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  • 99
    Thermo Fisher bca assay kit
    Bca Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phosphate Buffered Saline, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rabbit reticulocyte lysate
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    Promega tnt coupled reticulocyte lysate system
    Characterization of <t>NR4A1-induced</t> stimulation of the promoter activity of the TSHβ gene. A) To determine the region responsible for the NR4A1-induced stimulation of the TSHβ promoter activity, a series of deletion constructs of the human TSHβ promoter were established. The consensus sequence of the NR4A1 response element (NurRE) was identified in the region between -1091 and -1083 from the transcription start site. The right figure shows the fold-stimulation by NR4A in each deletion mutant of the TSHβ gene. Putative Pit1 and GATA2 binding sites, and the negative thyroid hormone response element (NRE) were indicated as pit1, GATA2 and NRE. All the deletion constructs including pA3TSHβ (−138 ∼ +37) -Luc showed similar stimulation by NR4A1, suggesting the region responsible to be within this area. Fold-increase in activity against that without TRH is shown. B) Chromatin-immunoprecipitation (ChIP) assays were performed with anti-NR4A1 antibody (NR4A1) and normal mouse IgG as a negative control (IgG). GH4C1 cells were transfected with pA3TSHβ (-1192 ∼ +37)-Luc in the presence or absence of 100 n M TRH. Amplified PCR products were stained with ethidium bromide in 2% agarose gels and scanned with a Molecular Imager FX. Chip assays demonstrated that NR4A1 was recruited to the region between -138 and +13 of the TSHβ promoter (-138/+13), but not the region containing NurRE (NuRE). Addition of TRH did not alter recruitment of NR4A1 on the gene (data not shown). All ChIP assays were repeated at least three times. C) The EMSA was performed using a fragment of the radiolabeled POMC promoter containing typical NurRE and a fragment containing the human TSHβ bp -123∼-87. There was no binding of NR4A1 to the TSHβ gene in the absence (−) or presence (+) of 100 nM TRH, while the POMC promoter fragment bound to NR4A1. Arrows indicate NR4A1 bound to the POMC promoter as monomers and dimers. NR4A1 was synthesized by the <t>TNT-coupled</t> reticulocyte lysate system (NR4A1). Lysate indicates un-programmed lysate. D) Effect of knockdown of NR4A1 on the TRH-induced stimulation of the promoter activity of the TSHβ gene. D-1. The NR4A1 mRNA levels were measured after transfection with siCONTROL or siNR4A1 in GH4C1 cells. Transfection of siNR4A1 led to an approximately 80% reduction of the mRNA level after 48 hr. Data are presented as the mean ± SEM from three experiments. **, p
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    Thermo Fisher bca protein assay
    Characterization of <t>NR4A1-induced</t> stimulation of the promoter activity of the TSHβ gene. A) To determine the region responsible for the NR4A1-induced stimulation of the TSHβ promoter activity, a series of deletion constructs of the human TSHβ promoter were established. The consensus sequence of the NR4A1 response element (NurRE) was identified in the region between -1091 and -1083 from the transcription start site. The right figure shows the fold-stimulation by NR4A in each deletion mutant of the TSHβ gene. Putative Pit1 and GATA2 binding sites, and the negative thyroid hormone response element (NRE) were indicated as pit1, GATA2 and NRE. All the deletion constructs including pA3TSHβ (−138 ∼ +37) -Luc showed similar stimulation by NR4A1, suggesting the region responsible to be within this area. Fold-increase in activity against that without TRH is shown. B) Chromatin-immunoprecipitation (ChIP) assays were performed with anti-NR4A1 antibody (NR4A1) and normal mouse IgG as a negative control (IgG). GH4C1 cells were transfected with pA3TSHβ (-1192 ∼ +37)-Luc in the presence or absence of 100 n M TRH. Amplified PCR products were stained with ethidium bromide in 2% agarose gels and scanned with a Molecular Imager FX. Chip assays demonstrated that NR4A1 was recruited to the region between -138 and +13 of the TSHβ promoter (-138/+13), but not the region containing NurRE (NuRE). Addition of TRH did not alter recruitment of NR4A1 on the gene (data not shown). All ChIP assays were repeated at least three times. C) The EMSA was performed using a fragment of the radiolabeled POMC promoter containing typical NurRE and a fragment containing the human TSHβ bp -123∼-87. There was no binding of NR4A1 to the TSHβ gene in the absence (−) or presence (+) of 100 nM TRH, while the POMC promoter fragment bound to NR4A1. Arrows indicate NR4A1 bound to the POMC promoter as monomers and dimers. NR4A1 was synthesized by the <t>TNT-coupled</t> reticulocyte lysate system (NR4A1). Lysate indicates un-programmed lysate. D) Effect of knockdown of NR4A1 on the TRH-induced stimulation of the promoter activity of the TSHβ gene. D-1. The NR4A1 mRNA levels were measured after transfection with siCONTROL or siNR4A1 in GH4C1 cells. Transfection of siNR4A1 led to an approximately 80% reduction of the mRNA level after 48 hr. Data are presented as the mean ± SEM from three experiments. **, p
    Bca Protein Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 42534 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pierce far western blot kit for biotinylated proteins
    GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. <t>Biotinylated</t> nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.
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    Thermo Fisher iblot gel transfer stacks
    GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. <t>Biotinylated</t> nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.
    Iblot Gel Transfer Stacks, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nitrocellulose pre cut blotting membranes
    GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. <t>Biotinylated</t> nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.
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    Millipore triton x 100
    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
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    Millipore amicon ultra 4 centrifugal filter
    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
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    Promega flexi rabbit reticulocyte lysate system
    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
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    Millipore bsa
    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
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    Thermo Fisher dntp mix
    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
    Dntp Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
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    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
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    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
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    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .
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    Characterization of NR4A1-induced stimulation of the promoter activity of the TSHβ gene. A) To determine the region responsible for the NR4A1-induced stimulation of the TSHβ promoter activity, a series of deletion constructs of the human TSHβ promoter were established. The consensus sequence of the NR4A1 response element (NurRE) was identified in the region between -1091 and -1083 from the transcription start site. The right figure shows the fold-stimulation by NR4A in each deletion mutant of the TSHβ gene. Putative Pit1 and GATA2 binding sites, and the negative thyroid hormone response element (NRE) were indicated as pit1, GATA2 and NRE. All the deletion constructs including pA3TSHβ (−138 ∼ +37) -Luc showed similar stimulation by NR4A1, suggesting the region responsible to be within this area. Fold-increase in activity against that without TRH is shown. B) Chromatin-immunoprecipitation (ChIP) assays were performed with anti-NR4A1 antibody (NR4A1) and normal mouse IgG as a negative control (IgG). GH4C1 cells were transfected with pA3TSHβ (-1192 ∼ +37)-Luc in the presence or absence of 100 n M TRH. Amplified PCR products were stained with ethidium bromide in 2% agarose gels and scanned with a Molecular Imager FX. Chip assays demonstrated that NR4A1 was recruited to the region between -138 and +13 of the TSHβ promoter (-138/+13), but not the region containing NurRE (NuRE). Addition of TRH did not alter recruitment of NR4A1 on the gene (data not shown). All ChIP assays were repeated at least three times. C) The EMSA was performed using a fragment of the radiolabeled POMC promoter containing typical NurRE and a fragment containing the human TSHβ bp -123∼-87. There was no binding of NR4A1 to the TSHβ gene in the absence (−) or presence (+) of 100 nM TRH, while the POMC promoter fragment bound to NR4A1. Arrows indicate NR4A1 bound to the POMC promoter as monomers and dimers. NR4A1 was synthesized by the TNT-coupled reticulocyte lysate system (NR4A1). Lysate indicates un-programmed lysate. D) Effect of knockdown of NR4A1 on the TRH-induced stimulation of the promoter activity of the TSHβ gene. D-1. The NR4A1 mRNA levels were measured after transfection with siCONTROL or siNR4A1 in GH4C1 cells. Transfection of siNR4A1 led to an approximately 80% reduction of the mRNA level after 48 hr. Data are presented as the mean ± SEM from three experiments. **, p

    Journal: PLoS ONE

    Article Title: NR4A1 (Nur77) Mediates Thyrotropin-Releasing Hormone-Induced Stimulation of Transcription of the Thyrotropin ? Gene: Analysis of TRH Knockout Mice

    doi: 10.1371/journal.pone.0040437

    Figure Lengend Snippet: Characterization of NR4A1-induced stimulation of the promoter activity of the TSHβ gene. A) To determine the region responsible for the NR4A1-induced stimulation of the TSHβ promoter activity, a series of deletion constructs of the human TSHβ promoter were established. The consensus sequence of the NR4A1 response element (NurRE) was identified in the region between -1091 and -1083 from the transcription start site. The right figure shows the fold-stimulation by NR4A in each deletion mutant of the TSHβ gene. Putative Pit1 and GATA2 binding sites, and the negative thyroid hormone response element (NRE) were indicated as pit1, GATA2 and NRE. All the deletion constructs including pA3TSHβ (−138 ∼ +37) -Luc showed similar stimulation by NR4A1, suggesting the region responsible to be within this area. Fold-increase in activity against that without TRH is shown. B) Chromatin-immunoprecipitation (ChIP) assays were performed with anti-NR4A1 antibody (NR4A1) and normal mouse IgG as a negative control (IgG). GH4C1 cells were transfected with pA3TSHβ (-1192 ∼ +37)-Luc in the presence or absence of 100 n M TRH. Amplified PCR products were stained with ethidium bromide in 2% agarose gels and scanned with a Molecular Imager FX. Chip assays demonstrated that NR4A1 was recruited to the region between -138 and +13 of the TSHβ promoter (-138/+13), but not the region containing NurRE (NuRE). Addition of TRH did not alter recruitment of NR4A1 on the gene (data not shown). All ChIP assays were repeated at least three times. C) The EMSA was performed using a fragment of the radiolabeled POMC promoter containing typical NurRE and a fragment containing the human TSHβ bp -123∼-87. There was no binding of NR4A1 to the TSHβ gene in the absence (−) or presence (+) of 100 nM TRH, while the POMC promoter fragment bound to NR4A1. Arrows indicate NR4A1 bound to the POMC promoter as monomers and dimers. NR4A1 was synthesized by the TNT-coupled reticulocyte lysate system (NR4A1). Lysate indicates un-programmed lysate. D) Effect of knockdown of NR4A1 on the TRH-induced stimulation of the promoter activity of the TSHβ gene. D-1. The NR4A1 mRNA levels were measured after transfection with siCONTROL or siNR4A1 in GH4C1 cells. Transfection of siNR4A1 led to an approximately 80% reduction of the mRNA level after 48 hr. Data are presented as the mean ± SEM from three experiments. **, p

    Article Snippet: Double-stranded oligonucleotides were labeled with [α32 P]dCTP by a fill-in reaction using a Klenow fragment of DNA polymerase I. NR4A1 was synthesized by in vitro transcription/translation from pcDNA3.1-NR4A1 using T7 RNA polymerase and the TNT-coupled reticulocyte lysate system (Promega Corporation).

    Techniques: Activity Assay, Construct, Sequencing, Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Transfection, Amplification, Polymerase Chain Reaction, Staining, Synthesized

    GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. Biotinylated nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.

    Journal: bioRxiv

    Article Title: GRP78 and Integrins Play Different Roles in Host Cell Invasion During Mucormycosis

    doi: 10.1101/2020.04.29.069666

    Figure Lengend Snippet: GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. Biotinylated nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.

    Article Snippet: Thus, far-western blot analysis using recombinant human GRP78 and anti-GRP78 antibodies was done to identify R.delemar ligand.

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

    The cytoplasmic interaction domain underlies Hrd1-dependent ERAD.  (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in  Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD  cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD  cells expressing Hrd1 mutants (L489A, R503L). (C) Representative  35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD  cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students  t -test is shown. ** P ≤0.01. (E) Representative  35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students  t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in  C. elegans  disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .

    Journal: Journal of Cell Science

    Article Title: Conserved cytoplasmic domains promote Hrd1 ubiquitin ligase complex formation for ER-associated degradation (ERAD)

    doi: 10.1242/jcs.206847

    Figure Lengend Snippet: The cytoplasmic interaction domain underlies Hrd1-dependent ERAD. (A) Degradation of CD147 monitored by CHX chase (0, 2, 4 h; 10 μg/ml) and western blotting in Flp -In T-REx 293 cells, and HEK293 Flp-In/Hrd1-KD cells stably reintroduced with S-tagged Hrd1 variants. Lysates of LMNG-solubilised post-nuclear fractions were separated by SDS-PAGE and the resulting blots probed with antibodies against CD147 and Hrd1 (S-tag). Black arrowheads indicate the immature, ER-localised form of CD147 while asterisks designate the post-ER, mature forms. (B) Degradation of CD147 monitored in HEK293 Flp-In/Hrd1-KD cells expressing Hrd1 mutants (L489A, R503L). (C) Representative 35 S-Met/Cys pulse-chase assays (0, 2 and 4 h) of the HA-tagged ERAD substrate NHK-HA expressed in HEK293 FlpIn/Hrd1-KD cells with or without DOX-induced expression of Hrd1-S (empty, FL, Δ480-535 or R503L). Radiolabelled substrates immunoprecipitated from 1% Triton X-100 lysates by anti-HA were separated by SDS-PAGE. (D) Quantification of biological replicates in C. Band intensities quantified by phosphorimaging (BioRad) were normalised to values at t=0 h. Results are presented as mean±s.e.m. for each ( n =3). Significance as determined by Students t -test is shown. ** P ≤0.01. (E) Representative 35 S-Met/Cys pulse-chase assays of NHK-HA transiently expressed in Flp-In T-REx 293 cells WT, ΔHrd1, ΔFAM8A1 and ΔHerp cells. Samples were collected and processed as in C. (F) Quantification of biological replicates in E. Band intensities quantified by phosphorimaging were normalised to values at t=0 h. Mean±s.e.m. are plotted for each ( n =3). Significance as determined by Students t -test is shown. *** P ≤0.001, **** P ≤0.0001. (G) Fluorescence images of the ERAD-substrate CPL-1W32A,Y35A-YFP in C. elegans disrupted for Hrd1 (sel-11), Herp (tag-353) or FAM8A1 (F48B9.8). (H) Western blot of CPL-1W32A,Y35A-YFP and tubulin from the mutants of Hrd1 complex. (I) Quantification of band intensities from I. Displayed as fold-change in YFP or tubulin with mean±s.e.m. are shown for each ( n =3). (J) Kaplan–Meyer survival curves of Hrd1 complex mutant worms. Calculated mean lifespans are: WT, 18.5 days; cup-2 and tag-353 (gk443)l, 18.9 days; F48B9.8 (gk272969), 20.2 days; sel-11 (nDf59) V, 10.5 days; sel-1 (e1948)V, 10.1 days. Raw data from biological replicates ( n .

    Article Snippet: Cells were lysed in buffer containing 1% Triton X-100 as above, and the detergent-soluble, post-nuclear lysates pre-cleared using mouse IgG conjugated to agarose beads followed by immunoprecipitation with anti-HA-agarose beads (Sigma).

    Techniques: Western Blot, Stable Transfection, SDS Page, Expressing, Pulse Chase, Immunoprecipitation, Fluorescence, Mutagenesis

    Hrd1 forms a complex with FAM8A1 through cytoplasmic and membrane interactions.  (A) Illustration of Hrd1 and FAM8A1 full-length (FL), transmembrane (TM) and cytoplasmic (CY) domain expression constructs. DFP, a non-fluorescent GFP variant appended to the FAM8A1 TM region (aa 229-413) was used to stabilise expression. (B) S-tagged FAM8A1 (FL, TM and CY) and Hrd1–Myc (FL, TM and CY) co-expressed in HEK293 Flp-In/Hrd1-KD  cells were solubilised in 1% LMNG and complexes affinity purified (AP) by S-Ag. Input (20%) and AP are shown in resulting blots probed with anti-S-tag (FAM8A1) and anti-Myc (Hrd1) with GAPDH serving as a loading control. (C) Hrd1-S (FL, TM and CY) co-expressed with Myc–FAM8A1 (FL) in HEK293 Flp-In/Hrd1-KD  cells lysed in either 1% LMNG (left) or 1% Triton X-100 (right) and isolated using S-Ag. Input and AP are shown in resulting blots probed for S-tag (Hrd1), Myc (FAM8A1) and SEL1L. (D) Myc–FAM8A1 (FL, TM and CY) co-expressed with Hrd1-S (FL, TM and CY). Western blots were also probed for SEL1L, Herp and Ube2j1; GAPDH is presented as a loading control.

    Journal: Journal of Cell Science

    Article Title: Conserved cytoplasmic domains promote Hrd1 ubiquitin ligase complex formation for ER-associated degradation (ERAD)

    doi: 10.1242/jcs.206847

    Figure Lengend Snippet: Hrd1 forms a complex with FAM8A1 through cytoplasmic and membrane interactions. (A) Illustration of Hrd1 and FAM8A1 full-length (FL), transmembrane (TM) and cytoplasmic (CY) domain expression constructs. DFP, a non-fluorescent GFP variant appended to the FAM8A1 TM region (aa 229-413) was used to stabilise expression. (B) S-tagged FAM8A1 (FL, TM and CY) and Hrd1–Myc (FL, TM and CY) co-expressed in HEK293 Flp-In/Hrd1-KD cells were solubilised in 1% LMNG and complexes affinity purified (AP) by S-Ag. Input (20%) and AP are shown in resulting blots probed with anti-S-tag (FAM8A1) and anti-Myc (Hrd1) with GAPDH serving as a loading control. (C) Hrd1-S (FL, TM and CY) co-expressed with Myc–FAM8A1 (FL) in HEK293 Flp-In/Hrd1-KD cells lysed in either 1% LMNG (left) or 1% Triton X-100 (right) and isolated using S-Ag. Input and AP are shown in resulting blots probed for S-tag (Hrd1), Myc (FAM8A1) and SEL1L. (D) Myc–FAM8A1 (FL, TM and CY) co-expressed with Hrd1-S (FL, TM and CY). Western blots were also probed for SEL1L, Herp and Ube2j1; GAPDH is presented as a loading control.

    Article Snippet: Cells were lysed in buffer containing 1% Triton X-100 as above, and the detergent-soluble, post-nuclear lysates pre-cleared using mouse IgG conjugated to agarose beads followed by immunoprecipitation with anti-HA-agarose beads (Sigma).

    Techniques: Expressing, Construct, Variant Assay, Affinity Purification, Isolation, Western Blot