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nature research | reporting summary
October 20181:1000), MLKL (Mus) (home made, 1:1000).Flow cytometry and intracellular cytokine staining:
CD3-APC-H7 (BD Biosciences, 560176, clone SK7, lot 9022755, 1:20), CD14-PE-CY7 (BD Biosciences, 557742, clone M5E2, lot
3291733, 1:20), CD4-FITC (BD Biosciences, 555346, clone RPA-T4, lot 8037703, 1:20), CD19-BB700 (BD Biosciences, 566396,
clone SJ25C, lot 8130761, 1:20), IL8-PE (Biolegend, 511408, clone E8N1, lot B230067,1:20), IL10-BV421 (Biolegend, 501421,
clone JES3-9D7, lot B256252, 1:20), IL6-Percp-cy5.5 (Biolegend, 501118, clone MQ2-13A5, lot B267970, 1:20), TNF-V450 (BD
Biosciences, 561311, clone Mab11, lot 7209966, 1:20).Alexa Fluor® 647-conjugated antibody against STAT3 phosphorylated at Y705 (BD Biosciences, 557815, clone 4/P-STAT3, lot
7346960, 1:10), Alexa Fluor® 488-conjugated antibody against NF-κB p65 phosphorylated at S529 (BD Biosciences, 558421, clone
K10-895.12.50, lot 8241506, 1:10), Alexa Fluor® 488-conjugated antibody against p38 phosphorylated at T180/Y182 (BD
Biosciences, 612594, clone 36/p38 (pT180/pY182), lot 8032972, 1:10).Isotype control antibodies:
Alexa Fluor® 647-conjugated mouse IgG1κ (BD Biosciences, 557714, clone MOPC-21, lot 7076782, 1:20), Alexa Fluor® 488-
Mouse IgG1κ (BD Biosciences, 557782, clone MOPC-21, lot 7102576, 1:20), BV421-Rat IgG1κ (Biolegend, 400439, clone
RTK2071, lot B272547, 1:20), PE- Mouse IgG1κ (Biolegend, 400140, clone MOPC-21, lot B272822, 1:20), Percp-cy5.5-Rat IgG1κ
(Biolegend, 400426, clone RTK2071, lot B255186, 1:20), V450- Mouse IgG1κ (BD Biosciences, 560373, clone MOPC-21, lot
6021915, 1:20).Validation All antibodies except MLKL (Mus, home made) are commercially available and have been verified by the manufacturers
according to the immunoblots and/or images on their websites.
β-Actin: https://www.cst-c.com.cn/products/primary-antibodies/b-tubulin-d3u1w-mouse-mab/86298,
β-Tubulin: https://www.cst-c.com.cn/products/primary-antibodies/b-tubulin-d3u1w-mouse-mab/86298,
GAPDH: https://www.cst-c.com.cn/products/primary-antibodies/gapdh-d16h11-xp-rabbit-mab/5174,
RIPK1: https://www.cst-c.com.cn/products/primary-antibodies/rip-d94c12-xp-rabbit-mab/3493,
p-RIPK1 (Ser166): https://www.cst-c.com.cn/products/primary-antibodies/phospho-rip-ser166-d1l3s-rabbit-mab/65746,
MLKL: https://www.cst-c.com.cn/products/primary-antibodies/mlkl-d2i6n-rabbit-mab/14993,
p-MLKL (Ser358): https://www.cst-c.com.cn/products/primary-antibodies/phospho-mlkl-ser358-d6h3v-rabbit-mab/91689,
p65: https://www.cst-c.com.cn/products/primary-antibodies/nf-kb-p65-d14e12-xp-rabbit-mab/8242,
p-p65 (Ser65): https://www.cst-c.com.cn/products/primary-antibodies/phospho-nf-kb-p65-ser536-93h1-rabbit-mab/3033,
IKKα: https://www.cst-c.com.cn/products/primary-antibodies/ikka-3g12-mouse-mab/11930,
IKKβ: https://www.cst-c.com.cn/products/primary-antibodies/ikkb-d30c6-rabbit-mab/8943,
p-IKKα/β (Ser176/180): https://www.cst-c.com.cn/products/primary-antibodies/phospho-ikka-b-ser176-180-16a6-rabbit-
mab/2697,
IκBα: https://www.cst-c.com.cn/products/primary-antibodies/ikba-l35a5-mouse-mab-amino-terminal-antigen/4814,
p-IκBα (Ser32): https://www.cst-c.com.cn/products/primary-antibodies/phospho-ikba-ser32-14d4-rabbit-mab/2859,
p38: https://www.cst-c.com.cn/products/primary-antibodies/p38-mapk-d13e1-xp-rabbit-mab/8690,
p-p38 (Thr180/Tyr182): https://www.cst-c.com.cn/products/primary-antibodies/phospho-p38-mapk-thr180-tyr182-d3f9-xp-
rabbit-mab/4511,
TNFR1: https://www.cst-c.com.cn/products/primary-antibodies/tnf-r1-c25c1-rabbit-mab/3736,
HA-Tag: https://www.cst-c.com.cn/products/primary-antibodies/ha-tag-c29f4-rabbit-mab/3724,
p-MLKL (Ser345): https://www.cst-c.com.cn/products/antibody-conjugates/phospho-mlkl-ser345-d6e3g-rabbit-mab/37333,
p-MLKL (Ser358): https://www.cst-c.com.cn/products/primary-antibodies/phospho-mlkl-ser358-d6h3v-rabbit-mab/91689,
caspase-8: https://www.cst-c.com.cn/products/primary-antibodies/caspase-8-1c12-mouse-mab/9746,
caspase-3:https://www.cst-c.com.cn/products/primary-antibodies/caspase-3-antibody/9662,
cleaved caspase-3 (Asp175): https://www.cst-c.com.cn/products/primary-antibodies/cleaved-caspase-3-asp175-antibody/9661,
SLC7A11: https://www.cst-c.com.cn/products/primary-antibodies/xct-slc7a11-d2m7a-rabbit-mab/12691,
HSP90: http://affbiotech.cn/goods-4354-BF9107-HSP90+beta+Antibody.html,
Cyclophilin A: https://www.abcam.com/cyclophilin-a-antibody-ab41684.html,
GPX4: https://www.abcam.com/glutathione-peroxidase-4-antibody-epncir144-ab125066.html,
Lamp-2A: https://www.abcam.com/lamp2a-antibody-epr42072-lysosome-marker-ab125068.html,
COX2: https://www.abcam.com/cox2--cyclooxygenase-2-antibody-ab15191.html,
p53: https://www.abcam.com/p53-antibody-e26-ab32389.html,
FADD: https://www.scbt.com/scbt/zh/product/fadd-antibody-m-19,
ACSL4: https://www.scbt.com/scbt/product/acsl4-antibody-a-5,
HSC70: https://www.ptglab.com/products/HSPA8-Antibody-10654-1-AP.html,
p-RIPK1 (Ser166) (Biolynx) : http://www.biolynx.cn/product/seDetail/722,
CD3-APC-H7: http://www.bdbiosciences.com/eu/applications/research/t-cell-immunology/th-1-cells/surface-markers/human/
apc-h7-mouse-anti-human-cd3-sk7-also-known-as-leu-4/p/560176,
CD14-PE-CY7: http://www.bdbiosciences.com/eu/applications/research/stem-cell-research/hematopoietic-stem-cell-markers/
human/negative-markers/pe-cy7-mouse-anti-human-cd14-m5e2/p/557742,
CD4-FITC: http://www.bdbiosciences.com/eu/applications/research/t-cell-immunology/th-1-cells/surface-markers/human/fitc-
mouse-anti-human-cd4-rpa-t4/p/555346,
CD19-BB700: http://www.bdbiosciences.com/eu/reagents/research/antibodies-buffers/immunology-reagents/anti-human-
antibodies/cell-surface-antigens/bb700-mouse-anti-human-cd19-sj25c1-also-known-as-sj25-c1/p/566396,
IL8-PE: https://www.biolegend.com/en-us/products/pe-anti-human-il-8-antibody-4131,
IL10-BV421: https://www.biolegend.com/en-us/products/brilliant-violet-421-anti-human-il-10-antibody-7156,
IL6-Percp-cy5.5: https://www.biolegend.com/en-us/products/percpcyanine55-anti-human-il-6-antibody-13095,
TNF-V450: http://www.bdbiosciences.com/eu/applications/research/t-cell-immunology/th-1-cells/intracellular-markers/
cytokines-and-chemokines/human/v450-mouse-anti-human-tnf-mab11/p/561311,
Alexa Fluor® 647-conjugated antibody against STAT3 phosphorylated at Y705: http://www.bdbiosciences.com/eu/reagents/
research/antibodies-buffers/cell-biology-reagents/cell-biology-antibodies/alexa-fluor-647-mouse-anti-stat3-py705-4p-stat3/