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  • 97
    Millipore monoclonal anti flag m2 antibody
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β actin
    Activation of mitosis and mitochondria by F3-T3 a , Immunoblot analysis of FGFR3 and p-FGFR3 in HA-F3-T3 treated with DMSO or PD173074, HA-F3-T3-K508M and HA-vector. <t>β-actin</t> is shown as loading control. Experiment was repeated at least five times with similar results. b , Heatmap of correlations among HA-F3-T3, HA-F3-T3 treated with PD173074, HA-vector and HA-F3-T3-K508M. Upper and right track colors represent sample type; left track color scale represents correlation between each sample and the F3-T3 group. HA-F3-T3, HA-F3-T3-PD173074 (n=5 biologically independent samples per group). HA-vector and HA-F3-T3-K508M (n=3 biologically independent samples per group). c , Enrichment map network of GO categories scoring as significant ( q -value
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Millipore
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    Millipore anti β actin
    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. <t>β-Actin</t> was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p
    Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β actin/product/Millipore
    Average 99 stars, based on 1 article reviews
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    Mouse Anti Mouse ATG4C Antibody 400 µl
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    Mouse Anti Mouse TSC2 Antibody 400 µl
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    Mouse Anti Mouse TLR3 Antibody 400 µl
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    Image Search Results


    Activation of mitosis and mitochondria by F3-T3 a , Immunoblot analysis of FGFR3 and p-FGFR3 in HA-F3-T3 treated with DMSO or PD173074, HA-F3-T3-K508M and HA-vector. β-actin is shown as loading control. Experiment was repeated at least five times with similar results. b , Heatmap of correlations among HA-F3-T3, HA-F3-T3 treated with PD173074, HA-vector and HA-F3-T3-K508M. Upper and right track colors represent sample type; left track color scale represents correlation between each sample and the F3-T3 group. HA-F3-T3, HA-F3-T3-PD173074 (n=5 biologically independent samples per group). HA-vector and HA-F3-T3-K508M (n=3 biologically independent samples per group). c , Enrichment map network of GO categories scoring as significant ( q -value

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: Activation of mitosis and mitochondria by F3-T3 a , Immunoblot analysis of FGFR3 and p-FGFR3 in HA-F3-T3 treated with DMSO or PD173074, HA-F3-T3-K508M and HA-vector. β-actin is shown as loading control. Experiment was repeated at least five times with similar results. b , Heatmap of correlations among HA-F3-T3, HA-F3-T3 treated with PD173074, HA-vector and HA-F3-T3-K508M. Upper and right track colors represent sample type; left track color scale represents correlation between each sample and the F3-T3 group. HA-F3-T3, HA-F3-T3-PD173074 (n=5 biologically independent samples per group). HA-vector and HA-F3-T3-K508M (n=3 biologically independent samples per group). c , Enrichment map network of GO categories scoring as significant ( q -value

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Activation Assay, Plasmid Preparation

    Acute expression of F3-T3 fusion induces peroxisome biogenesis through phosphorylation of Y122 of PIN4 a , Representative confocal microphotographs (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left panel) and pY122-PIN4 (pPIN4, red, middle panel) in vector and HA expressing F3-T3. Right panels show higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently two times with similar results. b , Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle panel) and pY122-PIN4 (red, right panel) in HA-F3-T3. Arrows indicate protein co-localization. Experiment was repeated independently two times with similar results. c , Co-immunoprecipitation from H1299 cells using PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analyzed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d , Western blot analysis of co-immunoprecipitation of exogenous FLAG-PEX1 in HA-F3-T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e , qRT-PCR for PEX1 in HA-F3-T3 and HA-vector. Data are Mean±s.d. (n=3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f , Western blot analysis of PEX1 expression in HA transduced with F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Experiment was repeated independently three times with similar results. g , Time course analysis of F3-T3 expression in HA by western blot. α-tubulin is shown as loading control. Experiment was repeated independently two times with similar results. h , Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in HA reconstituted with PIN4-WT or PIN4-Y122F after silencing of the endogenous PIN4 and acutely transduced with F3-T3 or vector. Representative bar plots (n=4 technical replicates) from one out of three independent experiments. P: *

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: Acute expression of F3-T3 fusion induces peroxisome biogenesis through phosphorylation of Y122 of PIN4 a , Representative confocal microphotographs (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left panel) and pY122-PIN4 (pPIN4, red, middle panel) in vector and HA expressing F3-T3. Right panels show higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently two times with similar results. b , Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle panel) and pY122-PIN4 (red, right panel) in HA-F3-T3. Arrows indicate protein co-localization. Experiment was repeated independently two times with similar results. c , Co-immunoprecipitation from H1299 cells using PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analyzed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d , Western blot analysis of co-immunoprecipitation of exogenous FLAG-PEX1 in HA-F3-T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e , qRT-PCR for PEX1 in HA-F3-T3 and HA-vector. Data are Mean±s.d. (n=3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f , Western blot analysis of PEX1 expression in HA transduced with F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Experiment was repeated independently three times with similar results. g , Time course analysis of F3-T3 expression in HA by western blot. α-tubulin is shown as loading control. Experiment was repeated independently two times with similar results. h , Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in HA reconstituted with PIN4-WT or PIN4-Y122F after silencing of the endogenous PIN4 and acutely transduced with F3-T3 or vector. Representative bar plots (n=4 technical replicates) from one out of three independent experiments. P: *

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Expressing, Immunofluorescence, Staining, Plasmid Preparation, Double Immunofluorescence Staining, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Transduction, Microscopy

    Phosphorylation of Tyr-122 of PIN4 by F3-T3 a , Amino acid sequence flanking Tyr-122 of PIN4 (in red) is evolutionarily conserved. b , Immunoprecipitation-western blot analysis of HA-F3-T3 or F3-T3-K508M with or without silencing of endogenous PIN4 . β-actin is shown as loading control. WCL, whole cell lysates. c , Immunoblot analysis of phosphotyrosine immunoprecipitates from U87 glioma cells expressing empty vector, FGFR3, F3-T3 or F3-T3-K508M using the indicated antibodies (left panels). WCL, right panels. The asterisk indicates a non-specific band. d , Immunoblot analysis of phosphotyrosine immunoprecipitates from HA expressing empty vector, F3-T3 or F3-T3-K508M using the indicated antibodies (left panels); WCL, right panels. FAK is shown as loading control. e , Immunoblot analysis of phosphotyrosine immunoprecipitates from GSC1123 harboring endogenous F3-T3 shows a decreased phosphorylation of F3-T3 substrates following treatment with AZD4547 for the indicated times (left panels); WCL, right panels. Paxillin is shown as loading control. f , Immunoblot analysis of canonical FGFR signaling proteins in GSC1123 treated with AZD4547 for the indicated time. β-actin is shown as loading control. g , Immunoblot analysis of phosphotyrosine immunoprecipitates from HA-F3-T3 or HA-vector transduced with WT or the unphosphorylable (Y/A) F3-T3 kinase substrate mutants. Paxillin is shown as loading control. The asterisk indicates a non-specific band. h , Confocal images of immunofluorescence staining using the pY122-PIN4 specific antibody (red) in HA transduced with vector or F3-T3 without or with silencing of endogenous PIN4 . Nuclei were stained with DAPI (blue). i , Immunoblot analysis of pY122-PIN4, total PIN4 and FGFR3 in SF126 cells transduced with FGFR3, F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Molecular weights are indicated in all panels. Experiments in b - g , and i were repeated independently three times with similar results. Experiment in h was repeated independently four times with similar results.

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: Phosphorylation of Tyr-122 of PIN4 by F3-T3 a , Amino acid sequence flanking Tyr-122 of PIN4 (in red) is evolutionarily conserved. b , Immunoprecipitation-western blot analysis of HA-F3-T3 or F3-T3-K508M with or without silencing of endogenous PIN4 . β-actin is shown as loading control. WCL, whole cell lysates. c , Immunoblot analysis of phosphotyrosine immunoprecipitates from U87 glioma cells expressing empty vector, FGFR3, F3-T3 or F3-T3-K508M using the indicated antibodies (left panels). WCL, right panels. The asterisk indicates a non-specific band. d , Immunoblot analysis of phosphotyrosine immunoprecipitates from HA expressing empty vector, F3-T3 or F3-T3-K508M using the indicated antibodies (left panels); WCL, right panels. FAK is shown as loading control. e , Immunoblot analysis of phosphotyrosine immunoprecipitates from GSC1123 harboring endogenous F3-T3 shows a decreased phosphorylation of F3-T3 substrates following treatment with AZD4547 for the indicated times (left panels); WCL, right panels. Paxillin is shown as loading control. f , Immunoblot analysis of canonical FGFR signaling proteins in GSC1123 treated with AZD4547 for the indicated time. β-actin is shown as loading control. g , Immunoblot analysis of phosphotyrosine immunoprecipitates from HA-F3-T3 or HA-vector transduced with WT or the unphosphorylable (Y/A) F3-T3 kinase substrate mutants. Paxillin is shown as loading control. The asterisk indicates a non-specific band. h , Confocal images of immunofluorescence staining using the pY122-PIN4 specific antibody (red) in HA transduced with vector or F3-T3 without or with silencing of endogenous PIN4 . Nuclei were stained with DAPI (blue). i , Immunoblot analysis of pY122-PIN4, total PIN4 and FGFR3 in SF126 cells transduced with FGFR3, F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Molecular weights are indicated in all panels. Experiments in b - g , and i were repeated independently three times with similar results. Experiment in h was repeated independently four times with similar results.

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Sequencing, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Transduction, Immunofluorescence, Staining

    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Immunoprecipitation, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Immunofluorescence, Staining, Immunostaining, Infection, Expressing, Construct, Mutagenesis

    Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( a ) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates ( b ) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates ( c ) from WT and Peli2 −/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( e ) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates ( f ) from WT and Peli2 −/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2 −/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( a ) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates ( b ) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates ( c ) from WT and Peli2 −/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( e ) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates ( f ) from WT and Peli2 −/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2 −/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Transfection, Isolation, Mouse Assay, Quantitative RT-PCR, Expressing

    Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2 −/− BMDMs were infected with a, b C. rodentium , c, d E.coli , or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1β, IL-18, and CXCL1 in medium from BMDMs infected for 6 h. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0–6 h. f Immunoblot analysis of IL-1β and Caspase-1 in lysates from cells infected with PAO1 for 3 h. β-Actin was used as loading controls. g ELISA of IL-1β, IL-18, and IL-6 in peritoneal lavage from WT and Peli2 −/− mice previously infected for 10 h by intraperitoneal injection of PAO1 (1.5 × 10 7 CFU). * p

    Journal: Nature Communications

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    doi: 10.1038/s41467-018-03669-z

    Figure Lengend Snippet: Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2 −/− BMDMs were infected with a, b C. rodentium , c, d E.coli , or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1β, IL-18, and CXCL1 in medium from BMDMs infected for 6 h. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0–6 h. f Immunoblot analysis of IL-1β and Caspase-1 in lysates from cells infected with PAO1 for 3 h. β-Actin was used as loading controls. g ELISA of IL-1β, IL-18, and IL-6 in peritoneal lavage from WT and Peli2 −/− mice previously infected for 10 h by intraperitoneal injection of PAO1 (1.5 × 10 7 CFU). * p

    Article Snippet: Anti-p-ERK (9101), anti-ERK (9102), anti-p-IκBα (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1β (AF-401-NA) was from R & D; anti-IκB-α (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-β-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega.

    Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection