rabbit irak4 antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: (A ): Analysis of the AML patient’s microarray database (source: http://vizome.org/aml2/expression_strat/ showed that IRAK4 mRNA is significantly overexpressed in initial diagnosis (p<0.0001, n=16 each), relapse (p<0.001, n=16 each), residual disease (p=0.031, n=16 each), residual (p=0.034, n=16 each), FLT-3-ITD positive (p<0.0001, n=16 each), prior MDS (p=0.03, n=16 each), prior MPN (p<0.0001, n=16 each) and MDS- MPN+ve patients (p<0.0001, n=16 each) compared to healthy pooled CD34+ cells. On the other hand, (B ): IRAK1 mRNA expression was not significantly upregulated among these AML conditions than healthy pooled CD34+ cells. ( C ): IRAK4 mRNA is significantly upregulated in T(15:17), Inv(16)/t(16:16) and T(11q23)/MLL AML patients (source: Bloodspot.org ). ( D ): Kaplan-Meier analysis showed that IRAK4 mRNA overexpressor patients face early and greater proportions of deaths compared to low expressor patients (Source: GEPIA, http://gepia.cancer-pku.cn/detail.php?gene=IRAK4&clicktag=survival ). ( E ): IRAK1 mRNA is significantly upregulated in complex and T(11q23)/MLL karyotype presenting AML patients (source: Bloodspot.org ). ( F ): Kaplan-Meier analysis showed that IRAK1-mRNA overexpressor patients face early and great proportions of deaths than low expressor patients (Source: GEPIA, http://gepia.cancer-pku.cn/detail.php?gene=IRAK1&clicktag=survival ).

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: Microarray, Expressing, Biomarker Discovery

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: In silico docking of PSP-0119 and PSP-0102 with CRBN and IRAK1 and IRAK4 complex. (A): In silico docking showed how glutarimide ring of lenalidomide interacts with CRBN and how pyrazolyl-pyridinyl portion engages with IRAK4. Red arrows point to both interactions. (B): Individual CRBN and IRAK4 residues interacting with PSP-0119 atoms are shown. (C): In silico docking showed how glutarimide ring of lenalidomide interacts with CRBN and how pyrazolyl-pyridinyl portion engages with IRAK1. (D): Individual CRBN and IRAK1 residues interacting with PSP-0102 atoms are shown.

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: In Silico

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: ( A ): Hotspot kinase assay shows that despite drastic modifications on UR241-2, the kinase inhibition activity of PSP-0102 and PSP-0119 was not lost and showed IRAK4 kinase inhibition at 2.33nM. ( B ): PSP-0119 inhibited NF-kβ reporter activity induced by IL1β. ( C ): PSP-0119 pretreatment blocked the IL1β induced IRAK4 phosphorylation.

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: Kinase Assay, Inhibition, Activity Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: Repeated experiments showed PSP-0119 treatment did not degrade IRAK4 in THP-1 (FLT-3 wild-type) ( A ), FLT-3 wild-type AML patient ( B ) and normal bone marrow cells ( C ). In immunoblotting of FLT-3 normal AML patients ( C) , total ERK1/2 was used as a loading control as Actin and GAPDH showed variabilities. ( D ): PSP-0119 treatment degraded IRAK4 in MOLM-13 and MV-4-11 ( E ) FLT-3 mutant AML cells between doses 20-40nM during 24 hrs. of treatment ( D and E ). Normalized (IRAK4/GAPDH) pixel density is written in numbers in between the bands. ( F ): IRAK4/GAPDH normalized curves and calculated DC 50 of PSP-0119 against MV-4-11 and MOLM-13 are shown. ( G ): PS-0119 did not degrade IRAK1 in MOLM-13 cells. ( H ): PS-0119 strongly degraded IRAK1 in MV-4-11 cells.

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: Western Blot, Control, Mutagenesis

Journal: bioRxiv

Article Title: PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

doi: 10.1101/2025.09.30.679569

Figure Lengend Snippet: ( A-B ): Bulk-sequencing of PSP-0119 treated MOLM-13 cells have identified gene affected by IRAK4 degradation. ( C ): Volcano plot of the upregulated and downregulated gene after IRAK1/4 degradation in MOLM-13 cells treated with PSP-0119 compared to vehicle. PS-0119 treatment led to downregulation of metabolic driver ENOS-1 gene in MOM-13 cells. Analysis of AML patient’s microarray data available at R2-Genomics and Visualization platform showed that ENOS-1 overexpression predicted poor prognoses in AML patients.

Article Snippet: The PVDF membrane containing the transferred protein was blocked in 5% fat free milk solution for 30 minutes and probed with rabbit IRAK4 antibody (Cell Signaling Tech, cat. no:4363, dilution: 1:1000), rabbit IRAK1 antibody (Cell Signaling Tech, cat. no:4504, dilution: 1:1000).

Techniques: Sequencing, Microarray, Over Expression

Journal: bioRxiv

Article Title: IL-1β/IRAK4 Axis Promotes Ovarian Tumor Development at the Mesothelium Injury Sites

doi: 10.1101/2025.03.18.643950

Figure Lengend Snippet: ( A ): Schema of IL1β/TLR/IRAK1/2/4 signaling pathway. ( B-D ): Analysis of ovarian cancer patient’s microarray data (TCGA-381-tpm-gencode36) using R2 Genomics Analysis and Visualization Platform tools showed that IRAK1, -2 and -4 mRNA overexpression predict poor survival. ( E ): IRAK4 mRNA was overexpressed in malignant than normal ovaries. The data present in GENT2 ovarian cancer databases were analyzed using the inbuilt tools. ( F ): Analysis of the EOC patient’s microarray data using GENT2 tools showed that IRAK4 mRNA expression was altered in various stages of disease. Two-sample T-test showed statistical differentiations: IA vs I (p=0.004); IA vs II (p=0.007); IA vs III (p<0.001); IC vs IIA (p=0.002); IC vs III (p<0.001); IIA vs I (p<0.001); IIA vs II (p<0.001); IIA vs III (p<0.001); IIA vs IIC (p=0.006); IIA vs IIIC (p=0.001); IIC vs III (p<0.001); III vs IIB (p=0.004); IIIA vs I (p=0.001); IIIA vs II (p=0.004); IIIA vs III (p<0.001); IIB vs I (p=0.008); IIIB vs III (p=<0.001); IV vs I (p=0.004); IV vs IIA (p=0.005); IV vs III (p=<0.001). ( http://gent2.appex.kr/gent2/ , date accessed 10/2/2023). ( G ): Compared to non-invasive EOC, tissues from invaded EOC phenotype showed greater IRAK4 mRNA. EOC patient microarray data (TCGA-541-custom-tcgaovag1) deposited at R2-Genomics Analysis and Visualization Platform were analyzed using their MegaSnitch tools. ( H ): Chemical structures of three representative IRAK4–kinase inhibitors undergoing clinical trials. ( I ): Structure-activity relationship (SAR) guided optimization of JH-I-25 scaffold, a literature described IRAK4 inhibitor leading to 3 potent novel analogs. The chemical structure of UR241-2 (sulfone), PSP-099 (sulfide) and PSP-100 (sulfoxide), from among the many analogs synthesized, are shown. ( J ): Calculated drug-likeness/physicochemical properties (LogP, cLogP and topological polar surface area-tPSA are shown. These properties were calculated using Chemdraw software. ( K ): Comparison of IRAK1-, IRAK2-, and IRAK4-kinase inhibitory IC50s of UR241-2 versus CA4948, PF-06650833, PSP-099 and PSP-100 are shown. Compounds were screened using HotSpot Kinase assay available in Reaction Biology Laboratories using 1μM ATP concentration under a 10-dose singlet screening program. ( L ): Dendrograms of global kinome activity of UR241-2 at 50- and 500nM doses. Red indicates kinase affected. At 50nM 13 of 682 kinases were significantly inhibited, whereas at 500nM 34 kinases, shown as red dots, from among 682 total kinases were inhibited. ( M ). A HEK293 cell-based NanoBret target Engagement screening assay was conducted to determine the selectivity of UR241-2 among 10 most affected kinases revealed by HotSpot kinase assay. The nanoBret assay showed that UR241-2 inhibits IRAK4 kinase activity only and other kinases including IRAK1 are affected at 10-100 folds higher doses, making UR241-2 as one of the most selective and specific IRAK4 kinase inhibitors known currently.

Article Snippet: Chemiluminescent detection was done with a Super Signal West FemtoLuninol/Enhancer (ThermoScientific, cat#1859022 and peroxide cat#1859023) or using Amersham ECL Prime (peroxide solution, cat#RPN2232V2 and enhancer cat#29018903; Cytiva) and probed with p-IRAK4 antibodies (Cell Signaling, cat#11927) (dilution: 1:500).

Techniques: Microarray, Over Expression, Expressing, Clinical Proteomics, Activity Assay, Synthesized, Software, Comparison, Kinase Assay, Concentration Assay, Drug discovery, Screening Assay

Journal: bioRxiv

Article Title: IL-1β/IRAK4 Axis Promotes Ovarian Tumor Development at the Mesothelium Injury Sites

doi: 10.1101/2025.03.18.643950

Figure Lengend Snippet: ( A ): JH-I-25 and its newer analogs UR241-2, PSP-099 and PSP-100 were docked individually to IRAK4 crystal structure using Gnina docking software, which is built on neural networks (CNNs) as a scoring function. ( B ): The interacting amino acid residues and their nature of interactions are shown. Green=aliphatic, magenta=aromatic, blue=basic, sky blue=polar, and yellow=sulfur. Red indicates unfavorable interactions. ( C ): JH-I-25, UR241-2, PSP-099 and PSP-100 analogs were docked together to IRAK4 protein using Gnina tools. ( D ): Root-mean square density (RMSD) simulations of JH-I-25, UR241-2, PSP-099 and PSP-100 ligands docked to IRAK4 falling within 2Å unit range are shown. RMSD-ligand is the root mean deviation of the bounded ligand compared to the ligand in the crystal structure. ( E ): Root-mean square density (RMSD) simulations of IRAK4 protein docked with JH-I-25, UR241-2, PSP-099 and PSP-100 ligands falling with 2Å unit range are shown. RMSD-Protein is the root mean deviation of the protein for each frame of the simulation compared to the crystal structure. Both D and E indicate that docking quality was acceptable. ( F ): RMSF (root mean square fluctuation) of JH-I-25, UR241-2, PSP-099 and PSP-100 ligands are shown. The ligands, when bound, induce the most notable structural changes in residues 172-181 of IRAK4 which reflected in the spike in the RMSF charts. RMSF is the mean fluctuation of IRAK4 protein residues after binding of ligands. RMSF indicates the conformational flexibility of the complex. The time/frame element is removed from RMSF in order to show an average fluctuation of the residues. ( H ): The affinity score measures affinity of a ligand (in that particular frame) binding to the protein. The frames represent different conformations that a ligand may adopt. The affinity is measured in KCal/mol and the lowest frame (lowest energy) is selected as the most stable binder. UR241-2 showed best affinity score (−11.95), hence was considered the most stable binder ligand of IRAK4 crystal structure.

Article Snippet: Chemiluminescent detection was done with a Super Signal West FemtoLuninol/Enhancer (ThermoScientific, cat#1859022 and peroxide cat#1859023) or using Amersham ECL Prime (peroxide solution, cat#RPN2232V2 and enhancer cat#29018903; Cytiva) and probed with p-IRAK4 antibodies (Cell Signaling, cat#11927) (dilution: 1:500).

Techniques: Software, Binding Assay

Journal: bioRxiv

Article Title: IL-1β/IRAK4 Axis Promotes Ovarian Tumor Development at the Mesothelium Injury Sites

doi: 10.1101/2025.03.18.643950

Figure Lengend Snippet: ( A ): UR241-2 (5-150nM) treatment for 4hrs blocks human and murine IL1β (5nM, 30 minutes) induced IRAK4-phosphorylation in both human (HCH-1) and murine high-grade serous EOC cells (HGS-1 and -3) cells. ( B ): Analysis of ovarian cancer patient’s microarray database (Kim_161-DESeq2_vst-ensh38e100) using R2 Genomics Analysis and Visualization Platform tools showed strong correlation of IRAK4 mRNA expression with NF-κβ mRNA expression in ovarian tumors (R=0.581, p=6.29e -016 ). Another database (TCGA-381-tpm-gencode36) showed similar correlation (R=0.523, p=5.28e -018 ) between IRAK4 and NF-κβ mRNA expression levels in ovarian tumors. ( C ): Analysis of ovarian cancer patient’s microarray showed using the GENT2 software showed that NF-κβ mRNA is overexpressed (Log2-Fold-change Log2FC=1.1) in malignant ovaries than normal (p<0.001). ( D ): NF-κβ mRNA overexpression showed significant risk of mortality among EOC patients (p=0.0012). The EOC patient’s microarray data was analyzed using the R2-Genomics Analysis and Visualization Platform tools. ( E-upper ): UR241-2 (2.5μM) treatment reduced NF-κβ-luciferase reporter activity in stably transfected OVCAR-3-NF-κβ-Luc cell-lines. (E-lower ): UR241-2 (2.5μM) treatment reduced NF-κB luciferase reporter activity induced by human TNF-α (3, 10 and 30nM) in stably transfected OVCAR-3-NF-κβ-Luc cell-lines. ( F ): UR241-2 (2.5μM) treatment, IL-1β (5ng), TNF-α (10ng), LPS (100nM) and R848 (TLR-7/8 agonist, 10μM). Ratio of positive/negative NF-κB nuclei are shown. NF-κβ (Green) nuclear migration was inhibited significantly in IL-1β and LPS stimulated cells. The number of NF-κβ nuclear positive cells were counted using ImageJ software. * indicates <0.05 (Student T-test).

Article Snippet: Chemiluminescent detection was done with a Super Signal West FemtoLuninol/Enhancer (ThermoScientific, cat#1859022 and peroxide cat#1859023) or using Amersham ECL Prime (peroxide solution, cat#RPN2232V2 and enhancer cat#29018903; Cytiva) and probed with p-IRAK4 antibodies (Cell Signaling, cat#11927) (dilution: 1:500).

Techniques: Phospho-proteomics, Microarray, Expressing, Software, Over Expression, Luciferase, Activity Assay, Stable Transfection, Transfection, Migration

Journal: bioRxiv

Article Title: IL-1β/IRAK4 Axis Promotes Ovarian Tumor Development at the Mesothelium Injury Sites

doi: 10.1101/2025.03.18.643950

Figure Lengend Snippet: IRAK4 inhibition via UR241-2 decreases tumor burden at the needle injury site and increases the number of MHCII expressing myeloid cells in the peritoneal cavity and tumor. ( A ): tumor weights on the needle injury site were significantly lower in the UR241-2 treatment group than vehicle treated animals. Statistical significance was determined using Mann-Whitney test, *p < 0.033, **p < 0.002, ***p < 0.001. ( B ): Tumor weights on omentum did not differ between UR241-2 treatment group than vehicle treated animals (NS). Two of the three replicates are combined and shown. IRAK4 inhibition via UR241-2 increases the number of MHCII expressing myeloid cells and decreases MHCII low and CD206+ macrophages in the peritoneal cavity . Leukocytes were isolated from the peritoneal lavage or tumor of HGS-3-tumor bearing mice and stained for flow cytometry. ( C ) Representative flow cytometry plots of F4/80 lo MHCII hi M1 macrophages and F4/80 hi MHCII Low macrophages from the peritoneal lavage of HGS-3 tumor bearing mice treated with Vehicle or UR241-2. ( D ) Percentage and cell number of F4/80 lo MHCII hi M1 macrophages and F4/80 hi MHCII Low macrophages from C. (E) Representative flow cytometry plots of F4/80 hi CD206 + macrophages from the peritoneal lavage of HGS-3 tumor bearing mice treated with Vehicle (Vx) or UR241-2. ( E-right ) Percentage and cell number of F4/80 hi CD206 + macrophages. IRAK4 inhibition reduces neutrophil numbers in the peritoneal cavity and tumors of HGS3 bearing mic e: (F ): Flow cytometry plots showing CD11b + Ly6G + neutrophils and MHCII + neutrophils in the HGS-3 tumor. Percentage and cell number per gram are quantified to the right. ( G ): Flow cytometry plots showing CD11b + Ly6G + and MHCII + neutrophils in the peritoneal lavage of HGS-3 tumor bearing mice. Percentage and cell number are quantified to the right. N= 9 mice per group from two independent experiments.

Article Snippet: Chemiluminescent detection was done with a Super Signal West FemtoLuninol/Enhancer (ThermoScientific, cat#1859022 and peroxide cat#1859023) or using Amersham ECL Prime (peroxide solution, cat#RPN2232V2 and enhancer cat#29018903; Cytiva) and probed with p-IRAK4 antibodies (Cell Signaling, cat#11927) (dilution: 1:500).

Techniques: Inhibition, Expressing, MANN-WHITNEY, Isolation, Staining, Flow Cytometry