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ALSV proteins regulate <t>MDA5-induced</t> IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with HA-MDA5 to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting, and the relative levels of phosphorylated IRF3 normalized to IRF3 are shown in the right. ( B ) HEK293T cells were treated as indicated in ( A ), and cells were subjected to luciferase activity assays. ( C ) HEK293T cells were transfected with HA-MDA5 and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA and IFNB1 were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the MDA5-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).
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ALSV proteins regulate <t>MDA5-induced</t> IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with HA-MDA5 to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting, and the relative levels of phosphorylated IRF3 normalized to IRF3 are shown in the right. ( B ) HEK293T cells were treated as indicated in ( A ), and cells were subjected to luciferase activity assays. ( C ) HEK293T cells were transfected with HA-MDA5 and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA and IFNB1 were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the MDA5-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).
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ALSV proteins regulate <t>MDA5-induced</t> IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with HA-MDA5 to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting, and the relative levels of phosphorylated IRF3 normalized to IRF3 are shown in the right. ( B ) HEK293T cells were treated as indicated in ( A ), and cells were subjected to luciferase activity assays. ( C ) HEK293T cells were transfected with HA-MDA5 and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA and IFNB1 were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the MDA5-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).
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ALSV proteins regulate MDA5-induced IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with HA-MDA5 to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting, and the relative levels of phosphorylated IRF3 normalized to IRF3 are shown in the right. ( B ) HEK293T cells were treated as indicated in ( A ), and cells were subjected to luciferase activity assays. ( C ) HEK293T cells were transfected with HA-MDA5 and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA and IFNB1 were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the MDA5-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV proteins regulate MDA5-induced IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with HA-MDA5 to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting, and the relative levels of phosphorylated IRF3 normalized to IRF3 are shown in the right. ( B ) HEK293T cells were treated as indicated in ( A ), and cells were subjected to luciferase activity assays. ( C ) HEK293T cells were transfected with HA-MDA5 and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA and IFNB1 were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the MDA5-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Luciferase, Activity Assay, Comparison

ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot