14 3 3 beta alpha  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 14 3 3 beta alpha
    14 3 3 Beta Alpha, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/14 3 3 beta alpha/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    14 3 3 beta alpha  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 14 3 3 beta alpha
    14 3 3 Beta Alpha, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    anti human 14 3 3ζ  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti human 14 3 3ζ
    Anti Human 14 3 3ζ, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti isg15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti isg15
    A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of <t>ISG15,</t> Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
    Anti Isg15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    anti isg15 - by Bioz Stars, 2023-01
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    1) Product Images from "Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity"

    Article Title: Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity

    Journal: bioRxiv

    doi: 10.1101/2021.09.29.462388

    A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of ISG15, Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
    Figure Legend Snippet: A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of ISG15, Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection, Produced, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control

    A. il1b, tnfa, il12, cxcl10, ifnb, and isg15 mRNA levels were assessed by qPCR in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. B. il10 mRNA levels were assessed by qPCR in the lungs of infected wild-type (WT), sarm1 -/- , and Sarm1 em1.1Tft mice for 24. C. Percentage of immune cells in the lungs of wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. D. PhenoGraph cluster analysis of immune populations in the lungs wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. E. Heat map showing relative signal intensities of the indicated markers on neutrophils of clusters 13, 15 found in the lungs of infected wild-type mice, and clusters 11 and 13 detected in the lungs of sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. F. Heat map showing relative signal intensities of the indicated markers on alveolar macrophages of clusters 5 and 6 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. G. Heat map showing relative signal intensities of the indicated markers on interstitial macrophages of clusters 16 and 17 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. H. Bacterial load in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. I. Bacterial load in the livers and spleens of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. In panels A, B, H and I values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; **P≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
    Figure Legend Snippet: A. il1b, tnfa, il12, cxcl10, ifnb, and isg15 mRNA levels were assessed by qPCR in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. B. il10 mRNA levels were assessed by qPCR in the lungs of infected wild-type (WT), sarm1 -/- , and Sarm1 em1.1Tft mice for 24. C. Percentage of immune cells in the lungs of wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. D. PhenoGraph cluster analysis of immune populations in the lungs wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. E. Heat map showing relative signal intensities of the indicated markers on neutrophils of clusters 13, 15 found in the lungs of infected wild-type mice, and clusters 11 and 13 detected in the lungs of sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. F. Heat map showing relative signal intensities of the indicated markers on alveolar macrophages of clusters 5 and 6 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. G. Heat map showing relative signal intensities of the indicated markers on interstitial macrophages of clusters 16 and 17 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. H. Bacterial load in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. I. Bacterial load in the livers and spleens of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. In panels A, B, H and I values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; **P≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

    Techniques Used: Infection

    anti 14 3 3 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti 14 3 3 antibody
    Anti 14 3 3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    anti 14 3 3 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti 14 3 3 antibody
    Anti 14 3 3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti 14 3 3 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    anti 14 3 3 β α  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti 14 3 3 β α
    Anti 14 3 3 β α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti 14 3 3 β α/product/Cell Signaling Technology Inc
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    rabbit polyclonal anti 14 3 3 γ  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti 14 3 3 γ
    KEY RESOURCES TABLE
    Rabbit Polyclonal Anti 14 3 3 γ, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti 14 3 3 γ/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    1) Product Images from "Decoding the Function of Expansion Segments in Ribosomes"

    Article Title: Decoding the Function of Expansion Segments in Ribosomes

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.11.023

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, SYBR Green Assay, Reporter Assay, Plasmid Preparation, Software, Negative Control

    primary rabbit polyclonal antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc primary rabbit polyclonal antibodies
    Primary Rabbit Polyclonal Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/primary rabbit polyclonal antibodies/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    primary rabbit polyclonal antibodies - by Bioz Stars, 2023-01
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    anti we14  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti we14
    Anti We14, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti 14 3 3β  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti 14 3 3β
    FAM163A promoted the phosphorylation of the ERK/p90RSK signaling pathway by interacting with 14-3-3β. ( A ) The expression of 14-3-3β, 14-3-3γ, 14-3-3ζ/δ and 14-3-3ε were evaluated by Western blotting after transfecting with FAM163A cDNA and interfering by siRNA in LK2 and SK-MES-1 cell lines. ( B ) We designed 3 individual siRNA for 14-3-3β in which si14-3-3β#3 was shown significantly downregulated the protein level of 14-3-3β and was chosen to perform the subsequent experiments. ( C ) The levels of FAM163A, 14-3-3β, p-ERK, ERK, p -p90RSK and p90RSK were tested by Western blots in LK2 and SK-MES-1 cells after transfected with the indicated pretreatments. ( D ) The lysates of SK-MES-1 cell stably transfected with FAM163A were immunoprecipitated overnight at 4°C with anti-FAM163A. The immunoprecipitated proteins were analyzed by Western blots using anti-FAM163A and <t>anti-14-3-3β</t> antibody, respectively. ( E ) The expression of FAM163A and 14-3-3β in SK-MES-1 cells were detected by the indicated antibodies and stained with different secondary antibodies: TRITC (red) and FITC (green), respectively. The locations were detected by immunofluorescence. The colony formation ability ( F-G ) and cell viability ( H ) in LK2 and SK-MES-1 cells transfected with the indicated pretreatments were evaluated by colony formation assay and MTT assays. Error bars represent SEM, * P< 0.05, scale bar =20 μM.
    Anti 14 3 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti 14 3 3β - by Bioz Stars, 2023-01
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    1) Product Images from "FAM163A, a positive regulator of ERK signaling pathway, interacts with 14-3-3β and promotes cell proliferation in squamous cell lung carcinoma"

    Article Title: FAM163A, a positive regulator of ERK signaling pathway, interacts with 14-3-3β and promotes cell proliferation in squamous cell lung carcinoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S214731

    FAM163A promoted the phosphorylation of the ERK/p90RSK signaling pathway by interacting with 14-3-3β. ( A ) The expression of 14-3-3β, 14-3-3γ, 14-3-3ζ/δ and 14-3-3ε were evaluated by Western blotting after transfecting with FAM163A cDNA and interfering by siRNA in LK2 and SK-MES-1 cell lines. ( B ) We designed 3 individual siRNA for 14-3-3β in which si14-3-3β#3 was shown significantly downregulated the protein level of 14-3-3β and was chosen to perform the subsequent experiments. ( C ) The levels of FAM163A, 14-3-3β, p-ERK, ERK, p -p90RSK and p90RSK were tested by Western blots in LK2 and SK-MES-1 cells after transfected with the indicated pretreatments. ( D ) The lysates of SK-MES-1 cell stably transfected with FAM163A were immunoprecipitated overnight at 4°C with anti-FAM163A. The immunoprecipitated proteins were analyzed by Western blots using anti-FAM163A and anti-14-3-3β antibody, respectively. ( E ) The expression of FAM163A and 14-3-3β in SK-MES-1 cells were detected by the indicated antibodies and stained with different secondary antibodies: TRITC (red) and FITC (green), respectively. The locations were detected by immunofluorescence. The colony formation ability ( F-G ) and cell viability ( H ) in LK2 and SK-MES-1 cells transfected with the indicated pretreatments were evaluated by colony formation assay and MTT assays. Error bars represent SEM, * P< 0.05, scale bar =20 μM.
    Figure Legend Snippet: FAM163A promoted the phosphorylation of the ERK/p90RSK signaling pathway by interacting with 14-3-3β. ( A ) The expression of 14-3-3β, 14-3-3γ, 14-3-3ζ/δ and 14-3-3ε were evaluated by Western blotting after transfecting with FAM163A cDNA and interfering by siRNA in LK2 and SK-MES-1 cell lines. ( B ) We designed 3 individual siRNA for 14-3-3β in which si14-3-3β#3 was shown significantly downregulated the protein level of 14-3-3β and was chosen to perform the subsequent experiments. ( C ) The levels of FAM163A, 14-3-3β, p-ERK, ERK, p -p90RSK and p90RSK were tested by Western blots in LK2 and SK-MES-1 cells after transfected with the indicated pretreatments. ( D ) The lysates of SK-MES-1 cell stably transfected with FAM163A were immunoprecipitated overnight at 4°C with anti-FAM163A. The immunoprecipitated proteins were analyzed by Western blots using anti-FAM163A and anti-14-3-3β antibody, respectively. ( E ) The expression of FAM163A and 14-3-3β in SK-MES-1 cells were detected by the indicated antibodies and stained with different secondary antibodies: TRITC (red) and FITC (green), respectively. The locations were detected by immunofluorescence. The colony formation ability ( F-G ) and cell viability ( H ) in LK2 and SK-MES-1 cells transfected with the indicated pretreatments were evaluated by colony formation assay and MTT assays. Error bars represent SEM, * P< 0.05, scale bar =20 μM.

    Techniques Used: Expressing, Western Blot, Transfection, Stable Transfection, Immunoprecipitation, Staining, Immunofluorescence, Colony Assay

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    Cell Signaling Technology Inc 14 3 3 beta alpha
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    A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of <t>ISG15,</t> Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
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    A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of <t>ISG15,</t> Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
    Anti 14 3 3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti 14 3 3 β α
    A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of <t>ISG15,</t> Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
    Anti 14 3 3 β α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc primary rabbit polyclonal antibodies
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    Cell Signaling Technology Inc anti 14 3 3β
    FAM163A promoted the phosphorylation of the ERK/p90RSK signaling pathway by interacting with 14-3-3β. ( A ) The expression of 14-3-3β, 14-3-3γ, 14-3-3ζ/δ and 14-3-3ε were evaluated by Western blotting after transfecting with FAM163A cDNA and interfering by siRNA in LK2 and SK-MES-1 cell lines. ( B ) We designed 3 individual siRNA for 14-3-3β in which si14-3-3β#3 was shown significantly downregulated the protein level of 14-3-3β and was chosen to perform the subsequent experiments. ( C ) The levels of FAM163A, 14-3-3β, p-ERK, ERK, p -p90RSK and p90RSK were tested by Western blots in LK2 and SK-MES-1 cells after transfected with the indicated pretreatments. ( D ) The lysates of SK-MES-1 cell stably transfected with FAM163A were immunoprecipitated overnight at 4°C with anti-FAM163A. The immunoprecipitated proteins were analyzed by Western blots using anti-FAM163A and <t>anti-14-3-3β</t> antibody, respectively. ( E ) The expression of FAM163A and 14-3-3β in SK-MES-1 cells were detected by the indicated antibodies and stained with different secondary antibodies: TRITC (red) and FITC (green), respectively. The locations were detected by immunofluorescence. The colony formation ability ( F-G ) and cell viability ( H ) in LK2 and SK-MES-1 cells transfected with the indicated pretreatments were evaluated by colony formation assay and MTT assays. Error bars represent SEM, * P< 0.05, scale bar =20 μM.
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    Image Search Results


    A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of ISG15, Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

    Journal: bioRxiv

    Article Title: Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity

    doi: 10.1101/2021.09.29.462388

    Figure Lengend Snippet: A. ELISA of TNFα, IL1β, CXCL10 secreted by wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. Type I IFN levels determined in the supernatants of macrophages 16 h post infection. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as OD 655 . After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. B. Immunoblot analysis of ISG15, Viperin and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. C. ELISA of TNFα , IL1β, CXCL10 secreted by wild-type (WT) macrophages, and retrovirally transfected sarm1 -/- cells with FLAG-SARM1 or control vector (EV) non-infected (ni) or infected with Kp52145 (Kp) for 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. D. il1b, tnfa, cxcl10, isg15, ifit1 , and mx1 mRNA levels were assessed by qPCR, in wild-type (WT) and sarm1 -/- macrophages non-infected (ni) or infected with Kp52145 for 6 and 16 h. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. E. Immunoblot analysis of phosphorylated Iκκα/β (P-Iκκ), phosphorylated IκBα (P-IκBα), total IκBα (IκBα) and tubulin levels in lysates of wild-type (WT) and sarm1-/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. F. Immunoblot analysis of phosphorylated TBK1 (P-TBK1), phosphorylated Irf3 (P-IRF3) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. After 1 h contact, the medium was replaced with medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. G. Sarm1 FLAG macrophages were transfected with a MyD88-HA or TRIF-HA plasmids, and the following day infected with Kp52145. Cells were lysed in RIPA buffer, and lysates immunoprecipitated using anti-FLAG antibody. Preimmune mouse IgG served as negative control. H. Immunoblot analysis of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK phosphorylated p38 (P-p38) and tubulin levels in lysates of wild-type (WT) and sarm1 -/- macrophages non-infected (NI) or infected with Kp52145 for the indicated time points. In panels B, E, F, G, and H images are representative of three independent experiments. In panels A, C and D values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 for the indicated comparisons determined using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

    Article Snippet: The following antibodies were used: anti-IL-1β (anti-goat, 1:1000; # AF-401-NA, R&D Systems), anti-caspase-1 (anti-rabbit, 1:1000; #24232, Cell Signaling), anti-AIM2 (anti-rabbit, 1:1000; sc- 515895, Santa Cruz), anti-NLRP3 (anti-mouse, 1:1000; #15101, Cell Signaling), anti-Gasdermin-D (anti-rabbit, 1:1000; #93709, Cell Signaling), anti-Viperin (anti-rabbit, 1:1000 # NBP2-03971, Novus Biologicals), anti-ISG15 (1:1000; #9636, Cell Signaling), anti-phospho-STAT3 (anti-rabbit, 1:1,000; #9145, Cell Signaling), anti- κBα (anti-rabbit, 1:1,000; #4814, Cell Signaling), anti- phospho-Iκ Bα (Ser32) (anti-goat, 1:1,000; #9246, Cell Signaling), anti-phospho-AKT1/2/3 (Ser 473) (anti-rabbit, 1:1000; sc-33437, Santa Cruz), anti-phospho-IKKα/β (Ser176/180)(16A6) (anti- rabbit, 1:1000; #2697,Cell Signaling), anti-phospho-IRF3 (Ser 396) (anti-rabbit, 1:1000; #4947, Cell Signaling), anti-phospho-p-TBK-1/NAK (Ser172) (D52C2) (anti-rabbit, 1:1000; #5483, Cell Signaling), anti-phospho-JNK (anti-rabbit, 1:1000; #9251S, Cell Signaling), anti-phospho-ERK (anti-rabbit, 1:1000; #9101, Cell Signaling), anti-phospho-p38 MAPK (Thr180/Tyr182) (D3F9) (anti-rabbit, 1:1000; #4511, Cell Signaling), anti-SARM1 (anti-chicken, 1:70; generated by Icosagen by immunizing chicken with the TIR domain of human SARM1), anti-Flag M2 (1 μ Sigma F3165), anti-HA (1:1000, Santa Cruz Biotechnology sc-805).

    Techniques: Enzyme-linked Immunosorbent Assay, Infection, Produced, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control

    A. il1b, tnfa, il12, cxcl10, ifnb, and isg15 mRNA levels were assessed by qPCR in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. B. il10 mRNA levels were assessed by qPCR in the lungs of infected wild-type (WT), sarm1 -/- , and Sarm1 em1.1Tft mice for 24. C. Percentage of immune cells in the lungs of wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. D. PhenoGraph cluster analysis of immune populations in the lungs wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. E. Heat map showing relative signal intensities of the indicated markers on neutrophils of clusters 13, 15 found in the lungs of infected wild-type mice, and clusters 11 and 13 detected in the lungs of sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. F. Heat map showing relative signal intensities of the indicated markers on alveolar macrophages of clusters 5 and 6 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. G. Heat map showing relative signal intensities of the indicated markers on interstitial macrophages of clusters 16 and 17 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. H. Bacterial load in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. I. Bacterial load in the livers and spleens of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. In panels A, B, H and I values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; **P≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

    Journal: bioRxiv

    Article Title: Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity

    doi: 10.1101/2021.09.29.462388

    Figure Lengend Snippet: A. il1b, tnfa, il12, cxcl10, ifnb, and isg15 mRNA levels were assessed by qPCR in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. B. il10 mRNA levels were assessed by qPCR in the lungs of infected wild-type (WT), sarm1 -/- , and Sarm1 em1.1Tft mice for 24. C. Percentage of immune cells in the lungs of wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. D. PhenoGraph cluster analysis of immune populations in the lungs wild-type (WT), and sarm1 -/- mice non-infected (ni) or infected intranasally with Kp52145 for 24. Results are based on data from three mice per group. E. Heat map showing relative signal intensities of the indicated markers on neutrophils of clusters 13, 15 found in the lungs of infected wild-type mice, and clusters 11 and 13 detected in the lungs of sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. F. Heat map showing relative signal intensities of the indicated markers on alveolar macrophages of clusters 5 and 6 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. G. Heat map showing relative signal intensities of the indicated markers on interstitial macrophages of clusters 16 and 17 found in the lungs of infected wild-type and sarm1 -/- mice. The heat map is coloured based on signal intensity of the indicated markers. Results are based on data from three mice per group. H. Bacterial load in the lungs of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. I. Bacterial load in the livers and spleens of infected wild-type mice (WT), sarm1 -/- , and Sarm1 em1.1Tft for 24. Each dot represents a different mouse. In panels A, B, H and I values are presented as the mean ± SD of three independent experiments measured in duplicate. ****P ≤ 0.0001; ***P ≤ 0.001; **P≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

    Article Snippet: The following antibodies were used: anti-IL-1β (anti-goat, 1:1000; # AF-401-NA, R&D Systems), anti-caspase-1 (anti-rabbit, 1:1000; #24232, Cell Signaling), anti-AIM2 (anti-rabbit, 1:1000; sc- 515895, Santa Cruz), anti-NLRP3 (anti-mouse, 1:1000; #15101, Cell Signaling), anti-Gasdermin-D (anti-rabbit, 1:1000; #93709, Cell Signaling), anti-Viperin (anti-rabbit, 1:1000 # NBP2-03971, Novus Biologicals), anti-ISG15 (1:1000; #9636, Cell Signaling), anti-phospho-STAT3 (anti-rabbit, 1:1,000; #9145, Cell Signaling), anti- κBα (anti-rabbit, 1:1,000; #4814, Cell Signaling), anti- phospho-Iκ Bα (Ser32) (anti-goat, 1:1,000; #9246, Cell Signaling), anti-phospho-AKT1/2/3 (Ser 473) (anti-rabbit, 1:1000; sc-33437, Santa Cruz), anti-phospho-IKKα/β (Ser176/180)(16A6) (anti- rabbit, 1:1000; #2697,Cell Signaling), anti-phospho-IRF3 (Ser 396) (anti-rabbit, 1:1000; #4947, Cell Signaling), anti-phospho-p-TBK-1/NAK (Ser172) (D52C2) (anti-rabbit, 1:1000; #5483, Cell Signaling), anti-phospho-JNK (anti-rabbit, 1:1000; #9251S, Cell Signaling), anti-phospho-ERK (anti-rabbit, 1:1000; #9101, Cell Signaling), anti-phospho-p38 MAPK (Thr180/Tyr182) (D3F9) (anti-rabbit, 1:1000; #4511, Cell Signaling), anti-SARM1 (anti-chicken, 1:70; generated by Icosagen by immunizing chicken with the TIR domain of human SARM1), anti-Flag M2 (1 μ Sigma F3165), anti-HA (1:1000, Santa Cruz Biotechnology sc-805).

    Techniques: Infection

    KEY RESOURCES TABLE

    Journal: Molecular cell

    Article Title: Decoding the Function of Expansion Segments in Ribosomes

    doi: 10.1016/j.molcel.2018.11.023

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit polyclonal anti-14-3-3 γ , Cell Signaling technology , Cat# D15B7, RRID: AB_10827887.

    Techniques: Recombinant, SYBR Green Assay, Reporter Assay, Plasmid Preparation, Software, Negative Control

    FAM163A promoted the phosphorylation of the ERK/p90RSK signaling pathway by interacting with 14-3-3β. ( A ) The expression of 14-3-3β, 14-3-3γ, 14-3-3ζ/δ and 14-3-3ε were evaluated by Western blotting after transfecting with FAM163A cDNA and interfering by siRNA in LK2 and SK-MES-1 cell lines. ( B ) We designed 3 individual siRNA for 14-3-3β in which si14-3-3β#3 was shown significantly downregulated the protein level of 14-3-3β and was chosen to perform the subsequent experiments. ( C ) The levels of FAM163A, 14-3-3β, p-ERK, ERK, p -p90RSK and p90RSK were tested by Western blots in LK2 and SK-MES-1 cells after transfected with the indicated pretreatments. ( D ) The lysates of SK-MES-1 cell stably transfected with FAM163A were immunoprecipitated overnight at 4°C with anti-FAM163A. The immunoprecipitated proteins were analyzed by Western blots using anti-FAM163A and anti-14-3-3β antibody, respectively. ( E ) The expression of FAM163A and 14-3-3β in SK-MES-1 cells were detected by the indicated antibodies and stained with different secondary antibodies: TRITC (red) and FITC (green), respectively. The locations were detected by immunofluorescence. The colony formation ability ( F-G ) and cell viability ( H ) in LK2 and SK-MES-1 cells transfected with the indicated pretreatments were evaluated by colony formation assay and MTT assays. Error bars represent SEM, * P< 0.05, scale bar =20 μM.

    Journal: OncoTargets and therapy

    Article Title: FAM163A, a positive regulator of ERK signaling pathway, interacts with 14-3-3β and promotes cell proliferation in squamous cell lung carcinoma

    doi: 10.2147/OTT.S214731

    Figure Lengend Snippet: FAM163A promoted the phosphorylation of the ERK/p90RSK signaling pathway by interacting with 14-3-3β. ( A ) The expression of 14-3-3β, 14-3-3γ, 14-3-3ζ/δ and 14-3-3ε were evaluated by Western blotting after transfecting with FAM163A cDNA and interfering by siRNA in LK2 and SK-MES-1 cell lines. ( B ) We designed 3 individual siRNA for 14-3-3β in which si14-3-3β#3 was shown significantly downregulated the protein level of 14-3-3β and was chosen to perform the subsequent experiments. ( C ) The levels of FAM163A, 14-3-3β, p-ERK, ERK, p -p90RSK and p90RSK were tested by Western blots in LK2 and SK-MES-1 cells after transfected with the indicated pretreatments. ( D ) The lysates of SK-MES-1 cell stably transfected with FAM163A were immunoprecipitated overnight at 4°C with anti-FAM163A. The immunoprecipitated proteins were analyzed by Western blots using anti-FAM163A and anti-14-3-3β antibody, respectively. ( E ) The expression of FAM163A and 14-3-3β in SK-MES-1 cells were detected by the indicated antibodies and stained with different secondary antibodies: TRITC (red) and FITC (green), respectively. The locations were detected by immunofluorescence. The colony formation ability ( F-G ) and cell viability ( H ) in LK2 and SK-MES-1 cells transfected with the indicated pretreatments were evaluated by colony formation assay and MTT assays. Error bars represent SEM, * P< 0.05, scale bar =20 μM.

    Article Snippet: The HBE, LK2 and SK-MES-1 cells were fixed with 4% paraformaldehyde, blocked with 1% bovine serum albumin (BSA) and then incubated with the FAM163A (1:50, sc-390936, Santa Cruz Biotechnology), anti-ERK (#4695, Cell Signaling Technology) and anti-14-3-3β (#9636, Cell Signaling Technology) overnight at 4°C, followed by incubation with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:50, Zhongshan Golden Bridge, Beijing, China) at 37°C.

    Techniques: Expressing, Western Blot, Transfection, Stable Transfection, Immunoprecipitation, Staining, Immunofluorescence, Colony Assay