Journal: Journal of Biological Chemistry

Article Title: Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers

doi: 10.1074/jbc.ra117.001014

Figure Lengend Snippet: Figure 1. Assembly of the MUC5B N termini at low pH and high Ca 2. A, the MUC5B monomer consists of the following domains: D1 (orange), D2 (yel- low), D’ (light blue), D3 (blue), D4 (light gray), CysD (red), the mucin domain (PTS, green), von Willebrand C (VWC), (C1 (medium gray) and C2 (dark gray)), and the cystine knot (CK) domain (black). The MUC5B-N recombinant protein included a His6 tag and the D1, D2, D’, and D3 domains. B, MUC5B-N was analyzed by gel filtration chromatography on a Superose 6 3.2/300 column previously equilibrated in 50 mM MES (pH 6.2), 150 mM NaCl (green) or 50 mM MES (pH 6.2) 150 mM NaCl, 4 mM CaCl2 (blue). D means covalent MUC5B-N and Dx2 its non-covalent dimer (tetramer). mAU, milliabsorbance units. C, SDS- PAGE stained by Coomassie Blue of the purified MUC5B-N under reducing (left) and non-reducing conditions (right). Molecular mass standards marked in kilodalton are shown to the left of the gel.

Article Snippet: For size exclusion chromatography analysis, MUC5B-N was incubated for 0 or 1 h at room temperature in 50 mM MES, 150 mM NaCl, pH 6.2 or 50 mM MES, 150 mM NaCl, and 4 mM CaCl2 (pH 6.2) and then applied to a Superose 6 3.2/300 column (GE Healthcare) previously equilibrated in the same buffer using an Ettan LC (GE Healthcare).

Techniques: Recombinant, Filtration, Chromatography, SDS Page, Staining, Purification

Journal: Journal of Biological Chemistry

Article Title: Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers

doi: 10.1074/jbc.ra117.001014

Figure Lengend Snippet: Figure 5. Packing of the MUC5B mucin in the intracellular storage gran- ule and release into the gland lumen. A, MUC5B-N was analyzed by gel filtration chromatography on a Superose 6 3.2/300 column previously equil- ibrated in 50 mM MES (pH 6.2) and 150 mM NaCl (green) or incubated for 1 h in 50 mM MES (pH 6.2), 150 mM NaCl, and 4 mM CaCl2 (red). MUC5B-N without calcium showed one peak corresponding to the dimer; with calcium incuba- tion, most of the protein eluted in the void volume corresponding to high- order oligomers, suggesting heterogeneous oligomerization ((Dx2)n). mAU, milliabsorbance units. B, MUC5B-N incubated at pH 6.2 with 20 mM Ca2 and viewed by negatively stained TEM. Blue arrow, single Dx2 particle; red arrow, linear arrangement of particles ((Dx2)n). C, proximal gland duct filled with linear material (green arrow), likely MUC5B mucin bundles (TEM). C’, C at higher magnification. D, mucus strands moving through a human submucosal gland tract, frame from Movie S2. E, immunofluorescence in a paraffin section from pig trachea. Shown is MUC5B in bundles secreted from the submucosal gland (green). The staining was repeated in sections from five different pigs.

Article Snippet: For size exclusion chromatography analysis, MUC5B-N was incubated for 0 or 1 h at room temperature in 50 mM MES, 150 mM NaCl, pH 6.2 or 50 mM MES, 150 mM NaCl, and 4 mM CaCl2 (pH 6.2) and then applied to a Superose 6 3.2/300 column (GE Healthcare) previously equilibrated in the same buffer using an Ettan LC (GE Healthcare).

Techniques: Filtration, Chromatography, Incubation, Staining, Immunofluorescence, Paraffin Section

Journal:

Article Title: Self-Assembled Hydrogels from Poly[ N -(2-hydroxypropyl)methacrylamide] Grafted with ?-Sheet Peptides

doi: 10.1021/bm9005084

Figure Lengend Snippet: Synthesis of poly(HPMA)-g-CGGBeta11 graft copolymer and its self-assembly into a hydrogel via association of pendant β-sheet peptide strands.

Article Snippet: The number-average molecular weight and molecular weight distribution of the copolymer were measured by size exclusion chromatography (SEC) on an ÄKTA FPLC system (Amersham Pharmacia Biotech) equipped with UV and RI detectors, using a Superose 6 column, previously calibrated with poly(HPMA) fractions, and phosphate buffer solution (PBS, pH = 7.2) as eluent.

Techniques:

Journal:

Article Title: Self-Assembled Hydrogels from Poly[ N -(2-hydroxypropyl)methacrylamide] Grafted with ?-Sheet Peptides

doi: 10.1021/bm9005084

Figure Lengend Snippet: A. Temperature-dependent CD spectra of Beta11 peptide (the arrow indicates the change of spectrum upon temperature increase); B. Temperature-dependent CD spectra of poly(HPMA)-g-CGGBeta11 graft copolymer; C. Temperature effect on the secondary structure of Beta11 and poly(HPMA)-g-CGGBeta11, recorded as change in ellipticity at 218 nm; D. pH-Dependent CD spectra of Beta11 peptide; E. pH-Dependent CD spectra of poly(HPMA)-g-CGGBeta11 graft copolymer.

Article Snippet: The number-average molecular weight and molecular weight distribution of the copolymer were measured by size exclusion chromatography (SEC) on an ÄKTA FPLC system (Amersham Pharmacia Biotech) equipped with UV and RI detectors, using a Superose 6 column, previously calibrated with poly(HPMA) fractions, and phosphate buffer solution (PBS, pH = 7.2) as eluent.

Techniques:

Journal:

Article Title: Self-Assembled Hydrogels from Poly[ N -(2-hydroxypropyl)methacrylamide] Grafted with ?-Sheet Peptides

doi: 10.1021/bm9005084

Figure Lengend Snippet: FTIR spectra (solid) and second-derivatives (dotted) of A. Beta11 peptide; B. poly(HPMA)-g-CGGBeta11 graft copolymer.

Article Snippet: The number-average molecular weight and molecular weight distribution of the copolymer were measured by size exclusion chromatography (SEC) on an ÄKTA FPLC system (Amersham Pharmacia Biotech) equipped with UV and RI detectors, using a Superose 6 column, previously calibrated with poly(HPMA) fractions, and phosphate buffer solution (PBS, pH = 7.2) as eluent.

Techniques:

Journal:

Article Title: Self-Assembled Hydrogels from Poly[ N -(2-hydroxypropyl)methacrylamide] Grafted with ?-Sheet Peptides

doi: 10.1021/bm9005084

Figure Lengend Snippet: A. Fluorescence emission spectra (λexc = 440 nm) of Beta11 peptide and poly(HPMA)-g-CGGBeta11 graft copolymer after addition of 50 µM ThT. ThT alone was used as blank; B. CR binding assay; C. Differential spectra of (Beta11/CR)-CR and (poly(HPMA)-g-CGGBeta11/CR)-CR showing the points of maximum absorption.

Article Snippet: The number-average molecular weight and molecular weight distribution of the copolymer were measured by size exclusion chromatography (SEC) on an ÄKTA FPLC system (Amersham Pharmacia Biotech) equipped with UV and RI detectors, using a Superose 6 column, previously calibrated with poly(HPMA) fractions, and phosphate buffer solution (PBS, pH = 7.2) as eluent.

Techniques: Fluorescence, Binding Assay

Journal:

Article Title: Self-Assembled Hydrogels from Poly[ N -(2-hydroxypropyl)methacrylamide] Grafted with ?-Sheet Peptides

doi: 10.1021/bm9005084

Figure Lengend Snippet: A. TEM image of negatively stained Beta11 fibrils; B. TEM image of negatively stained poly(HPMA)-g-CGGBeta11 fibrils; C. SEM image of freeze-dried 0.4 wt % Beta11 gel; D. SEM image of freeze-dried 0.4 wt % poly(HPMA)-g-CGGBeta11 gel (the inset is the image of the cross-sectioned copolymer gel).

Article Snippet: The number-average molecular weight and molecular weight distribution of the copolymer were measured by size exclusion chromatography (SEC) on an ÄKTA FPLC system (Amersham Pharmacia Biotech) equipped with UV and RI detectors, using a Superose 6 column, previously calibrated with poly(HPMA) fractions, and phosphate buffer solution (PBS, pH = 7.2) as eluent.

Techniques: Staining

Journal:

Article Title: Self-Assembled Hydrogels from Poly[ N -(2-hydroxypropyl)methacrylamide] Grafted with ?-Sheet Peptides

doi: 10.1021/bm9005084

Figure Lengend Snippet: Mean square displacement (MSD) as a function of lag time for 0.52 µm amidine-modified PS particles in water solutions/gels at different concentrations of A. Beta11 peptide; B. poly(HPMA)-g-CGGBeta11 graft copolymer.

Article Snippet: The number-average molecular weight and molecular weight distribution of the copolymer were measured by size exclusion chromatography (SEC) on an ÄKTA FPLC system (Amersham Pharmacia Biotech) equipped with UV and RI detectors, using a Superose 6 column, previously calibrated with poly(HPMA) fractions, and phosphate buffer solution (PBS, pH = 7.2) as eluent.

Techniques: Modification

Journal:

Article Title: Self-Assembled Hydrogels from Poly[ N -(2-hydroxypropyl)methacrylamide] Grafted with ?-Sheet Peptides

doi: 10.1021/bm9005084

Figure Lengend Snippet: Frequency-dependent linear viscoelastic moduli for 0.52 µm amidine-modified PS particles in water solutions/gels of A. Beta11 peptide (solid symbols for loss modulus, G”, and half-open red symbols for storage modulus, G’); B. poly(HPMA)-g-CGGBeta11 graft copolymer (open symbols for loss modulus, G”, and half-solid red symbols for storage modulus, G’).

Article Snippet: The number-average molecular weight and molecular weight distribution of the copolymer were measured by size exclusion chromatography (SEC) on an ÄKTA FPLC system (Amersham Pharmacia Biotech) equipped with UV and RI detectors, using a Superose 6 column, previously calibrated with poly(HPMA) fractions, and phosphate buffer solution (PBS, pH = 7.2) as eluent.

Techniques: Modification