Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hydrangea serrata extract attenuates PM-exacerbated airway inflammation in the CARAS model by modulating the IL-33/ST2/NF-κB signaling pathway.

doi: 10.1016/j.biopha.2024.116596

Figure Lengend Snippet: Fig. 6. HSE regulated IL-4, IL-5, and histamine in PM-exacerbated CARAS mice. (A,B) Levels of IL-4 and IL-5 in NLF. (C,D) Levels of IL-4 and IL-5 in BALF. (E) Levels of histamine in BALF. (G) Levels of IL-33 in BALF. All levels were determined using ELISA kits. Values are presented as mean ± SEM. Significant differences at ###p<0.001 and ##p<0.01 compared with the control group; ***p<0.001, **p<0.01, and *p<0.05 compared with the CARAS/PM group. CARAS, combined allergic rhinitis and asthma syndrome; Dexa, dexamethasone; HSE, Hydrangea serrata extract; OVA, ovalbumin; PM, particulate matter.

Article Snippet: Thus, IL-33 in BALF was measured using an ELISA kit (M33000, R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s guidelines.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: HSP70 is a chaperone for IL-33 secretion and function in chronic airway disease

doi: 10.1101/2025.01.30.635799

Figure Lengend Snippet: A) SDS-PAGE gel demonstrating co-purification of a high molecular weight band (>65 kDa, MW markers as indicated) that co-purified with all IL-33 isoforms on tandem anti-FLAG, NiNTA affinity purification. B) Mass Spectrometry analysis of contaminating band isolated from Expi293 expression cells and HBE-1 airway cell line identified as HSP70 (and HSC70) with coverage and relative # peptides reported. C) Recombinant purified human IL-33 Δ34 and HSP70 interact by Co-Immunoprecipitation of biotinylated IL-33 protein compared to oxidation resistant Cys->Ser x4 mutant IL-33 Δ34SX4 . D) Intracellular interaction of these proteins verified based on overexpression of miniTurboID-IL-33 Δ34 proximity ligase fusion protein with robust biotinylation of HSP70 in HBE-1 cells but not in U937 cells. E) Differential proximity ligation comparison for IL-33 Δ34 and IL-33 Δ34SX4 miniTurboID fusions in HBE-1 cells. Heatmap of top 20 most abundant proteins and additional heat shock intermediates of relevance. Peptide number indicated by color mapping blue (0), white (25), red (300), replicates 1-3 as shown.

Article Snippet: Non-biotinylated IL-33 Δ34 (500 ng/ml) +/− HSP70 (500 ng/mL) were bound at room temp for 30 mins, plates washed 3x and bound IL-33 Δ34 was detected using R&D DuoSet Human IL-33 biotinylated detection antibody following manufacturer protocols and developed using streptavidin HRP (R&D Systems) and TMB peroxidase substrate (SeraCare).

Techniques: SDS Page, Copurification, High Molecular Weight, Purification, Affinity Purification, Mass Spectrometry, Isolation, Expressing, Recombinant, Immunoprecipitation, Mutagenesis, Over Expression, Ligation, Comparison

Journal: bioRxiv

Article Title: HSP70 is a chaperone for IL-33 secretion and function in chronic airway disease

doi: 10.1101/2025.01.30.635799

Figure Lengend Snippet: A) Phosphatidylserine (PS) binding for IL-33 Δ34(SX4) mutant (C208S, C228S, C232S, C259S) and B) phosphatidylcholine (PC) for IL-33 Δ34 binding ELISAs under conditions of HSP70 (500 ng/ml) and GW4869 (20 μM) in competition. Detection was performed using biotinylated anti-IL-33 secondary antibody (R&D Systems) and Streptavidin HRP, and TMB substrate followed by A450 absorbance measurements. C, D) Secreted and total Flag-IL-33 Δ34 -His and endogenous HSP70 in HBE-1 cells under conditions of GW4869 (20 μM) and GGA (1 μM) treatment, detection using anti-IL-33 and HSP70 Duosets (R&D Systems) and TMB substrate. E) Data points displayed with mean ± SEM. P -value limits: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Non-biotinylated IL-33 Δ34 (500 ng/ml) +/− HSP70 (500 ng/mL) were bound at room temp for 30 mins, plates washed 3x and bound IL-33 Δ34 was detected using R&D DuoSet Human IL-33 biotinylated detection antibody following manufacturer protocols and developed using streptavidin HRP (R&D Systems) and TMB peroxidase substrate (SeraCare).

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: HSP70 is a chaperone for IL-33 secretion and function in chronic airway disease

doi: 10.1101/2025.01.30.635799

Figure Lengend Snippet: A and B) Protein levels for IL-33 and HSP70 in lung tissue and lavage fluid measured by ELISA. C) HSP70 and HSP90 protein levels measured by ELISA in SPIROMICS BAL specimens. D) Schematic for EV isolation by size exclusion chromatography from bronchial wash (BW) and volcano plots for differential protein expression in COPD IV and COPD I-III specimens compared to nonsmoker control. Proteins annotated are associated with heat shock or proteostasis systems and were biotinylated by IL-33 in miniTurboID experiment . Inclusion in bronchial wash EV proteome was demonstrated for HSP70, HSC70 and HSP90. E) Transcript expression of molecular chaperonin TCP1 subunits CCT5 , CCT7 and CCT8 in lung tissue displayed as fold-change by ΔΔCt method with normalization to GAPDH . Data points displayed with mean ± SEM. P -value limits: * P < 0.05, ** P < 0.01, *** P < 0.001, *** P < 0.0001.

Article Snippet: Non-biotinylated IL-33 Δ34 (500 ng/ml) +/− HSP70 (500 ng/mL) were bound at room temp for 30 mins, plates washed 3x and bound IL-33 Δ34 was detected using R&D DuoSet Human IL-33 biotinylated detection antibody following manufacturer protocols and developed using streptavidin HRP (R&D Systems) and TMB peroxidase substrate (SeraCare).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Size-exclusion Chromatography, Expressing, Control