Journal: JCI Insight

Article Title: The MUC5B promoter variant results in proteomic changes in the nonfibrotic lung

doi: 10.1172/jci.insight.189636

Figure Lengend Snippet: ( A and B ) Volcano plots comparing the MUC5B promoter variant and WT allele in nonfibrotic control ( A ) small airways and ( B ) alveoli, showing the negative natural log of adjusted P values (0.05) for each protein. ( C ) Representative images for the IHC against phosphorylated STAT5A at Tyr694 on nonfibrotic control lungs. Scale bars: 50 μm. ( D ) Quantification of C showing the percentage of positive cells ( n = 12, balanced for MUC5B; 5 fields of view per patient with a total of 22,999 counted nuclei). ** P < 0.005 by unpaired, 2-tailed t test.

Article Snippet: Antibodies against the following proteins were used: p-eIF2α (Cell Signaling Technology, 3398S; 1:50 in EDTA), cytokeratin 17 (Abcam, ab53707; 1:16,000 in HIER), ATF4 (Proteintech, 10835-1-AP; 1:16,000 in HIER), ATF6 (Abcam, ab227830; 1:1,500 in HIER), MUC5B (Novus Bio, NBP2-50522; 1:8,000 in HIER), p-STAT5A Tyr694 (Cell Signaling Technology, 9359S; 1:500 in HIER), CD9 (Abcam, ab2215; 1:80,000 in HIER), and MYOF (Invitrogen, PA5-53134; 1:8,000 in HIER).

Techniques: Variant Assay, Control

Journal: JCI Insight

Article Title: The MUC5B promoter variant results in proteomic changes in the nonfibrotic lung

doi: 10.1172/jci.insight.189636

Figure Lengend Snippet: A hierarchical cluster analysis based on the top 1,000 variable proteins. For IPF specimens ( n = 12), we collected 12 small airway, 11 honeycomb cyst, 12 epithelia overlying fibroblastic foci (FF), 12 alveoli, and 12 FF samples from the same 12 IPF specimens. We further collected an additional 8 FF samples (a total of 20 FF samples) from 8 additional IPF specimens. For nonfibrotic control specimens ( n = 12), we collected 12 small airway and 12 alveoli samples from the same specimen. The samples were balanced for the MUC5B promoter variant.

Article Snippet: Antibodies against the following proteins were used: p-eIF2α (Cell Signaling Technology, 3398S; 1:50 in EDTA), cytokeratin 17 (Abcam, ab53707; 1:16,000 in HIER), ATF4 (Proteintech, 10835-1-AP; 1:16,000 in HIER), ATF6 (Abcam, ab227830; 1:1,500 in HIER), MUC5B (Novus Bio, NBP2-50522; 1:8,000 in HIER), p-STAT5A Tyr694 (Cell Signaling Technology, 9359S; 1:500 in HIER), CD9 (Abcam, ab2215; 1:80,000 in HIER), and MYOF (Invitrogen, PA5-53134; 1:8,000 in HIER).

Techniques: Control, Variant Assay

Journal: JCI Insight

Article Title: The MUC5B promoter variant results in proteomic changes in the nonfibrotic lung

doi: 10.1172/jci.insight.189636

Figure Lengend Snippet: ( A and C ) Volcano plots comparing ( A ) IPF small airway and nonfibrotic control small airways and ( C ) IPF alveoli and nonfibrotic control alveoli, showing the negative natural log of adjusted P values plotted against the log 2 (fold change) for each protein. ( B and D ) Focusing on the significantly changed proteins from A and C , we reanalyzed the data for the MUC5B promoter variant and show volcano plots for ( B ) IPF small airways and ( D ) IPF alveoli. n = 12 per group (6 WT and 6 MUC5B promoter variant).

Article Snippet: Antibodies against the following proteins were used: p-eIF2α (Cell Signaling Technology, 3398S; 1:50 in EDTA), cytokeratin 17 (Abcam, ab53707; 1:16,000 in HIER), ATF4 (Proteintech, 10835-1-AP; 1:16,000 in HIER), ATF6 (Abcam, ab227830; 1:1,500 in HIER), MUC5B (Novus Bio, NBP2-50522; 1:8,000 in HIER), p-STAT5A Tyr694 (Cell Signaling Technology, 9359S; 1:500 in HIER), CD9 (Abcam, ab2215; 1:80,000 in HIER), and MYOF (Invitrogen, PA5-53134; 1:8,000 in HIER).

Techniques: Control, Variant Assay

Journal: JCI Insight

Article Title: The MUC5B promoter variant results in proteomic changes in the nonfibrotic lung

doi: 10.1172/jci.insight.189636

Figure Lengend Snippet: ( A ) Volcano plot comparing IPF honeycomb cyst (HC) and IPF small airways. ( B ) The subset of proteins associated with “remodeling of epithelial adherens junctions,” showing the negative natural log of adjusted P values plotted against the log 2 (fold change) for each protein. ( C ) A heatmap displaying z scores for the significantly changed “remodeling of epithelial adherens junctions” proteins, comparing IPF HC, IPF small airways, and nonfibrotic control small airways. ( D and E ) Volcano plots comparing MUC5B promoter variant and WT allele in ( D ) the subset of significantly changed IPF HC proteins in A and ( E ) the subset of all significantly changed airway proteins in IPF and nonfibrotic controls while controlling for region. n = 12 per group (6 WT and 6 MUC5B promoter variant).

Article Snippet: Antibodies against the following proteins were used: p-eIF2α (Cell Signaling Technology, 3398S; 1:50 in EDTA), cytokeratin 17 (Abcam, ab53707; 1:16,000 in HIER), ATF4 (Proteintech, 10835-1-AP; 1:16,000 in HIER), ATF6 (Abcam, ab227830; 1:1,500 in HIER), MUC5B (Novus Bio, NBP2-50522; 1:8,000 in HIER), p-STAT5A Tyr694 (Cell Signaling Technology, 9359S; 1:500 in HIER), CD9 (Abcam, ab2215; 1:80,000 in HIER), and MYOF (Invitrogen, PA5-53134; 1:8,000 in HIER).

Techniques: Control, Variant Assay

Journal: JCI Insight

Article Title: The MUC5B promoter variant results in proteomic changes in the nonfibrotic lung

doi: 10.1172/jci.insight.189636

Figure Lengend Snippet: Representative IHC staining for mucin 5B (MUC5B), CD9, and myoferlin (MYOF) in ( A ) IPF honeycomb cyst (red arrows), ( B ) IPF small airway, and ( C ) nonfibrotic control small airway. Scale bars: 20 μm. Quantification of the IHC staining for ( D ) CD9 and ( E ) MYOF positivity per airway ( n = 6 nonfibrotic control and n = 6 IPF specimens). ** P < 0.005; *** P < 0.0005; **** P < 0.0001 by 1-way ANOVA.

Article Snippet: Antibodies against the following proteins were used: p-eIF2α (Cell Signaling Technology, 3398S; 1:50 in EDTA), cytokeratin 17 (Abcam, ab53707; 1:16,000 in HIER), ATF4 (Proteintech, 10835-1-AP; 1:16,000 in HIER), ATF6 (Abcam, ab227830; 1:1,500 in HIER), MUC5B (Novus Bio, NBP2-50522; 1:8,000 in HIER), p-STAT5A Tyr694 (Cell Signaling Technology, 9359S; 1:500 in HIER), CD9 (Abcam, ab2215; 1:80,000 in HIER), and MYOF (Invitrogen, PA5-53134; 1:8,000 in HIER).

Techniques: Immunohistochemistry, Control

Journal: JCI Insight

Article Title: The MUC5B promoter variant results in proteomic changes in the nonfibrotic lung

doi: 10.1172/jci.insight.189636

Figure Lengend Snippet: ( A , B , and D ) Volcano plots comparing ( A ) IPF FF versus IPF alveoli, ( B ) the subset of proteins associated with “eukaryotic translation initiation,” and ( D ) the subset of significantly changed proteins from A reanalyzed for the MUC5B promoter variant showing the negative natural log of adjusted P values plotted against the log 2 (fold change) for each protein. ( C ) A heatmap displaying z scores of the significantly changed “eukaryotic translation initiation” proteins comparing IPF FF, IPF alveoli, and nonfibrotic control alveoli. n = 12 per group ( n = 20 IPF FF, balanced for the MUC5B promoter variant).

Article Snippet: Antibodies against the following proteins were used: p-eIF2α (Cell Signaling Technology, 3398S; 1:50 in EDTA), cytokeratin 17 (Abcam, ab53707; 1:16,000 in HIER), ATF4 (Proteintech, 10835-1-AP; 1:16,000 in HIER), ATF6 (Abcam, ab227830; 1:1,500 in HIER), MUC5B (Novus Bio, NBP2-50522; 1:8,000 in HIER), p-STAT5A Tyr694 (Cell Signaling Technology, 9359S; 1:500 in HIER), CD9 (Abcam, ab2215; 1:80,000 in HIER), and MYOF (Invitrogen, PA5-53134; 1:8,000 in HIER).

Techniques: Variant Assay, Control

Journal: JCI Insight

Article Title: The MUC5B promoter variant results in proteomic changes in the nonfibrotic lung

doi: 10.1172/jci.insight.189636

Figure Lengend Snippet: ( A ) Venn diagram of detected proteins in the IPF epithelia overlying FF as compared with IPF and nonfibrotic control alveoli. ( B , C , and E ) Volcano plots comparing ( B ) IPF epithelia overlying the FF and IPF alveoli, ( C ) the subset of proteins associated with “eIF2 signaling,” and ( E ) the subset of significantly changed proteins in B reassessed for the MUC5B promoter variant, showing the negative natural log of adjusted P values plotted against the log 2 (fold change) for each protein. ( D ) A heatmap displaying z scores of the significantly changed “eIF2 signaling” proteins comparing IPF epithelia overlying FF, IPF alveoli, and nonfibrotic control alveoli. n = 12 per group (6 WT and 6 MUC5B promoter variant).

Article Snippet: Antibodies against the following proteins were used: p-eIF2α (Cell Signaling Technology, 3398S; 1:50 in EDTA), cytokeratin 17 (Abcam, ab53707; 1:16,000 in HIER), ATF4 (Proteintech, 10835-1-AP; 1:16,000 in HIER), ATF6 (Abcam, ab227830; 1:1,500 in HIER), MUC5B (Novus Bio, NBP2-50522; 1:8,000 in HIER), p-STAT5A Tyr694 (Cell Signaling Technology, 9359S; 1:500 in HIER), CD9 (Abcam, ab2215; 1:80,000 in HIER), and MYOF (Invitrogen, PA5-53134; 1:8,000 in HIER).

Techniques: Control, Variant Assay

Journal: Cell stem cell

Article Title: Human airway submucosal gland organoids to study respiratory inflammation and infection.

doi: 10.1016/j.stem.2025.05.013

Figure Lengend Snippet: Figure 1. Establishment and characterization of human bronchus SMG organoids (A) Schematic of tissue processing procedures to isolate SAE and SMG compartments from human bronchus tissue. (B) Culture media compositions for SAE and SMG organoids. (C) Time-lapse bright-field images of SMG and SAE organoids established from human bronchus tissues (donor: #OHLT019). Note: SAE organoids were replated on day 7 to support continued growth and spatial expansion. (D) H&E staining, goblet cells (PAS), SMG-specific mucous cells (MUC5B), and SAE ciliated cells (acetylated α-tubulin) in SMG and SAE organoids detected by immunohistochemistry (IHC) staining (donor: #OHLT019). (E) Representative immunofluorescence staining of SMG-specific mucous cells (MUC5B, green), SAE-specific mucous cells (MUC5AC, red), and nuclei (DAPI, cyan) in human bronchus tissue and organoids (donor: #0704). See also Figure S1.

Article Snippet: The membranes were then blocked with 5% w/v non-fat dry milk in TBST, followed by staining with primary antibodies specific to MUC5B (Atlas Antibodies) or MUC5AC (Thermo Fisher Scientific).

Techniques: Staining, Immunohistochemistry, Immunofluorescence

Journal: Cell stem cell

Article Title: Human airway submucosal gland organoids to study respiratory inflammation and infection.

doi: 10.1016/j.stem.2025.05.013

Figure Lengend Snippet: Figure 4. HCoV-229E primarily targets SMG secretory cells and causes cellular stress (A) Left: diagram depicting transwell culture for SMG and SAE organoids. Right: representative immunofluorescence staining of SMG mucous cells (MUC5B, red), basal cells (KRT14, green), ciliated cells (acetylated α-tubulin, white), and nuclei (DAPI, blue) in transwell cultures (donor: #0704).

Article Snippet: The membranes were then blocked with 5% w/v non-fat dry milk in TBST, followed by staining with primary antibodies specific to MUC5B (Atlas Antibodies) or MUC5AC (Thermo Fisher Scientific).

Techniques: Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Reduced sialylation of airway mucin impairs mucus transport by altering the biophysical properties of mucin

doi: 10.1038/s41598-024-66510-2

Figure Lengend Snippet: Reducing Sialylation of MUC5B Impairs Mucin Linearization. ( A , B ) Natively purified MUC5B was treated with either vehicle or sialidase at pH 7.4 or 10 mM CaCl 2 at a pH of 5. 10 mM EGTA at pH 7.4 was added to vehicle and sialidase groups prior to ON incubation at 4 °C. MUC5B polymers were subsequently visualized by negative stain TEM. ( A ) representative TEM images of MUC5B polymers treated with either vehicle, sialidase, or calcium CaCl 2 /pH 5. Imaging was performed to capture 72–85 polymers per group (vehicle, N = 73; sialidase, N = 72; and 10 mM CaCl 2 /pH 5, N = 85). After blinding, polymers were counted and categorized into 3 groups for each condition: linear, entangled, or condensed. White arrows indicate a linearized polymer in the vehicle group, an entangled polymer in the sialidase group, and a condensed polymer in the CaCl 2 /pH 5 group. ( B ) Quantification showing the percentage total polymers that were linear, entangled, or condensed for each group. N = 72–85 per group. ** P < 0.01, **** P < 0.0001 by Chi Square. Scale bars, 200 nm. ( C , D ) Untreated MUC5B was separated on 10–35% Sucrose gradient by rate zonal centrifugation to separate MUC5B by shape; more compact mucins sediment faster. Gradient fractions were slot blotted and probed for MUC5B and sialic acid content (SIAFIND™ Pan Lectenz). ( C ) Intensities were quantified as a percentage of whole for both MUC5B and sialic acid. ( D ) Representative slot blots of MUC5B and sialic acid, showing a higher percentage of sialic acid content compared to MUC5B in the early (more linear) fractions. ( E ) Fractions were pooled to represent 4 different sedimentation rates across the gradient and imaged via TEM. Scale bars, 200 nm.

Article Snippet: Separate, identical blots were incubated with either MUC5B primary antibody (4A10-H2, Novus Biologicals, Centennial, CO) at 1:500 dilution followed by mouse anti-IgG horse radish peroxidase (HRP) conjugated secondary (31,340, Invitrogen, Waltham, MA) at 1:3000 dilution or 1 μg/mL biotinylated WGA (B-1025–5, Vector Labs, Newark, CA) followed by Vectastain ABC-HRP reagent at 1:2000 dilution.

Techniques: Purification, Incubation, Staining, Imaging, Polymer, Centrifugation, Sedimentation

Journal: Scientific Reports

Article Title: Reduced sialylation of airway mucin impairs mucus transport by altering the biophysical properties of mucin

doi: 10.1038/s41598-024-66510-2

Figure Lengend Snippet: MCT and ST3Gal1 expression are decreased in CF and increased with CFTR modulation in HBECs. ( A ) Representative μOCT images of non-CF HBECs treated with vehicle and CF HBECs treated with either vehicle or ETI triple modulators for 72 h demonstrate decreased ASL (yellow bar) and PCL (red bar) in CF HBECs that were increased with ETI treatment. ( B ) Reprocessed M-mode (e.g., x vs time) µOCT images show tracks of mucus particles above the epithelial surface of non-CF HBECs treated with vehicle and CF HBECs treated with vehicle or ETI modulators; the more horizontal direction of particle streaks (yellow arrow) indicates more rapid transport. Summary data shows the differences in ( C ) ASL and ( D ) PCL depths and ( E ) MCT between non-CF and CF and ( F – H ) CF after ETI correction. Regions of interest from 4 different filters were measured and averaged for each HBEC donor. ( I ) Representative western blot of cell lysates probed for ST3Gal1 from non-CF HBECs treated with vehicle and CF HBECs treated with either vehicle or ETI triple modulators for 72 h, demonstrate decreased expression of ST3Gal1 in CF HBECs that was partially restored after ETI treatment. ( J ) Representative western blot of cell lysates probed for ST6GalNAC1 from non-CF HBECs treated with vehicle and CF HBECs treated with either vehicle or ETI triple modulators for 72 h, demonstrate no differences in expression of ST6GalNAC1. Quantification by densitometry of ST3Gal1 expression showing ( K ) a significant decrease in ST3Gal1 expression in CF HBECs compared to non-CF and ( L ) an increase in paired ETI treated CF HBECs. Quantification by densitometry of ST6GalNAC1 expression showing no differences in ST6GalNAC1 expression between ( M ) non-CF and CF or ( N ) paired ETI treated CF HBECs. ( O ) Slot blot and accompanying densitometry of secreted, isolated MUC5B probed for sialic acid shows decreased MUC5B sialylation in CF and an increase in sialylation after ETI treatment. N = 5–6 donors/ condition. ns P > 0.05, * P < 0.05, ** P < 0.01 by unpaired or paired T-test as appropriate. Scale bars, 20 μm. Western blot densitometry is presented normalized to βactin. Slot blots were normalized to MUC5B prior to loading and represent pooled mucin from 3 HBEC filters per 5 separate donors for each group. Blots were cropped to improve clarity and conciseness; the original, uncropped blots are presented in SI Fig. D-E.

Article Snippet: Separate, identical blots were incubated with either MUC5B primary antibody (4A10-H2, Novus Biologicals, Centennial, CO) at 1:500 dilution followed by mouse anti-IgG horse radish peroxidase (HRP) conjugated secondary (31,340, Invitrogen, Waltham, MA) at 1:3000 dilution or 1 μg/mL biotinylated WGA (B-1025–5, Vector Labs, Newark, CA) followed by Vectastain ABC-HRP reagent at 1:2000 dilution.

Techniques: Expressing, Western Blot, Dot Blot, Isolation

Journal: Journal of Oral Microbiology

Article Title: Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B

doi: 10.1080/20002297.2021.1967636

Figure Lengend Snippet: Comparison of proteomes in planktonic and biofilm cells (grown on uncoated and MUC5B- or LDP-coated surface). Urcl : Urease cluster protein, LeuC : 3-isopropylmalate dehydratase (large subunit), SGO_1479 : Uncharacterized protein, PurD : Phosphoribosylamine–glycine ligase, PurN : Phosphoribosylglycinamide formyltransferase, PurK : N5-carboxyaminoimidazole ribonucleotide synthase, AbpB : Membrane dipeptidase (amylase binding protein), MsmF : Multiple sugar metabolism transmembrane permease, SGO_0965 : Uncharacterized protein

Article Snippet: Antibodies for the central domain of the MUC5B polypeptide backbone (6F10-E4, Novus Biological) and lysozyme as a representative LDP (EC 3.2.1.17) were used for ELISA to detect the fractions enriched with the proteins of interest.

Techniques: Comparison, Membrane, Binding Assay

Journal: Journal of Oral Microbiology

Article Title: Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B

doi: 10.1080/20002297.2021.1967636

Figure Lengend Snippet: Cellular functions associated with uniquely expressed proteins in planktonic and biofilm cells

Article Snippet: Antibodies for the central domain of the MUC5B polypeptide backbone (6F10-E4, Novus Biological) and lysozyme as a representative LDP (EC 3.2.1.17) were used for ELISA to detect the fractions enriched with the proteins of interest.

Techniques: Membrane, Binding Assay, Activation Assay, Inhibition

Journal: Journal of Oral Microbiology

Article Title: Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B

doi: 10.1080/20002297.2021.1967636

Figure Lengend Snippet: Volcano plot representation of proteins in S. gordonii DL1 biofilm cells grown on salivary MUC5B-coated surface versus LDP-coated surface from LC-MS/MS data. The lines indicate arbitrary cut-off values for enrichment (fold change > 2) and significance (p < 0.05). Proteins with significant difference in abundance are indicated in the figure and listed in

Article Snippet: Antibodies for the central domain of the MUC5B polypeptide backbone (6F10-E4, Novus Biological) and lysozyme as a representative LDP (EC 3.2.1.17) were used for ELISA to detect the fractions enriched with the proteins of interest.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Oral Microbiology

Article Title: Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B

doi: 10.1080/20002297.2021.1967636

Figure Lengend Snippet: Protein abundances in MUC5B biofilms compared to LDP biofilms (p < 0.05, FC > 2)

Article Snippet: Antibodies for the central domain of the MUC5B polypeptide backbone (6F10-E4, Novus Biological) and lysozyme as a representative LDP (EC 3.2.1.17) were used for ELISA to detect the fractions enriched with the proteins of interest.

Techniques:

Journal: Journal of Oral Microbiology

Article Title: Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B

doi: 10.1080/20002297.2021.1967636

Figure Lengend Snippet: S. gordonii DL1 and S. mutans UA159 biofilms on LDP- and MUC5B-coated Ibidi VI flow cell IbiTreat surfaces after 2 h of flow. Differences in % coverage were calculated between all four groups using one-way ANOVA, * = p < 0.05. The experiment was performed in triplicate using independent bacterial cultures

Article Snippet: Antibodies for the central domain of the MUC5B polypeptide backbone (6F10-E4, Novus Biological) and lysozyme as a representative LDP (EC 3.2.1.17) were used for ELISA to detect the fractions enriched with the proteins of interest.

Techniques: