For the immunoprecipitation of NLRP3 and ZBP1, RIPK3, or RIPK1 in the overexpression system, HEK293T cells were seeded into six-well plates and transfected with 600 ng each of pcDNA3-N-FLAG-NLRP3 (Addgene, #75127), RIPK3-GFP (Addgene #41382), pCMV-mRIPK1, or pcDNA-mCherry-ZBP1 expression plasmids for 30 h. For the immunoprecipitation of the complex by RIPK3, HEK293T cells in a 10 cm dish were co-transfected with 600 ng each of pCMV6-mASC-turboGFP (Origene MG201872), pCDH-CMV-CASP8, pcDNA3-N-FLAG-NLRP3 (Addgene, #75127), RIPK3-GFP (Addgene #41382), pCMV-mRIPK1, and pcDNA-mCherry-ZBP1 expression plasmids for 30 h. The pan-caspase inhibitor zVAD-fmk was used to inhibit cell death due to the overexpression of these molecules. Subsequently, cells were lysed in NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM HEPES, and protease inhibitor cocktail) for 20–30 min. Cell lysates were then centrifuged at 13,000 × g for 10 min. Supernatants were collected and incubated with 1–2 μg of the indicated antibody overnight at 4°C. Protein A/G-plus agarose beads (Santa Cruz sc-2003) were added into the lysates and incubated for 1–2 h at 4°C. After incubation, the beads were washed 4 times with lysis buffer and boiled in 2 × SDS loading buffer at 100°C for 5 min. Immunoprecipitates in sample buffer were subjected to immunoblotting analysis.
For immunoprecipitation in the endogenous system, fully differentiated primary wild-type C57BL/6 BMDMs were seeded 24 h prior to stimulations. BMDMs were stimulated with LPS (100 ng/mL) alone, LPS + TAK1-inhibitor (5Z-7-oxozeaenol [0.1 μM]) or LPS + TAK1-inhibior + zVAD (30 mM). After 4 h of stimulation, BMDMs were lysed in NP-40 lysis buffer. Whole cell lysates were harvested and incubated with 3 mg of indicated primary antibodies overnight at 4°C. Protein A/G plus agarose beads were added to these samples and incubated at 4°C for another 2 h. Agarose beads were centrifuged and washed in NP-40 lysis buffer three times, and immunoprecipitates were eluted by adding sample buffer. Immunoprecipitates in sample buffer were then subjected to immunoblotting analysis.
For immunoprecipitation in the endogenous system, fully differentiated primary wild-type C57BL/6 BMDMs were seeded 24 h prior to stimulations. BMDMs were stimulated with LPS (100 ng/mL) alone, LPS + TAK1-inhibitor (5Z-7-oxozeaenol [0.1 μM]) or LPS + TAK1-inhibior + zVAD (30 mM). After 4 h of stimulation, BMDMs were lysed in NP-40 lysis buffer. Whole cell lysates were harvested and incubated with 3 mg of indicated primary antibodies overnight at 4°C. Protein A/G plus agarose beads were added to these samples and incubated at 4°C for another 2 h. Agarose beads were centrifuged and washed in NP-40 lysis buffer three times, and immunoprecipitates were eluted by adding sample buffer. Immunoprecipitates in sample buffer were then subjected to immunoblotting analysis.
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