polyclonal rabbit anti-human muc5b  (Millipore)

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: 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: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Cluster identification. SAE cell types were identified from Dropseq single cell RNA sequencing datasets from six individuals (three healthy nonsmokers and three healthy smokers) using established cell specific markers including KRT5 for basal cells, SCGB1A1 for club cells, MUC5AC for goblet cells, and FOXJ1/DNAI1 for ciliated cells . Club cells were identified by the presence of SCGB1A1, low levels of the basal marker KRT5, and the absence of the differentiated goblet cell marker MUC5AC. Club cells underwent Seurat unsupervised clustering which identified three unique subsets of club cells: club cells 1–3 (CC1, CC2, CC3, respectively). One nonsmoker sample was enriched in pseudogenes ( arrow ); the pseudogene expression data was not used in subsequent analyses. A three-dimensional representation helps to discern the distinct localization of the three clusters in the tSNE plot. The distribution of CC1, 2 and 3 within the original low resolution cluster map of the airway cells is shown in Supplementary Fig. . PNEC pulmonary neuroendocrine cells, APC antigen-presenting cells; NCL high represents a distinct cluster of cells identified in Zuo et al. 2020 . The relative size of the populations shown on the tSNE plot is not representative of the proportions of cells found in the tissue in vivo. The single-cell RNA-seq process involves separation of the epithelium into single, live cells, a process that incurs greater loss and cell death among larger cells such as ciliated cells and favors preservation of smaller cells such as basal cells. It is potentially possible for a cell type or subcluster to be under- or over-represented in this analysis. b Quantification of the average percent of club cells in each club cell subset relative to all club cells. c Heatmap of the top ten differentially expressed genes in each subcluster highlighting the differences among the three subclusters. Yellow represents increased expression levels. For the identity of genes contributing to this heatmap, see Supplementary Fig. . d Representation of each of the club cell subsets in each of the six study subjects. Note the presence of the pseudogene expression in the first nonsmoker subject ( arrow ) that was marked in panel a and excluded from subsequent analysis .

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: RNA Sequencing, Marker, Expressing, In Vivo, Preserving

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Airway in adult normal nonsmoker human lung was analyzed for club cell subtypes. Club cells were identified as SCGB1A1 + cells (green) and subsets were identified using various markers in the single cell sequencing. a Secretoglobin family 3A member 1 (SCGB3A1) (red) and secretory leukocyte protease inhibitor (SLPI) (purple). b SCGB1A1 (green), mucin 5B (MUC5B; purple). Club cells that only express SCGB1A1 but no secondary markers are denoted with a white arrow. Club cells expressing multiple markers are denoted with a white arrowhead. Scale bar = 20 μm.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Sequencing, Protease Inhibitor, Expressing

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Differentially expressed genes in progenitor vs. effector club cells a .

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Most commonly enriched genes in each club cell subcluster.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Expressing, Binding Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Nonsmoker primary small airway epithelium (SAE) basal cells were cultured in air–liquid interface (ALI) culture for 28 days. a Morphology of the cultures at day 7 and 28. Shown are cross sections of the ALI culture, hematoxylin and eosin stain. b , c Multicolor immunofluorescence assessment of club cell subtypes. All club cells were identified using SCGB1A1 marker (green); effector club cells were further identified by the co-expression of MUC5B (panel b red) or SCGB3A1 (panel c red). DAPI identifies the nucleus of all cells. The number of days after establishment of the air liquid interface is noted. Green arrowheads mark cells that are SCGB1A1 + club cells that lack other markers. Orange arrowheads mark cells that are positive for SCGB1A1 and either MUC5B or SCGB3A1. Red arrowheads mark cells that are positive for MUC5B or SCGB3A1, but express little if any SCGB1A1. Scale bars are 50 μm. d Gene expression on ALI over time. TaqMan probes for genes enriched in effector club cells were assessed by qPCR and normalized to an 18S rRNA control. Probes were tested at days 0, 7, 14, and 28 of SAE nonsmoker cells differentiated in ALI. Error bars represent standard deviation among three different ALI cultures. Genes assessed for expression include MUC5B, SCGB1A1, SLPI, PIGR, and LYZ.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Cell Culture, H&E Stain, Immunofluorescence, Marker, Expressing, Gene Expression, Control, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Smoking-related reprogramming of small airway epithelium club cells.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Numbers of club cells in smokers vs. nonsmokers. Club cells from nonsmoker and smoker human small airway epithelium (SAE) cytopreps, identified by SCGB1A1 + immunostaining and absence of KRT5, were quantified and compared against total cells, quantified by DAPI staining, per cytoprep. Three samples of each phenotype were evaluated by a blinded observer, with over 500 total cells per sample; plots show mean ± standard error. b Proportion of club cells exhibiting characteristics of subclusters 1 (progenitor), 2 (proliferative), and 3 (effector) in the population of club cells derived from nonsmokers and smokers. c Effect of cigarette smoke extract (CSE) exposure on differentiation of small airway epithelial club cells. Exposure of basal cells differentiated on air liquid interface (ALI) to cigarette smoke extract (3% Marlboro Red) led to a decrease in defense-related transcript in cells: CYP1A1, positive control demonstrating exposure to cigarette smoke extract; MUC5B; PIGR; SLPI; LYZ; and MUC1. Values are expressed as normalized expression relative to 18S rRNA. Each point represents one well of an experiment done in triplicate; plot shows mean ± standard deviation; p values are from a two-sided unequal variance Student’s t -test. All data is from ALI day 28.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Immunostaining, Staining, Derivative Assay, Positive Control, Expressing, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Immunofluorescence analysis of cytopreps containing club cells (SCGB1A1 + ) expressing the effector club cell genes PIGR or MUC5B. White arrowheads indicate dual-labeled cells. White arrows indicate SCGB1A1 + club cells that lack significant expression of a PIGR or MUC5B. Black arrows mark examples of SCGB1A1-negative non-club cells in the cytoprep. Bar = 20 µm. b Total number of dual labeled (SCGB1A1 + PIGR + or SCGB1A1 + MUC5B + ) cells, or SCGB1A1 single labeled cells were quantified by a blinded observer in nonsmokers and compared with smokers. Plot shows mean of three experiments ± standard error; p values are from a two-sided unequal variance Student’s t -test.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Immunofluorescence, Expressing, Labeling