mouse anti human aat monoclonal polymer protein recognize antibody  (Hycult Biotech)


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    Hycult Biotech mouse anti human aat monoclonal polymer protein recognize antibody
    Mouse Anti Human Aat Monoclonal Polymer Protein Recognize Antibody, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti human aat monoclonal polymer protein recognize antibody  (Hycult Biotech)


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    Hycult Biotech mouse anti human aat monoclonal polymer protein recognize antibody
    Mouse Anti Human Aat Monoclonal Polymer Protein Recognize Antibody, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse monoclonal anti aat polymers  (Hycult Biotech)


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    Hycult Biotech mouse monoclonal anti aat polymers
    (A) Targeting strategy for the SERPINA1 <t>(AAT)</t> locus. (B) Schematic of directed differentiation protocol for generating iHeps. (C) Representative flow cytometry plots of fixed, permeabilized ZZ, MZ, and MM iHeps. (D) MFI of intracellular AAT protein in ZZ, MZ and MM iHeps. (E and F) Immunostaining of ZZ, MZ, and MM iHeps for AAT, <t>2C1,</t> and HNF4α; scale bar, 10 μm. (G and H) Secreted total (G) AAT and (H) ZAAT protein in ZZ, MZ, and MM iHep supernatants. (I) Assay of anti-neutrophil elastase inhibition in concentrated iHep supernatants. (J) Representative quantification of AAT secretion kinetics using 35 S-Met/Cys-labeled AAT protein from intracellular protein lysates and supernatants. (K) Kinetic of aggregated AAT-labeled cell lysate and supernatants from (J). n = 3 independent experiments from each of the syngeneic backgrounds (D and G–I). n = 1 independent experiment from each of the syngeneic backgrounds (K). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test using MZ as control. Neutrophil elastase inhibition ZZ versus MZ **p < 0.01, ZZ versus MM ### p < 0.001, MZ versus MM $$$ p < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test.
    Mouse Monoclonal Anti Aat Polymers, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity"

    Article Title: Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.111775

    (A) Targeting strategy for the SERPINA1 (AAT) locus. (B) Schematic of directed differentiation protocol for generating iHeps. (C) Representative flow cytometry plots of fixed, permeabilized ZZ, MZ, and MM iHeps. (D) MFI of intracellular AAT protein in ZZ, MZ and MM iHeps. (E and F) Immunostaining of ZZ, MZ, and MM iHeps for AAT, 2C1, and HNF4α; scale bar, 10 μm. (G and H) Secreted total (G) AAT and (H) ZAAT protein in ZZ, MZ, and MM iHep supernatants. (I) Assay of anti-neutrophil elastase inhibition in concentrated iHep supernatants. (J) Representative quantification of AAT secretion kinetics using 35 S-Met/Cys-labeled AAT protein from intracellular protein lysates and supernatants. (K) Kinetic of aggregated AAT-labeled cell lysate and supernatants from (J). n = 3 independent experiments from each of the syngeneic backgrounds (D and G–I). n = 1 independent experiment from each of the syngeneic backgrounds (K). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test using MZ as control. Neutrophil elastase inhibition ZZ versus MZ **p < 0.01, ZZ versus MM ### p < 0.001, MZ versus MM $$$ p < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test.
    Figure Legend Snippet: (A) Targeting strategy for the SERPINA1 (AAT) locus. (B) Schematic of directed differentiation protocol for generating iHeps. (C) Representative flow cytometry plots of fixed, permeabilized ZZ, MZ, and MM iHeps. (D) MFI of intracellular AAT protein in ZZ, MZ and MM iHeps. (E and F) Immunostaining of ZZ, MZ, and MM iHeps for AAT, 2C1, and HNF4α; scale bar, 10 μm. (G and H) Secreted total (G) AAT and (H) ZAAT protein in ZZ, MZ, and MM iHep supernatants. (I) Assay of anti-neutrophil elastase inhibition in concentrated iHep supernatants. (J) Representative quantification of AAT secretion kinetics using 35 S-Met/Cys-labeled AAT protein from intracellular protein lysates and supernatants. (K) Kinetic of aggregated AAT-labeled cell lysate and supernatants from (J). n = 3 independent experiments from each of the syngeneic backgrounds (D and G–I). n = 1 independent experiment from each of the syngeneic backgrounds (K). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test using MZ as control. Neutrophil elastase inhibition ZZ versus MZ **p < 0.01, ZZ versus MM ### p < 0.001, MZ versus MM $$$ p < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test.

    Techniques Used: Flow Cytometry, Immunostaining, Inhibition, Labeling


    Figure Legend Snippet:

    Techniques Used: Recombinant, Electron Microscopy, Lysis, Lactate Assay, Pyruvate Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Sequencing, Mutagenesis, Software, Modification

    aat polymer  (Hycult Biotech)


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    Hycult Biotech aat polymer
    ( A ) Shown is control WT monomeric and heat-treated polymeric <t>AAT</t> on native gel (left <t>panel).</t> <t>Monoclonal</t> antibody 16f8 generated shows strong interaction with WT monomeric AAT but not to heat-treated polymeric AAT (middle panel). Monoclonal antibody 2C1 shows a strong interaction responding to the heated polymeric AAT but not WT monomeric AAT (right panel). ( B ) A schematic figure showing the high-throughput assays used to measure the intracellular and secreted monomer or intracellular and secreted polymer pools using conformational dependent antibodies (see Methods ). The activity of secreted AAT is determined by using a fluorogenic substrate of NE (see Methods ). ( C ) The fluorescence of the NE substrate is dependent on the protein level of AAT.
    Aat Polymer, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Profiling Genetic Diversity Reveals the Molecular Basis for Balancing Function with Misfolding in Alpha-1 Antitrypsin"

    Article Title: Profiling Genetic Diversity Reveals the Molecular Basis for Balancing Function with Misfolding in Alpha-1 Antitrypsin

    Journal: bioRxiv

    doi: 10.1101/2022.03.04.483066

    ( A ) Shown is control WT monomeric and heat-treated polymeric AAT on native gel (left panel). Monoclonal antibody 16f8 generated shows strong interaction with WT monomeric AAT but not to heat-treated polymeric AAT (middle panel). Monoclonal antibody 2C1 shows a strong interaction responding to the heated polymeric AAT but not WT monomeric AAT (right panel). ( B ) A schematic figure showing the high-throughput assays used to measure the intracellular and secreted monomer or intracellular and secreted polymer pools using conformational dependent antibodies (see Methods ). The activity of secreted AAT is determined by using a fluorogenic substrate of NE (see Methods ). ( C ) The fluorescence of the NE substrate is dependent on the protein level of AAT.
    Figure Legend Snippet: ( A ) Shown is control WT monomeric and heat-treated polymeric AAT on native gel (left panel). Monoclonal antibody 16f8 generated shows strong interaction with WT monomeric AAT but not to heat-treated polymeric AAT (middle panel). Monoclonal antibody 2C1 shows a strong interaction responding to the heated polymeric AAT but not WT monomeric AAT (right panel). ( B ) A schematic figure showing the high-throughput assays used to measure the intracellular and secreted monomer or intracellular and secreted polymer pools using conformational dependent antibodies (see Methods ). The activity of secreted AAT is determined by using a fluorogenic substrate of NE (see Methods ). ( C ) The fluorescence of the NE substrate is dependent on the protein level of AAT.

    Techniques Used: Generated, High Throughput Screening Assay, Activity Assay, Fluorescence

    ( A ) Phenotype landscape linking secreted monomer ( y -axis) to intracellular polymer (color scale, z-axis) across the entire AAT polypeptide sequence ( x- axis). The N1, M2 and C3 regions that were identified by low NE inhibitory activity are indicated in the landscape. The top 25% confidence interval is defined by contour lines. ( B ) The IVW averaged intracellular polymer levels for each residue are presented from N-terminal to C-terminal of the AAT polypeptide as a barcode with the color scale from red to green representing high (Z-variant AAT) to low intracellular (WT AAT) polymer levels. ( C-G ) The IVW averaged intracellular polymer levels for each residue are mapped on the 3D structure of AAT ( C , left panel; PDB:3NE4 ) or AAT intracellular polymer ( C , right panel; PDB:3T1P , ) with the structural regions N1 ( D ), M2 ( E ), C3 ( F ) and β-sheet B ( G ) highlighted.
    Figure Legend Snippet: ( A ) Phenotype landscape linking secreted monomer ( y -axis) to intracellular polymer (color scale, z-axis) across the entire AAT polypeptide sequence ( x- axis). The N1, M2 and C3 regions that were identified by low NE inhibitory activity are indicated in the landscape. The top 25% confidence interval is defined by contour lines. ( B ) The IVW averaged intracellular polymer levels for each residue are presented from N-terminal to C-terminal of the AAT polypeptide as a barcode with the color scale from red to green representing high (Z-variant AAT) to low intracellular (WT AAT) polymer levels. ( C-G ) The IVW averaged intracellular polymer levels for each residue are mapped on the 3D structure of AAT ( C , left panel; PDB:3NE4 ) or AAT intracellular polymer ( C , right panel; PDB:3T1P , ) with the structural regions N1 ( D ), M2 ( E ), C3 ( F ) and β-sheet B ( G ) highlighted.

    Techniques Used: Sequencing, Activity Assay, Variant Assay

    mouse monoclonal anti aat polymer antibody  (Hycult Biotech)


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    Hycult Biotech mouse monoclonal anti aat polymer antibody
    Pulmonary function of 52 ZZ-AATD patients was measured 15 min after the inhalation of 400 µg of salbutamol followed by spirometry and a CO diffusion gas transfer. Levels of plasma <t>Z-AAT</t> polymers (µg/mL) were measured by ELISA based on <t>LG96</t> mAb (see materials and methods). There was no correlation between plasma Z-AAT polymer values and FEV1 % pred, or Kco % pred.
    Mouse Monoclonal Anti Aat Polymer Antibody, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Relationship between Plasma Alpha-1-Antitrypsin Polymers and Lung or Liver Function in ZZ Alpha-1-Antitrypsin-Deficient Patients"

    Article Title: The Relationship between Plasma Alpha-1-Antitrypsin Polymers and Lung or Liver Function in ZZ Alpha-1-Antitrypsin-Deficient Patients

    Journal: Biomolecules

    doi: 10.3390/biom12030380

    Pulmonary function of 52 ZZ-AATD patients was measured 15 min after the inhalation of 400 µg of salbutamol followed by spirometry and a CO diffusion gas transfer. Levels of plasma Z-AAT polymers (µg/mL) were measured by ELISA based on LG96 mAb (see materials and methods). There was no correlation between plasma Z-AAT polymer values and FEV1 % pred, or Kco % pred.
    Figure Legend Snippet: Pulmonary function of 52 ZZ-AATD patients was measured 15 min after the inhalation of 400 µg of salbutamol followed by spirometry and a CO diffusion gas transfer. Levels of plasma Z-AAT polymers (µg/mL) were measured by ELISA based on LG96 mAb (see materials and methods). There was no correlation between plasma Z-AAT polymer values and FEV1 % pred, or Kco % pred.

    Techniques Used: Diffusion-based Assay, Enzyme-linked Immunosorbent Assay

    Z-AAT polymer analysis by Western blots using two different monoclonal antibodies. Equal amounts of plasma samples from seven ZZ-AATD individuals were electrophoretically separated using 7.5% native polyacrylamide gels followed by western blots. Purified polymeric Z-AAT isolated from pooled ZZ plasma samples was used as a positive control. The western blots were probed against mouse monoclonal anti-AAT polymer antibodies LG96 ( A ) and 2C1 ( B )(both diluted 1:700). The image demonstrates that both antibodies recognize the Z-AAT polymers, but the profiles of recognized polymers differ. This blot is the representative from n = 2 independent repeats.
    Figure Legend Snippet: Z-AAT polymer analysis by Western blots using two different monoclonal antibodies. Equal amounts of plasma samples from seven ZZ-AATD individuals were electrophoretically separated using 7.5% native polyacrylamide gels followed by western blots. Purified polymeric Z-AAT isolated from pooled ZZ plasma samples was used as a positive control. The western blots were probed against mouse monoclonal anti-AAT polymer antibodies LG96 ( A ) and 2C1 ( B )(both diluted 1:700). The image demonstrates that both antibodies recognize the Z-AAT polymers, but the profiles of recognized polymers differ. This blot is the representative from n = 2 independent repeats.

    Techniques Used: Western Blot, Purification, Isolation, Positive Control

    mouse monoclonal anti aat polymer antibody  (Hycult Biotech)


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    Hycult Biotech mouse monoclonal anti aat polymer antibody
    Pulmonary function of 52 ZZ-AATD patients was measured 15 min after the inhalation of 400 µg of salbutamol followed by spirometry and a CO diffusion gas transfer. Levels of plasma <t>Z-AAT</t> polymers (µg/mL) were measured by ELISA based on <t>LG96</t> mAb (see materials and methods). There was no correlation between plasma Z-AAT polymer values and FEV1 % pred, or Kco % pred.
    Mouse Monoclonal Anti Aat Polymer Antibody, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Relationship between Plasma Alpha-1-Antitrypsin Polymers and Lung or Liver Function in ZZ Alpha-1-Antitrypsin-Deficient Patients"

    Article Title: The Relationship between Plasma Alpha-1-Antitrypsin Polymers and Lung or Liver Function in ZZ Alpha-1-Antitrypsin-Deficient Patients

    Journal: Biomolecules

    doi: 10.3390/biom12030380

    Pulmonary function of 52 ZZ-AATD patients was measured 15 min after the inhalation of 400 µg of salbutamol followed by spirometry and a CO diffusion gas transfer. Levels of plasma Z-AAT polymers (µg/mL) were measured by ELISA based on LG96 mAb (see materials and methods). There was no correlation between plasma Z-AAT polymer values and FEV1 % pred, or Kco % pred.
    Figure Legend Snippet: Pulmonary function of 52 ZZ-AATD patients was measured 15 min after the inhalation of 400 µg of salbutamol followed by spirometry and a CO diffusion gas transfer. Levels of plasma Z-AAT polymers (µg/mL) were measured by ELISA based on LG96 mAb (see materials and methods). There was no correlation between plasma Z-AAT polymer values and FEV1 % pred, or Kco % pred.

    Techniques Used: Diffusion-based Assay, Enzyme-linked Immunosorbent Assay

    Z-AAT polymer analysis by Western blots using two different monoclonal antibodies. Equal amounts of plasma samples from seven ZZ-AATD individuals were electrophoretically separated using 7.5% native polyacrylamide gels followed by western blots. Purified polymeric Z-AAT isolated from pooled ZZ plasma samples was used as a positive control. The western blots were probed against mouse monoclonal anti-AAT polymer antibodies LG96 ( A ) and 2C1 ( B )(both diluted 1:700). The image demonstrates that both antibodies recognize the Z-AAT polymers, but the profiles of recognized polymers differ. This blot is the representative from n = 2 independent repeats.
    Figure Legend Snippet: Z-AAT polymer analysis by Western blots using two different monoclonal antibodies. Equal amounts of plasma samples from seven ZZ-AATD individuals were electrophoretically separated using 7.5% native polyacrylamide gels followed by western blots. Purified polymeric Z-AAT isolated from pooled ZZ plasma samples was used as a positive control. The western blots were probed against mouse monoclonal anti-AAT polymer antibodies LG96 ( A ) and 2C1 ( B )(both diluted 1:700). The image demonstrates that both antibodies recognize the Z-AAT polymers, but the profiles of recognized polymers differ. This blot is the representative from n = 2 independent repeats.

    Techniques Used: Western Blot, Purification, Isolation, Positive Control

    mouse monoclonal anti aat polymer antibody  (Hycult Biotech)


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    Hycult Biotech mouse monoclonal anti aat polymer antibody
    <t>CX3CR1</t> gene expression levels in peripheral blood mononuclear cells (PBMCs) related to alpha-1 antitrypsin-deficiency (AATD) and plasma concentrations of CX3CR1 ligand (CX3CL1) and <t>Z-AAT</t> polymer. ( A )PBMCs were isolated from AATD subjects and non-AATD controls. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Measurements were carried out in duplicates. Data are presented as median (IQR) in boxplots, lines represent medians. Outliers are defined as data points located outside the whiskers. p-Value was calculated by Mann-Whitney U test. ( B ) Plasma levels of CX3CL1 in AATD (plasma available for n = 38 AATD) and non-AATD individuals measured by ELISA. Measurements were carried out in triplicates. Data are presented as median (IQR) in boxplots with whiskers. Outliers are defined as data points located outside the whiskers. ( C ) Negative correlation of CX3CR1 mRNA in PBMCs and plasma Z-AAT polymer levels from ZZ AATD individuals from graph ( B ). Pearson’s correlation test, r 2 = −0.313, p=0.055, n = 38. Figure 1—source data 1. Source files, containing original data for , to document CX3CR1 expression ( A ), and plasma levels of CX3CL1 in alpha-1 antitrypsin-deficient (AATD) and non-AATD individuals ( B ).
    Mouse Monoclonal Anti Aat Polymer Antibody, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti aat polymer antibody/product/Hycult Biotech
    Average 86 stars, based on 1 article reviews
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    1) Product Images from "Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs"

    Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

    Journal: eLife

    doi: 10.7554/eLife.64881

    CX3CR1 gene expression levels in peripheral blood mononuclear cells (PBMCs) related to alpha-1 antitrypsin-deficiency (AATD) and plasma concentrations of CX3CR1 ligand (CX3CL1) and Z-AAT polymer. ( A )PBMCs were isolated from AATD subjects and non-AATD controls. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Measurements were carried out in duplicates. Data are presented as median (IQR) in boxplots, lines represent medians. Outliers are defined as data points located outside the whiskers. p-Value was calculated by Mann-Whitney U test. ( B ) Plasma levels of CX3CL1 in AATD (plasma available for n = 38 AATD) and non-AATD individuals measured by ELISA. Measurements were carried out in triplicates. Data are presented as median (IQR) in boxplots with whiskers. Outliers are defined as data points located outside the whiskers. ( C ) Negative correlation of CX3CR1 mRNA in PBMCs and plasma Z-AAT polymer levels from ZZ AATD individuals from graph ( B ). Pearson’s correlation test, r 2 = −0.313, p=0.055, n = 38. Figure 1—source data 1. Source files, containing original data for , to document CX3CR1 expression ( A ), and plasma levels of CX3CL1 in alpha-1 antitrypsin-deficient (AATD) and non-AATD individuals ( B ).
    Figure Legend Snippet: CX3CR1 gene expression levels in peripheral blood mononuclear cells (PBMCs) related to alpha-1 antitrypsin-deficiency (AATD) and plasma concentrations of CX3CR1 ligand (CX3CL1) and Z-AAT polymer. ( A )PBMCs were isolated from AATD subjects and non-AATD controls. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Measurements were carried out in duplicates. Data are presented as median (IQR) in boxplots, lines represent medians. Outliers are defined as data points located outside the whiskers. p-Value was calculated by Mann-Whitney U test. ( B ) Plasma levels of CX3CL1 in AATD (plasma available for n = 38 AATD) and non-AATD individuals measured by ELISA. Measurements were carried out in triplicates. Data are presented as median (IQR) in boxplots with whiskers. Outliers are defined as data points located outside the whiskers. ( C ) Negative correlation of CX3CR1 mRNA in PBMCs and plasma Z-AAT polymer levels from ZZ AATD individuals from graph ( B ). Pearson’s correlation test, r 2 = −0.313, p=0.055, n = 38. Figure 1—source data 1. Source files, containing original data for , to document CX3CR1 expression ( A ), and plasma levels of CX3CL1 in alpha-1 antitrypsin-deficient (AATD) and non-AATD individuals ( B ).

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

    PBMCs were incubated for 18 hr with plasma-derived Z-AAT in the concentrations as indicated. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Curves show results from two independent experiments. Each point represents mean of two repeats.
    Figure Legend Snippet: PBMCs were incubated for 18 hr with plasma-derived Z-AAT in the concentrations as indicated. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Curves show results from two independent experiments. Each point represents mean of two repeats.

    Techniques Used: Incubation, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

    ( A ) CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr with plasma-derived Z-AAT, lipopolysaccharide (LPS), or M-AAT in the concentrations as indicated, or with RPMI medium alone (control). The data from n = 6 independent experiments are presented as median (IQR) in box and whisker plot format; lines represent medians in each box. Measurements were carried out in duplicates. p-Value was calculated by nonparametric Kruskal-Wallis test. ( B ) Representative uncut Western blot (n = 3 independent experiments) of CX3CR1 in RIPA lysates prepared from PBMCs incubated for 18 hr alone or with LPS (1 µg/ml), M-AAT (1 mg/ml), or Z-AAT (0.5 mg/ml). For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. Relative intensities were calculated for each band using the ratio relative to β-actin, as a loading control, and then normalized by the experimental control. ( C ) For analysis of cellular AAT, the same lysates were separated under non-reducing conditions. Western blots were probed with polyclonal rabbit anti-human AAT recognizing monomeric, polymeric, or truncated forms of AAT. One representative blot from n = 3 independent experiments is shown. β-Actin was used for a loading control. ( D ) and ( E ) Co-distribution of Z-AAT polymers with CX3CR1 in human total PBMCs incubated with 0.5 mg/ml Z-AAT polymers for 18 hr. ( D ) Immunofluorescence microscopy revealed co-localization of Z-AAT polymers ( red ) with CX3CR1-positive structures ( green ). Arrows point areas of co-localization. Scale bar, 10 µm. ( E ) Confocal microscopy 3D stack with orthogonal reconstruction shows an aggregate of Z-AAT polymers ( red ) surrounded by CX3CR1-positive ( green ) cellular extensions forming a cap-like structure (arrowhead). Scale bar, 5 µm. The images with indicated channels merged and the corresponding differential interference contrast (DIC) image are presented. 4', 6- Diamidino 2-phenylindole (DAPI) was used for nuclei staining ( blue ). Figure 2—source data 1. Source file, containing original data for , to document reduced CX3CR1 expression in peripheral blood mononuclear cells (PBMCs) treated with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) ( A ).
    Figure Legend Snippet: ( A ) CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr with plasma-derived Z-AAT, lipopolysaccharide (LPS), or M-AAT in the concentrations as indicated, or with RPMI medium alone (control). The data from n = 6 independent experiments are presented as median (IQR) in box and whisker plot format; lines represent medians in each box. Measurements were carried out in duplicates. p-Value was calculated by nonparametric Kruskal-Wallis test. ( B ) Representative uncut Western blot (n = 3 independent experiments) of CX3CR1 in RIPA lysates prepared from PBMCs incubated for 18 hr alone or with LPS (1 µg/ml), M-AAT (1 mg/ml), or Z-AAT (0.5 mg/ml). For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. Relative intensities were calculated for each band using the ratio relative to β-actin, as a loading control, and then normalized by the experimental control. ( C ) For analysis of cellular AAT, the same lysates were separated under non-reducing conditions. Western blots were probed with polyclonal rabbit anti-human AAT recognizing monomeric, polymeric, or truncated forms of AAT. One representative blot from n = 3 independent experiments is shown. β-Actin was used for a loading control. ( D ) and ( E ) Co-distribution of Z-AAT polymers with CX3CR1 in human total PBMCs incubated with 0.5 mg/ml Z-AAT polymers for 18 hr. ( D ) Immunofluorescence microscopy revealed co-localization of Z-AAT polymers ( red ) with CX3CR1-positive structures ( green ). Arrows point areas of co-localization. Scale bar, 10 µm. ( E ) Confocal microscopy 3D stack with orthogonal reconstruction shows an aggregate of Z-AAT polymers ( red ) surrounded by CX3CR1-positive ( green ) cellular extensions forming a cap-like structure (arrowhead). Scale bar, 5 µm. The images with indicated channels merged and the corresponding differential interference contrast (DIC) image are presented. 4', 6- Diamidino 2-phenylindole (DAPI) was used for nuclei staining ( blue ). Figure 2—source data 1. Source file, containing original data for , to document reduced CX3CR1 expression in peripheral blood mononuclear cells (PBMCs) treated with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) ( A ).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Derivative Assay, Whisker Assay, Western Blot, SDS Page, Immunofluorescence, Microscopy, Confocal Microscopy, Staining

    CX3CR1-expressing cells were found in the monocyte gate ( A ) and in the NK cell gate (CD56 lo CD16 + ) ( B ). Histograms show representative results and bars represent mean (SD) of n = 4 independent biological repeats each measured one time. After incubation with Z-AAT or LPS, monocytes and NK cells show significantly reduced CX3CR1 surface expression in comparison to untreated control cells. p-Values were calculated by one-way ANOVA. Figure 3—source data 1. Source files, containing original data for , to document reduced CX3CR1 surface expression in monocytes ( A ) and NK cells ( B ) after treatment with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) ( A ).
    Figure Legend Snippet: CX3CR1-expressing cells were found in the monocyte gate ( A ) and in the NK cell gate (CD56 lo CD16 + ) ( B ). Histograms show representative results and bars represent mean (SD) of n = 4 independent biological repeats each measured one time. After incubation with Z-AAT or LPS, monocytes and NK cells show significantly reduced CX3CR1 surface expression in comparison to untreated control cells. p-Values were calculated by one-way ANOVA. Figure 3—source data 1. Source files, containing original data for , to document reduced CX3CR1 surface expression in monocytes ( A ) and NK cells ( B ) after treatment with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) ( A ).

    Techniques Used: Expressing, Incubation

    Lipid rafts were solubilized from membrane fractions with UltraRIPA kit. ( a ) For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. One representative blot from n = 3 independent experiments is shown. ( b ) For analysis of lipid raft associated AAT polymers, the same samples were separated under non-reducing conditions. The Western blot was probed with monoclonal antibody (2C1) recognizing polymeric AAT. One representative blot from n = 3 independent experiments is shown.
    Figure Legend Snippet: Lipid rafts were solubilized from membrane fractions with UltraRIPA kit. ( a ) For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. One representative blot from n = 3 independent experiments is shown. ( b ) For analysis of lipid raft associated AAT polymers, the same samples were separated under non-reducing conditions. The Western blot was probed with monoclonal antibody (2C1) recognizing polymeric AAT. One representative blot from n = 3 independent experiments is shown.

    Techniques Used: SDS Page, Western Blot

    ( A ) CX3CR1 mRNA expression relative to HPRT1 was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr in RPMI medium alone or with addition of Z-AAT monomer or Z-AAT polymer (each 0.5 mg/ml), or lipopolysaccharide (LPS) (1 µg/ml) or native M-AAT (0.5 mg/ml). Measurements were carried out in duplicates. Data are represented as bars from four independent experiments. ( B ) Levels of CX3CR1 lysates prepared in RIPA buffer from PBMCs incubated for 18 hr with RPMI (control) and with Z-AAT monomer (0.5 mg/ml) or LPS (1 µg/ml) (used as a positive control). For analysis of CX3CR1, equal amounts of protein were separated by 10% SDS-PAGE under reducing conditions followed by Western blotting. Representative uncut Western blot (n = 2 independent experiments) is shown. β-Actin was used for a loading control. ( C ) CX3CR1 mRNA expression relative to HPRT1 was determined by real-time PCR using Taqman gene expression assays. PBMCs were incubated for 18 hr in RPMI medium alone or with different concentrations of heat-induced M-AAT polymers (60°C, for 3 hr). Measurements were carried out in duplicates. Data are represented as curves from two independent donors.
    Figure Legend Snippet: ( A ) CX3CR1 mRNA expression relative to HPRT1 was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr in RPMI medium alone or with addition of Z-AAT monomer or Z-AAT polymer (each 0.5 mg/ml), or lipopolysaccharide (LPS) (1 µg/ml) or native M-AAT (0.5 mg/ml). Measurements were carried out in duplicates. Data are represented as bars from four independent experiments. ( B ) Levels of CX3CR1 lysates prepared in RIPA buffer from PBMCs incubated for 18 hr with RPMI (control) and with Z-AAT monomer (0.5 mg/ml) or LPS (1 µg/ml) (used as a positive control). For analysis of CX3CR1, equal amounts of protein were separated by 10% SDS-PAGE under reducing conditions followed by Western blotting. Representative uncut Western blot (n = 2 independent experiments) is shown. β-Actin was used for a loading control. ( C ) CX3CR1 mRNA expression relative to HPRT1 was determined by real-time PCR using Taqman gene expression assays. PBMCs were incubated for 18 hr in RPMI medium alone or with different concentrations of heat-induced M-AAT polymers (60°C, for 3 hr). Measurements were carried out in duplicates. Data are represented as curves from two independent donors.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Positive Control, SDS Page, Western Blot

    PBMCs were incubated for 18 hr with plasma-derived Z-AAT in the concentrations as indicated, or with RPMI medium alone (control). CX3CR1 and CD14 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Curves represent two independent experiments.
    Figure Legend Snippet: PBMCs were incubated for 18 hr with plasma-derived Z-AAT in the concentrations as indicated, or with RPMI medium alone (control). CX3CR1 and CD14 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Curves represent two independent experiments.

    Techniques Used: Incubation, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction


    Figure Legend Snippet:

    Techniques Used: TaqMan Assay, Purification, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Recombinant, Software

    antibody anti aat polymer  (Hycult Biotech)


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    Antibody Anti Aat Polymer, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse monoclonal anti aat polymer antibody  (Hycult Biotech)


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    Hycult Biotech mouse monoclonal anti aat polymer antibody
    Mouse Monoclonal Anti Aat Polymer Antibody, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hycult Biotech anti aat polymer
    Lipid rafts were solubilized from membrane fractions with UltraRIPA kit. ( a ) For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. One representative blot from n = 3 independent experiments is shown. ( b ) For analysis of lipid raft associated <t>AAT</t> polymers, the same samples were separated under non-reducing conditions. The Western blot was probed <t>with</t> <t>monoclonal</t> antibody (2C1) recognizing polymeric AAT. One representative blot from n = 3 independent experiments is shown.
    Anti Aat Polymer, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs"

    Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

    Journal: eLife

    doi: 10.7554/eLife.64881

    Lipid rafts were solubilized from membrane fractions with UltraRIPA kit. ( a ) For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. One representative blot from n = 3 independent experiments is shown. ( b ) For analysis of lipid raft associated AAT polymers, the same samples were separated under non-reducing conditions. The Western blot was probed with monoclonal antibody (2C1) recognizing polymeric AAT. One representative blot from n = 3 independent experiments is shown.
    Figure Legend Snippet: Lipid rafts were solubilized from membrane fractions with UltraRIPA kit. ( a ) For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. One representative blot from n = 3 independent experiments is shown. ( b ) For analysis of lipid raft associated AAT polymers, the same samples were separated under non-reducing conditions. The Western blot was probed with monoclonal antibody (2C1) recognizing polymeric AAT. One representative blot from n = 3 independent experiments is shown.

    Techniques Used: SDS Page, Western Blot


    Figure Legend Snippet:

    Techniques Used: TaqMan Assay, Purification, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Recombinant, Software

    anti human aat polymer selective  (Hycult Biotech)


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    <t>CRT</t> enhances ATZ trafficking in K42 mouse embryonic fibroblasts, and this effect is partially dependent on glycan binding. A , EM structure of CRT (PDB ID 6ENY ) depicting the glycan-binding site which includes residue Tyr-92 within the globular domain. CRT's acidic domain is helical and the P-domain forms an extended β-hairpin structure. B , workflow for the estimation of media and cellular fluorescence in , , , , and . Briefly, media was collected 48 h post-transfection and cleared of debris by high-speed centrifugation. eYFP fluorescence was measured at an excitation wavelength of 514 nm and emission wavelength of 527 nm. Cellular fluorescence (post-fixation) was measured in the FITC channel on a flow cytometer. C , representative flow cytometry dot plots of cellular eYFP fluorescence in untransfected, <t>eYFP-AAT–transfected,</t> or eYFP-ATZ–transfected K42 CRT −/− and CRT WT or CRT Y92A cells. D , percentage live cells of all cells (pre-gated on forward and side scatter) and percentage of eYFP + cells identified from the total live cell population. E , total media fluorescence and cell MFI values were normalized relative to corresponding values from CRT −/− cells in eYFP-AAT– or eYFP-ATZ–transfected cells. F , the ratio between the media and cell fluorescence values calculated as M e d i a f l u o r e s c e n c e C e l l u l a r e Y F P M F I × N u m b e r o f e Y F P + c e l l s . For D – F , data were obtained from nine independent transfections of the indicated K42 cells and are shown as mean ± S.D. ( error bars ). Repeated measures (RM) one-way ANOVA analysis was performed for each set of measurements, comparing CRT −/− , CRT WT, and CRT Y92A. Because the data in panel E is normalized, for this panel, RM one-way ANOVA analysis was performed on the log-transformed data. Only significant comparisons are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001. See also for additional replicates comparing K42 CRT −/− and CRT WT cells and for individual experimental trends of the eYFP-ATZ data.
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    1) Product Images from "Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin"

    Article Title: Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.014372

    CRT enhances ATZ trafficking in K42 mouse embryonic fibroblasts, and this effect is partially dependent on glycan binding. A , EM structure of CRT (PDB ID 6ENY ) depicting the glycan-binding site which includes residue Tyr-92 within the globular domain. CRT's acidic domain is helical and the P-domain forms an extended β-hairpin structure. B , workflow for the estimation of media and cellular fluorescence in , , , , and . Briefly, media was collected 48 h post-transfection and cleared of debris by high-speed centrifugation. eYFP fluorescence was measured at an excitation wavelength of 514 nm and emission wavelength of 527 nm. Cellular fluorescence (post-fixation) was measured in the FITC channel on a flow cytometer. C , representative flow cytometry dot plots of cellular eYFP fluorescence in untransfected, eYFP-AAT–transfected, or eYFP-ATZ–transfected K42 CRT −/− and CRT WT or CRT Y92A cells. D , percentage live cells of all cells (pre-gated on forward and side scatter) and percentage of eYFP + cells identified from the total live cell population. E , total media fluorescence and cell MFI values were normalized relative to corresponding values from CRT −/− cells in eYFP-AAT– or eYFP-ATZ–transfected cells. F , the ratio between the media and cell fluorescence values calculated as M e d i a f l u o r e s c e n c e C e l l u l a r e Y F P M F I × N u m b e r o f e Y F P + c e l l s . For D – F , data were obtained from nine independent transfections of the indicated K42 cells and are shown as mean ± S.D. ( error bars ). Repeated measures (RM) one-way ANOVA analysis was performed for each set of measurements, comparing CRT −/− , CRT WT, and CRT Y92A. Because the data in panel E is normalized, for this panel, RM one-way ANOVA analysis was performed on the log-transformed data. Only significant comparisons are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001. See also for additional replicates comparing K42 CRT −/− and CRT WT cells and for individual experimental trends of the eYFP-ATZ data.
    Figure Legend Snippet: CRT enhances ATZ trafficking in K42 mouse embryonic fibroblasts, and this effect is partially dependent on glycan binding. A , EM structure of CRT (PDB ID 6ENY ) depicting the glycan-binding site which includes residue Tyr-92 within the globular domain. CRT's acidic domain is helical and the P-domain forms an extended β-hairpin structure. B , workflow for the estimation of media and cellular fluorescence in , , , , and . Briefly, media was collected 48 h post-transfection and cleared of debris by high-speed centrifugation. eYFP fluorescence was measured at an excitation wavelength of 514 nm and emission wavelength of 527 nm. Cellular fluorescence (post-fixation) was measured in the FITC channel on a flow cytometer. C , representative flow cytometry dot plots of cellular eYFP fluorescence in untransfected, eYFP-AAT–transfected, or eYFP-ATZ–transfected K42 CRT −/− and CRT WT or CRT Y92A cells. D , percentage live cells of all cells (pre-gated on forward and side scatter) and percentage of eYFP + cells identified from the total live cell population. E , total media fluorescence and cell MFI values were normalized relative to corresponding values from CRT −/− cells in eYFP-AAT– or eYFP-ATZ–transfected cells. F , the ratio between the media and cell fluorescence values calculated as M e d i a f l u o r e s c e n c e C e l l u l a r e Y F P M F I × N u m b e r o f e Y F P + c e l l s . For D – F , data were obtained from nine independent transfections of the indicated K42 cells and are shown as mean ± S.D. ( error bars ). Repeated measures (RM) one-way ANOVA analysis was performed for each set of measurements, comparing CRT −/− , CRT WT, and CRT Y92A. Because the data in panel E is normalized, for this panel, RM one-way ANOVA analysis was performed on the log-transformed data. Only significant comparisons are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001. See also for additional replicates comparing K42 CRT −/− and CRT WT cells and for individual experimental trends of the eYFP-ATZ data.

    Techniques Used: Binding Assay, Fluorescence, Transfection, Centrifugation, Flow Cytometry, Transformation Assay

    CRT promotes the secretory trafficking of ATZ in Huh7.5 cells. A , representative flow cytometry dot plots of cellular eYFP fluorescence in untransfected, eYFP-AAT–transfected, or eYFP-ATZ–transfected Huh7.5 CRT-knockout (CRT KO) and WT (control vector) cells. B , total live cells expressed as a percentage of all cells (pre-gated on forward and side scatter), and percentage of eYFP + cells identified from the total live cell population. C , total media fluorescence and cell MFI values were normalized relative to corresponding values from CRT KO cells in eYFP-AAT–transfected or eYFP-ATZ–transfected cells. D , the ratio between the media and cell fluorescence was obtained, as in . Data in ( B – D ) were obtained from 16 independent transfections of the Huh7.5 CRT KO and WT lines and are shown as mean ± S.D. Nine replicates were done in parallel with CNX data in . Paired two-tailed t tests were performed comparing CRT KO with WT conditions for each measurement. Because the data in panel C is normalized, for this panel, t tests were performed on log-transformed data. * p <0.05, **** p <0.0001. See also for individual experimental trends of the eYFP-ATZ data.
    Figure Legend Snippet: CRT promotes the secretory trafficking of ATZ in Huh7.5 cells. A , representative flow cytometry dot plots of cellular eYFP fluorescence in untransfected, eYFP-AAT–transfected, or eYFP-ATZ–transfected Huh7.5 CRT-knockout (CRT KO) and WT (control vector) cells. B , total live cells expressed as a percentage of all cells (pre-gated on forward and side scatter), and percentage of eYFP + cells identified from the total live cell population. C , total media fluorescence and cell MFI values were normalized relative to corresponding values from CRT KO cells in eYFP-AAT–transfected or eYFP-ATZ–transfected cells. D , the ratio between the media and cell fluorescence was obtained, as in . Data in ( B – D ) were obtained from 16 independent transfections of the Huh7.5 CRT KO and WT lines and are shown as mean ± S.D. Nine replicates were done in parallel with CNX data in . Paired two-tailed t tests were performed comparing CRT KO with WT conditions for each measurement. Because the data in panel C is normalized, for this panel, t tests were performed on log-transformed data. * p <0.05, **** p <0.0001. See also for individual experimental trends of the eYFP-ATZ data.

    Techniques Used: Flow Cytometry, Fluorescence, Transfection, Knock-Out, Plasmid Preparation, Two Tailed Test, Transformation Assay

    Small influences of CRT and CNX on ATZ degradation and polymeric ATZ accumulation. A – D , Huh7.5 CRT KO, CNX KO, or corresponding WT cells as indicated were transfected with eYFP-ATZ–encoding plasmids and treated with either 100 n m bafilomycin or 10 µg/ml MG132 or left untreated at 20 h post-transfection. At 24 h post-transfection, the cells were harvested, stained with 2C1, and analyzed. The polymeric ATZ (2C1) gate was determined by gating on forward and side scatter, live cells, then eYFP + cells. The secondary antibody staining control was used as the cutoff for setting the polymeric ATZ gate. For each cell type and drug-treatment condition, ratios of signals from drug-treated/untreated cells were measured to calculate eYFP MFI ratios ( A and B ) or 2C1 MFI ratios ( C and D ). Data for CRT KO and WT were obtained from 14 independent replicates, eight of which were conducted in parallel with CNX KO. Data for CNX KO and WT were obtained from eight independent replicates. All data are shown as mean ± S.D. (error bars ). RM one-way ANOVA analysis was performed, and p -values are reported for comparisons of KO and WT conditions for each drug treatment. E and F , Huh7.5 CRT KO, CNX KO, or corresponding WT cells as indicated were transfected with eYFP-AAT– or eYFP-ATZ–encoding plasmids and stained with the polymer-selective antibody 2C1 at 48 h post-transfection. 2C1 MFI (of eYFP + populations) is shown (gated as described in A – D ). Data quantified over nine independent experiments, conducted in parallel, are shown as mean ± S.D. ( error bars ). Data are normalized relative to the WT eYFP-AAT transfected signals. RM one-way ANOVA analysis was performed on the log-transformed data in E and F . * p < 0.05, *** p < 0.001.
    Figure Legend Snippet: Small influences of CRT and CNX on ATZ degradation and polymeric ATZ accumulation. A – D , Huh7.5 CRT KO, CNX KO, or corresponding WT cells as indicated were transfected with eYFP-ATZ–encoding plasmids and treated with either 100 n m bafilomycin or 10 µg/ml MG132 or left untreated at 20 h post-transfection. At 24 h post-transfection, the cells were harvested, stained with 2C1, and analyzed. The polymeric ATZ (2C1) gate was determined by gating on forward and side scatter, live cells, then eYFP + cells. The secondary antibody staining control was used as the cutoff for setting the polymeric ATZ gate. For each cell type and drug-treatment condition, ratios of signals from drug-treated/untreated cells were measured to calculate eYFP MFI ratios ( A and B ) or 2C1 MFI ratios ( C and D ). Data for CRT KO and WT were obtained from 14 independent replicates, eight of which were conducted in parallel with CNX KO. Data for CNX KO and WT were obtained from eight independent replicates. All data are shown as mean ± S.D. (error bars ). RM one-way ANOVA analysis was performed, and p -values are reported for comparisons of KO and WT conditions for each drug treatment. E and F , Huh7.5 CRT KO, CNX KO, or corresponding WT cells as indicated were transfected with eYFP-AAT– or eYFP-ATZ–encoding plasmids and stained with the polymer-selective antibody 2C1 at 48 h post-transfection. 2C1 MFI (of eYFP + populations) is shown (gated as described in A – D ). Data quantified over nine independent experiments, conducted in parallel, are shown as mean ± S.D. ( error bars ). Data are normalized relative to the WT eYFP-AAT transfected signals. RM one-way ANOVA analysis was performed on the log-transformed data in E and F . * p < 0.05, *** p < 0.001.

    Techniques Used: Transfection, Staining, Transformation Assay

    CRT deficiency alters the distributions of ATZ complexes with ER chaperones. A , ( top ) BiP–eYFP-AAT or BiP–eYFP-ATZ interactions in K42 cells were visualized by immunoprecipitation following 1% digitonin lysis. Vinculin was used as a loading control. CRT interactions were not detectable in parallel IPs. (Bottom) densitometric quantification for total BiP and eYFP-AAT or eYFP-ATZ–co-immunoprecipitated BiP levels in CRT −/− and CRT WT cells. Total BiP levels were calculated by dividing raw BiP intensities by their corresponding loading control intensities, whereas immunoprecipitated BiP levels were calculated by dividing immunoprecipitated BiP intensities by their corresponding immunoprecipitated ATZ intensities. The ratios were then normalized to CRT −/− cells. Data were obtained from four independent transfections of one retroviral transduction and are shown as mean ± S.D. Paired one-sample t tests were performed on log-transformed data, comparing CRT −/− and CRT WT conditions. * p < 0.05, ** p < 0.01. B , ( top ) BiP–eYFP-ATZ interactions in Huh7.5 CRT KO (−) and WT (+) cells were visualized by indicated immunoprecipitation following 1% Triton lysis. GAPDH was used as a loading control. The asterisk indicates the BiP bands. ( Bottom ) densitometric quantifications for total BiP and eYFP-ATZ–co-immunoprecipitated BiP levels in CRT KO and WT cells as described in ( A ). Data were obtained from three independent transfections and are shown as mean ± S.D. Paired one-sample t tests were performed on log-transformed data comparing CRT KO and WT conditions. C , ( left ) CNX-eYFP-ATZ interactions in Huh7.5 CRT KO (−) and WT (+) cells were visualized by indicated immunoprecipitation following 1% Triton lysis. GAPDH was used as a loading control. The asterisk indicates the ATZ bands. (Right) densitometric quantifications for total CNX and eYFP-ATZ–co-immunoprecipitated CNX levels in CRT KO and WT cells as described in ( A ). Data were obtained from four independent transfections and are shown as mean ± S.D. Paired one-sample t tests were performed on log-transformed data comparing CRT KO and WT conditions. * p < 0.05. D , ( top and middle ) PNGase F and Endo H digestions in untransfected or eYFP-ATZ–transfected Huh7.5 cells. The two asterisks indicate two Endo H resistant bands of endogenous AAT in eYFP-ATZ–transfected cells. (Lower) densitometric quantification for Endo H resistant endogenous AAT of the total AAT levels in untransfected or eYFP-ATZ–transfected cells, or Endo H resistant eYFP-ATZ levels of the total ATZ levels in CRT KO and WT cells. The ratios were normalized to WT cells. Data were obtained from three independent experiments and are shown as mean ± S.D. Paired one-sample t tests were performed on log-transformed data comparing CRT KO and WT conditions.
    Figure Legend Snippet: CRT deficiency alters the distributions of ATZ complexes with ER chaperones. A , ( top ) BiP–eYFP-AAT or BiP–eYFP-ATZ interactions in K42 cells were visualized by immunoprecipitation following 1% digitonin lysis. Vinculin was used as a loading control. CRT interactions were not detectable in parallel IPs. (Bottom) densitometric quantification for total BiP and eYFP-AAT or eYFP-ATZ–co-immunoprecipitated BiP levels in CRT −/− and CRT WT cells. Total BiP levels were calculated by dividing raw BiP intensities by their corresponding loading control intensities, whereas immunoprecipitated BiP levels were calculated by dividing immunoprecipitated BiP intensities by their corresponding immunoprecipitated ATZ intensities. The ratios were then normalized to CRT −/− cells. Data were obtained from four independent transfections of one retroviral transduction and are shown as mean ± S.D. Paired one-sample t tests were performed on log-transformed data, comparing CRT −/− and CRT WT conditions. * p < 0.05, ** p < 0.01. B , ( top ) BiP–eYFP-ATZ interactions in Huh7.5 CRT KO (−) and WT (+) cells were visualized by indicated immunoprecipitation following 1% Triton lysis. GAPDH was used as a loading control. The asterisk indicates the BiP bands. ( Bottom ) densitometric quantifications for total BiP and eYFP-ATZ–co-immunoprecipitated BiP levels in CRT KO and WT cells as described in ( A ). Data were obtained from three independent transfections and are shown as mean ± S.D. Paired one-sample t tests were performed on log-transformed data comparing CRT KO and WT conditions. C , ( left ) CNX-eYFP-ATZ interactions in Huh7.5 CRT KO (−) and WT (+) cells were visualized by indicated immunoprecipitation following 1% Triton lysis. GAPDH was used as a loading control. The asterisk indicates the ATZ bands. (Right) densitometric quantifications for total CNX and eYFP-ATZ–co-immunoprecipitated CNX levels in CRT KO and WT cells as described in ( A ). Data were obtained from four independent transfections and are shown as mean ± S.D. Paired one-sample t tests were performed on log-transformed data comparing CRT KO and WT conditions. * p < 0.05. D , ( top and middle ) PNGase F and Endo H digestions in untransfected or eYFP-ATZ–transfected Huh7.5 cells. The two asterisks indicate two Endo H resistant bands of endogenous AAT in eYFP-ATZ–transfected cells. (Lower) densitometric quantification for Endo H resistant endogenous AAT of the total AAT levels in untransfected or eYFP-ATZ–transfected cells, or Endo H resistant eYFP-ATZ levels of the total ATZ levels in CRT KO and WT cells. The ratios were normalized to WT cells. Data were obtained from three independent experiments and are shown as mean ± S.D. Paired one-sample t tests were performed on log-transformed data comparing CRT KO and WT conditions.

    Techniques Used: Immunoprecipitation, Lysis, Transfection, Transduction, Transformation Assay

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