf3
Promega
Stainings
To pro 3
Ulk1
Yvad fmk
Corresponding Organization : University of New Mexico
Other organizations : UiT The Arctic University of Norway, University of Queensland
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Variable analysis
independent variables
- Bafilomycin A1
- Ultrapure LPS
- IFN-γ
- Autophagic activity
- Cell viability (measured by LDH assay)
- Apoptosis (measured by FLICA staining)
- Immunoblotting and immunostaining conditions (as previously described)
- FAM-YVAD-FMK staining (FLICA) protocol
- Not explicitly mentioned
- Not explicitly mentioned
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