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thpd komyd thp1  (InvivoGen)
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InvivoGen thpd komyd thp1
STING1 is required for LPS-induced ACOD1 expression (A–D) RAW264.7 and <t>THP1</t> cells were treated with LPS (50-5000 ng/mL) in the absence or presence of 3′3′-cGAMP (10 μg/mL) for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (E and F) RAW264.7 and THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 2′2′-cGAMP, 2′3′-cGAMP, c-di-AMP, or c-di-GMP at 10 μg/mL for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; p < 0.01 versus LPS along group; two-way ANOVA with Tukey’s multiple comparisons test). (G–J) WT and V155M-THP1 cells were treated with indicated LPS for 6 h, and then IFNA1 mRNA, IL6 mRNA, ACOD1 mRNA, and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (K) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) for 6 h. (L) In parallel, intracellular itaconate concentration was assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).
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STING1 is required for LPS-induced ACOD1 expression (A–D) RAW264.7 and THP1 cells were treated with LPS (50-5000 ng/mL) in the absence or presence of 3′3′-cGAMP (10 μg/mL) for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (E and F) RAW264.7 and THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 2′2′-cGAMP, 2′3′-cGAMP, c-di-AMP, or c-di-GMP at 10 μg/mL for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; p < 0.01 versus LPS along group; two-way ANOVA with Tukey’s multiple comparisons test). (G–J) WT and V155M-THP1 cells were treated with indicated LPS for 6 h, and then IFNA1 mRNA, IL6 mRNA, ACOD1 mRNA, and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (K) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) for 6 h. (L) In parallel, intracellular itaconate concentration was assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: STING1 is required for LPS-induced ACOD1 expression (A–D) RAW264.7 and THP1 cells were treated with LPS (50-5000 ng/mL) in the absence or presence of 3′3′-cGAMP (10 μg/mL) for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (E and F) RAW264.7 and THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 2′2′-cGAMP, 2′3′-cGAMP, c-di-AMP, or c-di-GMP at 10 μg/mL for 6 h, and then Acod1/ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; p < 0.01 versus LPS along group; two-way ANOVA with Tukey’s multiple comparisons test). (G–J) WT and V155M-THP1 cells were treated with indicated LPS for 6 h, and then IFNA1 mRNA, IL6 mRNA, ACOD1 mRNA, and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (K) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) for 6 h. (L) In parallel, intracellular itaconate concentration was assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: The STING1 −/− (thpd-kostg), IRF3 −/− (thpd-koirf3; rawl-koirf3), STING1-V155M (thpd-m155), CGAS −/− (thpd-kocgas), and MYD88 −/− (thpd-komyd) THP1 or RAW264.7 cell lines were obtained from InvivoGen.

Techniques: Expressing, Concentration Assay, Western Blot

MYD88, but not CGAS, mediates LPS-induced ACOD1 expression (A–C) Indicated THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 3′3′-cGAMP (10 μg/mL), G3-YSD (1 μg/mL), G3-YSD-Ctrl (1 μg/mL), or LyoVec for 6 h, and then ACOD1 or IFNA1 mRNA was assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D and E) Indicated THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 3′3′-cGAMP, 2′2′-cGAMP, 2′3′-cGAMP, c-di-AMP, or c-di-GMP at 10 μg/mL for 6 h, and then ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (F) Western blot analysis of protein expression in indicated V155M-THP1 cells following treatment with LPS (500 ng/mL) for 6 h. (G and H) Analysis of ACOD1 mRNA and intracellular itaconate concentration in indicated V155M-THP1 cells following treatment with LPS (50-5000 ng/mL) for 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: MYD88, but not CGAS, mediates LPS-induced ACOD1 expression (A–C) Indicated THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 3′3′-cGAMP (10 μg/mL), G3-YSD (1 μg/mL), G3-YSD-Ctrl (1 μg/mL), or LyoVec for 6 h, and then ACOD1 or IFNA1 mRNA was assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D and E) Indicated THP1 cells were treated with LPS (500 ng/mL) in the absence or presence of 3′3′-cGAMP, 2′2′-cGAMP, 2′3′-cGAMP, c-di-AMP, or c-di-GMP at 10 μg/mL for 6 h, and then ACOD1 mRNA and intracellular itaconate concentration were assayed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (F) Western blot analysis of protein expression in indicated V155M-THP1 cells following treatment with LPS (500 ng/mL) for 6 h. (G and H) Analysis of ACOD1 mRNA and intracellular itaconate concentration in indicated V155M-THP1 cells following treatment with LPS (50-5000 ng/mL) for 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: The STING1 −/− (thpd-kostg), IRF3 −/− (thpd-koirf3; rawl-koirf3), STING1-V155M (thpd-m155), CGAS −/− (thpd-kocgas), and MYD88 −/− (thpd-komyd) THP1 or RAW264.7 cell lines were obtained from InvivoGen.

Techniques: Expressing, Concentration Assay, Western Blot

The MYD88-STING1 protein complex prevents autophagic degradation of STING1 (A) IP analysis of the MYD88-STING1 protein complex in THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) for 6 h. (B) Representative colocalization images of MYD88 and STING1 in RAW264.7 cells in the presence or absence of LPS (500 ng/mL) and 3′3'-cGAMP (10 μg/mL) for 6 h. Bar = 10 μm. (C–F) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) in the absence or presence of chloroquine (50 μM) or MG132 (5 μM) for 6 h. (G) qPCR analysis of ACOD1 mRNA expression in indicated THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) in the absence or presence of chloroquine (CQ; 50 μM) for 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: The MYD88-STING1 protein complex prevents autophagic degradation of STING1 (A) IP analysis of the MYD88-STING1 protein complex in THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) for 6 h. (B) Representative colocalization images of MYD88 and STING1 in RAW264.7 cells in the presence or absence of LPS (500 ng/mL) and 3′3'-cGAMP (10 μg/mL) for 6 h. Bar = 10 μm. (C–F) Western blot analysis of protein expression in indicated THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) in the absence or presence of chloroquine (50 μM) or MG132 (5 μM) for 6 h. (G) qPCR analysis of ACOD1 mRNA expression in indicated THP1 cells following treatment with LPS (500 ng/mL) and 3′3′-cGAMP (10 μg/mL) in the absence or presence of chloroquine (CQ; 50 μM) for 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: The STING1 −/− (thpd-kostg), IRF3 −/− (thpd-koirf3; rawl-koirf3), STING1-V155M (thpd-m155), CGAS −/− (thpd-kocgas), and MYD88 −/− (thpd-komyd) THP1 or RAW264.7 cell lines were obtained from InvivoGen.

Techniques: Western Blot, Expressing

STING1 and MYD88 selectively mediate TLR signaling to induce ACOD1 expression (A) Indicated THP1 cells were stimulated with pam3CSK4 (1 ng/mL), HKLM (10 7 cells/mL), poly (I:C) (10 μg/mL), LPS (500 ng/mL), FLA-ST (100 ng/mL), FSL1 (0.1 ng/mL), imiquimod (5 μg/mL), ssRNA40 (5 μg/mL), or ODN2006 (10 μg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (B) Wild-type THP1 cells were stimulated with indicated TLR ligands for 3 h and the mRNA level of TNF was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples). (C) Wild-type THP1 cells were stimulated with indicated TLR ligands in the absence or presence of 3′3′-cGAMP (10 μg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D) Wild-type and V115M THP1 cells were stimulated with indicated TLR ligands for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). The TLR ligand concentration used in panels B-D is the same as for panel A.

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: STING1 and MYD88 selectively mediate TLR signaling to induce ACOD1 expression (A) Indicated THP1 cells were stimulated with pam3CSK4 (1 ng/mL), HKLM (10 7 cells/mL), poly (I:C) (10 μg/mL), LPS (500 ng/mL), FLA-ST (100 ng/mL), FSL1 (0.1 ng/mL), imiquimod (5 μg/mL), ssRNA40 (5 μg/mL), or ODN2006 (10 μg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (B) Wild-type THP1 cells were stimulated with indicated TLR ligands for 3 h and the mRNA level of TNF was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples). (C) Wild-type THP1 cells were stimulated with indicated TLR ligands in the absence or presence of 3′3′-cGAMP (10 μg/mL) for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D) Wild-type and V115M THP1 cells were stimulated with indicated TLR ligands for 6 h and the mRNA level of ACOD1 was assessed (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). The TLR ligand concentration used in panels B-D is the same as for panel A.

Article Snippet: The STING1 −/− (thpd-kostg), IRF3 −/− (thpd-koirf3; rawl-koirf3), STING1-V155M (thpd-m155), CGAS −/− (thpd-kocgas), and MYD88 −/− (thpd-komyd) THP1 or RAW264.7 cell lines were obtained from InvivoGen.

Techniques: Expressing, Concentration Assay

The IRF3-JUN transcription factor complex is required for ACOD1 upregulation (A) Western blot analysis of p-p65 and p -IRF3 in indicated THP1 cells following stimulation with 3′3'-cGAMP/LPS for 6 h. (B and C) In parallel, the activity of IRF or NF-κB was assayed by luciferase reporter gene assay (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D and E) Analysis of ACOD1 mRNA in indicated THP1 or RAW264.7 cells following stimulation with 3′3′-cGAMP/LPS for 3 and 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (F) Heatmap of kinase phosphorylation changes in indicated THP1 cells following 3′3'-cGAMP/LPS stimulation for 6 h. (G) IP analysis of IRF3-JUN protein complex in THP1 cells following stimulation with 3′3′-cGAMP/LPS for 6 h. (H and I) ChIP-qPCR analysis of the binding of IRF3 and JUN on the promoter of ACOD1 in THP1 cells following stimulation with 3′3′-cGAMP/LPS for 3 and 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; t test). (J) Western blot analysis of ACOD1 in indicated THP1 cells following stimulation with 3′3′-cGAMP/LPS for 6 h.

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet: The IRF3-JUN transcription factor complex is required for ACOD1 upregulation (A) Western blot analysis of p-p65 and p -IRF3 in indicated THP1 cells following stimulation with 3′3'-cGAMP/LPS for 6 h. (B and C) In parallel, the activity of IRF or NF-κB was assayed by luciferase reporter gene assay (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (D and E) Analysis of ACOD1 mRNA in indicated THP1 or RAW264.7 cells following stimulation with 3′3′-cGAMP/LPS for 3 and 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test). (F) Heatmap of kinase phosphorylation changes in indicated THP1 cells following 3′3'-cGAMP/LPS stimulation for 6 h. (G) IP analysis of IRF3-JUN protein complex in THP1 cells following stimulation with 3′3′-cGAMP/LPS for 6 h. (H and I) ChIP-qPCR analysis of the binding of IRF3 and JUN on the promoter of ACOD1 in THP1 cells following stimulation with 3′3′-cGAMP/LPS for 3 and 6 h (Data are presented as mean ± SD; n = 3 biologically independent samples; t test). (J) Western blot analysis of ACOD1 in indicated THP1 cells following stimulation with 3′3′-cGAMP/LPS for 6 h.

Article Snippet: The STING1 −/− (thpd-kostg), IRF3 −/− (thpd-koirf3; rawl-koirf3), STING1-V155M (thpd-m155), CGAS −/− (thpd-kocgas), and MYD88 −/− (thpd-komyd) THP1 or RAW264.7 cell lines were obtained from InvivoGen.

Techniques: Western Blot, Activity Assay, Luciferase, Reporter Gene Assay, Binding Assay

Journal: iScience

Article Title: The STING1-MYD88 complex drives ACOD1/IRG1 expression and function in lethal innate immunity

doi: 10.1016/j.isci.2022.104561

Figure Lengend Snippet:

Article Snippet: The STING1 −/− (thpd-kostg), IRF3 −/− (thpd-koirf3; rawl-koirf3), STING1-V155M (thpd-m155), CGAS −/− (thpd-kocgas), and MYD88 −/− (thpd-komyd) THP1 or RAW264.7 cell lines were obtained from InvivoGen.

Techniques: Recombinant, Control, Lysis, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Magnetic Beads, Chromatin Immunoprecipitation, Purification, CCK-8 Assay, shRNA, Software, Western Blot