polyclonal anti muc5b antibody  (Proteintech)

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Cervical MUC5B and MUC5AC are Barriers to Ascending Pathogens During Pregnancy.

doi: 10.1210/clinem/dgac545

Figure Lengend Snippet: Figure 3. Localization and quantification of MUC5B and MUC5AC in the human uterus during pregnancy and after trachelectomy. (A) Schema of uterine section. (B-G) Representative MUC5B and MUC5AC expression in the human uterus. Right panels are magnified images. A nonpregnant woman (B and E), a pregnant woman at 35 weeks of gestation (C and F), and a nonpregnant woman after trachelectomy (D and G). (H) The areas of cervical glands in 6 normal pregnant women compared with those in 9 nonpregnant women. (I and J) Quantification of MUC5B- and MUC5AC-positive areas in 6 normal pregnant women and 4 nonpregnant women after trachelectomy compared with those in 9 nonpregnant women. Data are represented as the median ± interquartile range. **P < .01, ***P < .001 using a 2-tailed Mann–Whitney U test. Cx, cervix; Ub, uterine body; Va, vagina; Scale bars, 1 mm.

Article Snippet: These sections were incubated with rabbit polyclonal antibody against MUC5B (Novus Biologicals, #NBP1-92151, RRID:AB 467559, 1:500) or mouse monoclonal antibody against MUC5AC (Novus Biologicals, #NBP2-15196, RRID:AB 2894883, 1:100) overnight at 4 °C, followed by incubation with biotinylated goat antirabbit or rabbit antimouse secondary antibody (#424032 or #424022, Nichirei) at room temperature for 30 minutes.

Techniques: Expressing, MANN-WHITNEY

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Cervical MUC5B and MUC5AC are Barriers to Ascending Pathogens During Pregnancy.

doi: 10.1210/clinem/dgac545

Figure Lengend Snippet: Figure 4. Regulation of MUC5B and MUC5AC in primary epithelial cells isolated from the human endocervix. (A) Representative immunofluorescence for E-cadherin, vimentin, and DAPI (upper panels) and CK18, CK14, and DAPI (lower panels) of the primary epithelial cells isolated from the human endocervix. (B, C) Changes in the mRNA expression of MUC5B and MUC5AC by estradiol and progesterone. Primary human endocervical epithelial cells were treated with estradiol (10 nM) and progesterone (100 nM) at physiological concentrations during pregnancy. Data are represented as the mean ± SD from 3 biological replicate experiments. ***P < .001 using ANOVA with Dunnett’s multiple comparisons test. E2, estradiol; CK, cytokeratin; P4, progesterone; Scale bars, 50 μm.

Article Snippet: These sections were incubated with rabbit polyclonal antibody against MUC5B (Novus Biologicals, #NBP1-92151, RRID:AB 467559, 1:500) or mouse monoclonal antibody against MUC5AC (Novus Biologicals, #NBP2-15196, RRID:AB 2894883, 1:100) overnight at 4 °C, followed by incubation with biotinylated goat antirabbit or rabbit antimouse secondary antibody (#424032 or #424022, Nichirei) at room temperature for 30 minutes.

Techniques: Isolation, Immunofluorescence, Expressing

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Cervical MUC5B and MUC5AC are Barriers to Ascending Pathogens During Pregnancy.

doi: 10.1210/clinem/dgac545

Figure Lengend Snippet: Figure 6. Localization of Escherichia coli, neutrophils, and mucins in the cervical canal of a pregnant mouse model of ascending infection. (A) A schematic illustration of the experimental design. (B and C) Hematoxylin and eosin (H&E) and immunofluorescence staining for MUC5B, MUC5AC, E. coli, and Ly6G on an axial section of murine cervix. Representative images of mice treated with PBS (B) (P1 mouse) or E. coli (C) (E1 mouse). Scale bars, 200 μm. (D, E) H&E and immunofluorescence staining for E. coli and Ly6G on an axial section of the cervix in other mice. Treatment with PBS (D) (P2, P3, P4 mice) or E. coli (E) (E2, E3, E4 mice). The arrowhead represents E. coli antigens. Scale bars, 200 μm. (F) Enlarged views indicated by arrowheads in (E) (E2, E3 mice). Scale bars, 10 μm. (G) The number of Ly6G-positive cells in the cervical mucins per field. Data are represented as the median ± interquartile range. *P < .05. Two-tailed Mann–Whitney U tests. (H-K) mRNA expression of Tnf, Il1b, Muc5b, and Muc5ac in murine cervices after E. coli treatment. Data are represented as the mean ± SD. *P < .05, **P < .01 using a 2-tailed unpaired t test.

Article Snippet: These sections were incubated with rabbit polyclonal antibody against MUC5B (Novus Biologicals, #NBP1-92151, RRID:AB 467559, 1:500) or mouse monoclonal antibody against MUC5AC (Novus Biologicals, #NBP2-15196, RRID:AB 2894883, 1:100) overnight at 4 °C, followed by incubation with biotinylated goat antirabbit or rabbit antimouse secondary antibody (#424032 or #424022, Nichirei) at room temperature for 30 minutes.

Techniques: Infection, Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Integrated multiomic analysis identifies TRIP13 as a mediator of alveolar epithelial type II cell dysfunction in idiopathic pulmonary fibrosis

doi: 10.1016/j.bbadis.2024.167572

Figure Lengend Snippet: Key resources.

Article Snippet: Rabbit IgG anti-human MUC5B polyclonal, unconjugated; 1:100 , Santa Cruz Biotech , Cat# sc-20,119 RRID: AB_2282256.

Techniques: Plasmid Preparation, Magnetic Beads, Recombinant, Blocking Assay, Lysis, Protease Inhibitor, Red Blood Cell Lysis, Software, Microscopy, Fluorescence, Imaging

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Generation of human MUC5B [rs35705950] transgenic mice. Preparation of a construct of human MUC5B [rs35705950] recombinant bacterial artificial chromosome (BAC) using the Red/ET recombination strategy ( A ). Sequence analysis of the junction DNA of the recombinant BAC clone ( B ). The expression construct linearized with PI-SceI (Proteinase Intein-Scel Endonuclease I) was separated by pulsed-field gel electrophoresis ( C ). The gel containing the expression construct within the agarose gel was cut out without UV irradiation (upper figure of panel C ). The DNA fragments purified by electrophoretic elution and dialysis were applied to pulsed-field gel electrophoresis to confirm that the long-chain DNA fragments were purified without fragmentation (middle figure of panel C ). The DNA concentration was determined using a NanoDrop spectrophotometer (Shimazu biotech, Kyoto, Japan) lower figure of panel C ). The linearized recombinant BAC clone was microinjected into fertilized eggs of the C57BL/6J strain and the fertilized eggs were transplanted into the oviducts of pseudo-pregnant mice to get the founders ( D – F ). The marker used was M: NEB Low Range PFG marker. Red/ET, Red recombinase system (RedαRedβ proteins)/Electroporation-Transformation-cloning. Arrows indicate the bands.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Transgenic Assay, Construct, Recombinant, Sequencing, Expressing, Pulsed-Field Gel, Electrophoresis, Agarose Gel Electrophoresis, Irradiation, Purification, Concentration Assay, Spectrophotometry, Marker, Electroporation, Transformation Assay, Cloning

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Expression of the human MUC5B [rs35705950] transgene. Southern blot screening for the transgene in the offspring, identifying three transgenic mouse founders (red arrows in ( A )). Analysis of relative gene expression of the human MUC5B [rs35705950] transgene in various tissues and organs by PCR ( B ).

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Southern Blot, Transgenic Assay, Gene Expression

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Sequential body weight changes and significant expression of the human MUC5B transgene rs35705950 in the proximal airways in the bleomycin-induced lung fibrosis model. Lung fibrosis was induced in wild-type (WT/BLM) and human MUC5B rs35705950 transgenic (h-rs35705950-Tg/BLM) mice through continuous subcutaneous administration of BLM. Control groups, consisting of WT (WT/SAL) and human MUC5B rs35705950 transgenic (h-rs35705950-Tg/SAL) mice, similarly received sterile physiological saline. Sequential body weight changes in the four groups of mice ( A ). Relative mRNA expression of the transgene in the experimental mouse groups ( B ). Immunostaining for MUC5B protein was performed using an anti-MUC5B polyclonal antibody, which cross-reacts with both human and mouse MUC5B ( C , D ). Additionally, MUC5B protein staining was conducted using an anti-human MUC5B monoclonal antibody ( E , F ). Scale bars indicate 100 µm. Data are expressed as the mean ± SD. Statistical analysis was performed using ANOVA with the Neuman-Keuls test. * p < 0.05; *** p < 0.001; **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Transgenic Assay, Control, Sterility, Saline, Immunostaining, Staining

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Reduced infiltration of inflammatory cells in human MUC5B rs35705950 transgenic mice with lung fibrosis. ( A , B ) Lung fibrosis was induced in wild-type (WT/BLM) and human MUC5B rs35705950 transgenic (h-rs35705950-Tg/BLM) mice through continuous subcutaneous administration of BLM. Control groups, consisting of WT (WT/SAL) and human MUC5B rs35705950 transgenic (h-rs35705950-Tg/SAL) mice, similarly received sterile physiological saline. On the 22nd day following BLM administration, bronchoalveolar lavage fluid was collected under profound anesthesia. The total cell count and differential cell count were then assessed. Scale bars indicate 200 μm. Data are expressed as the mean ± SD. Statistical analysis was performed by ANOVA with Neuman-Keuls test. * p < 0.05, *** p < 0.001, **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Transgenic Assay, Control, Sterility, Saline, Cell Counting

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Reduced expression of inflammatory cytokines in human MUC5B rs35705950 transgenic mice with lung fibrosis. Inflammatory cytokines were measured in bronchoalveolar lavage fluid ( A ) and lung tissue homogenate ( B ) by immunoassays using commercially available kits and following the protocols of the manufacturers. Data are expressed as the mean ± SD. Statistical analysis was performed by ANOVA with Neuman-Keuls test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline; ns, not significant; OPN, osteopontin.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Transgenic Assay, Saline

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Decreased expression of growth factors in human MUC5B rs35705950 transgenic mice with lung fibrosis. Growth factors were measured in bronchoalveolar lavage fluid ( A ) and lung tissue homogenate ( B ) by immunoassays using commercially available kits and following the protocols of the manufacturers. Data are expressed as the mean ± SD. Statistical analysis was performed by ANOVA with Neuman-Keuls test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline; ns, not significant.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Transgenic Assay, Saline

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Reduced expression of extracellular matrix markers in human MUC5B rs35705950 transgenic mice with lung fibrosis. The relative mRNA expression of extracellular matrix markers was assessed by polymerase-chain reaction and the levels of collagen I was assessed by enzyme immunoassays using commercially available kits following the protocol of the manufacturers. Data are expressed as the mean ± SD. Statistical analysis was performed by ANOVA with Neuman-Keuls test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline; ns, not significant.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Transgenic Assay, Polymerase Chain Reaction, Enzyme Immunoassay, Saline

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Reduced lung fibrosis in human MUC5B rs35705950 transgenic mice. Ashcroft scoring was performed in lung tissue stained with hematoxylin & eosin by blinded experts for the treatment groups. The number of mice in: WT/SAL, n = 8, WT/BLM, n = 18, h-rs35705950-TG/SAL, n = 10, h-rs35705950-TG/BLM, n = 16 ( A , B ). Collagen deposition was evaluated after trichrome staining, and the total lung collagen volume fraction was calculated. The number of mice: WT/SAL, n = 3, WT/BLM, n = 5, h-rs35705950-TG/SAL, n = 3, h-rs35705950-TG/BLM, n = 5 ( C , D ). The lung tissue hydroxyproline content was measured by a colorimetric assay. The number of mice: WT/SAL, n = 8, WT/BLM, n = 18, h-rs35705950-TG/SAL, n = 10, h-rs35705950-TG/BLM, n = 16 ( E ). Scale bars indicate 200 µm. Data are the mean ± S.D. Statistical analysis by ANOVA with Newman-Keuls test. * p < 0.05, ** p < 0.001; **** p < 0.001. WT, wild-type; SAL, saline; BLM, bleomycin.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Transgenic Assay, Staining, Colorimetric Assay, Saline

Journal: bioRxiv

Article Title: Structural mechanism of MUC5AC mucin net-like polymer formation and its SNP variability that affect risk of the lung diseases COPD and IPF

doi: 10.1101/2024.08.02.606332

Figure Lengend Snippet: (A) MUC5AC-D3 sequence. VWD3 sequence is showed in green, C8-3 in blue, TIL3 in pink and E3 in orange. The residues affected by the SNPs rs36189285 (R996) and rs878913005 (R1201) are highlighted in blue and yellow respectively. (B) Schematic sketch of the domains of MUC5AC mucin with the N-terminal region (VWD1 (orange), VWD2 (yellow), VWD’ (light blue) and VWD3 (dark blue)), nine CysD domains (red) surrounded by PTS sequences densely O -glycosylated to form mucin domains (green) and the C-terminal region (VWD4 (light grey), VWCs (dark grey) and CK (black)). The fragments analyzed are enlarged. (C) SDS-PAGE analysis of reduced and non-reduced D3 (1), D3-CysD (2) and D’-D3-CysD (3) reveals the formation of reducible dimers in all three fragments. (D) MUC5AC D3 assembly cryoEM density map and cartoon representation showing the disulfide bonds. The map and model of the two monomers are shown in green and cyan. The Ca 2+ ions are shown as green spheres. The top left figure represents the top view of the molecule. It is rotated anticlockwise by 90°around the x-axis in the top right figure, and rotated clockwise by 45°around the y-axis and enlarged by 50% in the bottom figure showing the details of the front view. Putative intermolecular disulfide bonds are marked by red starts (Cys1132-Cys1132’ bond seems to be reduced). N-terminal (N) and C-terminal (C) of each monomer are marked. (E) Detail of the MUC5AC-D3 covalent dimerization interface zoomed in from (D) showing the interaction between C8-3 domains. (F) Detail of the MUC5AC-D3 covalent dimerization interface zoomed in from (D) showing the TIL3-TIL3’ interaction.

Article Snippet: Stainings were performed with sequential incubation with custom made polyclonal rabbit anti-human MUC5B antibodies (1:200) in block solution ( Fakih et al ., 2020 ) overnight at 4°C and monoclonal mouse anti-human MUC5AC ( Lidell et al ., 2008 )(1:200) in block solution over night at 4°C (Cat# ab3649, Abcam, Cambridge, UK, RRID:AB_2146844).

Techniques: Sequencing, SDS Page

Journal: bioRxiv

Article Title: Structural mechanism of MUC5AC mucin net-like polymer formation and its SNP variability that affect risk of the lung diseases COPD and IPF

doi: 10.1101/2024.08.02.606332

Figure Lengend Snippet: (A) Front view of MUC5AC-D3 assembly dimer from cryoEM density map and model. The two VWD3 domains are shown in dark and light green, C8-3 domains in dark and light blue, and TIL3 domains in dark and light pink. (B) Structural alignment of MUC5AC-D3 (blue) and MUC2-D3 (pink. PDB code: 6rbf). Left image is presented in the same orientation as (A). Right image is rotated clockwise 45°. Regions with high variability are marked by black arrows. The N -glycosylated Asn1154 in lateral chain of MUC2 is marked with a black star. (C) Detail of TIL3 structurally aligned of MUC5AC-D3 (blue), MUC2-D3 (pink. PDB code: 6rbf) and VWF-D3 (orange. PDB code: 6n29). The TIL3 β1-β2 loop is highlighted in brighter colors. To the left, superposition of all three structures showing the larger distance between the TIL3 and VWD3 domains in MUC5AC. The distinct disulfide bonds are marked by stars, in red the one connecting the TIL3 domain with C8-3 domain and in blue the internal TIL3 β1-β2 loop disulfide bond. All three structures are shown separately showing the TIL3 β1-β2 loop and interfacing residues of lateral chains. The cysteines involved in the distinctive disulfide pattern are labeled. Hydrogen bonds between VWD3 and TIL3 are showed with dashed black lines and the residues involved are annotated. In MUC5AC, the residues affected by SNP variation at the amino acids R996 and R1201 are marked. (D) Amino acid sequence alignment of MUC5AC, MUC5B, MUC2 and VWF TIL3 domains. Disulfide bonds are marked. Stars mark distinct disulfide bonds as in (C). Cysteines are colored yellow and arginines in blue.

Article Snippet: Stainings were performed with sequential incubation with custom made polyclonal rabbit anti-human MUC5B antibodies (1:200) in block solution ( Fakih et al ., 2020 ) overnight at 4°C and monoclonal mouse anti-human MUC5AC ( Lidell et al ., 2008 )(1:200) in block solution over night at 4°C (Cat# ab3649, Abcam, Cambridge, UK, RRID:AB_2146844).

Techniques: Labeling, Sequencing

Journal: bioRxiv

Article Title: Structural mechanism of MUC5AC mucin net-like polymer formation and its SNP variability that affect risk of the lung diseases COPD and IPF

doi: 10.1101/2024.08.02.606332

Figure Lengend Snippet: (A) CryoEM 2D classes, box size 220Å. The top figure shows the closed conformation 2D classes from the high-resolution structure shown in and . The discarded particles from an initial 3D classification were further 2D classified. These 2D classes are shown in the bottom panel, open conformation. (B) CryoEM low-resolution map generated using the particles from (A) bottom panel. The top figure shows the fitting on the closed conformation model and the bottom the proposed model for the open conformation. The VWD3 domain of one monomer is shown in light green, C8-3 and TIL3 domains in dark blue and the connecting loop in magenta. The VWD3 domain of the other monomer is shown in cyan, C8-3 and TIL3 in blue and the connecting loop in orange. The black arrows show the densities not covered by the models. The non-occupied densities in the closed form are explained by the movement of VWD3 as shown by the cyan arrows. The C-terminal of TIL3 points toward the marked densities in the open conformation as they could represent E3 and/or CysD. (C) Surface representation of the closed (left) and open (right) conformation colored by molecular lipophilicity potential (MPL) from dark cyan (most hydrophilic) via white to dark goldenrod (most lipophilic). The newly exposed hydrophobic pocket residues in the open conformation are labeled. (D) Detail of proposed model for MUC5AC-D3 open conformation. VWD3 is colored in green, C8-3 in blue and TIL3 in pink. Putative salt bridges between K962 and E1148, and E981 and R1201, and the hydrogen bond between K979 and V1149 are represented by dashed lines. These residues and the interfacing histidines His977 and His1109 are labeled. (E) MUC5AC-D3 closed and open conformation and FCGBP D10 alignment ( Yeshaya et al ., 2024 ). In the left figure MUC5AC-D3 closed (C8-3 in cyan and TIL3 in light pink) and open (C8-3 in blue and TIL3 in dark pink) conformation and FCGBP D10 (C8-10 in orange) were aligned to the VWD domain (green). The N-terminal (N) of VWD and C-terminal (C) of the different C8 domains are marked following the same color code. In the right figure MUC5AC-D3 closed (cyan) and open (green) conformation and FCGBP D10 (orange) were aligned by the C8 (blue) and TIL (pink) domains.

Article Snippet: Stainings were performed with sequential incubation with custom made polyclonal rabbit anti-human MUC5B antibodies (1:200) in block solution ( Fakih et al ., 2020 ) overnight at 4°C and monoclonal mouse anti-human MUC5AC ( Lidell et al ., 2008 )(1:200) in block solution over night at 4°C (Cat# ab3649, Abcam, Cambridge, UK, RRID:AB_2146844).

Techniques: Generated, Labeling

Journal: bioRxiv

Article Title: Structural mechanism of MUC5AC mucin net-like polymer formation and its SNP variability that affect risk of the lung diseases COPD and IPF

doi: 10.1101/2024.08.02.606332

Figure Lengend Snippet: (A) Detail of TIL3-VWD3 interface mutants aligned with the MUC5AC-D3 WT (dark grey). The left figure shows the Arg996Gln mutant in cyan, the mid shows the Arg1201Trp mutant in yellow, and the right shows the double mutant Arg996Gln-Arg1201Trp in green. The mutations are pointed by pink arrows. (B) CryoEM density map and cartoon representation of the MUC5AC-D3 dimeric assemblies WT (grey), Arg996Gln (cyan), Arg1201Trp (yellow) and Arg996Gln-Arg1201Trp (green) at two 45° angles. (C) CryoEM 2D classes of higher order oligomers. Box sizes are specified for every group of classes. The groups of closed conformation oligomers are marked with “C” and the open conformation with “O”. (D) MUC5AC-D3 Arg996Gln tetrameric assembly cryoEM density map and cartoon representation. One covalent dimer is shown in yellow (chain A) and magenta (chain B) and the other in green (chain C) and cyan (chain D). The His-tag density is shown in white. The top image shows a lateral view of the tetramer. It is rotated clockwise by 90° around y-axis and reduced 1.5 times in the middle-left figure, rotated clockwise by 90° around y-axis again in the middle-right figure, and rotated anticlockwise by 90°around x-axis and rescaled to the original size in the bottom figure. (E) Detail of the MUC5AC-D3 non-covalent tetramerization interface zoomed from (D). The predicted salt bridges and hydrogens bonds are shown as dashed cyan lines. (F) Structural alignment of the tetramer chain B (pink) and chain D (blue) against the R996Q dimer chain A (grey).

Article Snippet: Stainings were performed with sequential incubation with custom made polyclonal rabbit anti-human MUC5B antibodies (1:200) in block solution ( Fakih et al ., 2020 ) overnight at 4°C and monoclonal mouse anti-human MUC5AC ( Lidell et al ., 2008 )(1:200) in block solution over night at 4°C (Cat# ab3649, Abcam, Cambridge, UK, RRID:AB_2146844).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Structural mechanism of MUC5AC mucin net-like polymer formation and its SNP variability that affect risk of the lung diseases COPD and IPF

doi: 10.1101/2024.08.02.606332

Figure Lengend Snippet: (A) MUC5AC-D3 Arg996Gln tetrameric assembly cryoEM density map and cartoon representation. One covalent dimer is shown in yellow (chain A) and magenta (chain B) and the other in green (chain C) and cyan (chain D). The PTS domains are schematically represented as a polyalanine straighten chain protruding from the TIL3 C-termini. The PTS from each covalent dimer extend in opposite directions forming an angle of about 40° with the PTS from the other dimer. (B) Ideal schematic representation of the MUC5AC network generated by repetitions of (A) linked by covalent dimerization at the end of the PTS domains (cystine-knot domain). (C) Carnoy fixed human stomach biopsy paraffin section stained with a monoclonal anti-human MUC5AC antibody (45M1; red) and Hoechst (nuclei; blue). The enlarged white square shows stratified surface mucus positive for MUC5AC. (D) Scanning electron micrograph of a piglet airway showing a MUC5B bundled strand, MUC5AC mucus attached to the bundle, and cilia. (E) Frequency of SNPs increases in COPD (left) and IPF (right). AC M stands for mutant MUC5AC Arg1201Trp (rs878913005), B M for mutant MUC5B promotor (rs35705950), AC Wt for wild type in MUC5AC Arg1201 position and B Wt for wild type MUC5B promotor in the position for rs35705950. Significance with the Fisher exact test is shown by three stars (p<0.001), one star (p<0.05) or a triangle (p<0.1). The bottom table shows the raw values used for the frequency calculations. (F) Linkage disequilibrium between MUC5AC Arg1201Trp (rs878913005) and MUC5B promotor (rs35705950) in (E) control, COPD and IPF groups. The graphic shows the frequency increase of both mutations appearing in the same subject in relation to the expected frequency if both mutations were independent. (G) Genomic organization of the MUC5AC and MUC5B gene locus och chromosome 11. (H) Formalin fixed paraffin section from an IPF lung explanted at lung transplantation, stained with a polyclonal anti-human MUC5B antibody (green), a monoclonal anti-human MUC5AC antibody (45M1; red) and Hoechst (nuclei; blue).

Article Snippet: Stainings were performed with sequential incubation with custom made polyclonal rabbit anti-human MUC5B antibodies (1:200) in block solution ( Fakih et al ., 2020 ) overnight at 4°C and monoclonal mouse anti-human MUC5AC ( Lidell et al ., 2008 )(1:200) in block solution over night at 4°C (Cat# ab3649, Abcam, Cambridge, UK, RRID:AB_2146844).

Techniques: Generated, Paraffin Section, Staining, Mutagenesis, Control, Transplantation Assay

Journal: Cell genomics

Article Title: Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma

doi: 10.1016/j.xgen.2022.100229

Figure Lengend Snippet: (A) Scheme for culturing primary human bronchial epithelial cells (HBECs) at an air-liquid interface (ALI) with IL-13 stimulation. On day 0, cells are predominantly basal cells (orange). ALI culture allows differentiation into ciliated (magenta) and secretory cells (cyan). The mucin 5B (MUC5B)-rich mucus gel layer (red) changes to a MUC5AC-rich gel (green) upon IL-13 stimulation. (B and C) Effects of IL-13 on secretory cells compared with basal cells (B) or ciliated cells (C). Each point represents a gene. Genes that were regulated differently between cell types are shown in black (log 2 fold change difference > 1 and FDR < 0.1 for interactions between cell type and cytokine effect), and other genes are shown in gray. R , Pearson correlation coefficient (p < 2.2 × 10 −16 for both comparisons). (D) Relative expression of 20 genes with the highest absolute IL-13-induced fold change in any of the three cell types. (E) Gene set enrichment analysis of IL-13-induced cell type-selective responses in HBECs. Relative enrichment coefficients were calculated for the full collection of 2,555 gene ontology biological process (GOBP) gene sets; results are shown for the five GOBP gene sets that were most highly induced by IL-13 in basal, ciliated, and secretory cells (p < 10 −5 in one cell type and enrichment coefficient < 0.5 in other cell types). (F) IL-13-induced genes in secretory cells are highly enriched for a previously defined set of goblet cell genes not included in GOBP. For comparison, unlabeled points represent values for the full collection of 2,555 GOBP gene sets. (G) Distribution of IL-13-enriched H3K27ac peaks (N = 387) by genomic region classification. (H) Bulk RNA-seq expression changes in genes closest to H3K27ac ChIP-seq peaks that were not significantly affected (p > 0.1) by IL-13 (no Δ, 26,875 peaks) or were significantly (FDR < 0.1) enriched (387 peaks) or depleted (215 peaks) after IL-13 stimulation. Shown are p values for comparison with unaffected peaks by two-sided Wilcoxon test. (I) The most highly enriched motif discovered in regions with enriched H3K27ac after IL-13 stimulation closely resembles a previously defined STAT6 binding motif. See also and and – .

Article Snippet: Immunofluorescence staining was done with following primary antibodies: mouse monoclonal anti-MUC5AC (45M1; Thermo Fisher Scientific; 1:200), rabbit polyclonal anti-MUC5B (H-300, sc-20119; Santa Cruz Biotechnology, Dallas, TX; 1:200), and mouse monoclonal anti acetylated alpha tubulin (6-11B-1, sc-23950; Santa Cruz Biotechnology; 1:200).

Techniques: Expressing, RNA Sequencing Assay, ChIP-sequencing, Binding Assay

Journal: Cell genomics

Article Title: Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma

doi: 10.1016/j.xgen.2022.100229

Figure Lengend Snippet: (A) Scheme of CRISPRi targeting of SPDEFe . (B–D) CRISPRi effects on gene expression. HBECs were transduced with lentiviruses driving expression of dCas9-KRAB and non-targeting (NT) control sgRNAs (gray/black) or sgRNAs targeting the SPDEF promoter (red), MUC5AC promoter (blue), or SPDEFe (orange). After differentiation, cells were left unstimulated or stimulated with IL-13 for 7 days, as indicated. Expression of SPDEF (B), FOXA3 (C), and MUC5AC (D) was measured by quantitative real-time PCR. mRNA levels are relative to IL-13-stimulated HBECs with NT control sgRNAs. (E) CRISPRi effects on intracellular MUC5AC were quantified by flow cytometry. Each point corresponds to a different gRNA targeting the indicated region, tested separately in a single culture well from the same donor (B–E). **p < 0.01, ****p < 0.0001 for comparison with IL-13-stimulated HBECs with NT control sgRNAs by one-way ANOVA with Dunnett’s post-test (B–E). (F) Effects of targeting SPDEFe and surrounding regions on IL-13-induced MUC5AC production. gRNAs used in (B)–(E) were compared with gRNAs targeting flanking regions ~2 and 4 kb away from SPDEFe (magenta) in a separate set of experiments (three donors, two replicates per donor). To combine results from two donors, values for MUC5AC-producing cells are shown as percentages of mean values for IL-13-stimulated cells with NT sgRNAs in the same donor. **p < 0.01, ****p < 0.0001 compared with IL-13-stimulated HBECs with NT-1 sgRNA by one-way ANOVA with Tukey’s post-test. p values for all comparisons are provided in . (G–J) CRISPRi effects on mucin staining and mucociliary transport. HBECs were treated as above using NT-2 gRNA, SPDEF -TSS(+34) gRNA, or SPDEFe (+87) gRNA. Sections (G) and extracellular mucus gels from whole-mount preparations (H) were stained for MUC5AC (cyan), MUC5B (red), and the ciliated cell marker ac-α-Tub (yellow). Nuclei were stained with DAPI (purple). Scale bars: 20 μm (G) and 100 μm (H). Images are representative of two experiments with different donors. Mucociliary transport rates were determined from trajectories of fluorescent microspheres placed on gels atop cells (I and J). Shown is superimposition of 10 images acquired at 1-s intervals; scale bars: 50 μm (I). Microsphere speeds were determined from three donors, one well per donor, four fields per well (J). Values represent median microsphere speed for each field. Boxes extend from the 25th to the 75th percentile, horizontal lines within the box indicate means, and whiskers represent minimum and maximum values. *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey’s post-test. See also and and .

Article Snippet: Immunofluorescence staining was done with following primary antibodies: mouse monoclonal anti-MUC5AC (45M1; Thermo Fisher Scientific; 1:200), rabbit polyclonal anti-MUC5B (H-300, sc-20119; Santa Cruz Biotechnology, Dallas, TX; 1:200), and mouse monoclonal anti acetylated alpha tubulin (6-11B-1, sc-23950; Santa Cruz Biotechnology; 1:200).

Techniques: Expressing, Transduction, Real-time Polymerase Chain Reaction, Flow Cytometry, Staining, Marker

Journal: Cell genomics

Article Title: Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma

doi: 10.1016/j.xgen.2022.100229

Figure Lengend Snippet: (A) Scheme of IL-13-responsive, secretory cell-specific, SPDEFe -driven CRISPRi. minP, minimal promoter. (B–H) A lentivirus containing a KRAB-dCas9 transgene driven by SPDEFe (or other enhancers) and a second lentivirus driving expression of sgRNA targeting the SPDEF (SPDEF-TSS(+34)) or MUC5AC (MUC5AC-TSS(+134)) promoter were used in combination. Transduced cells were ALI cultured without cytokine stimulation or with IL-13 (B–E) or IL-1β (F–H) stimulation for the last 7 days of culture, as indicated. Changes in expression of dCas9 (B and F), SPDEF (C and G), and MUC5AC (D and H) were measured by quantitative real-time PCR. MUC5AC-producing cells (E) were quantitated by flow cytometry. mRNA levels and MUC5AC-producing cells are relative to mRNA levels and MUC5AC-producing cells from IL-13-stimulated cells with SV40e-KRAB-dCas9 and NT-2 sgRNA in the same donor. IL-13 data (B–E) are from four donors. The IL-1β data (F–H) are from cells from three donors, including two used for the IL-13 experiment. *p < 0.05, **p < 0.01, ****p < 0.0001 compared with the unstimulated empty vector control for NT gRNA and IL-13-stimulated empty vector control for SPDEF and MUC5AC gRNAs by one-way ANOVA Tukey’s post-test (B–H).

Article Snippet: Immunofluorescence staining was done with following primary antibodies: mouse monoclonal anti-MUC5AC (45M1; Thermo Fisher Scientific; 1:200), rabbit polyclonal anti-MUC5B (H-300, sc-20119; Santa Cruz Biotechnology, Dallas, TX; 1:200), and mouse monoclonal anti acetylated alpha tubulin (6-11B-1, sc-23950; Santa Cruz Biotechnology; 1:200).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Plasmid Preparation

Journal: Cell genomics

Article Title: Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma

doi: 10.1016/j.xgen.2022.100229

Figure Lengend Snippet:

Article Snippet: Immunofluorescence staining was done with following primary antibodies: mouse monoclonal anti-MUC5AC (45M1; Thermo Fisher Scientific; 1:200), rabbit polyclonal anti-MUC5B (H-300, sc-20119; Santa Cruz Biotechnology, Dallas, TX; 1:200), and mouse monoclonal anti acetylated alpha tubulin (6-11B-1, sc-23950; Santa Cruz Biotechnology; 1:200).

Techniques: Recombinant, SYBR Green Assay, Software

Journal: Virology Journal

Article Title: Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

doi: 10.1186/1743-422X-7-269

Figure Lengend Snippet: Mucin toxicity

Article Snippet: Polyclonal goat anti-MUC5B (sc-23024), anti-MUC7 (sc-16918) and rabbit anti-goat HRPO linked secondary antibodies were from Santa Cruz (California).

Techniques:

Journal: Virology Journal

Article Title: Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

doi: 10.1186/1743-422X-7-269

Figure Lengend Snippet: Anti-HIV-1 activities of salivary MUC5B and MUC7 of different CD4 counts for unfiltered samples . Salivary MUC5B and MUC7 mucins (500 μl or 0.9 mg each) from patients with CD4 count (< 200, 200-400 and > 400) were incubated with subtype D HIV-1 for 60 min and filtered through 0.45 μm pore size cellulose acetate filter. As controls HIV-1 treated with media and heat inactivated HIV-1 were used. The unfiltered samples were then incubated with CEM SS cells at a concentration of 0.5 × 10 6 cells/ml for 30 min, 1 h and 2 h. After PBS wash cells were cultured and viral replication was measured by a qualitative p24 antigen assay. Letters A, B and C indicate the anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with CD4 counts < 200, 200-400 and > 400 respectively.

Article Snippet: Polyclonal goat anti-MUC5B (sc-23024), anti-MUC7 (sc-16918) and rabbit anti-goat HRPO linked secondary antibodies were from Santa Cruz (California).

Techniques: Incubation, Concentration Assay, Cell Culture, Antigen Assay, Activity Assay

Journal: Virology Journal

Article Title: Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

doi: 10.1186/1743-422X-7-269

Figure Lengend Snippet: Anti-HIV-1 activities of salivary MUC5B and MUC7 of different CD4 counts for filtrates . The rest is as for Fig 1.

Article Snippet: Polyclonal goat anti-MUC5B (sc-23024), anti-MUC7 (sc-16918) and rabbit anti-goat HRPO linked secondary antibodies were from Santa Cruz (California).

Techniques:

Journal: Virology Journal

Article Title: Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

doi: 10.1186/1743-422X-7-269

Figure Lengend Snippet: Gradient gel analysis of salivary MUC5B and MUC7 mucins from HIV negative and positive individuals . Freeze-dried samples (30 μg) of MUC5B from HIV negative individual (lane 1), MUC5B from HIV positive individuals with CD4 counts < 200 (lane 2), 200-400 (lane 3), > 400 (lane 4), MUC7 from HIV negative individual (lane 5), MUC7 from HIV positive individuals with CD4 counts < 200 (lane 6), 200-400 (lane 7) and > 400 (lane 8) were prepared in a gel loading buffer and separated in a 4-20% gradient gel. Following electrophoresis the gel was stained with PAS. While arrows in red indicates the two glycoforms of MUC7 on top of the running gel (MUC7a) and slightly entering the running gel (MUC7b), the arrow in black is at the start of running gel.

Article Snippet: Polyclonal goat anti-MUC5B (sc-23024), anti-MUC7 (sc-16918) and rabbit anti-goat HRPO linked secondary antibodies were from Santa Cruz (California).

Techniques: Electrophoresis, Staining

Journal: Virology Journal

Article Title: Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

doi: 10.1186/1743-422X-7-269

Figure Lengend Snippet: ELISA monitoring immunoreactivity of salivary MUC5B and MUC7 mucins from HIV negative and positive individuals . Plate (A) was coated with MUC5B from HIV negative individual (dark blue diamond), MUC5B from HIV positive individuals with CD4 counts < 200 (pink square), 200-400 (blue triangle) and > 400 (pale blue cross) and plate (B) was coated with MUC7 from HIV negative individual (dark blue diamond), MUC7 from HIV positive individuals with CD4 counts < 200 (pink square), 200-400 (blue triangle) and > 400 (pale blue cross). Plates were incubated with serial two-fold dilutions of goat anti-MUC5B (A) and goat anti-MUC7 (B) polyclonal antibodies at concentrations between 8 μg/ml and 0.25 μg/ml (goat anti-MUC5B) and 12 μg/ml and 0.375 μg/ml (goat anti-MUC7). Antibody binding was detected using rabbit anti-goat HRPO linked secondary antibody and visualized with TMB/H2O2 substrate. Absorbance values were read at 405 nm in a Titertek ELISA reader. Each point is the average absorbance of duplicate samples. As a negative control wells were coated with PBS (+).

Article Snippet: Polyclonal goat anti-MUC5B (sc-23024), anti-MUC7 (sc-16918) and rabbit anti-goat HRPO linked secondary antibodies were from Santa Cruz (California).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Negative Control

Journal: Virology Journal

Article Title: Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

doi: 10.1186/1743-422X-7-269

Figure Lengend Snippet: Western blotting of salivary MUC5B and MUC7 from HIV positive individuals with different CD4 counts . Lane 1, MUC5B from patients with CD4 > 400, lane 2, MUC5B from patients with CD4 200-400, lane 3, MUC5B from patients with CD4 < 200, lane 4, MUC5B from HIV negative individual, lane 5, crude saliva (positive control), lane 6, MUC7 (negative control), lane 7, MUC7 from patients with CD4 > 400, lane 8, MUC7 from patients with CD4 200-400, lane 9, MUC7 from patients with CD4 < 200, lane 10, MUC7 from HIV negative individuals, lane 11, crude saliva (positive control) and lane 12, MUC5B (negative control) were separated by a 1% agarose gel and transferred to nitrocellulose membrane. Following overnight blocking, the membranes were incubated for 2 h with rabbit anti-MUC5B polyclonal (lanes 1-6) and mouse anti-MUC7 monoclonal (lanes 7-12) antibodies diluted in 5% (m/v) low fat milk powder in TBST at 1 in 2000 (rabbit anti-MUC5B) and 1 in 1000 (mouse anti-MUC7). Membranes were then washed 3 × 5 min with TBST and incubated for 1 h with HRPO linked goat anti-rabbit (lanes 1-6) and goat anti-mouse (lanes 7-12) secondary antibodies diluted in 5% (m/v) low fat milk powder in TBST at dilutions of 1 in 5000 and 1 in 1500 respectively. After another TBST wash (3 × 5 min), bands were detected using an ECL detection kit.

Article Snippet: Polyclonal goat anti-MUC5B (sc-23024), anti-MUC7 (sc-16918) and rabbit anti-goat HRPO linked secondary antibodies were from Santa Cruz (California).

Techniques: Western Blot, Positive Control, Negative Control, Agarose Gel Electrophoresis, Blocking Assay, Incubation

Journal: PLoS ONE

Article Title: Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

doi: 10.1371/journal.pone.0050704

Figure Lengend Snippet: Primers.

Article Snippet: Kalamazoo, MI) and polyclonal anti-Muc5b (H300, Santa Cruz Biotechnology.

Techniques:

Journal: PLoS ONE

Article Title: Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

doi: 10.1371/journal.pone.0050704

Figure Lengend Snippet: Cornea (C), Conjunctiva (Cj), and Lacrimal gland (L) were dissected and total RNA was extracted for RT-PCR assay. M: molecular weight marker. Actin was used as a loading control. #1, 2, 3, 4, 5, 6 were different mice. WT: wild-type mice. KO: Muc5ac knockout mice.

Article Snippet: Kalamazoo, MI) and polyclonal anti-Muc5b (H300, Santa Cruz Biotechnology.

Techniques: Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Knock-Out

Journal: PLoS ONE

Article Title: Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

doi: 10.1371/journal.pone.0050704

Figure Lengend Snippet: Conjunctiva was dissected and total RNA was extracted for realtime PCR assay. n = 5, $, *: p<0.05. NS: not significant. WT: wild-type mice. KO: Muc5ac knockout mice.

Article Snippet: Kalamazoo, MI) and polyclonal anti-Muc5b (H300, Santa Cruz Biotechnology.

Techniques: Knock-Out

Journal: PLoS ONE

Article Title: Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

doi: 10.1371/journal.pone.0050704

Figure Lengend Snippet: Mouse conjunctiva tissues were harvested, fixed and the tissue sections were stained with AB/PAS or anti-Muc5ac and anti-Muc5b antibodies. A) Representative images (60X, 10 um scale bar was shown) from AB/PAS staining on mouse conjunctiva tissues. B) Statistical analysis of AB/PAS staining positive area per basement membrane length. n = 5, *: p<0.05. C) Representative images (60X, 10 um scale bar was shown) from dual staining of Muc5ac and Muc5b on mouse conjunctiva tissues. D) Statistical analysis of Muc5b staining positive area per basement membrane length. n = 5, *: p<0.05. WT: wild-type mice. KO: Muc5ac knockout mice.

Article Snippet: Kalamazoo, MI) and polyclonal anti-Muc5b (H300, Santa Cruz Biotechnology.

Techniques: Staining, Knock-Out

Journal: PLoS ONE

Article Title: Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

doi: 10.1371/journal.pone.0050704

Figure Lengend Snippet: Corneal opacification of various degrees was noted in both eyes of 4 out of 35 Muc5ac-deficient mice (KO), but not in any of the wild-type mice (WT).

Article Snippet: Kalamazoo, MI) and polyclonal anti-Muc5b (H300, Santa Cruz Biotechnology.

Techniques:

Journal: PLoS ONE

Article Title: Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

doi: 10.1371/journal.pone.0050704

Figure Lengend Snippet: This is determined by red phenol thread test. Bar graph shows the mean thread measurement in Muc5ac -deficient mice and wild-type. Three measurements (mm) were obtained with the thread placed in the lower lid for 15 seconds with the eyes open. These 3 values were averaged to generate the final measurement, representing the data presented. The mean values from total 31 KO and 15 WT were compared. These two groups were gender and age matched. (NS: Not statistically significant. p = 0.061, independent t-test, error bars indicate 95%CI).

Article Snippet: Kalamazoo, MI) and polyclonal anti-Muc5b (H300, Santa Cruz Biotechnology.

Techniques: