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: 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: Biomolecules

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

doi: 10.3390/biom12030380

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

Article Snippet: Membranes were blocked for 1 h with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4 °C with specific primary antibodies: mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), or mouse monoclonal anti-AAT polymer antibody (LG96, 1:500).

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

Journal: Biomolecules

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

doi: 10.3390/biom12030380

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

Article Snippet: Membranes were blocked for 1 h with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4 °C with specific primary antibodies: mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), or mouse monoclonal anti-AAT polymer antibody (LG96, 1:500).

Techniques: Western Blot, Purification, Isolation, Positive Control

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 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

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