With the application of BCA protein assay kit (Pierce Chemical), the prepared protein samples with the aid of Protein extraction kit was quantified. Afterwards, 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was adopted to divide and shift protein samples to polyvinylidene difluoride membranes (Sigma). Incubation in 5% skim milk for half an hour was to impede nonspecific binding. Subsequently, the membranes were probed with primary antibodies including anti‐HDAC4 (cat. no. ab235583; 1:1000; Abcam), anti‐MUC5AC (cat. no. ab198294; 1:20000; Abcam), anti‐SIRT1 (cat. no. ab189494; 1:1000; Abcam), anti‐phosphorylated (p)‐NF‐κB (cat. no. ab247871; 1:1000; Abcam), anti‐NF‐κB (cat. no. ab32360; 1:1000; Abcam), and anti‐GAPDH (cat. no. ab9485; 1:2500; Abcam) at 4°C overnight. The membranes were incubated with horseradish peroxidase (HRP)‐linked anti‐rabbit (cat. no. ab6721; 1:2000; Abcam) secondary antibody on the following day. The ECL detection system (Millipore, WBKLS0100) was utilized to visualize the protein bands and band intensity was determined on Image Lab™ Software (Bio‐Rad, Shanghai, China). GAPDH was used as a loading control.
Anti muc5ac
Manufactured by Abcam
Sourced in United States
Anti-MUC5AC is an antibody that targets the MUC5AC protein. MUC5AC is a secreted mucin glycoprotein that is primarily expressed in the epithelial cells of the respiratory and gastrointestinal tracts. This antibody can be used in various research applications to detect and study the MUC5AC protein.
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Lab products found in correlation
Anti gapdh, by Abcam (2 mentions)
Anti nf κb, by Abcam (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl detection system, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti sirt1, by Abcam (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti p p65, by Cell Signaling Technology (1 mentions)
Anti ap 1, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Tissue lysis extraction reagent, by Merck Group (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Anti muc2, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
7 protocols using anti muc5ac
1
Western Blot Analysis of Protein Expression
2
Lung Protein Quantification and Analysis
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Lung tissues were homogenized with a tissue lysis/extraction reagent (Sigma-Aldrich, Carlsbad, CA, United States). The concentration of proteins in each sample was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, United States). Equal amounts of cellular proteins (30 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated in blocking solution (5% skim milk, Millipore Co., Bedford, MA, United States), followed by overnight incubation at 4°C with the appropriate primary antibodies, as follows: anti-p65 (Cell Signaling, Denver, MA, United States), anti-p-p65 (Cell Signaling) anti-MUC5AC (Abcam), anti-AP-1 (Cell Signaling) and anti-β-actin (Cell Signaling) antibodies. The blots were washed with Tris-buffered saline containing Tween 20 (TBST), and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, United States) for 30 min at room temperature. The blots were washed again with TBST, and then, developed using an enhanced chemiluminescence kit (Thermo Scientific, San Diego, CA, United States). We evaluated the band expression values by ChemiDocTM (Bio-Rad, Hercules, CA, United States).
3
Evaluating Mucin Expression in Cells
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The effect of BR/NAC on mucin content of the cells was explored using Western blot analysis of the mucin expressions 48 hours post-treatment. Briefly, at the endpoints, cultured cells were homogenized and the protein concentrations were quantified by BioRad protein assay (Bio-Rad Hercules, CA, USA). After protein separation by SDS-PAGE and transfer to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), the following primary antibodies were applied to the membranes according to the manufacturers' protocols: mouse monoclonal anti-MUC1, anti-MUC2 and anti-MUC5AC (Abcam, Cambridge, MA, USA). The membranes were then treated with appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). A similar process was carried out for the GAPDH protein, as a loading control, using anti-GAPDH mouse monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA). The antigen-antibody reaction was digitized with ImageQuant LAS 4000 Biomolecular imager and ImageQuant software (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
4
Lung Histopathology and Mucin Expression
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The upper lobe of left lung without lavage was immediately fixed with 10% formaldehyde for 48 h, embedded in paraffin, sliced into 4 μm thick sections, and stained with hematoxylin and eosin solution (H&E) or Alcian blue periodic acid Schiff (AB/PAS) for histopathologic examination and goblet cell metaplasia of bronchial epithelium.
For analyzing the effects of GFDHP on mucus secretion, the Mucin5ac (Muc5ac) protein expression levels in the airway epithelium were performed by immunohistochemistry (IHC) analysis [18 (link)]. Change in the MAPK pathway in the airway was investigated on the basis of detecting Muc5ac regulatory proteins including extracellular signal-regulated kinase (ERK)/p-ERK/specificity protein1(SP1) through IHC. After antigen was repaired and endogenous peroxidase was inactivated and blocked, the sections were incubated overnight at 4°C with anti-Muc5ac (Abcam, USA, dilution 1 : 200), anti-ERK (dilution 1 : 200), anti-p-ERK (dilution 1 : 500), and anti-SP1 (dilution 1 : 200) antibodies (Proteintech, China) successively. The sections were then incubated with horseradish peroxidase- (HRP-) conjugated second antibodies and developed with 3, 3-diaminobenzidine tetrahydrochloride. The degree of brown stains reflected the expression levels of the target proteins.
For analyzing the effects of GFDHP on mucus secretion, the Mucin5ac (Muc5ac) protein expression levels in the airway epithelium were performed by immunohistochemistry (IHC) analysis [18 (link)]. Change in the MAPK pathway in the airway was investigated on the basis of detecting Muc5ac regulatory proteins including extracellular signal-regulated kinase (ERK)/p-ERK/specificity protein1(SP1) through IHC. After antigen was repaired and endogenous peroxidase was inactivated and blocked, the sections were incubated overnight at 4°C with anti-Muc5ac (Abcam, USA, dilution 1 : 200), anti-ERK (dilution 1 : 200), anti-p-ERK (dilution 1 : 500), and anti-SP1 (dilution 1 : 200) antibodies (Proteintech, China) successively. The sections were then incubated with horseradish peroxidase- (HRP-) conjugated second antibodies and developed with 3, 3-diaminobenzidine tetrahydrochloride. The degree of brown stains reflected the expression levels of the target proteins.
5
Western Blot Analysis of Mouse Lung Proteins
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Total proteins were extracted from part of the mouse left lung with radio immunoprecipitation assay (RIPA) lysis buffer (containing 1 mM PMSF). The protein levels of each sample were determined with a BCA assay kit. Each denatured protein sample (40 μg) was separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membranes. The membranes were preblocked with 5% bovine serum albumin in tris buffered saline tween (TBST) for 1 h at room temperature and then incubated overnight at 4°C with anti-Muc5ac (56 kDa, Abcam, USA), anti-GAPDH (36 kDa), anti-ERK (42/44 kDa), anti-p-ERK (42/44 kDa), anti-SP1 (95 kDa), and anti-Tubulin (52 kDa) antibodies (Proteintech, China) successively. After being washed three times with TBST, the membranes were incubated with HRP-conjugated second antibodies for 1 h at room temperature. The band of each protein was detected using Western blotting reagents and autoradiographed with 3300 Mini ChemiScope Series (Clinx Science Instruments, Shanghai, China). Band intensities were quantified with 3300 Mini ChemiScope image analysis software. The phosphorylation and expression levels of proteins in the Dex group were not analyzed by WB.
6
Protein Characterization Using Antibodies
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Chromatin immunoprecipitation (ChIP), immunoblotting, immunofluorescence, or immunohistochemistry were performed using the following antibodies: anti-α-SMA (#19245T; Cell Signaling); anti-β-Actin (#64225332; Bio-Rad), anti-amylase (#ab21156; Abcam), anti-chromogranin-A (#ab45179; Abcam), anti-cytokeratin 19 (#ab52625; Abcam), anti-E-cadherin (#3195S; Cell Signaling), anti-glucagon (#2760; Cell Signaling), anti-insulin (#4590; Cell Signaling), anti-JunB (#3753; Cell Signaling), anti-Muc5AC (#ab3649; Abcam), anti-Prdm16 (#ab202344 and #ab106410; Abcam), anti-Smad2 (#5339; Cell Signaling), anti-Smad3, (#9523; Cell Signaling), anti-Smad4, (#46535; Cell Signaling), anti-Smad4 (#sc-7966; Santa Cruz), anti-Smad2/3 (#8685; Cell Signaling), and anti-vimentin (#5741S; Cell Signaling).
7
Muc5ac and JAK/STAT Signaling in Lung Cells
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The total proteins from mice lung tissues and 16HBE cells were isolated, and the protein concentrations were determined using a bicinchoninic acid assay. The proteins from whole-cell lysates were used for Western blot using standard techniques as reported [11] . Anti-Muc5ac (Abcam, USA), anti-JAK2, anti-p-JAK2, anti-STAT6, anti-p-STAT6 (1:2000) (Cell Signaling Technology, USA) antibodies were used. Densitometry of bands from Western blots was done by ImageJ2x 2.1.4.7 (Wayne Rasband, National Institutes of Health, USA), and the density of the Muc5ac, JAK2, p-JAK2, STAT6, p-STAT6 proteins relative to β-actin was measured.
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