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Autophagy regulates inflammatory programmed cell death via turnover of RHIM-domain proteins

Research Article
Cite this article as: eLife 2019;8:e44452 doi: 10.7554/eLife.44452
8 figures, 1 table and 3 additional files

Figures

Figure 1 with 2 supplements
Defective autophagy enhances RIPK1-dependent and independent necroptosis.

(A, B) Cell death assayed by Propidium Iodide (PI) staining and live-cell imaging for 12–16 hr (n = 5). BMDMs from mice of the indicated genotypes were treated with combinations of TNF/zVAD/Nec-1 (A) or PolyI:C/zVAD/Nec-1 and LPS/zVAD/Nec-1 (B). (C) Immunoblots confirming deletion of autophagy genes in BMDMs of indicated genotypes using RNP electroporation. NTC = non targeting control gRNA. (D, E) Cell death assayed under combinations of PolyI:C/zVAD/Nec-1 (D) or LPS/zVAD/Nec-1 (E) treatment (n = 4). Data in (A, B) are representative of four independent experiments; (C–E) are representative of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Bar graphs depict mean.

https://doi.org/10.7554/eLife.44452.002
Figure 1—source data 1

Defective autophagy enhances RIPK1-dependent and independent necroptosis.

https://doi.org/10.7554/eLife.44452.005
Figure 1—figure supplement 1
Elevated cell death and cytokine production by Atg16l1-cKO BMDMs.

(A) Cell death assayed by PI staining and live-cell imaging for 12–16 hr following with combinations of Pam3CSK4/zVAD/Nec-1, R848/zVAD/Nec-1 or CpG-ODN 1826/zVAD/Nec-1 (n = 5). (B, C) ELISA measurements of IL-1β (B) and TNFα (C) in cell culture supernatants following treatment with combinations of LPS/zVAD/Nec-1 for 18 hr (n = 5). Data in (A) are representative of four independent experiments; (B, C) are representative of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Bar graphs depict mean.

https://doi.org/10.7554/eLife.44452.003
Figure 1—figure supplement 1—source data 1

Elevated cell death and cytokine production by Atg16l1-cKO BMDMs.

https://doi.org/10.7554/eLife.44452.006
Figure 1—figure supplement 2
CRISPR-mediated deletion of genes in primary BMDMs.

(A) Schematic of screening protocol to identify conditions for high efficiency eGFP deletion in monocytes and BMDMs using electroporation of CRISPR/Cas9-guide RNA(gRNA) ribonucleoprotein (RNP) complexes. (B) Flow cytometry plot demonstrating condition resulting in highly efficient eGFP loss. (C) Schematic illustrating CRISPR-mediated deletion of Ptprc/CD45. (D) Flow cytometry plots depicting Ptprc deletion and associated quantification of CD45 knockdown pooled from two independent experiments. Selected electroporation conditions were repeated at least three times with consistent results. Bar graphs depict mean. NTC = non targeting control gRNA.

https://doi.org/10.7554/eLife.44452.004
Figure 1—figure supplement 2—source data 2

CRISPR-mediated deletion of genes in primary BMDMs.

https://doi.org/10.7554/eLife.44452.007
Figure 2 with 2 supplements
RIPK3, MLKL and TRIF are required for RIPK1-independent necroptosis in Atg16l1-deficient BMDMs.

(A-E) Immunoblot (A, C) and cell death assays (B, D, E) of BMDMs from mice of indicated genotypes treated with combinations of LPS/zVAD/Nec-1 following CRISPR-mediated deletion of RIPK3, MLKL or GSDMD (A, B) (n = 4) or TRIF (C–E) (n = 6). Cell death assayed by PI staining and live-cell imaging for 12–16 hr. Data in (A, B) are representative of three independent experiments; (C, D, E) are representative of four independent experiments. **p<0.01, ***p<0.001, ****p<0.0001. Bar graphs depict mean. NTC = non targeting gRNA.

https://doi.org/10.7554/eLife.44452.008
Figure 2—source data 1

RIPK3, MLKL and TRIF are required for RIPK1-independent necroptosis in Atg16l1-deficient BMDMs.

https://doi.org/10.7554/eLife.44452.010
Figure 2—figure supplement 1
RIPK3 and MLKL drive RIPK1-dependent, TNF-mediated necroptosis; TRIF drives RIPK1-independent, PolyI:C-mediated necroptosis in Atg16l1-cKO BMDMs.

(A) Cell death assayed by PI staining and live-cell imaging for 12–16 hr of BMDMs treated with combinations of TNFα/zVAD/Nec-1 following CRISPR-mediated deletion of Ripk3, Mlkl or Gsdmd (n = 4). Gene deletion confirmed by immunoblots in Figure 2A. (B) Immunoblots confirming CRISPR-mediated deletion of Nlrp3 or Pycard in wild-type or Atg16l1-cKO BMDMs. (C) Cell death assayed by PI staining and live-cell imaging for 12–16 hr following CRISPR-mediated deletion of Nlrp3 or Pycard and treatment with PolyI:C/zVAD/Nec-1 or LPS/zVAD/Nec-1 (n = 5). Data in(A) are representative of three independent experiments; (B, C) are representative of two independent experiments. Bar graphs depict mean. *p<0.05, **p<0.01, ****p<0.0001. NTC = non targeting gRNA.

https://doi.org/10.7554/eLife.44452.009
Figure 2—figure supplement 1—source data 1

RIPK3 and MLKL drive RIPK1-dependent, TNF-mediated necroptosis; TRIF drives RIPK1-independent, PolyI:C mediated necroptosis in Atg16l1-cKO BMDMs.

https://doi.org/10.7554/eLife.44452.011
Figure 2—figure supplement 2
TNF and Type I interferon license necroptosis in BMDMs.

(A-F) Cell death assayed by PI staining and live-cell imaging for 12–16 hr of Atg16l1-WT and Atg16l1-cKO BMDMs pre-treated with TNFR2-Fc or α-IFNAR1 followed by TLR ligand or TNF mediated necroptosis (n = 5). Cells were pre-treated for 36 hr with 20 μg/mL α-Ragweed, TNFR2-Fc or α-IFNAR1 prior to addition of TLR ligands or TNF. (G) Immunoblots for phosphorylated STAT1 in BMDMs following LPS/zVAD treatment over 6 hr. Data in (A–F) are representative of four independent experiments; (G) are representative of two independent experiments. **p<0.01, ****p<0.0001. Bar graphs depict mean.

https://doi.org/10.7554/eLife.44452.013
Figure 2—figure supplement 2—source data 2

TNF and Type I interferon license necroptosis in BMDMs.

https://doi.org/10.7554/eLife.44452.012
Loss of Atg16l1 drives accumulation of detergent insoluble, high molecular weight TRIF, RIPK1, RIPK3 and enhances RIPK1/RIPK3 phosphorylation.

(A, B) Immunoblots of TRIF in Atg16l1-WT and Atg16l1-cKO BMDM lysates following 4 hr of treatment with indicated combinations of LPS/zVAD/Nec-1 and enrichment of NP-40 soluble (A) or insoluble (B) fractions. (C, D) immunoblots for autophosphorylated RIPK1 (Ser166/Thr169, p-RIPK1) and total RIPK1 in Atg16l1-WT and Atg16l1-cKO BMDM lysates following 4 hr of treatment with indicated combinations LPS/zVAD/Nec-1 and enrichment of NP-40 soluble (C) or insoluble (D) fractions. (E, F) immunoblot assay for autophosphorylated RIPK3 (Thr231/Ser232, p-RIPK3) and total RIPK3 in Atg16l1-WT and Atg16l1-cKO BMDM lysates following 4 hr of treatment with indicated combinations of LPS/zVAD/Nec-1 and enrichment of NP-40 soluble (E) or insoluble (F) fractions. Representative data shown from three independent experiments. In all immunoblots, CRISPR-mediated TRIF deletion was performed in Atg16l1-cKO BMDMs followed by LPS/zVAD treatment as a negative control. *=non specific bands (n.s.).

https://doi.org/10.7554/eLife.44452.014
Figure 4 with 2 supplements
Overabundance of TRIF, phosphorylated and ubiquitinated RIPK1 and RIPK3 coincides with accelerated necroptosis of Atg16l1 deficient BMDMs.

(A) Kinetic measurement of cell death over 18 hr of LPS/zVAD treatment (n = 5). (B) Immunoblot of TRIF in NP-40 insoluble fractions of BMDM lysates over 6 hr of LPS/zVAD treatment. (C, D) Immunoblots of autophosphorylated and total RIPK1 (C), RIPK3 (D) in NP-40 insoluble fractions of BMDM lysates treated as in (B). (E) Immunoblots of autophosphorylated RIPK1, RIPK3 and ubiquitin in BMDM lysates following immunoprecipitation of M1 or K63-ubiquitinated proteins after 4 hr of LPS/zVAD treatment. Data in (A) are representative of four independent experiments; (B–D) are representative of three independent experiments; (E) are representative of three independent experiments. *=P < 0.05.

https://doi.org/10.7554/eLife.44452.015
Figure 4—source data 1

Overabundance of TRIF, phosphorylated and ubiquitinated RIPK1 and RIPK3 coincides with accelerated necroptosis of ATG16L deficient BMDMs.

https://doi.org/10.7554/eLife.44452.018
Figure 4—figure supplement 1
Enhanced MLKL activation and accelerated cell death in Atg16l1 deficient BMDMs following LPS- or PolyI:C-mediated necroptosis.

(A) Immunoblots depicting p-MLKL Ser345 in NP-40 soluble and insoluble fractions of BMDM lysates 4 hr after indicated treatments. (B, C) Death of wild-type and Atg16l1-cKO BMDMs assayed by PI staining and live-cell imaging over 18 hr of LPS- (B) or PolyI:C- (C) mediated necroptosis. LPS +zVAD time-course is same as in Figure 4A. Data in (A) are representative of three independent experiments; (B, C) are representative of four independent experiments. Dots represent mean (n = 5)±S.D. *p<0.05, **p<0.01, ****p<0.0001.

https://doi.org/10.7554/eLife.44452.016
Figure 4—figure supplement 1—source data 1

Enhanced MLKL activation and accelerated cell death in ATG16L1 deficient BMDMs following LPS- or PolyI:C-mediated necroptosis.

https://doi.org/10.7554/eLife.44452.019
Figure 4—figure supplement 2
Lysosomal function and autophagic flux drive turnover of active TRIF, RIPK1 and RIPK3 during necroptosis.

(A-C) Immunoblots assaying levels of TRIF (A), RIPK1 (B) or RIPK3 (C) in total lysatse (bottom) and detergent- insoluble fractions (top) of BMDM lysates over 6 hr of LPS/zVAD treatment in the presence of Bafilomycin A1. (D–F) Immunoblots assaying basal turnover of TRIF (D), RIPK1 (E) or RIPK3 (F) by perturbation of proteasomal (MG132, 2 μM) or lysosomal (Bafilomycin A1, BafA1 100 nM) activity. Dot plots in (D–F) summarize protein abundance measured by immunoblot intensity normalized to 0 hr time point. Lines depict mean (n = 4). (G, H) Autophagic flux during LPS/zVAD-mediated necroptosis assayed by immunoblots of LC3-II and indicated autophagy receptors. Protein levels were assayed over 6 hr of necroptosis in presence of Bafilomycin A1 to halt autophagic flux. (G) total lysate; (H) detergent-insoluble fraction. Data in (A–F) are representative of three independent experiments; (G, H) are representative of two independent experiments.

https://doi.org/10.7554/eLife.44452.017
Figure 4—figure supplement 2—source data 2

Lysosomal function and autophagic flux drive turnover of active TRIF, RIPK1 and RIPK3 during necroptosis.

https://doi.org/10.7554/eLife.44452.020
The autophagy receptor TAX1BP1 protects against necroptosis by TLR3 or TLR4 ligands.

(A, B) Immunoblots of indicated autophagy receptors in total (A) or NP-40 insoluble fractions (B) of BMDM lysates over 6 hr of LPS/zVAD treatment. (C) Immunoblots confirming CRISPR-mediated deletion of indicated autophagy receptor genes in wild-type BMDMs. (D) Cell death assayed by PI staining and live-cell imaging for 12–16 hr following treatment with indicated ligands. Data in (A, B) are representative of three independent experiment; (C, D) are representative of four independent experiments. **p<0.01, ****p<0.0001. Bar graphs depict mean. NTC = non targeting control gRNA.

https://doi.org/10.7554/eLife.44452.021
Figure 5—source data 1

The autophagy receptor TAX1BP1 protects against necroptosis by TLR3 or TLR4 ligands.

https://doi.org/10.7554/eLife.44452.022
Figure 6 with 1 supplement
Elevated ZBP1 in Atg16l1-deficient BMDMs suppresses TRIF-mediated necroptosis.

(A) ZBP1 turnover in Atg16l1-WT and Atg16l1-cKO BMDMs following cycloheximide (CHX) treatment for indicated time points. Representative immunoblot (top), ZBP1 quantification by densitometry (bottom) normalized to ZBP1 band intensity in WT samples at 0 hr. (B, C) immunoblot (B) and cell death (C) assays of BMDMs from mice of indicated genotypes treated with combinations of LPS/zVAD/Nec-1 following CRISPR-mediated deletion of Zbp1, Ticam1 or both (n = 4). (D, E) cell death assayed in Atg16l1-WT or Atg16l1-cKO BMDMs following CRISPR-mediated Zbp1 deletion and a dose titration of LPS in the presence of 20 μM zVAD and/or 30 μM Nec-1 (n = 4). Dot-plots depict mean ±S.D. (F–H) immunoblots depicting accumulation of TRIF (F), autophosphorylated and total RIPK1 (G), autophosphorylated and total RIPK3 (H) in NP-40 insoluble lysates of BMDMs lacking both Atg16l1 and Zbp1 following induction of necroptosis via LPS/zVAD for 3 hr. Top panels represent short exposures; middle panels represent long exposures. *=non specific band. Data (A) are representative of four independent experiments, densitometry is pooled from four independent experiments. Data in (B, C) are representative of three independent experiments; (D–H) are representative of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Bar graphs depict mean. NTC = non targeting control gRNA.

https://doi.org/10.7554/eLife.44452.023
Figure 6—source data 1

Elevated ZBP1 in ATG16L1 deficient BMDMs suppresses TRIF-mediated necroptosis.

https://doi.org/10.7554/eLife.44452.025
Figure 6—figure supplement 1
Loss of Atg16l1 leads to ZBP1 accumulation; deletion of Zbp1 in Atg16l1-cKO BMDMs enhances TRIF-mediated necroptosis and RIPK3 activation.

(A) ZBP1 turnover in wild-type BMDMs measured by inhibition of lysosomal (Bafilomycin A1, BafA1 100 nM) or proteasomal (MG132 2 μM) function for indicated time points. Immunoblot is representative of three independent experiments, summarized in dot plot below as ZBP1 band intensity normalized to WT 0 hr time point. Lines depict mean (n = 3). (B) ZBP1 accumulation assayed by immunoblot in NP-40 insoluble and soluble fractions of Atg16l1-WT and Atg16l1-cKO BMDM lysates following treatment with indicated combinations of LPS/zVAD/Nec-1 for 4 hr. CRISPR-mediated deletion of Ticam1 in Atg16l1-cKO BMDMs followed by LPS/zVAD treatment is used as a negative control. (C) Cell death assayed by PI staining and live-cell imaging for 12–16 hr. BMDMs from mice of indicated genotypes treated with combinations of LPS/zVAD/Nec-1 following CRISPR-mediated deletion of Zbp1, Ticam1 or both (n = 4). (D) Cell death time-course of BMDMs of indicated genotypes following LPS/zVAD mediated necroptosis. Dots represent mean (n = 4)±S.D. All data are representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. NTC = non targeting gRNA.

https://doi.org/10.7554/eLife.44452.024
Figure 6—figure supplement 1—source data 1

Loss of Atg16l1 leads to ZBP1 accumulation; deletion of Zbp1 in Atg16l1-cKO BMDMs enhances TRIF-mediated necroptosis and RIPK3 activation.

https://doi.org/10.7554/eLife.44452.026
Figure 7 with 1 supplement
Combined loss of myeloid-specific Atg16l1 and Zbp1 accelerates LPS-mediated sepsis in mice.

(A) Kaplan-Meier survival plots for mice following challenge with 10 mg/kg LPS administered intraperitoneally. Statistical analysis Figure 7—figure supplement 1A was performed using log-rank test (Figure 7—figure supplement 1; Figure 7—figure supplement 1A). (B) Serum cytokine measurements of IL-1β and TNFα performed by ELISA following 4 hr of intraperitoneal LPS administration at 10 mg/kg. Data in A are representative of two independent experiments. Data in B are pooled from two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.44452.027
Figure 7—source data 1

Accelerated morbidity conferred by double deficiency of ATG16L1 and ZBP1 in myeloid cells following LPS-mediated sepsis in mice.

https://doi.org/10.7554/eLife.44452.029
Figure 7—figure supplement 1
Accelerated morbidity conferred by double deficiency of ATG16L1 and ZBP1 in myeloid cells following LPS-mediated sepsis in mice.

(A) Statistical analysis of Kaplan-Meier curve depicted in Figure 7. P-values are generated using log-rank test. (B) Inflammatory cytokines, death ligands and TLR ligands induce necroptotic signaling upon caspase-inhibition. Autophagy promotes turnover of TRIF, RIPK1 and RIPK3 to control necroptosis in healthy macrophages. (C) In the absence of autophagy, accumulation of active TRIF, RIPK1 and RIPK3 enhances necroptosis as well as inflammatory cytokine production. Accumulation of ZBP1 attenuates TRIF-mediated necroptosis during autophagy deficiency. During TLR3- or TLR4- activation, overabundance of TRIF drives RIPK3-dependent necroptosis that is resistant to RIPK1 inhibition. Autocrine signaling via non-TRIF TLRs may contribute to enhanced necroptosis. Dotted lines depict indirect signaling events; solid lines depict direct signaling events.

https://doi.org/10.7554/eLife.44452.028
Author response image 1
TRIF, autophosphorylated RIPK1 and autophosphorylated RIPK3 are ubiquitinated during necroptosis.

Immunoblots of TRIF, autophosphorylated RIPK1, autophosphorylated RIPK3 and ubiquitin in BMDM lysates following immunoprecipitation of M1 or K63-ubiquitinated proteins after 4 hours of LPS/zVAD treatment. Red arrow depicts specific high MW TRIF signal.

https://doi.org/10.7554/eLife.44452.034

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (M. musculus)Atg16l1loxP/loxPPMID: 24553140Dr. Aditya Murthy (Genentech, Inc)
Genetic reagent (M. musculus)Zbp1loxP/loxPNewton et al., 2016Dr. Kim Newton (Genentech, Inc)
Commercial assay or kitMouse monocyte isolation kitMiltenyi BiotecCat#: 130-100-629
Peptide, recombinant proteinCas9 V3IDTCat#: 108105810 μg per reaction
Peptide, recombinant proteinmurine TNFαPeprotechCat#: 315-01A50 ng/ml
Peptide, recombinant proteinPam3CSK4InvivogenCat#: tlrl-pms1 μg/ml
Peptide, recombinant proteinPolyI:C (LMW)InvivogenCat#: tlrl-picw10 μg/ml
Peptide, recombinant proteinLPS-EB ultrapure (E. coli O111:B4)InvivogenCat#: tlrl-3pelps100 ng/ml
Peptide, recombinant proteinR848 (Resiquimod)InvivogenCat#: tlrl-r8481 μg/ml
Peptide, recombinant proteinCpG-ODN 1826InvivogenCat#: tlrl-18265 μM
Peptide, recombinant proteinzVAD-fmkPromegaCat#: G723220 μM
Chemical compound, drugNecrostatin-1Enzo Life SciencesCat#: BML-AP309-010030 μM
Chemical compound, drugBafilomycin A1SigmaCat#: B1793100 nM
Chemical compound, drugMG132SigmaCat#: M74492 μM
Peptide, recombinant proteinFcR-BlockBD biosciencesCat#: 5331441
Chemical compound, drugFixable viability dye efluor780InvitrogenCat#: 65–0865
Antibodyanti-CD62L PerCP Cy5.5
Rat monoclonal
BD biosciencesCat#: 560513
RRID: AB_10611578
Flow cytometry
Antibodyanti-CCR2 APCR and D SystemsCat#: FAB5538A
RRID: AB_10645617
Flow cytometry
Antibodyanti-F4/80 efluor450
Rat monoclonal
eBioscienceCat#: 48-4801-82
RRID: AB_1548747
Flow cytometry
Antibodyanti-CSF1R BV711
Rat monoclonal
BiolegendCat#: 135515
RRID: AB_2562679
Flow cytometry
Antibodyanti-Ly6G BUV395
Rat monoclonal
BD biosciencesCat#: 565964
RRID: AB_2739417
Flow cytometry
Antibodyanti-CD11b BUV737
Rat monoclonal
BD biosciencesCat#: 564443
RRID: AB_2738811
Flow cytometry
Antibodyanti-MHCII (IA/IE) PE
Rat monoclonal
eBioscienceCat#: 12-5322-81
RRID: AB_465930
Flow cytometry
Antibodyanti-Ly6C-PECy7
Rat monoclonal
eBioscienceCat#: 25-5932-82
RRID: AB_2573503
Flow cytometry
Antibodyanti-CD45 FITC
Rat monoclonal
eBioscienceCat#: 11-0451-82
RRID: AB_465050
Flow cytometry
Antibodyanti-F4/80 BV421
Rat monoclonal
BiolegendCat#: 123131
RRID: AB_10901171
Flow cytometry
Antibodyanti-CD11b BUV395
Rat monoclonal
BD biosciencesCat#: 563553
RRID: AB_2738276
Flow cytometry
Antibodyanti-ATG16L1
Mouse monoclonal
MBL internationalCat#: M150-3
RRID: AB_1278758
Immunoblot
Antibodyanti-ATG14L
Rabbit polyclonal
MBL internationalCat#: PD026
RRID: AB_1953054
Immunoblot
Antibodyanti-FIP200
Rabbit monoclonal
Cell Signaling TechnologyCat#: 12436
RRID: AB_2797913
Immunoblot
Antibodyanti-Rubicon
Mouse monoclonal
MBL internationalCat#: M170-3
RRID: AB_10598340
Immunoblot
Antibodyanti-TRIF
Host: Rat
Genentech, IncCat#: 1.3.5Immunoblot
Antibodyanti-MLKL
Host: Rabbit
Genentech, IncCat#: 1G12Immunoblot
Antibodyanti-p-MLKL
Rabbit monoclonal
AbcamCat#: ab196436
RRID: AB_2687465
Immunoblot
Antibodyanti-RIPK1
Mouse monoclonal
BD biosciencesCat#: 610459
RRID: AB_397832
Immunoblot
Antibodyanti-p- RIPK1
Host: Rabbit
Genentech, IncCat#: GNE175.DP.B1Immunoblot
Antibodyanti-RIPK3
Rabbit polyclonal
Novus BiologicalsCat#: NBP1-77299
RRID: AB_11040928
Immunoblot
Antibodyanti-p-RIPK3
Host: Rabbit
Genentech, IncCat#: GEN-135-35-9Immunoblot
Antibodyanti-GSDMD
Host: Rat
Genentech, IncCat#: GN20-13Immunoblot
Antibodyanti-LC3B
Rabbit polyclonal
Cell Signaling TechnologyCat#: 2775
RRID: AB_915950
Immunoblot
Antibodyanti-CALCOCO1 Rabbit polyclonalProteintechCat#: 19843–1-AP
RRID: AB_10637265
Immunoblot
Antibodyanti-TAX1BP1
Rabbit monoclonal
AbcamCat#: ab176572Immunoblot
Antibodyanti-p62
Guinea pig polyclonal
Progen biotechnicCat#: gp62-c
RRID: AB_2687531
Immunoblot
Antibodyanti-NLRP3
Rabbit monoclonal
Cell Signaling TechnologyCat#: 15101
RRID: AB_2722591
Immunoblot
Antibodyanti-ASC
Rabbit monoclonal
Cell Signaling TechnologyCat#: 67824
RRID: AB_2799736
Immunoblot
Antibodyanti-STAT1
Rabbit monoclonal
Cell Signaling TechnologyCat#: 14995
RRID: AB_2716280
Immunoblot
Antibodyanti-p- STAT1
Rabbit monoclonal
Cell Signaling TechnologyCat#: 7649
RRID: AB_10950970
Immunoblot
Antibodyanti-M1-polyubiquitin linkage specific antibodyGenentech, IncN/AImmunoprecipitation
Antibodyanti-K63-polyubiquitin linkage specific antibodyGenentech, IncN/AImmunoprecipitation
AntibodyAnti-Ubiquitin
Mouse monoclonal
Cell Signaling TechnologyCat#: 3936
RRID: AB_331292
Immunoblot
Antibodyanti-beta ActinCell Signaling TechnologyCat#: 3700
RRID: AB_2242334
Immunoblot
Antibodyanti-rabbit IgG HRP
Goat polyclonal
Cell Signaling TechnologyCat#: 7074
RRID: AB_2099233
Immunoblot
Antibodyanti-mouse IgG HRP
Horse polyclonal
Cell Signaling TechnologyCat#: 7076
RRID: AB_330924
Immunoblot
Antibodyanti-rat IgG HRP
Goat polyclonal
Cell Signaling TechnologyCat#: 7077
RRID: AB_10694715
Immunoblot
AntibodyAnti-RagweedGenentech, IncN/AInhibition
AntibodyAnti-mIFNAR1
Mouse monoclonal
Leinco TechnologiesCat#: I-401
RRID: AB_2737538
Inhibition
AntibodymTNFR2-Fc
Mouse Fc
Genentech, IncN/AInhibition
Tools (software)Image JImmunoblot densitometry
Tools (software)Graphpad Prism 7GraphpadData visualization and statistics

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Additional files

Supplementary file 1

Perinatal lethality of Ripk1RHIM/RHIM mice is prevented by ZBP1 deficiency (Newton et al., 2016) but not by addition of a 3xFlag N-terminal tag to ZBP1.

https://doi.org/10.7554/eLife.44452.030
Supplementary file 2

crRNA targeting sequences used for CRISPR/Cas9 gene editing.

https://doi.org/10.7554/eLife.44452.031
Transparent reporting form
https://doi.org/10.7554/eLife.44452.032

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