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

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

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

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

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

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

Journal: The EMBO Journal

Article Title: N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

doi: 10.15252/embj.2020107240

Figure Lengend Snippet: N‐glycan. Glucoses, blue; mannoses, green; N‐acetylglucosamines, black. GI removes glucose 1. GII removes glucoses 2 and 3. UGGT1 adds‐back glucose 3. UGGT1 can only modify the terminal mannose on branch A (black circle). αMI and EDEM1, EDEM2, and EDEM3 remove light green, α 1,2 ‐bonded, mannose residues. CST inhibits GI and GII. KIF inhibits α 1,2 mannosidases. AAT NNN and ATZ NNN . N‐glycans in red. The E342K mutation of ATZ NNN is in blue. Control of polymerization propensity for the Halo‐tagged versions of AAT (lanes 1 and 4) and of ATZ (lanes 2 and 5). Polymers and monomers corresponding to (Miranda et al , ; Ronzoni et al , ) are seen with anti‐Halo (lanes 1‐3) or with the polymer‐specific 2C1 antibody (lanes 4‐6). Synthesis control in SDS‐polyacrylamide gel by visualization of tetramethylrhodamine (TMR) ligand‐labeled Halo‐AAT NNN and Halo‐ATZ NNN (lanes 7‐9). HaloTag pulse‐chase analysis in WT MEF monitoring fluorescent Halo‐AAT NNN during up to 6 h of chase in the presence of BafA1. The fluorescent JF646‐Halo ligand and the polymer‐specific 2C1 antibody stains are shown. Same as (D) for fluorescent Halo‐ATZ NNN . Quantification of accumulation of fluorescent Halo‐AAT NNN and Halo‐ATZ NNN within LAMP1‐positive endolysosomes. Mean ± SEM, n = 20, 11, 10, 13 for Halo‐AAT NNN , n = 18, 12, 12, 13 for Halo‐ATZ NNN . Two‐way ANOVA and Sidak’s multiple comparison test, ns P > 0.05, *** P < 0.001. Data information: Scale bars 10 µm. Source data are available online for this figure.

Article Snippet: Antibodies used in this study are the monoclonal 2C1 anti‐polymeric ATZ (Hycult Biotech) (Miranda et al , ), polyclonal anti‐HA (Sigma), monoclonal anti‐HA probe F7 (Santa Cruz Biotech.), anti‐LAMP1 (Hybridoma Bank, 1D4B deposited by J.T.

Techniques: Mutagenesis, Labeling, Pulse Chase

Journal: The EMBO Journal

Article Title: N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

doi: 10.15252/embj.2020107240

Figure Lengend Snippet: A Confocal laser scanning microscopy analyses in cells treated with BafA1. Total ATZ NNN ‐HA (red) or ATZ NNN polymers (magenta) in LAMP1‐positive endolysosome (green) in WT MEF. B Radiolabeled ATZ NNN enters in 2C1‐positive polymers that are immunoisolated at the end of the chase times. BafA1 inhibits polymers degradation. Quantification for n = 4. Unpaired two‐tailed t‐test, **** P < 0.0001. C Same as (A) in Cnx ‐KO MEF. D Same as (A) in WT MEF exposed to kifunensine (KIF). E Same as (A) in WT MEF exposed to Castanospermine (CST). F Same as (A) in sCNX MEF. G Same as (A) in Uggt1 ‐KO MEF. H, I (H) Quantification of HA‐positive or (I) of 2C1‐positive endolysosomes (mean, n = 18, 14, 17, 13, 12, 12 cells for panels (A, C–G), respectively). One‐way ANOVA and Dunnett’s multiple comparison test, ns P > 0.05, **** P < 0.0001. J Halo‐ATZ NNN in Uggt1 ‐KO MEF mock‐transfected (empty pcDNA3 plasmid). K, L (K) Same as (J), with a plasmid for expression of active or (L) inactive UGGT1. M Quantification of Halo‐ATZ NNN ‐positive endolysosomes (mean, n = 22, 10, 15 cells for panels (J–L), respectively). One‐way ANOVA and Dunnett’s multiple comparison test, ns P > 0.05, **** P < 0.0001. Data information: Scale bars 10 μm. Source data are available online for this figure.

Article Snippet: Antibodies used in this study are the monoclonal 2C1 anti‐polymeric ATZ (Hycult Biotech) (Miranda et al , ), polyclonal anti‐HA (Sigma), monoclonal anti‐HA probe F7 (Santa Cruz Biotech.), anti‐LAMP1 (Hybridoma Bank, 1D4B deposited by J.T.

Techniques: Confocal Laser Scanning Microscopy, Two Tailed Test, Transfection, Plasmid Preparation, Expressing

Journal: The EMBO Journal

Article Title: N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

doi: 10.15252/embj.2020107240

Figure Lengend Snippet: A Control of genetic Fam134B‐ , Sec62‐, and Tex264 ‐KO, respectively. * shows a non‐specific cross‐reaction of the antibody. B Same as Fig in cells edited with CRISPR‐Cas9 to delete Fam134B (Fregno et al , ) transfected with an empty pcDNA3 plasmid. C Same as (B) in cells back‐transfected with active FAM134B. D Same as (B) in cells back‐transfected with inactive FAM134B (FAM134BLIR), which cannot bind LC3. E Quantification of HA‐positive endolysosomes (B‐D) (mean, n = 17, 13 and 10). One‐way ANOVA and Dunnett’s multiple comparison test, ns P > 0.05, **** P < 0.0001. F HEK293 cells transfected with empty vector (lane 1), ATZ‐HA (2), FAM134B‐V5 (3), FAM134B‐V5 and ATZ‐HA (4), or FAM134BLIR‐V5 and ATZ‐HA (5), treated for 6 h with 100 nM BafA1 and then cross‐linked with DSP before lysis (see Materials and Methods). FAM134BLIR‐V5 interacts with CNX and ATZ‐HA, but not with LC3. G–I (G) Same as (B) in control CRISPR cells and (H) in cells edited with CRISPR/Cas9 to delete Sec62 (Fumagalli et al , ) or (I) Tex264 . J, K (J) Quantification of (G–I) for HA‐positive endolysosomes, (mean, n = 11, 11, 22) and (K) for 2C1‐positive endolysosomes (mean, n = 11, 9, 11). One‐way ANOVA and Dunnett’s multiple comparison test, ns P > 0.05. Data information: Scale bars 10 μm. Source data are available online for this figure.

Article Snippet: Antibodies used in this study are the monoclonal 2C1 anti‐polymeric ATZ (Hycult Biotech) (Miranda et al , ), polyclonal anti‐HA (Sigma), monoclonal anti‐HA probe F7 (Santa Cruz Biotech.), anti‐LAMP1 (Hybridoma Bank, 1D4B deposited by J.T.

Techniques: CRISPR, Transfection, Plasmid Preparation, Lysis

Journal: The EMBO Journal

Article Title: N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

doi: 10.15252/embj.2020107240

Figure Lengend Snippet: Flow cytometric analyses of intracellular ATZ NNN detected with anti‐HA ( X ‐axis) and anti‐2C1 antibody ( Y ‐axis). Cells containing ATZ NNN polymers are in the upper right quadrant being immunoreactive to both anti‐HA and 2C1 antibody. Analyses have been performed for the conditions and cell lines shown in Fig . Control of polymerization propensity for the ATZ NNN in WT (lanes 1, 2), Uggt1 ‐KO (lane 3), and Cnx‐ KO MEF (lane 4). Polymers and monomers are seen with anti‐HA (upper panel) or with the polymer‐specific 2C1 antibody (middle panel). Total protein in SDS‐polyacrylamide gels in lower panel. Same as (A) for cells expressing glycosylation mutants as in Fig . Same as (B) for the 4 glycosylation mutants shown in Fig , for AAT and for the non‐polymerogenic, disease‐causing NHK variant of AAT expressed in HEK293 cells. Source data are available online for this figure.

Article Snippet: Antibodies used in this study are the monoclonal 2C1 anti‐polymeric ATZ (Hycult Biotech) (Miranda et al , ), polyclonal anti‐HA (Sigma), monoclonal anti‐HA probe F7 (Santa Cruz Biotech.), anti‐LAMP1 (Hybridoma Bank, 1D4B deposited by J.T.

Techniques: Expressing, Variant Assay

Journal: The EMBO Journal

Article Title: N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

doi: 10.15252/embj.2020107240

Figure Lengend Snippet: A ATZ glycosylation mutants and their electrophoretic mobility. N is the acceptor site asparagine; Q is for the mutation to glutamine that prevents glycosylation. The E342K mutation of ATZ NNN is in blue. B–I Confocal laser scanning microscopy analyses (as in Fig ) in WT MEF treated with BafA1. J, K (J) Quantification of ATZ xxx ‐positive endolysosomes and (K) of ATZ xxx polymers‐positive endolysosomes (mean, n = 18, 30, 24, 14, 27, 22, 27, 18 cells for HA stain and n = 12, 14, 14, 11, 14, 14, 17, 11 cells for 2C1 stain of panels B‐I, respectively). One‐way ANOVA and Dunnett’s multiple comparison test, ns P > 0.05, **** P < 0.0001. Data information: Scale bars 10 μm. Source data are available online for this figure.

Article Snippet: Antibodies used in this study are the monoclonal 2C1 anti‐polymeric ATZ (Hycult Biotech) (Miranda et al , ), polyclonal anti‐HA (Sigma), monoclonal anti‐HA probe F7 (Santa Cruz Biotech.), anti‐LAMP1 (Hybridoma Bank, 1D4B deposited by J.T.

Techniques: Mutagenesis, Confocal Laser Scanning Microscopy, Staining

Journal: The EMBO Journal

Article Title: N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

doi: 10.15252/embj.2020107240

Figure Lengend Snippet: A–D Same as Fig in HEK293 cells. Endolysosomes are labeled with GFP‐RAB7. E Quantification of HA‐positive endolysosomes (A‐D) (mean, n = 18, 16, 16, 15 cells). F–I Same as Fig in HEPA1‐6 cells. J Quantification of (F–I) (mean, n = 13, 12, 12, 13 cells). K–N Same as Fig in HeLa cells. O (J) Quantification of (K–N) (mean, n = 16, 11, 14, 14 cells). P Same as Fig in CHO cells. The polymer‐specific 2C1 antibody stain is shown. Data information: One‐way ANOVA and Dunnett’s multiple comparison test, ns P > 0.05, **** P < 0.0001. Scale bars 10 µm.

Article Snippet: Antibodies used in this study are the monoclonal 2C1 anti‐polymeric ATZ (Hycult Biotech) (Miranda et al , ), polyclonal anti‐HA (Sigma), monoclonal anti‐HA probe F7 (Santa Cruz Biotech.), anti‐LAMP1 (Hybridoma Bank, 1D4B deposited by J.T.

Techniques: Labeling, Staining

Journal: The EMBO Journal

Article Title: N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

doi: 10.15252/embj.2020107240

Figure Lengend Snippet: Radiolabeled ATZ XXX enter in 2C1‐positive polymers that are immunoisolated at the end of the given chase times. Total ATZ NNN is immunoisolated with HA. Protein load and 35 S‐methionine/cysteine incorporated in nascent polypeptides are shown. Quantification of radiolabeled polymers. Green columns are for the ERLAD‐competent ATZ NNN and ATZ QNQ variants (see Fig and and ), red columns are for the ERLAD‐incompetent ATZ QQN and ATZ QQQ (see Fig ) (mean, n = 5 for ATZ NNN , n = 3 for ATZ QNQ , n = 4 ATZ QQN and n = 4 for ATZ QQQ ). Two‐way ANOVA and Sidak’s multiple comparison test, ns P > 0.05, * P < 0.005, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Co‐immunoprecipitation of ATZ XXX (Bait, lower panel) with CNX (upper panel) and BiP (middle panel). Protein content in the total cell extracts (TCE) is shown. Quantification of (C) for CNX association ( n = 3 ). Unpaired two‐tailed t ‐test, ** P < 0.01*** P < 0.001, **** P < 0.0001. Quantification of (C) for BiP association ( n = 3 for ATZ NNN and ATZ QQN ; n = 4 for ATZ QNQ and ATZ QQQ ). Unpaired two‐tailed t‐test, * P < 0.005, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Detergent‐insoluble fraction of (C). Confocal laser scanning microscopy in Flp‐In 3T3 cells stably expressing ATZ NNN in the absence of BafA1 (to allow clearance of misfolded model proteins delivered within endolysosomes). Co‐localization with Calnexin (CNX). Same as (G) for BiP co‐localization. Same as (G) for ATZ QQQ . Same as (H) for ATZ QQQ . HaloTag pulse‐chase analysis in MEF monitoring fluorescent Halo‐ATZ NNN during up to 24 h. Same as (K) for fluorescent Halo‐ATZ QQQ . Arrowheads show select clusters of JF646‐labeled Halo‐ATZ QQQ . Arrowheads show co‐localization Halo‐ATZ QQQ :BiP. Data information: Scale bars 10 μm. Source data are available online for this figure.

Article Snippet: Antibodies used in this study are the monoclonal 2C1 anti‐polymeric ATZ (Hycult Biotech) (Miranda et al , ), polyclonal anti‐HA (Sigma), monoclonal anti‐HA probe F7 (Santa Cruz Biotech.), anti‐LAMP1 (Hybridoma Bank, 1D4B deposited by J.T.

Techniques: Immunoprecipitation, Two Tailed Test, Confocal Laser Scanning Microscopy, Stable Transfection, Expressing, Pulse Chase, Labeling

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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