anti mda5 antibody  (Proteintech)

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: Advanced Science

Article Title: Slc44a2 Deficiency Unveils an IFN‐I–Dependent Feedback Control of pDC Egress

doi: 10.1002/advs.76325

Figure Lengend Snippet: Slc44a2 deficiency enhances type I interferon signaling in pDCs. (A) Heatmap illustrating differentially upregulated genes associated with pDC function in the RNA‐seq dataset of BM Sca‐1 + pDCs isolated from WT and cKO mice (n = 4). (B) Expression levels (FPKM value) of Irf7, Stat1 , Stat2 , and Zbp1 in BM Sca‐1 + pDCs from WT and cKO mice (n = 4). (C) Pathway enrichment analysis of differentially upregulated genes in the RNA‐seq dataset from BM Sca‐1 + pDCs of WT and cKO mice, with the top 15 enriched pathways listed. (D) Enriched type I interferon signaling pathway in cKO pDCs revealed by Gene Set Enrichment Analysis (GSEA). NES: Normalized Enrichment Score. (E) MFI of IRF‐7 expression in BM Sca‐1 + pDCs from WT and cKO mice (n = 3–4). (F) Expression (FPKM value) of Ifna2 and Opn in BM Sca‐1 + pDCs of WT and cKO mice (n = 4). (G) IFN‐α secretion by BM Sca‐1 + pDCs from WT and cKO mice was measured by ELISA under steady‐state conditions and following 24 h CpG‐A (1 µM) stimulation (n = 4–6). (H,I) Western blot analysis of IFIH1 and DDX58 in WT and cKO BM pDCs under the steady state (H) and following 2.5 h CpG‐A (1 µM) stimulation (I). (J) Intracellular staining and MFI of phosphorylated IRF7 (P‐IRF7) in BM pDCs from WT and cKO mice (n = 5). (K) Relative protein levels of STAT1 and STAT2 expression in BM pDCs from WT and cKO mice were analyzed using DIA‐based proteomics (n = 3). (L,M) Protein levels of STAT1, STAT2, P‐STAT1, and P‐STAT2 in BM pDCs from WT and cKO mice were analyzed by western blot under steady‐state conditions (L) and after 2.5 h CpG‐A (1 µM) stimulation (M). Data were expressed as mean ± SEM. Symbols represent individual mice. Statistical significance was determined by unpaired two‐tailed Student's t ‐test of n = 3 independent biological experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: MDA5 (1:1000, A2419) was purchased from ABclonal.

Techniques: RNA Sequencing, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Slc44a2 Deficiency Unveils an IFN‐I–Dependent Feedback Control of pDC Egress

doi: 10.1002/advs.76325

Figure Lengend Snippet: Slc44a2 restricts IFN‐I production by pDCs via regulating intracellular levels of threonine (T), asparagine (N), and glutamine (Q). (A) Metabolic profiling of BM pDCs from WT and cKO mice. (B) Pathway enrichment analysis of significantly upregulated metabolites identified through metabolomics (n = 3). (C) Concentration of IFN‐α secreted by BM pDCs upon CpG‐A (1 µM) stimulation in vitro for 20 h in the absence of the indicated amino acid in the culture medium. US: unstimulated; AA: complete medium. (D) Concentration of IFN‐α secreted by BM pDCs upon CpG‐A (1 µM) stimulation in vitro for 20 h with or without threonine/asparagine/glutamine (abbreviated as T/N/Q) in the culture medium (n = 3). (E) Concentration of IFN‐α secreted by BM pDCs upon CpG‐A (1 µM) stimulation in vitro for 20 h with gradually increased concentrations of threonine (T), asparagine (N), or glutamine (Q) in the culture medium (n = 3). (F) Expression of IFIH1, DDX58, STAT1, P‐STAT1, STAT2, and P‐STAT2 in BM pDCs upon the stimulation of CpG‐A (1 µM) with or without three amino acids T/N/Q. US: unstimulated. (G) Quantification of choline, T, N, and Q in BM pDCs from WT and cKO mice (n = 3). (H) Quantification of choline, T, and N levels in CD4 + T cells from Slc44a2 knockout mice (n = 3). (I) Quantification of choline, T, N, and Q in Slc44a2 ‐overexpressing HEK293T cells (n = 4). (J) HEK293T cells stably expressing empty vector (EV) or SLC44A2 ( Slc44a2 OE ) were stimulated with poly(I:C) (1 µg/mL) for the indicated times (0, 12, and 16 h). The mRNA levels of Slc44a2 , ISG15 , OAS1 , and MX1 were measured by qRT‐PCR and normalized to β‐actin (n = 3). (K) The potential binding pocket and residues in SLC44A2 with T, N, and Q in both human and mouse. (a) Overall structure of human and mouse SLC44A2, with membrane orientation predicted by the PPM 3.0 web server. The cytosolic side was indicated as “In,” and the extracellular or mitochondrial intermembrane space was labeled as “Out”. (b,c) Residues constituting the predicted largest‐volume binding pocket in human (light blue ribbons) and mouse (green ribbons) SLC44A2 were used to define the docking grid. (d–f) Close‐up view of human SLC44A2 bound to T, N, and Q, respectively. The polar interactions with the docked amino acids (magenta) and the binding site residues were shown as dashed violet lines. (h,i) Close‐up view of mouse SLC44A2 bound to T, N, and Q, respectively, with polar interactions similarly depicted. Data were presented as mean ± SEM, with individual data points indicated by symbols for each biological experiment. Statistical significance was determined by one‐way ANOVA (G–I) or unpaired two‐tailed Student's t ‐test (D,E). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: MDA5 (1:1000, A2419) was purchased from ABclonal.

Techniques: Concentration Assay, In Vitro, Expressing, Knock-Out, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Binding Assay, Membrane, Labeling, Two Tailed Test