anti mda5  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Multiple orthoflaviviruses secrete sfRNA in mosquito saliva to promote transmission by inhibiting MDA5-mediated early interferon response

doi: 10.1038/s41467-026-77288-4

Figure Lengend Snippet: a Scheme of RIG-I and MDA5-mediated antiviral interferon response. The methods used to disrupt the antiviral response are written in red. b – d WNV gRNA fold change post sfRNA transfection at 24 and 48 hpi in Huh7 cells treated with MRT67307 ( b ), in RIG-I-deficient Huh7.5 cells ( c ), and in Huh7.5 cells upon MDA5 silencing ( d ). n for ( b ) and ( d ) = 6 biological replicates. n for ( c ) = 9 biological replicates. Ctl., RNA control. Bars show geometric mean ± 95% C.I. Repeats are indicated by dots. Different letters show significant differences ( p < 0.05) according to post hoc Fisher’s LSD test. Souce data are provided as a Source data file.

Article Snippet: Membranes were blocked with 5% skimmed milk (ThermoScientific), incubated with 1:4000 anti-MDA5 antibody (21775-1-AP, Proteintech) or 1:400 anti-Actin (MA5-11869, Invitrogen) in blocking solution, washed with 0.05% tween-20 PBS, incubated with 1:500 anti-rabbit IgG HRP-linked antibody (Cell Signaling) in blocking solution, and washed.

Techniques: Transfection, Control

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A inhibits phosphorylation activation of IRF3 (A) RKO cells were treated with poly(I:C) and protein samples were collected at 0, 8, 16, and 24 h for Western blot (WB) analysis. (B) Quantification of p-IRF3, RIG-1, and MDA5 protein levels relative to control in the samples collected in (A). (C) IFN-β secretion levels at different time points from (A) were measured using ELISA. (D, E) RKO and HCT116 cells transfected with either an empty vector or a KMT5A overexpression plasmid were treated with poly(I:C), followed by WB analysis of whole-cell lysates. (F) RKO cells transduced with control shRNA (shNC) or KMT5A-specific shRNAs (#1 and #2) were treated with poly(I:C) and analyzed by WB using whole-cell lysates. (G) RKO cells from (F) were co-transfected with the IFN-β-Luc reporter plasmid and pRL-TK plasmid, and after 24 h, luciferase activity was measured using a dual-luciferase assay kit. (H) The relative mRNA levels of KMT5A and INF-β in RKO cells from F were quantified using qPCR. (I) IFN-β levels in the RKO cells from (F) were measured using ELISA. (J) RKO cells, either wild-type or stably expressing HA-KMT5A, were treated with poly(I:C) and whole-cell lysates were collected at 0, 8, 16, and 24 h for WB analysis. (K) Quantification of p-IRF3 protein levels relative to control in samples collected in (J). For (G)–(I), statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. For (K), two-way ANOVA with Tukey’s post-hoc test was used. Data are presented as mean ± SD. Statistical significance is indicated as ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All western blot analyses were performed independently three times, yielding consistent results.

Article Snippet: MDA5 Rabbit mAb , ABclonal , CAT#A2419; RRID: AB_3674625.

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Over Expression, Transduction, shRNA, Luciferase, Activity Assay, Stable Transfection, Expressing

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

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