hm2289  (Hycult Biotech)


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    Hycult Biotech hm2289

    Hm2289, 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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    Average 93 stars, based on 1 article reviews
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    hm2289 - by Bioz Stars, 2023-04
    93/100 stars

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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


    Figure Legend Snippet:

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

    hm2289  (Hycult Biotech)


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    Hycult Biotech hm2289

    Hm2289, 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
    https://www.bioz.com/result/hm2289/product/Hycult Biotech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hm2289 - by Bioz Stars, 2023-04
    93/100 stars

    Images

    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


    Figure Legend Snippet:

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

    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

    mab 2c1  (Hycult Biotech)


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    Hycult Biotech mab 2c1
    ( A ) A structural model of a predicted late folding intermediate of Z-α 1 -antitrypsin, showing three Glcα1–3Manα1–2Manα1–2Man glycans at residues N46, N83, and N247. The C-terminal region is shown in blue. ( B ) An enlargement of the C terminus shows hydrophobic amino acid side chains in red (and in inset peptide sequence). ( C ) Whole-cell lysates of CHO-K1 cells transiently transfected to express untagged M- or Z-α 1 -antitrypsin (M- or Z-A1AT) with mCherry-KDEL and Z-A1AT with either HaloTag-calreticulin (Halo-CRT) or HaloTag-calreticulin Y92A,W244A (Halo-CRT Y92A,W244A ) were separated by native-PAGE, and Western blots were probed with the α 1 -antitrypsin polymer-specific mAb <t>2C1</t> . The same samples were separated by SDS-PAGE and blotted for total α 1 -antitrypsin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Quantitation was performed on ( D ) total α 1 -antitrypsin on SDS-PAGE and ( E ) total lane intensity of native-PAGE mAb 2C1 signal, from three independent experiments, with means and SEs shown. ( F ) mAb 2C1 signal intensity quantification on the blot shown in (C) was profiled from bottom to top of the gel. The X axis reflects increasing polymer size. ( G ) CHO-K1 cell lysates prepared as in (C) were centrifuged at 16,100 g before both supernatant and pellet fractions were separated by native-PAGE. Western blots were probed with polymer-specific mAb 2C1 antiserum. Representative gel of three experiments.
    Mab 2c1, 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 "Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state"

    Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

    Journal: Science Advances

    doi: 10.1126/sciadv.abm2094

    ( A ) A structural model of a predicted late folding intermediate of Z-α 1 -antitrypsin, showing three Glcα1–3Manα1–2Manα1–2Man glycans at residues N46, N83, and N247. The C-terminal region is shown in blue. ( B ) An enlargement of the C terminus shows hydrophobic amino acid side chains in red (and in inset peptide sequence). ( C ) Whole-cell lysates of CHO-K1 cells transiently transfected to express untagged M- or Z-α 1 -antitrypsin (M- or Z-A1AT) with mCherry-KDEL and Z-A1AT with either HaloTag-calreticulin (Halo-CRT) or HaloTag-calreticulin Y92A,W244A (Halo-CRT Y92A,W244A ) were separated by native-PAGE, and Western blots were probed with the α 1 -antitrypsin polymer-specific mAb 2C1 . The same samples were separated by SDS-PAGE and blotted for total α 1 -antitrypsin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Quantitation was performed on ( D ) total α 1 -antitrypsin on SDS-PAGE and ( E ) total lane intensity of native-PAGE mAb 2C1 signal, from three independent experiments, with means and SEs shown. ( F ) mAb 2C1 signal intensity quantification on the blot shown in (C) was profiled from bottom to top of the gel. The X axis reflects increasing polymer size. ( G ) CHO-K1 cell lysates prepared as in (C) were centrifuged at 16,100 g before both supernatant and pellet fractions were separated by native-PAGE. Western blots were probed with polymer-specific mAb 2C1 antiserum. Representative gel of three experiments.
    Figure Legend Snippet: ( A ) A structural model of a predicted late folding intermediate of Z-α 1 -antitrypsin, showing three Glcα1–3Manα1–2Manα1–2Man glycans at residues N46, N83, and N247. The C-terminal region is shown in blue. ( B ) An enlargement of the C terminus shows hydrophobic amino acid side chains in red (and in inset peptide sequence). ( C ) Whole-cell lysates of CHO-K1 cells transiently transfected to express untagged M- or Z-α 1 -antitrypsin (M- or Z-A1AT) with mCherry-KDEL and Z-A1AT with either HaloTag-calreticulin (Halo-CRT) or HaloTag-calreticulin Y92A,W244A (Halo-CRT Y92A,W244A ) were separated by native-PAGE, and Western blots were probed with the α 1 -antitrypsin polymer-specific mAb 2C1 . The same samples were separated by SDS-PAGE and blotted for total α 1 -antitrypsin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Quantitation was performed on ( D ) total α 1 -antitrypsin on SDS-PAGE and ( E ) total lane intensity of native-PAGE mAb 2C1 signal, from three independent experiments, with means and SEs shown. ( F ) mAb 2C1 signal intensity quantification on the blot shown in (C) was profiled from bottom to top of the gel. The X axis reflects increasing polymer size. ( G ) CHO-K1 cell lysates prepared as in (C) were centrifuged at 16,100 g before both supernatant and pellet fractions were separated by native-PAGE. Western blots were probed with polymer-specific mAb 2C1 antiserum. Representative gel of three experiments.

    Techniques Used: Sequencing, Transfection, Clear Native PAGE, Western Blot, SDS Page, Quantitation Assay

    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: 93/100, 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

    anti aat polymer  (Hycult Biotech)


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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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    Average 93 stars, based on 1 article reviews
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    anti aat polymer - by Bioz Stars, 2023-04
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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

    hm2289  (Hycult Biotech)


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    Hycult Biotech hm2289

    Hm2289, 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
    https://www.bioz.com/result/hm2289/product/Hycult Biotech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hm2289 - by Bioz Stars, 2023-04
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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


    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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    Hycult Biotech anti human aat polymer selective
    <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.
    Anti Human Aat Polymer Selective, 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 "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

    polymeric a1at  (Hycult Biotech)


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    Structured Review

    Hycult Biotech polymeric a1at
    FA content in different <t> A1AT </t> preparations
    Polymeric A1at, 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 "Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression"

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1500740

    FA content in different  A1AT  preparations
    Figure Legend Snippet: FA content in different A1AT preparations

    Techniques Used: Affinity Purification

    Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.
    Figure Legend Snippet: Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.

    Techniques Used: Incubation

    FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.
    Figure Legend Snippet: FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.

    Techniques Used: Incubation

    Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.
    Figure Legend Snippet: Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.

    Techniques Used: SDS Page, Western Blot, Incubation, Affinity Purification

    Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.
    Figure Legend Snippet: Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.
    Figure Legend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.
    Figure Legend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Fractionation, Western Blot, Staining, Expressing, Flow Cytometry, Fluorescence

    Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.
    Figure Legend Snippet: Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.

    Techniques Used: Derivative Assay, Affinity Purification, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.
    Figure Legend Snippet: A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.

    Techniques Used: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.
    Figure Legend Snippet: (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.

    Techniques Used: Activation Assay, Western Blot, Staining, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction

    2c1 against human aat polymers  (Hycult Biotech)


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    Hycult Biotech 2c1 against human aat polymers
    Interaction of ERdj3 with ZAAT. (A) ZAAT co‐immunoprecipitated with ERdj3 with and without cross‐linker. <t>AAT</t> KO Huh 7.5 cells were transfected with NT siRNA or siERdj3. Then, 24 h post silencing, cells were transfected with ZAAT plasmid. AAT was pulled down, and ERdj3 bound to ZAAT was determined by Western blot. (B) ERdj3 co‐localizes with ZAAT polymers. ZAAT transiently transfected cells were immunostained using <t>2C1</t> antibody (Alexa 594; red) and anti‐ERdj3 (Alexa 488; green), and nuclei were stained using DAPI (blue). (C) Erdj3 overexpression increases ZAAT polymer formation. MAAT and ZAAT transiently transfected cells were transfected with control (CO) or ERdj3 plasmids 24 h after the first transfection. Then, 48 h post transfection, lysates were incubated for 1 h at 37°C or 60°C to form polymers. Lysates were loaded on non‐denaturing native gel.
    2c1 Against Human 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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    Images

    1) Product Images from "Erdj3 Has an Essential Role for Z Variant Alpha‐1‐Antitrypsin Degradation"

    Article Title: Erdj3 Has an Essential Role for Z Variant Alpha‐1‐Antitrypsin Degradation

    Journal: Journal of Cellular Biochemistry

    doi: 10.1002/jcb.26069

    Interaction of ERdj3 with ZAAT. (A) ZAAT co‐immunoprecipitated with ERdj3 with and without cross‐linker. AAT KO Huh 7.5 cells were transfected with NT siRNA or siERdj3. Then, 24 h post silencing, cells were transfected with ZAAT plasmid. AAT was pulled down, and ERdj3 bound to ZAAT was determined by Western blot. (B) ERdj3 co‐localizes with ZAAT polymers. ZAAT transiently transfected cells were immunostained using 2C1 antibody (Alexa 594; red) and anti‐ERdj3 (Alexa 488; green), and nuclei were stained using DAPI (blue). (C) Erdj3 overexpression increases ZAAT polymer formation. MAAT and ZAAT transiently transfected cells were transfected with control (CO) or ERdj3 plasmids 24 h after the first transfection. Then, 48 h post transfection, lysates were incubated for 1 h at 37°C or 60°C to form polymers. Lysates were loaded on non‐denaturing native gel.
    Figure Legend Snippet: Interaction of ERdj3 with ZAAT. (A) ZAAT co‐immunoprecipitated with ERdj3 with and without cross‐linker. AAT KO Huh 7.5 cells were transfected with NT siRNA or siERdj3. Then, 24 h post silencing, cells were transfected with ZAAT plasmid. AAT was pulled down, and ERdj3 bound to ZAAT was determined by Western blot. (B) ERdj3 co‐localizes with ZAAT polymers. ZAAT transiently transfected cells were immunostained using 2C1 antibody (Alexa 594; red) and anti‐ERdj3 (Alexa 488; green), and nuclei were stained using DAPI (blue). (C) Erdj3 overexpression increases ZAAT polymer formation. MAAT and ZAAT transiently transfected cells were transfected with control (CO) or ERdj3 plasmids 24 h after the first transfection. Then, 48 h post transfection, lysates were incubated for 1 h at 37°C or 60°C to form polymers. Lysates were loaded on non‐denaturing native gel.

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Staining, Over Expression, Incubation

    The effect of siERdj3 on ZAAT degradation. (A) siERdj3 increases ZAAT degradation. NT siRNA or siERdj3 (20 or 40 µM) were introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. The level of ZAAT inside the cells (IC) and in the media (EC) and the level of ERdj3 were measured by Western blot. GAPDH was used as loading control. (B) siERdj3 does not affect ZAAT secretion. By ELISA, total AAT was measured in the media from the same samples as in panel A experiment. Total AAT was shown in picomolar concentration. (C) siERdj3 accelerates ZAAT clearance from the ER of hepatocytes. NT siRNA or 20 nM of siERdj3 was introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. IC ZAAT and EC ZAAT from NT siRNA and siERdj3‐treated samples were shown after pulse‐chase radiolabeling. (D) ZAAT polymers are reduced following siERdj3 treatment. NT siRNA or 20 µM of siERdj3 were introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. ZAAT polymers were immunostained with 2C1 antibody (Alexa 594; red). (E) Integrated density of red fluorescent measurement from four images per sample.
    Figure Legend Snippet: The effect of siERdj3 on ZAAT degradation. (A) siERdj3 increases ZAAT degradation. NT siRNA or siERdj3 (20 or 40 µM) were introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. The level of ZAAT inside the cells (IC) and in the media (EC) and the level of ERdj3 were measured by Western blot. GAPDH was used as loading control. (B) siERdj3 does not affect ZAAT secretion. By ELISA, total AAT was measured in the media from the same samples as in panel A experiment. Total AAT was shown in picomolar concentration. (C) siERdj3 accelerates ZAAT clearance from the ER of hepatocytes. NT siRNA or 20 nM of siERdj3 was introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. IC ZAAT and EC ZAAT from NT siRNA and siERdj3‐treated samples were shown after pulse‐chase radiolabeling. (D) ZAAT polymers are reduced following siERdj3 treatment. NT siRNA or 20 µM of siERdj3 were introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. ZAAT polymers were immunostained with 2C1 antibody (Alexa 594; red). (E) Integrated density of red fluorescent measurement from four images per sample.

    Techniques Used: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Pulse Chase, Radioactivity

    a1at polymeric forms  (Hycult Biotech)


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    Structured Review

    Hycult Biotech a1at polymeric forms
    Summary of the nephelometric measurments of <t> A1AT </t> concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.
    A1at Polymeric Forms, 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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    Images

    1) Product Images from "How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?"

    Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135316

    Summary of the nephelometric measurments of  A1AT  concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.
    Figure Legend Snippet: Summary of the nephelometric measurments of A1AT concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.

    Techniques Used: Concentration Assay, Protein Electrophoresis

    Thresholds for suspicion of severe or intermediate A1ATD according to SPE.
    Figure Legend Snippet: Thresholds for suspicion of severe or intermediate A1ATD according to SPE.

    Techniques Used:

    The serum concentration of A1AT was determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% PAGE, and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration in the loaded sample per well is indicated at the bottom of the gels.
    Figure Legend Snippet: The serum concentration of A1AT was determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% PAGE, and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration in the loaded sample per well is indicated at the bottom of the gels.

    Techniques Used: Concentration Assay, Western Blot

    Serum concentrations of A1AT were determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% SDS-PAGE (without denaturing agents), and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration per well in the loaded serum sample is indicated at the bottom of the gels.
    Figure Legend Snippet: Serum concentrations of A1AT were determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% SDS-PAGE (without denaturing agents), and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration per well in the loaded serum sample is indicated at the bottom of the gels.

    Techniques Used: SDS Page, Western Blot, Concentration Assay

    a . Purified plasma A1AT (2.5 mg/ml) was heated at 60°C and aliquots were removed at indicated time points for analysis by a non-denaturing PAGE followed by Western blotting using rabbit polyclonal anti-A1AT antibody. b . For a more detailed analysis of the heat-treated A1AT a 4–15% gradient SDS-PAGE was used. This method is designed for separation of high molecular weight polymers. Electrophoretic separation of A1AT was followed by Western blot analysis with an anti-human A1AT antibody as above. The immunoblot patterns indicate that heat-treated A1AT typically comprises a mixture of a monomer and various sized polymers. The figure shows representative blots from 5 experiments. c . M A1AT heat-treated for 5 min exhibited a typical five band pattern in the IEF gel (lane 1). When M A1AT was heated for 15 min or longer t, the three prominent bands towards the anode were no longer detectable (lanes 2–9). Instead, a continuous smeared band occurred at the cathode (lane 3), these results paralleled those with heat-induced A1AT polymerization over time (Fig 3b). d . Initial concentrations of purified A1AT were 2.35 ± 0.042 mg/ml; n = 3. The same A1AT protein heated for a short period yielded a higher nephelometric signal than native protein. However, heating of A1AT for 45 min or longer, resulted in a remarkable decrease in the nephelometric signal relative to the native protein.
    Figure Legend Snippet: a . Purified plasma A1AT (2.5 mg/ml) was heated at 60°C and aliquots were removed at indicated time points for analysis by a non-denaturing PAGE followed by Western blotting using rabbit polyclonal anti-A1AT antibody. b . For a more detailed analysis of the heat-treated A1AT a 4–15% gradient SDS-PAGE was used. This method is designed for separation of high molecular weight polymers. Electrophoretic separation of A1AT was followed by Western blot analysis with an anti-human A1AT antibody as above. The immunoblot patterns indicate that heat-treated A1AT typically comprises a mixture of a monomer and various sized polymers. The figure shows representative blots from 5 experiments. c . M A1AT heat-treated for 5 min exhibited a typical five band pattern in the IEF gel (lane 1). When M A1AT was heated for 15 min or longer t, the three prominent bands towards the anode were no longer detectable (lanes 2–9). Instead, a continuous smeared band occurred at the cathode (lane 3), these results paralleled those with heat-induced A1AT polymerization over time (Fig 3b). d . Initial concentrations of purified A1AT were 2.35 ± 0.042 mg/ml; n = 3. The same A1AT protein heated for a short period yielded a higher nephelometric signal than native protein. However, heating of A1AT for 45 min or longer, resulted in a remarkable decrease in the nephelometric signal relative to the native protein.

    Techniques Used: Purification, Western Blot, SDS Page, Molecular Weight

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    Hycult Biotech hm2289

    Hm2289, 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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    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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    Hycult Biotech mab 2c1
    ( A ) A structural model of a predicted late folding intermediate of Z-α 1 -antitrypsin, showing three Glcα1–3Manα1–2Manα1–2Man glycans at residues N46, N83, and N247. The C-terminal region is shown in blue. ( B ) An enlargement of the C terminus shows hydrophobic amino acid side chains in red (and in inset peptide sequence). ( C ) Whole-cell lysates of CHO-K1 cells transiently transfected to express untagged M- or Z-α 1 -antitrypsin (M- or Z-A1AT) with mCherry-KDEL and Z-A1AT with either HaloTag-calreticulin (Halo-CRT) or HaloTag-calreticulin Y92A,W244A (Halo-CRT Y92A,W244A ) were separated by native-PAGE, and Western blots were probed with the α 1 -antitrypsin polymer-specific mAb <t>2C1</t> . The same samples were separated by SDS-PAGE and blotted for total α 1 -antitrypsin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Quantitation was performed on ( D ) total α 1 -antitrypsin on SDS-PAGE and ( E ) total lane intensity of native-PAGE mAb 2C1 signal, from three independent experiments, with means and SEs shown. ( F ) mAb 2C1 signal intensity quantification on the blot shown in (C) was profiled from bottom to top of the gel. The X axis reflects increasing polymer size. ( G ) CHO-K1 cell lysates prepared as in (C) were centrifuged at 16,100 g before both supernatant and pellet fractions were separated by native-PAGE. Western blots were probed with polymer-specific mAb 2C1 antiserum. Representative gel of three experiments.
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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: 93/100, 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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    Hycult Biotech anti human aat polymer selective
    <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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    Hycult Biotech polymeric a1at
    FA content in different <t> A1AT </t> preparations
    Polymeric A1at, 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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    Hycult Biotech 2c1 against human aat polymers
    Interaction of ERdj3 with ZAAT. (A) ZAAT co‐immunoprecipitated with ERdj3 with and without cross‐linker. <t>AAT</t> KO Huh 7.5 cells were transfected with NT siRNA or siERdj3. Then, 24 h post silencing, cells were transfected with ZAAT plasmid. AAT was pulled down, and ERdj3 bound to ZAAT was determined by Western blot. (B) ERdj3 co‐localizes with ZAAT polymers. ZAAT transiently transfected cells were immunostained using <t>2C1</t> antibody (Alexa 594; red) and anti‐ERdj3 (Alexa 488; green), and nuclei were stained using DAPI (blue). (C) Erdj3 overexpression increases ZAAT polymer formation. MAAT and ZAAT transiently transfected cells were transfected with control (CO) or ERdj3 plasmids 24 h after the first transfection. Then, 48 h post transfection, lysates were incubated for 1 h at 37°C or 60°C to form polymers. Lysates were loaded on non‐denaturing native gel.
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    Hycult Biotech a1at polymeric forms
    Summary of the nephelometric measurments of <t> A1AT </t> concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.
    A1at Polymeric Forms, 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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    Image Search Results


    Journal: Cell reports

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

    doi: 10.1016/j.celrep.2022.111775

    Figure Lengend Snippet:

    Article Snippet: Mouse monoclonal anti-AAT polymers (clone 2C1) , Hycult Biotech , HM2289.

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

    (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.

    Journal: Cell reports

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

    doi: 10.1016/j.celrep.2022.111775

    Figure Lengend 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.

    Article Snippet: Mouse monoclonal anti-AAT polymers (clone 2C1) , Hycult Biotech , HM2289.

    Techniques: Flow Cytometry, Immunostaining, Inhibition, Labeling

    Journal: Cell reports

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

    doi: 10.1016/j.celrep.2022.111775

    Figure Lengend Snippet:

    Article Snippet: Mouse monoclonal anti-AAT polymers (clone 2C1) , Hycult Biotech , HM2289.

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

    ( A ) A structural model of a predicted late folding intermediate of Z-α 1 -antitrypsin, showing three Glcα1–3Manα1–2Manα1–2Man glycans at residues N46, N83, and N247. The C-terminal region is shown in blue. ( B ) An enlargement of the C terminus shows hydrophobic amino acid side chains in red (and in inset peptide sequence). ( C ) Whole-cell lysates of CHO-K1 cells transiently transfected to express untagged M- or Z-α 1 -antitrypsin (M- or Z-A1AT) with mCherry-KDEL and Z-A1AT with either HaloTag-calreticulin (Halo-CRT) or HaloTag-calreticulin Y92A,W244A (Halo-CRT Y92A,W244A ) were separated by native-PAGE, and Western blots were probed with the α 1 -antitrypsin polymer-specific mAb 2C1 . The same samples were separated by SDS-PAGE and blotted for total α 1 -antitrypsin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Quantitation was performed on ( D ) total α 1 -antitrypsin on SDS-PAGE and ( E ) total lane intensity of native-PAGE mAb 2C1 signal, from three independent experiments, with means and SEs shown. ( F ) mAb 2C1 signal intensity quantification on the blot shown in (C) was profiled from bottom to top of the gel. The X axis reflects increasing polymer size. ( G ) CHO-K1 cell lysates prepared as in (C) were centrifuged at 16,100 g before both supernatant and pellet fractions were separated by native-PAGE. Western blots were probed with polymer-specific mAb 2C1 antiserum. Representative gel of three experiments.

    Journal: Science Advances

    Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

    doi: 10.1126/sciadv.abm2094

    Figure Lengend Snippet: ( A ) A structural model of a predicted late folding intermediate of Z-α 1 -antitrypsin, showing three Glcα1–3Manα1–2Manα1–2Man glycans at residues N46, N83, and N247. The C-terminal region is shown in blue. ( B ) An enlargement of the C terminus shows hydrophobic amino acid side chains in red (and in inset peptide sequence). ( C ) Whole-cell lysates of CHO-K1 cells transiently transfected to express untagged M- or Z-α 1 -antitrypsin (M- or Z-A1AT) with mCherry-KDEL and Z-A1AT with either HaloTag-calreticulin (Halo-CRT) or HaloTag-calreticulin Y92A,W244A (Halo-CRT Y92A,W244A ) were separated by native-PAGE, and Western blots were probed with the α 1 -antitrypsin polymer-specific mAb 2C1 . The same samples were separated by SDS-PAGE and blotted for total α 1 -antitrypsin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Quantitation was performed on ( D ) total α 1 -antitrypsin on SDS-PAGE and ( E ) total lane intensity of native-PAGE mAb 2C1 signal, from three independent experiments, with means and SEs shown. ( F ) mAb 2C1 signal intensity quantification on the blot shown in (C) was profiled from bottom to top of the gel. The X axis reflects increasing polymer size. ( G ) CHO-K1 cell lysates prepared as in (C) were centrifuged at 16,100 g before both supernatant and pellet fractions were separated by native-PAGE. Western blots were probed with polymer-specific mAb 2C1 antiserum. Representative gel of three experiments.

    Article Snippet: Antibodies used in this study were raised against total α 1 -antitrypsin (A0409, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), and the α 1 -antitrypsin polymer–specific mAb 2C1 (HM2289, Hycult Biotech).

    Techniques: Sequencing, Transfection, Clear Native PAGE, Western Blot, SDS Page, Quantitation Assay

    ( 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.

    Journal: bioRxiv

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

    doi: 10.1101/2022.03.04.483066

    Figure Lengend 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.

    Article Snippet: A mouse anti-human monoclonal AAT antibody 2C1 that preferentially recognizes AAT polymer were purchased from Hycult biotech (Wayne, PA).

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2022.03.04.483066

    Figure Lengend 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.

    Article Snippet: A mouse anti-human monoclonal AAT antibody 2C1 that preferentially recognizes AAT polymer were purchased from Hycult biotech (Wayne, PA).

    Techniques: Sequencing, Activity Assay, Variant Assay

    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.

    Journal: eLife

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

    doi: 10.7554/eLife.64881

    Figure Lengend 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.

    Article Snippet: Antibody , Anti-AAT polymer (mouse monoclonal) , Hycult Biotech , Clone 2C1; HM2289 , (1:500).

    Techniques: SDS Page, Western Blot

    Journal: eLife

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

    doi: 10.7554/eLife.64881

    Figure Lengend Snippet:

    Article Snippet: Antibody , Anti-AAT polymer (mouse monoclonal) , Hycult Biotech , Clone 2C1; HM2289 , (1:500).

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

    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.

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.RA120.014372

    Figure Lengend 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.

    Article Snippet: Primary antibodies used were anti-AAT (rabbit polyclonal IgG, Agilent Dako, A0012), anti-human AAT (polymer-selective) (2C1; mouse monoclonal IgG1, Hycult Biotech, HM2289), anti-CRT (rabbit polyclonal IgG, Thermo Fisher Scientific, PA3-900), anti-CNX (rabbit polyclonal IgG, Enzo Life Sciences, ADI-SPA-860), anti-CNX (C5C9; rabbit monoclonal, Cell Signaling Technology, 2679), anti-GAPDH (14C10; rabbit monoclonal IgG, Cell Signaling Technology, 2118), anti-GFP (GF28R; mouse monoclonal IgG1, Thermo Fisher Scientific, MA5-15256), anti-GRP78/BiP (rabbit polyclonal IgG, Abcam, ab21685), anti-ubiquitin (P4D1; mouse monoclonal IgG, Cell Signaling Technology, 3936), anti-Vinculin (E1E9V; rabbit monoclonal IgG, Cell Signaling Technology, 13901), anti-Sec31A (mouse monoclonal IgG1, BD Biosciences, 612350), anti-Sec24A (rabbit, Cell Signaling Technology, 9678), and anti-Sec24C (D9M4N, rabbit monoclonal IgG, Cell Signaling Technology, 14676).

    Techniques: 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.

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.RA120.014372

    Figure Lengend 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.

    Article Snippet: Primary antibodies used were anti-AAT (rabbit polyclonal IgG, Agilent Dako, A0012), anti-human AAT (polymer-selective) (2C1; mouse monoclonal IgG1, Hycult Biotech, HM2289), anti-CRT (rabbit polyclonal IgG, Thermo Fisher Scientific, PA3-900), anti-CNX (rabbit polyclonal IgG, Enzo Life Sciences, ADI-SPA-860), anti-CNX (C5C9; rabbit monoclonal, Cell Signaling Technology, 2679), anti-GAPDH (14C10; rabbit monoclonal IgG, Cell Signaling Technology, 2118), anti-GFP (GF28R; mouse monoclonal IgG1, Thermo Fisher Scientific, MA5-15256), anti-GRP78/BiP (rabbit polyclonal IgG, Abcam, ab21685), anti-ubiquitin (P4D1; mouse monoclonal IgG, Cell Signaling Technology, 3936), anti-Vinculin (E1E9V; rabbit monoclonal IgG, Cell Signaling Technology, 13901), anti-Sec31A (mouse monoclonal IgG1, BD Biosciences, 612350), anti-Sec24A (rabbit, Cell Signaling Technology, 9678), and anti-Sec24C (D9M4N, rabbit monoclonal IgG, Cell Signaling Technology, 14676).

    Techniques: 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.

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.RA120.014372

    Figure Lengend 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.

    Article Snippet: Primary antibodies used were anti-AAT (rabbit polyclonal IgG, Agilent Dako, A0012), anti-human AAT (polymer-selective) (2C1; mouse monoclonal IgG1, Hycult Biotech, HM2289), anti-CRT (rabbit polyclonal IgG, Thermo Fisher Scientific, PA3-900), anti-CNX (rabbit polyclonal IgG, Enzo Life Sciences, ADI-SPA-860), anti-CNX (C5C9; rabbit monoclonal, Cell Signaling Technology, 2679), anti-GAPDH (14C10; rabbit monoclonal IgG, Cell Signaling Technology, 2118), anti-GFP (GF28R; mouse monoclonal IgG1, Thermo Fisher Scientific, MA5-15256), anti-GRP78/BiP (rabbit polyclonal IgG, Abcam, ab21685), anti-ubiquitin (P4D1; mouse monoclonal IgG, Cell Signaling Technology, 3936), anti-Vinculin (E1E9V; rabbit monoclonal IgG, Cell Signaling Technology, 13901), anti-Sec31A (mouse monoclonal IgG1, BD Biosciences, 612350), anti-Sec24A (rabbit, Cell Signaling Technology, 9678), and anti-Sec24C (D9M4N, rabbit monoclonal IgG, Cell Signaling Technology, 14676).

    Techniques: 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.

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.RA120.014372

    Figure Lengend 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.

    Article Snippet: Primary antibodies used were anti-AAT (rabbit polyclonal IgG, Agilent Dako, A0012), anti-human AAT (polymer-selective) (2C1; mouse monoclonal IgG1, Hycult Biotech, HM2289), anti-CRT (rabbit polyclonal IgG, Thermo Fisher Scientific, PA3-900), anti-CNX (rabbit polyclonal IgG, Enzo Life Sciences, ADI-SPA-860), anti-CNX (C5C9; rabbit monoclonal, Cell Signaling Technology, 2679), anti-GAPDH (14C10; rabbit monoclonal IgG, Cell Signaling Technology, 2118), anti-GFP (GF28R; mouse monoclonal IgG1, Thermo Fisher Scientific, MA5-15256), anti-GRP78/BiP (rabbit polyclonal IgG, Abcam, ab21685), anti-ubiquitin (P4D1; mouse monoclonal IgG, Cell Signaling Technology, 3936), anti-Vinculin (E1E9V; rabbit monoclonal IgG, Cell Signaling Technology, 13901), anti-Sec31A (mouse monoclonal IgG1, BD Biosciences, 612350), anti-Sec24A (rabbit, Cell Signaling Technology, 9678), and anti-Sec24C (D9M4N, rabbit monoclonal IgG, Cell Signaling Technology, 14676).

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

    FA content in different  A1AT  preparations

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: FA content in different A1AT preparations

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Affinity Purification

    Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Pull down of FA-free HSA-0 and A1AT-0 by FA-coupled agarose beads. (A) Fatty acid free HSA-0 or A1AT-0 (Zemaira) was mixed on a rotating mixer with ω-aminohexyl-agarose beads coupled to LA, OA or control (co.) beads exposed to coupling reaction in absence of FA. After incubation for 2h, the beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. (B) For competition experiments target protein was mixed with competing protein in the concentrations as indicated in the figure before addition of beads. One representative blot out of 3 independent experiments is shown. Densities were evaluated using ImageJ. Results are given in mean±SD.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Incubation

    FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: FA pull down of A1AT and HSA from PiMM and PiZZ serum. Pooled serum samples were pre-diluted with 3 volumes of PBS and incubated with LA-, OA- or control (co.) beads for 2 h on a rotating mixer. Beads were washed thoroughly with PBS to remove unbound proteins, SDS sample buffer was added and bead-bound proteins were released by heating at 95°C for 5 min. Sample load was 15 μl for A1AT blot and 5 μl for HSA blot. To facilitate comparability of the blots 200 ng target proteins were applied. Antibodies don't show any cross reactivity with competing proteins.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Incubation

    Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Qualitative analysis of molecular forms of A1AT found in A1AT preparations by non-denaturing SDS-PAGE following Western blot analysis using rabbit polyclonal anti-A1AT and mouse monoclonal anti-A1AT polymer (2C1) antibodies. (A) A1AT-LA and –OA complexes were prepared by 3 h incubation at 37°C following separation from free FA with centricon-10 (MWCO 10 kDa). Each blot is representative out of n=3 independent experiments. (B) M- and Z-A1AT pools from Alph1-Antitrypsin Select affinity purification were analyzed by Western Blot. Each blot is representative out of n=3 independent experiments.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: SDS Page, Western Blot, Incubation, Affinity Purification

    Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Time-dependent effect of A1AT-0 and A1AT-LA on Angptl4 and FABP4 expression of human adherent blood monocytes. Cells were treated with A1AT -0 or A1AT-LA (1 mg/ml) for different periods of time. Gene expression levels of Angptl4 (A) and FABP4 (B) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ± SD of n=3 independent experiments, each with 3 repeats.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4 and related gene expression in adherent blood monocytes compared to those of A1AT-0 and FAs alone. Cells were treated with A1AT-0, complexes of A1AT-LA and A1AT-OA (1 mg/ml) or with LA and OA preparations alone for 6 h. Gene expression levels of Angptl4 (A), FABP4 (B) and CD36 (C) were assessed by RT-PCR and normalized to GAPDH. Each bar represents mean ± SD of n=6 independent experiments in case of A1AT-0, A1AT-LA and LA and n=3 in case of A1AT-OA and OA. Measurements were performed in triplicates.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Effects of A1AT-LA and A1AT-OA complexes on Angptl4, FABP4 and CD36 protein levels. Adherent monocytes were kept untreated for the control or treated with A1AT-0, A1AT-LA or A1AT-OA complexes (1 mg/ml) for 6 h. (A) Supernatants were collected and analyzed for released Angptl4 by ARP4 ELISA. (B) Cells were fractionated into cytoplasmic and nuclear fractions using Cytoplasmic and Nuclear Fractionation kit. FABP4 protein levels in the fractions were determined by Western Blot analysis. β-Actin was stained for a loading control. Presented blot is representative of n=3 repeated experiments. Cell surface expression of CD36 was analyzed by flow cytometry (C). Cells were stained with anti CD36-FITC or respective FITC isotype. Mean fluorescence intensities of ungated cells were determined. Each bar represents mean ±SD of n=3 independent experiments, each performed in duplicates.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Enzyme-linked Immunosorbent Assay, Fractionation, Western Blot, Staining, Expressing, Flow Cytometry, Fluorescence

    Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: Effects of serum derived affinity purified M- and Z-A1AT on Angptl4 and related gene expression. Adherent blood monocytes were treated with 0.5 mg/ml of M- and Z-A1AT, isolated from pooled PiMM and PiZZ serum, for a total of 6 h. mRNA levels of Angptl4 (A), FABP4 (C) and CD36 (D) were analyzed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=4 independent experiments, each with 3 repeats. (B) Concentration of Angptl4 in cell culture supernatants was determined by Duoset ELISA. Each bar represents mean ± SD of n=4 independent experiments, each with 2 repeats.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Derivative Assay, Affinity Purification, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: A1AT-LA induced expression of Angptl4 and FABP4 is related to PPARγ and PPARβ/δ activity. Cells were pretreated for 30 min with 10 μM GW9662, an irreversible PPARγ antagonist (A, B), or with 1 μM ST247, a selective and inverse agonist of PPARβ/δ (C, D), prior to addition of 1 mg/ml A1AT-0, A1AT-LA, LA preparation or Prolastin® for another 6 h. Expression levels of Angptl4 (A, C) and FABP4 (B, D) were determined by RT-PCR and normalized to GAPDH. In (A, B) each point represents mean ±SD of n=3 independent experiments, each with 3 repeats. In (C, D) each point represents mean ±SD of n=2 independent experiments, each with 3 repeats.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Alpha1-Antitrypsin combines with plasma fatty acids and induces angiopoietin-like protein 4 expression

    doi: 10.4049/jimmunol.1500740

    Figure Lengend Snippet: (A) Transient activation of MEK-ERK1/2 pathway is independent on A1AT complexation with LA. Western blot of cell lysates prepared from adherent blood monocytes illustrates phosphorylation of ERK1/2 at 30 min in response to treatment with 1 mg/ml A1AT-0 or A1AT-LA. Blots were stained for phosphorylated and total ERK1/2. β-Actin was used as a loading control. Pre-incubation of cells with MEK/ERK1/2 inhibitor UO126 (10 μM), completely abolished ERK1/2 activation in all samples. Presented blot is representative of n=4 repeated experiments. (B) Effect of ERK1/2 phosphorylation on A1AT-LA-induced Angptl4 expression. Adherent PBMCs were pre incubated for 30 min with UO126 (10 μM) prior to addition of 1 mg/ml A1AT-0 or A1AT-LA for another 6 h. Angptl4 mRNA levels were assessed by RT-PCR and normalized to GAPDH. Each point represents mean ±SD of n=3 independent experiments, each with 3 repeats.

    Article Snippet: For specific detection, the following primary antibodies were used: rabbit polyclonal anti A1AT (DAKO), mouse monoclonal anti A1AT (clone B9, Santa Cruz Biotechnology), mouse monoclonal anti polymeric A1AT (Clone 2C1, Hycult biotech), mouse monoclonal anti FABP4 (clone 1105CT1-1-1, Antibodies-Online GmbH), rabbit polyclonal anti ERK1/2 and mouse monoclonal anti p-ERK1/2 (both from Sigma-Aldrich), monoclonal anti β-actin (AC-15, Sigma-Aldrich).

    Techniques: Activation Assay, Western Blot, Staining, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction

    Interaction of ERdj3 with ZAAT. (A) ZAAT co‐immunoprecipitated with ERdj3 with and without cross‐linker. AAT KO Huh 7.5 cells were transfected with NT siRNA or siERdj3. Then, 24 h post silencing, cells were transfected with ZAAT plasmid. AAT was pulled down, and ERdj3 bound to ZAAT was determined by Western blot. (B) ERdj3 co‐localizes with ZAAT polymers. ZAAT transiently transfected cells were immunostained using 2C1 antibody (Alexa 594; red) and anti‐ERdj3 (Alexa 488; green), and nuclei were stained using DAPI (blue). (C) Erdj3 overexpression increases ZAAT polymer formation. MAAT and ZAAT transiently transfected cells were transfected with control (CO) or ERdj3 plasmids 24 h after the first transfection. Then, 48 h post transfection, lysates were incubated for 1 h at 37°C or 60°C to form polymers. Lysates were loaded on non‐denaturing native gel.

    Journal: Journal of Cellular Biochemistry

    Article Title: Erdj3 Has an Essential Role for Z Variant Alpha‐1‐Antitrypsin Degradation

    doi: 10.1002/jcb.26069

    Figure Lengend Snippet: Interaction of ERdj3 with ZAAT. (A) ZAAT co‐immunoprecipitated with ERdj3 with and without cross‐linker. AAT KO Huh 7.5 cells were transfected with NT siRNA or siERdj3. Then, 24 h post silencing, cells were transfected with ZAAT plasmid. AAT was pulled down, and ERdj3 bound to ZAAT was determined by Western blot. (B) ERdj3 co‐localizes with ZAAT polymers. ZAAT transiently transfected cells were immunostained using 2C1 antibody (Alexa 594; red) and anti‐ERdj3 (Alexa 488; green), and nuclei were stained using DAPI (blue). (C) Erdj3 overexpression increases ZAAT polymer formation. MAAT and ZAAT transiently transfected cells were transfected with control (CO) or ERdj3 plasmids 24 h after the first transfection. Then, 48 h post transfection, lysates were incubated for 1 h at 37°C or 60°C to form polymers. Lysates were loaded on non‐denaturing native gel.

    Article Snippet: Bafilomycin A1, MG132, Brefeldin A, and mouse monoclonal anti‐β‐actin were purchased from Sigma (St. Louis, MO), and 2C1 against human AAT polymers was purchased from Hycult biotech (Netherlands).

    Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Staining, Over Expression, Incubation

    The effect of siERdj3 on ZAAT degradation. (A) siERdj3 increases ZAAT degradation. NT siRNA or siERdj3 (20 or 40 µM) were introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. The level of ZAAT inside the cells (IC) and in the media (EC) and the level of ERdj3 were measured by Western blot. GAPDH was used as loading control. (B) siERdj3 does not affect ZAAT secretion. By ELISA, total AAT was measured in the media from the same samples as in panel A experiment. Total AAT was shown in picomolar concentration. (C) siERdj3 accelerates ZAAT clearance from the ER of hepatocytes. NT siRNA or 20 nM of siERdj3 was introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. IC ZAAT and EC ZAAT from NT siRNA and siERdj3‐treated samples were shown after pulse‐chase radiolabeling. (D) ZAAT polymers are reduced following siERdj3 treatment. NT siRNA or 20 µM of siERdj3 were introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. ZAAT polymers were immunostained with 2C1 antibody (Alexa 594; red). (E) Integrated density of red fluorescent measurement from four images per sample.

    Journal: Journal of Cellular Biochemistry

    Article Title: Erdj3 Has an Essential Role for Z Variant Alpha‐1‐Antitrypsin Degradation

    doi: 10.1002/jcb.26069

    Figure Lengend Snippet: The effect of siERdj3 on ZAAT degradation. (A) siERdj3 increases ZAAT degradation. NT siRNA or siERdj3 (20 or 40 µM) were introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. The level of ZAAT inside the cells (IC) and in the media (EC) and the level of ERdj3 were measured by Western blot. GAPDH was used as loading control. (B) siERdj3 does not affect ZAAT secretion. By ELISA, total AAT was measured in the media from the same samples as in panel A experiment. Total AAT was shown in picomolar concentration. (C) siERdj3 accelerates ZAAT clearance from the ER of hepatocytes. NT siRNA or 20 nM of siERdj3 was introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. IC ZAAT and EC ZAAT from NT siRNA and siERdj3‐treated samples were shown after pulse‐chase radiolabeling. (D) ZAAT polymers are reduced following siERdj3 treatment. NT siRNA or 20 µM of siERdj3 were introduced 24 h post ZAAT transfection to AAT KO Huh 7.5 cells. ZAAT polymers were immunostained with 2C1 antibody (Alexa 594; red). (E) Integrated density of red fluorescent measurement from four images per sample.

    Article Snippet: Bafilomycin A1, MG132, Brefeldin A, and mouse monoclonal anti‐β‐actin were purchased from Sigma (St. Louis, MO), and 2C1 against human AAT polymers was purchased from Hycult biotech (Netherlands).

    Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Pulse Chase, Radioactivity

    Summary of the nephelometric measurments of  A1AT  concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.

    Journal: PLoS ONE

    Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

    doi: 10.1371/journal.pone.0135316

    Figure Lengend Snippet: Summary of the nephelometric measurments of A1AT concentration in 966 samples analysed using routine serum protein electrophoresis (SPE) methods.

    Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

    Techniques: Concentration Assay, Protein Electrophoresis

    Thresholds for suspicion of severe or intermediate A1ATD according to SPE.

    Journal: PLoS ONE

    Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

    doi: 10.1371/journal.pone.0135316

    Figure Lengend Snippet: Thresholds for suspicion of severe or intermediate A1ATD according to SPE.

    Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

    Techniques:

    The serum concentration of A1AT was determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% PAGE, and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration in the loaded sample per well is indicated at the bottom of the gels.

    Journal: PLoS ONE

    Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

    doi: 10.1371/journal.pone.0135316

    Figure Lengend Snippet: The serum concentration of A1AT was determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% PAGE, and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration in the loaded sample per well is indicated at the bottom of the gels.

    Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

    Techniques: Concentration Assay, Western Blot

    Serum concentrations of A1AT were determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% SDS-PAGE (without denaturing agents), and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration per well in the loaded serum sample is indicated at the bottom of the gels.

    Journal: PLoS ONE

    Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

    doi: 10.1371/journal.pone.0135316

    Figure Lengend Snippet: Serum concentrations of A1AT were determined using routine nephelometric analysis. Equal volumes of diluted serum samples (1:100 in sterile 0.15M NaCl) were loaded and separated on the 7.5% SDS-PAGE (without denaturing agents), and Western blots were performed using specific anti-A1AT antibodies: a) mouse monoclonal against human A1AT; b) mouse monoclonal antibody, ATZ11, recognizing Z-type polymers of A1AT and c) mouse monoclonal 2C1 specific against A1AT polymers. Relative A1AI concentration per well in the loaded serum sample is indicated at the bottom of the gels.

    Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

    Techniques: SDS Page, Western Blot, Concentration Assay

    a . Purified plasma A1AT (2.5 mg/ml) was heated at 60°C and aliquots were removed at indicated time points for analysis by a non-denaturing PAGE followed by Western blotting using rabbit polyclonal anti-A1AT antibody. b . For a more detailed analysis of the heat-treated A1AT a 4–15% gradient SDS-PAGE was used. This method is designed for separation of high molecular weight polymers. Electrophoretic separation of A1AT was followed by Western blot analysis with an anti-human A1AT antibody as above. The immunoblot patterns indicate that heat-treated A1AT typically comprises a mixture of a monomer and various sized polymers. The figure shows representative blots from 5 experiments. c . M A1AT heat-treated for 5 min exhibited a typical five band pattern in the IEF gel (lane 1). When M A1AT was heated for 15 min or longer t, the three prominent bands towards the anode were no longer detectable (lanes 2–9). Instead, a continuous smeared band occurred at the cathode (lane 3), these results paralleled those with heat-induced A1AT polymerization over time (Fig 3b). d . Initial concentrations of purified A1AT were 2.35 ± 0.042 mg/ml; n = 3. The same A1AT protein heated for a short period yielded a higher nephelometric signal than native protein. However, heating of A1AT for 45 min or longer, resulted in a remarkable decrease in the nephelometric signal relative to the native protein.

    Journal: PLoS ONE

    Article Title: How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

    doi: 10.1371/journal.pone.0135316

    Figure Lengend Snippet: a . Purified plasma A1AT (2.5 mg/ml) was heated at 60°C and aliquots were removed at indicated time points for analysis by a non-denaturing PAGE followed by Western blotting using rabbit polyclonal anti-A1AT antibody. b . For a more detailed analysis of the heat-treated A1AT a 4–15% gradient SDS-PAGE was used. This method is designed for separation of high molecular weight polymers. Electrophoretic separation of A1AT was followed by Western blot analysis with an anti-human A1AT antibody as above. The immunoblot patterns indicate that heat-treated A1AT typically comprises a mixture of a monomer and various sized polymers. The figure shows representative blots from 5 experiments. c . M A1AT heat-treated for 5 min exhibited a typical five band pattern in the IEF gel (lane 1). When M A1AT was heated for 15 min or longer t, the three prominent bands towards the anode were no longer detectable (lanes 2–9). Instead, a continuous smeared band occurred at the cathode (lane 3), these results paralleled those with heat-induced A1AT polymerization over time (Fig 3b). d . Initial concentrations of purified A1AT were 2.35 ± 0.042 mg/ml; n = 3. The same A1AT protein heated for a short period yielded a higher nephelometric signal than native protein. However, heating of A1AT for 45 min or longer, resulted in a remarkable decrease in the nephelometric signal relative to the native protein.

    Article Snippet: Western blots were accomplished using mouse monoclonal anti-A1AT (1:500; B9, sc-59438, SantaCruz, USA), mouse monoclonal antibody, ATZ11, which recognizes both polymerized and elastase-complexed forms of A1AT (1:100, our own) and mouse monoclonal 2C1 that specifically recognizes A1AT polymeric forms (1:100, Catalog #: HM2289, Hycult Biotech, The Netherlands).

    Techniques: Purification, Western Blot, SDS Page, Molecular Weight