anti human aat polymer selective  (Hycult Biotech)


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    Structured Review

    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
    https://www.bioz.com/result/anti human aat polymer selective/product/Hycult Biotech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human aat polymer selective - by Bioz Stars, 2024-07
    93/100 stars

    Images

    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

    anti human aat polymer selective  (Hycult Biotech)


    Bioz Verified Symbol Hycult Biotech is a verified supplier
    Bioz Manufacturer Symbol Hycult Biotech manufactures this product  
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  • 93

    Structured Review

    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
    https://www.bioz.com/result/anti human aat polymer selective/product/Hycult Biotech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human aat polymer selective - by Bioz Stars, 2024-07
    93/100 stars

    Images

    1) Product Images from "Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin"

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.014372

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

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

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

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

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

    Techniques Used: Transfection, Staining, Transformation Assay

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

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

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    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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    anti human aat polymer selective - by Bioz Stars, 2024-07
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    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