mab rea462 apc miltenyi biotec  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: The role of extracellular vesicle fusion with target cells in triggering systemic inflammation

doi: 10.1038/s41467-024-45125-1

Figure Lengend Snippet: A Human blood (Healthy donors: n = 5) subjected to flow cytometry analysis post LPS-challenge are presented (left panel) . The translocation efficiency of CD42b, a platelet activation marker, to blood cells from healthy and LPS-treated blood is depicted (right panel) . Statistical analysis utilized ordinary one-way ANOVA with Tukey’s multiple comparisons test, results presented as box-and-whisker plots from minimum value and up to the maximum value, The error bars represent the standard error of the mean (SEM). B The schematic diagram illustrates the quantification of EV outer bound proteins and inner packed proteins, including their phosphorylation pattern (Created with BioRender.com). C THP-1 cells, stimulated with LPS (10 μg/ml) or PBS, underwent multi-cytokine membrane array analysis (upper panel) . Mean pixel densities, calculated via image software, identified up-regulated cytokines ( n = 1 , measurement) denoted in a bar graph (lower panel) . D LPS-stimulated THP-1 cells were subjected to multiplex immunoassay before and after EV lysis, revealing changes in selected inflammatory mediators. Statistical analysis employed ordinary one-way ANOVA unpaired t-test with Mann-Whitney test (The error bars represent the SEM; n = 9 of different measurements). E THP-1 cells, stimulated with LPS, were analyzed over time. Cell and EV lysates, separated on SDS-PAGE, were stained for phosphoproteins (left panel) and total protein content (right panel) . The data represent a representative experiment from two independent experiments. F Lysates from LPS-stimulated THP-1 cells and LPS-EVs were separated on SDS-PAGE and then probed with anti-phospho-tyrosine antibody. The data represent a representative experiment from three independent experiments. G Immunoprecipitation of IL1R1 receptor from LPS-stimulated THP-1 cell lysates and LPS-EV lysates was performed. Samples were probed for pIRAK4 and MyD88 antibodies, indicating receptor-associated signaling. The data represent a representative experiment from three independent experiments. H Lysates from LPS-stimulated THP-1 cells and LPS-EVs were separated on SDS-PAGE and probed for pIRAK4, MyD88, pTRAF2, and TRADD antibodies, revealing in vitro signaling dynamics. The data represent a representative experiment from two independent experiments.

Article Snippet: For Western blot analysis, proteins were transferred onto PVDF-membrane (Immobilon®-P Membrane, EMD Millipore Corporation, USA), blocked with 3% non-fat dry milk or 3% BSA in phosphate-buffered saline (PBS) containing 0.05% Tween-20 for 60 min at RT and incubated with monoclonal rabbit anti-human pIRAK4, (Cat#11927; Cell signaling Technology®), pTRAF2 antibodies (Cat#13908; Cell signaling Technology®), anti-human MyD88 (Cat#4283; Cell signaling Technology®), and TRADD antibodies (Cat# sc-46653; Santa Cruz), anti-Munc 18-1 or STXBP1 (Cat#ab3451; Abcam) diluted 1:1000 in blocking solution for 60 min at RT.

Techniques: Flow Cytometry, Translocation Assay, Activation Assay, Marker, Whisker Assay, Phospho-proteomics, Membrane, Software, Multiplex Assay, Lysis, MANN-WHITNEY, SDS Page, Staining, Immunoprecipitation, In Vitro

Journal: Cold Spring Harbor Molecular Case Studies

Article Title: Clinical IRAK4 deficiency caused by homozygosity for the novel IRAK4 (c.1049delG, p.Gly350Glufs*15) variant

doi: 10.1101/mcs.a005298

Figure Lengend Snippet: Pedigree and experimental analysis. ( A ) Pedigree depicting the presence of the c.1049delG, p.(Gly350Glufs*15) IRAK4 variant. The arrow denotes the index patient. ( B ) Sanger sequencing of the IRAK4 gene depicting the single-base-pair deletion in the affected family but not seen in the healthy control (HC). ( C ) Immunoblot showing lack of IRAK4 protein expression compared to a HC, as well as the predicted molecular weight (MW) and position of the truncated IRAK4 due to the variant ( n = 3). ( D ) Simplified schematic overview of the TLR7 signaling pathway resulting in NF-κB activation and TNF-α transcription emphasizing the central role of the IRAK4 protein in signal transduction. ( E ) Cytometric bead array (CBA) showing no TNF-α response to imiquimod (IMQ) stimulation of TLR7 in the IRAK4-deficient cells compared to a HC. PMA/ionomycin serves as a positive control confirming cell viability. Data are represented as the mean ( n = 3), and error bars represent the standard error of the mean (SEM). P -value calculated by the two-tailed Student's t -test with a Bonferroni correction.

Article Snippet: The primary antibodies for immunoblotting detection of human IRAK4 (Lys41; #4363, 1:1000) and β-actin (#3700) were from Cell Signaling Technology.

Techniques: Variant Assay, Sequencing, Control, Western Blot, Expressing, Molecular Weight, Activation Assay, Transduction, Positive Control, Two Tailed Test

Journal: Cold Spring Harbor Molecular Case Studies

Article Title: Clinical IRAK4 deficiency caused by homozygosity for the novel IRAK4 (c.1049delG, p.Gly350Glufs*15) variant

doi: 10.1101/mcs.a005298

Figure Lengend Snippet: Genetic findings

Article Snippet: The primary antibodies for immunoblotting detection of human IRAK4 (Lys41; #4363, 1:1000) and β-actin (#3700) were from Cell Signaling Technology.

Techniques: Variant Assay, Functional Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88.

doi: 10.2119/molmed.2015.00197

Figure Lengend Snippet: Figure 3. SQV inhibits the interaction between cathepsin V and the TLR4-Myddosome recep- tor signaling complex. (A,B) HEK293 cells transfected with HA tagged TLR4 were immunopre- cipitated for HA:TLR4 or cathepsin V followed by subsequent cathepsin V and or TLR4:HA im- munobloting. (C) Recombinant human TLR4 and cathepsin V binding was measured by surface plasmon resonance. SQV at 0.625, 1.25, 2.5, 5 μmol/L dose dependently inhibited binding of TLR4 to cathepsin V. (D) Human monocyte-derived macrophages were pre- treated with SQV (30 μmol/L for 1h) followed by the addition of HMGB1 for 30 and 60 min. Representative immunoblot of immunoprecipitated cathepsin V or MYD88 blotted against TLR4 and IRAK4. (E) Representative immunoblot of TLR4, IRAK4, cathepsin V and MYD88 total protein expression used as input for the experiments in (D). (F) Histogram of quantitation of multiple experiments from (D). (G) Effect of SQV on HMGB1-induced IRAK-1 protein expres- sion compared with β-actin as determined by Western blot analysis.

Article Snippet: Anti-human cathepsin V (MAB10801, R&D Systems), anti-HA (ab-hatag, InvivoGen), anti-human TLR4 (IMG-578A, IMGENEX), anti-human MyD88 (4283, Cell Signaling), anti-human IRAK4 (4363, Cell Signaling ), anti-human IRAK1 (4504, Cell Signaling).

Techniques: Transfection, Recombinant, Binding Assay, SPR Assay, Derivative Assay, Western Blot, Immunoprecipitation, Expressing, Quantitation Assay