anti p irf3 ser396  (Cell Signaling Technology Inc)

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 interacts with IRF3 and is associated with colorectal cancer (A and B) Whole-cell lysates (WCL) from RKO and HCT116 cells were collected for IP using anti-IRF3 or anti-KMT5A antibodies, followed by IB analysis. (C) Cytoplasmic and nuclear proteins from RKO were collected for IP using anti-KMT5A antibodies, followed by IB analysis. (D) Boxplots were used to compare KMT5A mRNA expression in COAD and READ, with Student’s two-tailed t test, p < 0.0001. (E) Disease-specific survival analysis of TCGA-COAD and READ based on KMT5A expression levels. (F) Protein expression of KMT5A was analyzed by IB in colorectal tumor and paired adjacent normal tissues. (G) mRNA was extracted from colorectal tumors and paired adjacent normal tissues, and KMT5A mRNA levels were analyzed by qPCR. (H) The levels of IFN-β colorectal tumor and paired adjacent normal tissues were measured using ELISA. For (D)–(H) statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Data are presented as mean ± SD. ns p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. All immunoblotting experiments were performed independently three times with similar results.

Article Snippet: Phospho-IRF3-S386 Rabbit mAb , ABclonal , CAT#AP0623; RRID: AB_2771210.

Techniques: Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot

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: Phospho-IRF3-S386 Rabbit mAb , ABclonal , CAT#AP0623; RRID: AB_2771210.

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: 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 induces mono-methylation of lysine 193 on IRF3 (A) HEK293T cells were transfected with FLAG-IRF3 and/or HA-KMT5A plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads. Subsequent analysis was conducted via IB. (B) Whole-cell lysates were extracted from RKO cells with either control (shNC) or KMT5A shRNA (#1 and #2) silencing. IP was performed using anti-IRF3 antibodies, followed by WB analysis. (C) RKO cells were transfected with HA-KMT5A WT or HA-KMT5A D338A, IP was performed using anti-IRF3 antibodies, followed by WB analysis. (D) RKO cells were treated with either DMSO or UNC0379. IP was performed using anti-IRF3 antibodies, followed by IB analysis. (E) RKO cells were treated with either DMSO or varying concentrations of UNC0379. IP was conducted using anti-IRF3 antibodies, followed by IB analysis. (F) The levels of IFN-β in RKO cells from experiment (E) were quantified using ELISA. Data were analyzed using one-way ANOVA with Tukey’s post-hoc test, presented as mean ± SD. Statistical significance was defined as ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. (G) In vitro methylation assays were conducted by incubating purified His-IRF3 with KMT5A in the presence of S-adenosyl-L-methionine, followed by IB analysis. (H) Secondary mass spectrometry results of IRF3 K193 methylation were obtained. (I) HEK293T cells were transfected with FLAG-IRF3 wild-type or mutant plasmids, followed by transfection with either a vector or HA-KMT5A. Whole-cell lysates were collected, and IP was performed using anti-FLAG magnetic beads, followed by IB analysis. (J) Amino acid sequences at the K193 site of IRF3 were compared across different species. All immunoblotting experiments were conducted independently in triplicate, yielding consistent results.

Article Snippet: Phospho-IRF3-S386 Rabbit mAb , ABclonal , CAT#AP0623; RRID: AB_2771210.

Techniques: Methylation, Transfection, Magnetic Beads, Control, shRNA, Enzyme-linked Immunosorbent Assay, In Vitro, Purification, Mass Spectrometry, Mutagenesis, Plasmid Preparation, Western Blot

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 hindering production of IFN-β depends on IRF3 K193 methylation (A) HEK293T cells were transfected with vector, FLAG-IRF3 wild-type, or K193R mutant plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads, and subsequent analysis was performed via IB. (B) KMT5A-knockout RKO cells were transfected with FLAG-IRF3 wild-type or K193R mutant plasmids. The cells were treated with poly(I) or UNC0379, then whole-cell lysates were collected, and IP was conducted using anti-FLAG magnetic beads, followed by IB analysis. (C) RKO cells from experiment (B) were co-transfected with reporter plasmids IFN-β-Luc and pRL-TK. After 24 h, the cells were harvested, and luciferase activity was measured using a dual-luciferase reporter assay kit. (D) The relative mRNA levels of INF-β in RKO cells from experiment (B) were quantified using qPCR. (E) KMT5A knockout RKO cells were transfected with HA-KMT5A and FLAG-IRF3 wild-type or K193R mutant plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads, and subsequent IB analysis was performed. (F) Relative quantification of phosphorylated IRF3 (p-IRF3) protein was conducted in experiment (E). (G) The relative mRNA levels of INF-β in RKO cells from experiment F were quantified using qPCR. (C) and (D) were analyzed using one-way ANOVA with Tukey’s post-hoc test, with data presented as mean ± SD. (F) and (G) were analyzed using two-way ANOVA with Tukey’s post-hoc test. Statistical significance was defined as ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, while ns indicates no statistical difference. All immunoblotting experiments were performed independently in triplicate, yielding consistent results.

Article Snippet: Phospho-IRF3-S386 Rabbit mAb , ABclonal , CAT#AP0623; RRID: AB_2771210.

Techniques: Methylation, Transfection, Plasmid Preparation, Mutagenesis, Magnetic Beads, Knock-Out, Luciferase, Activity Assay, Reporter Assay, Quantitative Proteomics, Western Blot

Journal: Research

Article Title: PDHA1 Hyperactivation Orchestrates Metabolic Reprogramming Promoting Endothelial Senescence

doi: 10.34133/research.1398

Figure Lengend Snippet: Decreased lactylation of pyruvate dehydrogenase E1 component subunit alpha (PDHA1) at Lys336 facilitates its dephosphorylation and drives a metabolic shift. (A) Pulmonary vascular endothelial cells (PVECs) were treated with D-galactose (D-gal; 30 mg/ml) for 24 h. Cell lysates were immunoprecipitated with PDHA1 antibody-conjugated Protein A/G Agarose, followed by Western blot for the lactylation of PDHA1 using anti-pan-lactyl antibodies. (B) PDHA1-K336R mutation increased pyruvate dehydrogenase complex (PDHC) activity in mouse primary PVECs. (C) Primary murine PVECs were cotransfected with wild-type PDHA1 WT or PDHA1-K336R mutant. Cell lysates were immunoprecipitated with PDHA1 antibody-conjugated Protein A/G Agarose, followed by a Western blot for the phosphorylation of PDHA1 using pan p-serine antibodies. (D and E) Lactyl-lysine protein level and the phosphorylation level at PDHA1 S293 were determined. (F and G) Quantification of intracellular and extracellular lactate levels. (H and I) The oxygen consumption rate (OCR) of PVECs transfected with the PDHA1-K336R mutant was measured using Seahorse XF24 Analyzer. (J and K) Statistical analyses of adenosine triphosphate (ATP)-linked respiration and maximal respiration. (L and M) The expression of oxidative phosphorylation (OXPHOS)-related proteins in PVECs transfected with the PDHA1-K336R mutant was detected by Western blot analysis. (N) Senescence-associated β-galactosidase (SA-β-gal) staining was assessed in mouse primary PVECs with PDHA1 WT or PDHA1 K336R (scale bar = 20 μm). (O and P) p53, p21, p16, and phosphorylated histone H2AX (γ-H2AX) protein levels in PVECs with PDHA1 WT or PDHA1 K336R were detected by Western blot. n = 3. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Rabbit anti-p-IRF3 S396 antibody , ABclonal , AP0623 , 1: 1,000.

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Mutagenesis, Activity Assay, Phospho-proteomics, Transfection, Expressing, Staining

Journal: Research

Article Title: PDHA1 Hyperactivation Orchestrates Metabolic Reprogramming Promoting Endothelial Senescence

doi: 10.34133/research.1398

Figure Lengend Snippet: Pyruvate dehydrogenase E1 component subunit alpha (PDHA1)-activation-driven endothelial senescence via the cyclic GMP-AMP synthase (cGAS) signaling pathway. Cellular senescence in pulmonary vascular endothelial cells (PVECs) was induced by treatment with AZD7545. (A) The mitochondrial reactive oxygen species (mtROS) in AZD7545-treated PVECs were detected by an ROS kit (scale bar = 20 μm). (B and C) JC-1 staining showing mitochondrial membrane potential in endothelial cells (scale bar = 20 μm). (D) Quantification of cytosolic mitochondrial DNA (mtDNA) for the Nd1 , Cytb , and D-loop in AZD7545-treated PVECs was performed by real-time polymerase chain reaction (PCR). (E and F) Western blot and quantification for cGAS, stimulator of interferon genes (STING), p-TBK1 Ser172 , TBK1, p-IRF3 Ser396 , and IRF3 protein expression. (G) Primary PVECs isolated from Cgas WT and Cgas KO mice were stimulated with AZD7545 (40 μM) for 24 h. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to detect AZD7545-induced senescence in primary PVECs from Cgas WT and Cgas KO mice; scale bar = 20 μm. (H) Ki67 staining was performed in AZD7545-treated primary PVECs from Cgas KO mice; scale bar = 50 μm. (I and J) The expression of senescence-related proteins (cGAS, p53, p21, p16, and phosphorylated histone H2AX [γ-H2AX]) was examined by Western blot in AZD7545-treated primary PVECs isolated from Cgas WT and Cgas KO mice. α-Tubulin was used as the internal control. n = 3. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Rabbit anti-p-IRF3 S396 antibody , ABclonal , AP0623 , 1: 1,000.

Techniques: Activation Assay, Staining, Membrane, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Isolation, Control

Journal: Research

Article Title: PDHA1 Hyperactivation Orchestrates Metabolic Reprogramming Promoting Endothelial Senescence

doi: 10.34133/research.1398

Figure Lengend Snippet: Schematic illustration. Pyruvate dehydrogenase E1 component subunit alpha (PDHA1) activation, driven by S293 dephosphorylation resulting from reduced lactylation at K336, disrupts mitochondrial homeostasis, leading to mitochondrial reactive oxygen species (mtROS)-mediated mitochondrial DNA (mtDNA) release and ultimately triggering cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS–STING)-dependent senescence.

Article Snippet: Rabbit anti-p-IRF3 S396 antibody , ABclonal , AP0623 , 1: 1,000.

Techniques: Activation Assay, De-Phosphorylation Assay

Journal: Research

Article Title: PDHA1 Hyperactivation Orchestrates Metabolic Reprogramming Promoting Endothelial Senescence

doi: 10.34133/research.1398

Figure Lengend Snippet: Antibody sources and dilutions

Article Snippet: Rabbit anti-p-IRF3 S396 antibody , ABclonal , AP0623 , 1: 1,000.

Techniques: Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Interferon-λ drives renal fibrosis by coordinating epithelial–fibroblast crosstalk

doi: 10.1084/jem.20251858

Figure Lengend Snippet: Renal TECs generate IFN-λ upon RIG-I/MAVS signaling activation. (A) Multiplex immunofluorescence staining for IFN-λ2/3 (red), TECs (E-cadherin, white), fibroblasts (vimentin, cyan), and immune cells (CD45, yellow) in kidney sections at day 7 from sham-operated mice (sham) and UUO mice. Scale bars = 50 μm. Quantification of IFN-λ2/3–positive cells are shown on the right panel ( n = 6). (B–D) Flow cytometry plots showing the purity of isolated E-cadherin + TECs (B), CD45 + immune cells (C), and vimentin + fibroblasts (D) from mouse kidneys ( n = 3). (E) RT-qPCR analysis of Ifn-λ2 and Ifn-λ3 mRNA levels in the indicated purified renal cell populations from sham and UUO mice at day 7 ( n = 4). (F) Western blot analysis of indicated pathway proteins in renal TECs of sham and UUO mice on day 7 ( n = 5 per group). (G–K) WT, Sting –/– (G), Myd88 −/− (H), Trl3 −/− (I), Mda5 −/− (J), and Mavs –/– (K) mice were subjected to sham or UUO surgery ( n = 5 per group). Renal TECs were isolated on day 7 after surgery and analyzed for IFN-λ2/3 and pathway protein expression by western blot (left panels) and Ifn-λ2 and Ifn-λ3 mRNA by RT-qPCR (right panels). (L) Renal TECs from sham and UUO treated with PBS or 50 mg/kg of cFP were analyzed for IFN-λ2/3, p-IRF3, and IRF3 protein levels by western blot (left panel) and Ifn-λ2 and Ifn-λ3 mRNA by RT-qPCR (right panel) ( n = 5 per group). (M) Immunofluorescence staining for IFN-λ2/3 or p-SMAD2/3 in renal TECs (E-cadherin) in kidneys of sham and UUO mice treated with PBS or 50 mg/kg of cFP on day 7 after surgery. Nuclei were counterstained with DAPI. Quantification is shown on the right. n = 4 per group, scale bars = 50 μm. (N) Representative images and quantitative analysis of IFN-λ2/3 or p-SMAD2/3 in renal TECs (E-cadherin) in kidneys of WT and Mavs –/– UUO mice at day 7. n = 4 per group, scale bar = 50 μm. (O) Representative images and quantitative analysis of dsRNA (J2) in renal TECs (E-cadherin) in kidneys of sham and UUO mice at day 7. n = 4 per group, scale bars = 50 μm. (P) ELISA analysis of dsRNA levels in isolated renal TECs. (Q and R) Primary renal TECs were treated with or without 2.5 mM cFP for 6 h, followed by 1 μg/ml of poly(I:C) transfection for 12 h ( n = 3). (Q) Western blot analysis of RIG-I, MAVS, p-IRF3, and IRF3 protein levels. (R) RT-qPCR and ELISA were used to evaluate IFN-λ2/3 mRNA and protein levels, respectively. (S and T) Renal TECs isolated from WT and Mavs –/– mice were transfected with 1 μg/ml of poly(I:C) for 12 h ( n = 3). (S) Western blot analysis of MAVS, p-IRF3, and IRF3 protein levels. (T) IFN-λ2/3 mRNA and protein levels were determined by RT-qPCR and ELISA. Data in A, E, and G–P are pooled from two independent experiments. Data in B–D are pooled from three independent experiments. Data in F and Q–T are representative of three independent experiments. Data are presented as mean ± SEM. ***P < 0.001, ****P < 0.0001, by unpaired two-tailed Student’s t test (A–F and O–P), two-way ANOVA with Tukey’s multiple-comparison test (G–K, N, R, and T), and one-way ANOVA with Tukey’s multiple-comparison test (L and M). ns, no significant difference. Source data are available for this figure: .

Article Snippet: The membrane was washed and blocked in TBS plus Tween (TBST) (1 × TBS with 0.05% Tween-20) supplemented with 5% skim milk powder for 1 h at room temperature with gentle shaking, then incubated overnight at 4°C with the following primary antibodies: anti-IFN-λ2/3 (ab191426; Abcam), anti-α-SMA antibody (ab5694; Abcam), anti-fibronectin antibody (ab2413; Abcam), anti-vimentin (A19607; ABclonal), p-STAT1 (9167; Cell Signaling Technology), STAT1 (66545-1-Ig; Proteintech), TGF-β (81746-2-RR; Proteintech), p-SMAD2 (18338; Cell Signaling Technology), p-SMAD3 (9520; Cell Signaling Technology), SMAD2/3 (8685; Cell Signaling Technology), SMAD4 (38454; Cell Signaling Technology), SMAD7 (25840-1-AP; Proteintech), RIG-I (ab302778; Abcam), MAVS (A25005; ABclonal), p-IRF3 (AP0623; ABclonal), IRF3 (MA5-32348; Invitrogen), p-ERK (9101; Cell Signaling Technology), ERK (9102; Cell Signaling Technology), p-JNK (9251; Cell Signaling Technology), JNK (9252; Cell Signaling Technology), p-mTOR (2971; Cell Signaling Technology), mTOR (2983; Cell Signaling Technology), p-p38 (9215; Cell Signaling Technology), p38 (9212; Cell Signaling Technology), p-PI3K (17366; Cell Signaling Technology), PI3K (4257; Cell Signaling Technology), MDA5 (ab315242; Abcam), p-STING (72971; Cell Signaling Tehcnology), STING (13647; Cell Signaling Technology), TLR3 (HA724070; HUABIO), Myd88 (AF7524; Beyotime), and GAPDH (81640-5-RR; Proteintech).

Techniques: Activation Assay, Multiplex Assay, Immunofluorescence, Staining, Flow Cytometry, Isolation, Quantitative RT-PCR, Purification, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test, Comparison

Journal: Journal of Virology

Article Title: African swine fever virus pM448R protein promotes STUB1-mediated ubiquitin-proteasome degradation of IRF1 to attenuate type III interferon induction

doi: 10.1128/jvi.00580-26

Figure Lengend Snippet: pM448R inhibits the production of type III interferons by targeting the IRF1 signaling pathway. ( A and B ) Effect of pM448R on the phosphorylation of IRF3 and NF-κB. IPEC-J2 cells were transfected with a Flag-M448R or a control plasmid. At 24 h post-transfection, the cells were stimulated with either poly(I:C) (1 μg/mL) for 1 h or SeV (MOI = 0.1) for 2 h. Whole-cell lysates were then subjected to western blot analysis using the indicated antibodies. ( C–H ) Effect of pM448R on IRF3 and NF-κB activation. IPEC-J2 cells were co-transfected with the Flag-M448R plasmid (or empty vector control), a firefly luciferase reporter plasmid (driven by the IFN-λ1, IFN-λ3, Mut-IRF, IRF3, IRF1, or NF-κB promoter), and the control plasmid. At 24 h post-transfection, the cells were either mock-treated or transfected with poly(I:C) (1 μg/mL) for 12 h. The activation of the respective promoters was analyzed using a dual-luciferase assay. For dual-luciferase reporter gene assays, three independent experiments were performed with three technical replicates. * , P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Antibodies against IRF3 (A2172), phosphorylated IRF3 (AP0995), FLAG (AE092), HA (AE105), and Myc (AE070) were obtained from ABclonal.

Techniques: Phospho-proteomics, Transfection, Control, Plasmid Preparation, Western Blot, Activation Assay, Luciferase