nonidet p 40  (Thermo Fisher)


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    Name:
    NP 40 Surfact Amps Detergent Solution
    Description:
    Thermo Scientific Surfact Amps NP 40 Detergent Solution is highly purified NP 40 detergent stabilized as a 10 solution in your choice of either 10 mL glass ampules or two sizes of HDPE plastic bottles Features of Surfact Amps NP 40 Detergent Solution • NP 40 a nonionic detergent for use in various protein methods • Accurate precise 10 detergent solutions in ultrapure water • Easy to use solutions are simple to dispense and dilute for use • Exceptionally pure less than 1 0µeq mL peroxides and carbonyls • Stable packaged under inert nitrogen gas in glass ampules This is an easy to use 10 w v solution of purified NP 40 detergent for use in routine and high demand protein research methods and molecular biology techniques NP 40 is a nonionic polyoxyethylene surfactant that is most frequently used as a component of cell lysis buffers or other solutions intended to extract and solubilize proteins Surfact Amps Detergent Solutions 10 w v provide unsurpassed purity quality and stability Unlike neat detergents which are extremely viscous Surfact Amps 10 Solutions are easy to pipette and accurately dispense The surfactant solutions are carefully prepared and packaged under nitrogen in glass ampules or non leaching HDPE bottles ensuring their stability and eliminating the accumulation of peroxides and degradation products Properties of NP 40 Detergent • Molecular Weight 617g • Detergent Class Nonionic • Aggregation Number 149 • Micelle Molecular Weight 90 000g • Critical Micelle Concentration CMC 0 29 mM 0 0179 w v • Cloud Point 80°C • Dialyzable No Specifications for NP 40 Surfact Amps Detergent Solution • Visual Clear colorless solution free of particulate matter • Concentration 10 0 1 0 • Oxidants 1 0µeq mL • Carbonyls 1 0µeq mL • Suspended Solids Residue present must not exceed Residue Reference For added safety when opening glass ampules consider using our Ampule Breakers which are disposable safety devices that protect the fingers when breaking open a glass ampule Related Products Surfact Amps Detergent Sampler
    Catalog Number:
    28324
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    Cell Lysis & Fractionation|Detergents for Protein Solubilization|Protein Biology|Protein Purification & Isolation
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    Structured Review

    Thermo Fisher nonidet p 40
    Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4  PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.
    Thermo Scientific Surfact Amps NP 40 Detergent Solution is highly purified NP 40 detergent stabilized as a 10 solution in your choice of either 10 mL glass ampules or two sizes of HDPE plastic bottles Features of Surfact Amps NP 40 Detergent Solution • NP 40 a nonionic detergent for use in various protein methods • Accurate precise 10 detergent solutions in ultrapure water • Easy to use solutions are simple to dispense and dilute for use • Exceptionally pure less than 1 0µeq mL peroxides and carbonyls • Stable packaged under inert nitrogen gas in glass ampules This is an easy to use 10 w v solution of purified NP 40 detergent for use in routine and high demand protein research methods and molecular biology techniques NP 40 is a nonionic polyoxyethylene surfactant that is most frequently used as a component of cell lysis buffers or other solutions intended to extract and solubilize proteins Surfact Amps Detergent Solutions 10 w v provide unsurpassed purity quality and stability Unlike neat detergents which are extremely viscous Surfact Amps 10 Solutions are easy to pipette and accurately dispense The surfactant solutions are carefully prepared and packaged under nitrogen in glass ampules or non leaching HDPE bottles ensuring their stability and eliminating the accumulation of peroxides and degradation products Properties of NP 40 Detergent • Molecular Weight 617g • Detergent Class Nonionic • Aggregation Number 149 • Micelle Molecular Weight 90 000g • Critical Micelle Concentration CMC 0 29 mM 0 0179 w v • Cloud Point 80°C • Dialyzable No Specifications for NP 40 Surfact Amps Detergent Solution • Visual Clear colorless solution free of particulate matter • Concentration 10 0 1 0 • Oxidants 1 0µeq mL • Carbonyls 1 0µeq mL • Suspended Solids Residue present must not exceed Residue Reference For added safety when opening glass ampules consider using our Ampule Breakers which are disposable safety devices that protect the fingers when breaking open a glass ampule Related Products Surfact Amps Detergent Sampler
    https://www.bioz.com/result/nonidet p 40/product/Thermo Fisher
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    Images

    1) Product Images from "Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM"

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

    Journal: Journal of Virology

    doi:

    Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4  PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.
    Figure Legend Snippet: Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4 PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.

    Techniques Used: Neutralization, Purification, Incubation, IA, Infection, Activity Assay

    2) Product Images from "The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination"

    Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21176315

    Importance of ALG-2-binding to SARAF for Ca 2+ -dependent interaction between the SARAF CytDs. ( A ) Schematic diagram of expressed proteins and co-IP/HiBiT assay. ( B ) After HEK293 cells in 6-cm dishes had been co-transfected with expression plasmids for full-length HiBiTHA-SARAF (WT or F228S mutant) and for either StrepHA-SGFP2 (Control) or StrepHA-SARAF_CytD-SGFP2 (WT or F228S mutant) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing 1% Nonidet P-40 supplemented with protease inhibitors, 10 mM NEM and 10 μM CaCl 2  ( Ca ) or 5 mM EGTA ( Eg ). SGFP2-fused proteins were immunoprecipitated with anti-GFP pAb, and the amounts of co-immunoprecipitated HiBiTHA-SARAF were estimated by the lytic HiBiT assay with LgBiT. The amounts of immunoprecipitated SGFP2-fused proteins were normalized by the StrepTactin-AP method as described in Materials and Methods. The relative amount of HiBiTHA-SARAF WT co-immunoprecipitated with StrepHA-SARAF_CytD-SGFP2 WT was expressed as 100% of relative interaction activity. The co-IP/HiBiT assay was performed in duplicate and repeated three times. Data are expressed as mean ± SEM ( n  = 3). Statistical significance by Tukey’s test is indicated in three different ways: asterisks for comparison between conditions of plus and minus Ca 2+ ;  a  for comparison with WT in the presence of Ca 2+ ;  b  for comparison with the control in the presence of Ca 2+ .  p  values were below 0.001 for all cases indicated (***,  a , and  b ). n.s., not significant. ( C ) Enhancement of the Ca 2+ -dependent CytD-to-CytD interaction of SARAF in HEK293 ALG-2KO cells by co-overexpression of FLAG-ALG-2. HEK293 ALG-2KO cells were co-transfected with expression plasmids for (i) full-length HiBiTHA-SARAF (WT), (ii) StrepHA-SARAF_CytD-SGFP2 (WT) and (iii) FLAG empty vector or FLAG-ALG-2 (WT, F85A or Y180A) and cultured for 24 h. The cells were lysed and subjected to HiBiT assay as described above. Relative interaction by co-overexpression of ALG-2 WT in the presence of Ca 2+  was expressed as 100% activity. Data are expressed as mean ± SEM ( n  = 3). Statistical significance by Tukey’s test is indicated by asterisks,  a , and  b  as described in ( B ) for comparison with the presence or absence of Ca 2+  (asterisks), WT ( a ) and control ( b ).
    Figure Legend Snippet: Importance of ALG-2-binding to SARAF for Ca 2+ -dependent interaction between the SARAF CytDs. ( A ) Schematic diagram of expressed proteins and co-IP/HiBiT assay. ( B ) After HEK293 cells in 6-cm dishes had been co-transfected with expression plasmids for full-length HiBiTHA-SARAF (WT or F228S mutant) and for either StrepHA-SGFP2 (Control) or StrepHA-SARAF_CytD-SGFP2 (WT or F228S mutant) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing 1% Nonidet P-40 supplemented with protease inhibitors, 10 mM NEM and 10 μM CaCl 2 ( Ca ) or 5 mM EGTA ( Eg ). SGFP2-fused proteins were immunoprecipitated with anti-GFP pAb, and the amounts of co-immunoprecipitated HiBiTHA-SARAF were estimated by the lytic HiBiT assay with LgBiT. The amounts of immunoprecipitated SGFP2-fused proteins were normalized by the StrepTactin-AP method as described in Materials and Methods. The relative amount of HiBiTHA-SARAF WT co-immunoprecipitated with StrepHA-SARAF_CytD-SGFP2 WT was expressed as 100% of relative interaction activity. The co-IP/HiBiT assay was performed in duplicate and repeated three times. Data are expressed as mean ± SEM ( n = 3). Statistical significance by Tukey’s test is indicated in three different ways: asterisks for comparison between conditions of plus and minus Ca 2+ ; a for comparison with WT in the presence of Ca 2+ ; b for comparison with the control in the presence of Ca 2+ . p values were below 0.001 for all cases indicated (***, a , and b ). n.s., not significant. ( C ) Enhancement of the Ca 2+ -dependent CytD-to-CytD interaction of SARAF in HEK293 ALG-2KO cells by co-overexpression of FLAG-ALG-2. HEK293 ALG-2KO cells were co-transfected with expression plasmids for (i) full-length HiBiTHA-SARAF (WT), (ii) StrepHA-SARAF_CytD-SGFP2 (WT) and (iii) FLAG empty vector or FLAG-ALG-2 (WT, F85A or Y180A) and cultured for 24 h. The cells were lysed and subjected to HiBiT assay as described above. Relative interaction by co-overexpression of ALG-2 WT in the presence of Ca 2+ was expressed as 100% activity. Data are expressed as mean ± SEM ( n = 3). Statistical significance by Tukey’s test is indicated by asterisks, a , and b as described in ( B ) for comparison with the presence or absence of Ca 2+ (asterisks), WT ( a ) and control ( b ).

    Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Expressing, Mutagenesis, Cell Culture, Lysis, Immunoprecipitation, Activity Assay, Over Expression, Plasmid Preparation

    Deletion mutation of the SARAF cytosolic domain (CytD) affecting ALG-2 binding and ubiquitination. ( A ) Schematic diagram of the SARAF CytD, which contains three distinct motifs rich in Pro and aromatic residues (designated regions 1, 2 and 3). Expression plasmids for the SARAF CytDs of WT and various deletion mutants that were fused with Strep-tag II and 3xHA (StrepHA) at the  N -terminus and SGFP2 at the C-terminus were constructed. ( B ) Strep-pulldown assay. After HEK293 cells had been transfected with pStrepHA-SARAF_CytD-SGFP2 (WT and deletion mutants) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing protease inhibitors, 0.2% Nonidet P-40 and 10 μM CaCl 2 . The cleared cell lysates ( Input ) were subjected to Strep-pulldown followed by WB with anti-ALG-2 and anti-HA as indicated.
    Figure Legend Snippet: Deletion mutation of the SARAF cytosolic domain (CytD) affecting ALG-2 binding and ubiquitination. ( A ) Schematic diagram of the SARAF CytD, which contains three distinct motifs rich in Pro and aromatic residues (designated regions 1, 2 and 3). Expression plasmids for the SARAF CytDs of WT and various deletion mutants that were fused with Strep-tag II and 3xHA (StrepHA) at the N -terminus and SGFP2 at the C-terminus were constructed. ( B ) Strep-pulldown assay. After HEK293 cells had been transfected with pStrepHA-SARAF_CytD-SGFP2 (WT and deletion mutants) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing protease inhibitors, 0.2% Nonidet P-40 and 10 μM CaCl 2 . The cleared cell lysates ( Input ) were subjected to Strep-pulldown followed by WB with anti-ALG-2 and anti-HA as indicated.

    Techniques Used: Mutagenesis, Binding Assay, Expressing, Strep-tag, Construct, Transfection, Cell Culture, Lysis, Western Blot

    Ubiquitination of SARAF by WWP1 ubiquitin E3 ligase. ( A ) Schematic diagrams of SGFP2-fused NEDD4 family ubiquitin E3 ligases investigated in this study. The E3 ligases contain the Ca 2+ -binding C2 domain, four repeats of WW domains, and the catalytic HECT domain. The catalytic Cys residues in each E3 ligases are shown in red lines. The active site Cys of WWP1 was substituted with Ala (C890A) and the HECT domain was deleted (ΔHECT). ( B ) Schematic diagrams representing StrepHA-SARAF mutants of Pro-to-Ala substitutions at PP X Y motifs and the CytD truncation. ( C ) StrepHA-SARAF was co-expressed in HEK293 cells with SGFP2-fused E3 ligase WWP1 (WT or C890A mutant), WWP2, ITCH or with a control vector ( Ctrl ). The cells were lysed in lysis buffer HK containing 1% Nonidet P-40 and E3 ligase inhibitors (2 mM EDTA, 10 mM NEM) as well as the protease inhibitor cocktail supplemented with 20 μM MG132. The cleared cell lysate ( Input ) was subjected to Strep-pulldown followed by WB with anti-HA and anti-GFP mAbs. Unmodified and ubiquitinated StrepHA-SARAF bands are marked with an arrowhead and unfilled arrowheads, respectively. ( D ) Strep-pulldown assays were performed using StrepHA-SARAF mutants and E3 ligase-defective mutants of SGFP2-WWP1 (C890A or ΔHECT) to determine important regions in SARAF for interaction with WWP1.
    Figure Legend Snippet: Ubiquitination of SARAF by WWP1 ubiquitin E3 ligase. ( A ) Schematic diagrams of SGFP2-fused NEDD4 family ubiquitin E3 ligases investigated in this study. The E3 ligases contain the Ca 2+ -binding C2 domain, four repeats of WW domains, and the catalytic HECT domain. The catalytic Cys residues in each E3 ligases are shown in red lines. The active site Cys of WWP1 was substituted with Ala (C890A) and the HECT domain was deleted (ΔHECT). ( B ) Schematic diagrams representing StrepHA-SARAF mutants of Pro-to-Ala substitutions at PP X Y motifs and the CytD truncation. ( C ) StrepHA-SARAF was co-expressed in HEK293 cells with SGFP2-fused E3 ligase WWP1 (WT or C890A mutant), WWP2, ITCH or with a control vector ( Ctrl ). The cells were lysed in lysis buffer HK containing 1% Nonidet P-40 and E3 ligase inhibitors (2 mM EDTA, 10 mM NEM) as well as the protease inhibitor cocktail supplemented with 20 μM MG132. The cleared cell lysate ( Input ) was subjected to Strep-pulldown followed by WB with anti-HA and anti-GFP mAbs. Unmodified and ubiquitinated StrepHA-SARAF bands are marked with an arrowhead and unfilled arrowheads, respectively. ( D ) Strep-pulldown assays were performed using StrepHA-SARAF mutants and E3 ligase-defective mutants of SGFP2-WWP1 (C890A or ΔHECT) to determine important regions in SARAF for interaction with WWP1.

    Techniques Used: Binding Assay, Mutagenesis, Plasmid Preparation, Lysis, Protease Inhibitor, Western Blot

    3) Product Images from "Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *"

    Article Title: Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.173781

    Direct interaction of PAI-1 with proteasome. Panel A  shows restriction analysis of five pGAD10 plasmids isolated from  His + LacZ +  colonies after yeast two-hybrid system screening. The encoded cDNA sequence, cloned into an EcoRI site, corresponded to the entire α3 subunit of human proteasome. Direct binding of PAI-1 to the proteasome was then confirmed by immunoprecipitation of the  125 I- PAI-1-proteasome complexes ( panel B ). Immunoprecipitated complexes of  125 I-labeled PAI-1 with proteasome were separated by SDS-PAGE (10% gels) under reducing conditions. The  first  and  third lanes  show complexes of  125 I-labeled PAI-1 with vitronectin ( Vn ) and proteasome, respectively. The  second  and  fourth lanes  show corresponding control immunoprecipitates.  Panel C  shows direct interaction of PAI-1 with proteasome analyzed by solid phase binding assay. Increasing doses of PAI-1 were incubated for 1 h at 37 °C in wells coated with proteasome. The unbound PAI-1 was washed away, and the bound PAI-1 was evaluated by ELISA using antibodies to PAI-1. Nonspecific binding observed in the absence of proteasome was subtracted. Data represent the mean ± S.D. obtained during three separate experiments.  Panel D  shows pulldown experiments using the His-tagged α3 proteasome subunit immobilized on NHS-Sepharose beads. EA.hy926 cells, control, or stimulated either with TNF (5 ng/ml and 20 ng/ml) or LPS (5 μg/ml and 20 μg/ml) were used as a source of PAI-1. PAI-1 was identified by Western immunoblotting with monoclonal anti-PAI-1 antibodies. Inputs of PAI-1 and β actin are shown. Direct interaction of both proteins in endothelial cells was further demonstrated by coimmunoprecipitation ( Panel E ). EA.hy926 cells, control and stimulated with TNF (5 ng/ml and 20 ng/ml) or LPS (5 μg/ml and 20 μg/ml), were lysed with 1% Nonidet P-40 lysis buffer and precipitated with anti-PAI-1 ( left ) or anti-α3 proteasome subunit ( right ) antibodies. Next, protein-protein complexes were captured by 100 μl of protein A/G-agarose beads. Bound proteins were solubilized by SDS sample buffer and subjected to SDS-PAGE and Western immunoblotting ( IB ) analysis. Coimmunoprecipitated ( IP ) proteins were detected with specific anti-α3 or anti-PAI-1 monoclonal antibodies. Inputs of α3 subunit and PAI-1 as well as blotting with nonimmune IgG and anti-actin antibodies are shown as well.
    Figure Legend Snippet: Direct interaction of PAI-1 with proteasome. Panel A shows restriction analysis of five pGAD10 plasmids isolated from His + LacZ + colonies after yeast two-hybrid system screening. The encoded cDNA sequence, cloned into an EcoRI site, corresponded to the entire α3 subunit of human proteasome. Direct binding of PAI-1 to the proteasome was then confirmed by immunoprecipitation of the 125 I- PAI-1-proteasome complexes ( panel B ). Immunoprecipitated complexes of 125 I-labeled PAI-1 with proteasome were separated by SDS-PAGE (10% gels) under reducing conditions. The first and third lanes show complexes of 125 I-labeled PAI-1 with vitronectin ( Vn ) and proteasome, respectively. The second and fourth lanes show corresponding control immunoprecipitates. Panel C shows direct interaction of PAI-1 with proteasome analyzed by solid phase binding assay. Increasing doses of PAI-1 were incubated for 1 h at 37 °C in wells coated with proteasome. The unbound PAI-1 was washed away, and the bound PAI-1 was evaluated by ELISA using antibodies to PAI-1. Nonspecific binding observed in the absence of proteasome was subtracted. Data represent the mean ± S.D. obtained during three separate experiments. Panel D shows pulldown experiments using the His-tagged α3 proteasome subunit immobilized on NHS-Sepharose beads. EA.hy926 cells, control, or stimulated either with TNF (5 ng/ml and 20 ng/ml) or LPS (5 μg/ml and 20 μg/ml) were used as a source of PAI-1. PAI-1 was identified by Western immunoblotting with monoclonal anti-PAI-1 antibodies. Inputs of PAI-1 and β actin are shown. Direct interaction of both proteins in endothelial cells was further demonstrated by coimmunoprecipitation ( Panel E ). EA.hy926 cells, control and stimulated with TNF (5 ng/ml and 20 ng/ml) or LPS (5 μg/ml and 20 μg/ml), were lysed with 1% Nonidet P-40 lysis buffer and precipitated with anti-PAI-1 ( left ) or anti-α3 proteasome subunit ( right ) antibodies. Next, protein-protein complexes were captured by 100 μl of protein A/G-agarose beads. Bound proteins were solubilized by SDS sample buffer and subjected to SDS-PAGE and Western immunoblotting ( IB ) analysis. Coimmunoprecipitated ( IP ) proteins were detected with specific anti-α3 or anti-PAI-1 monoclonal antibodies. Inputs of α3 subunit and PAI-1 as well as blotting with nonimmune IgG and anti-actin antibodies are shown as well.

    Techniques Used: Isolation, Sequencing, Clone Assay, Binding Assay, Immunoprecipitation, Labeling, SDS Page, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Lysis

    Interaction of PAI-1 with 20 S but not 26 S proteasome.  HeLa cells, which do not express endogenous PAI-1, were transfected with control pCMV or pCMV-PAI-1 for 24 and 48 h, lysed with 1% Nonidet P-40 buffer, and used to precipitate PAI-1 with anti PAI-1 antibodies. Then immunoprecipitates ( IP ) were probed for the proteasome 20 S (α3 and β1) or 26 S subunits (S4). To confirm the presence of the PAI-1-proteasome subunit complex, PVDF membranes were stripped and reprobed with anti-PAI-1 antibodies ( panel A ). In  panel B  samples of entire cell extracts were analyzed by Western blotting ( IB ) using antibodies to different proteasome subunits, namely α3, β1, and S4, as well as to β actin. In addition, normal mouse IgG was used as a control. Data are representative of those obtained during three separate experiments.
    Figure Legend Snippet: Interaction of PAI-1 with 20 S but not 26 S proteasome. HeLa cells, which do not express endogenous PAI-1, were transfected with control pCMV or pCMV-PAI-1 for 24 and 48 h, lysed with 1% Nonidet P-40 buffer, and used to precipitate PAI-1 with anti PAI-1 antibodies. Then immunoprecipitates ( IP ) were probed for the proteasome 20 S (α3 and β1) or 26 S subunits (S4). To confirm the presence of the PAI-1-proteasome subunit complex, PVDF membranes were stripped and reprobed with anti-PAI-1 antibodies ( panel A ). In panel B samples of entire cell extracts were analyzed by Western blotting ( IB ) using antibodies to different proteasome subunits, namely α3, β1, and S4, as well as to β actin. In addition, normal mouse IgG was used as a control. Data are representative of those obtained during three separate experiments.

    Techniques Used: Transfection, Western Blot

    4) Product Images from "Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes"

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1604258113

    Isolation of transition zones. ( A ) Live cells expressing eYFP::FTZC at the TZ (green) were ( B ) treated with 1% Nonidet P-40 to extract the soluble material. ( C ) Washed cytoskeletons were sonicated to shear the TZs from other flagellar material. Tagged
    Figure Legend Snippet: Isolation of transition zones. ( A ) Live cells expressing eYFP::FTZC at the TZ (green) were ( B ) treated with 1% Nonidet P-40 to extract the soluble material. ( C ) Washed cytoskeletons were sonicated to shear the TZs from other flagellar material. Tagged

    Techniques Used: Isolation, Expressing, Sonication

    5) Product Images from "Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM"

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

    Journal: Journal of Virology

    doi:

    Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4  PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.
    Figure Legend Snippet: Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4 PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.

    Techniques Used: Neutralization, Purification, Incubation, IA, Infection, Activity Assay

    6) Product Images from "A comparison of three approaches for the discovery of novel tripartite attachment complex proteins in Trypanosoma brucei"

    Article Title: A comparison of three approaches for the discovery of novel tripartite attachment complex proteins in Trypanosoma brucei

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0008568

    TAC102 enrichment and immunoprecipitation. The experiments were done in three independent replicates and all showed similar results. ( A ) Western blot analysis of the digitonin fractionation and subsequent lysis of the pellet fraction with 1% (v/v) Nonidet P-40 of whole cell protein from procyclic cells. T: total; S: supernatant; P: pellet. In orange: the pellet from the digitonin fractionation was used for the subsequent Nonidet P-40 lysis. The elongation factor EF1α served as a control. ( B ) Western blot analysis of the elutions (E1 and E2) of TAC102 immunoprecipitation replicates. ( C ) Silver stained SDS PAGE of TAC102 immunoprecipitation fractions. On the left are the molecular weights in kDa. Under each lane are written the equivalence of cells loaded. TAC102, indicated by a white arrow, was detected in E1. FT: flow-through; W1-W2-W3: washes; E1-E2: elutions. ( D ) Volcano plot representing the significance versus enrichment of the results from mass spectrometry analysis of TAC102 immunoprecipitation. A total of 775 proteins were identified from which 100 proteins were significantly enriched in the TAC102 immunoprecipitation (p ≤ 0.01). The two dotted lines represent the cut-off used (p ≤ 0.01 and enrichment  >  3). The protein the most enriched in the condition was TAC102. Three other TAC components were also highly enriched (p166, TAC40 and TAC60).
    Figure Legend Snippet: TAC102 enrichment and immunoprecipitation. The experiments were done in three independent replicates and all showed similar results. ( A ) Western blot analysis of the digitonin fractionation and subsequent lysis of the pellet fraction with 1% (v/v) Nonidet P-40 of whole cell protein from procyclic cells. T: total; S: supernatant; P: pellet. In orange: the pellet from the digitonin fractionation was used for the subsequent Nonidet P-40 lysis. The elongation factor EF1α served as a control. ( B ) Western blot analysis of the elutions (E1 and E2) of TAC102 immunoprecipitation replicates. ( C ) Silver stained SDS PAGE of TAC102 immunoprecipitation fractions. On the left are the molecular weights in kDa. Under each lane are written the equivalence of cells loaded. TAC102, indicated by a white arrow, was detected in E1. FT: flow-through; W1-W2-W3: washes; E1-E2: elutions. ( D ) Volcano plot representing the significance versus enrichment of the results from mass spectrometry analysis of TAC102 immunoprecipitation. A total of 775 proteins were identified from which 100 proteins were significantly enriched in the TAC102 immunoprecipitation (p ≤ 0.01). The two dotted lines represent the cut-off used (p ≤ 0.01 and enrichment > 3). The protein the most enriched in the condition was TAC102. Three other TAC components were also highly enriched (p166, TAC40 and TAC60).

    Techniques Used: Immunoprecipitation, Western Blot, Fractionation, Lysis, Staining, SDS Page, Mass Spectrometry

    7) Product Images from "Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation"

    Article Title: Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.121592298

    ( A ) Latent dbpB is associated with RNA in nonstimulated EC. Cytosolic extracts were prepared from either control bovine aortic EC or EC treated with bovine α-thrombin (10 units/ml, 2 h), as described in  Experimental Procedures . Extracts from control EC were incubated with RNase A (1 mg/ml) or micrococcal nuclease (10,000 units/ml) for 30 min at 37°C before EMSA with the thrombin-response element (CCACCCACC) oligonucleotide as a probe. Arrow shows the position of dbpB activated by thrombin treatment of intact cells or ribonuclease treatment of extracts from untreated EC; McNase, micrococcal nuclease. ( B ) Latent dbpB coimmunoprecipitates with RNA. Cytosolic extracts prepared from control EC were incubated with preimmune serum or anti-dbpB antiserum raised against a C-terminal peptide of dbpB, followed by precipitation with protein A/G Sepharose. Protein was eluted by heat treatment. Aliquots of the eluants were treated with RNase A to release dbpB coimmunoprecipitated in association with RNA, and EMSA was performed. −Ab, preimmune serum used as a control; +Ab, immune serum used for precipitation of dbpB; RNase A, sample treated with ribonuclease A. ( C ) Oligo(dT) affinity chromatography of dbpB. ( Left ) EC cytosolic extracts were prepared from control and thrombin-treated EC as described in  Experimental Procedures  and dialyzed overnight in 0.2 M NaCl/0.01 M Tris⋅HCl, pH 7.5/0.002 M EDTA/0.2% Nonidet P-40/0.005 M β-mercaptoethanol. Oligo(dT)–biotin complex was added to the extract, and the mixture was applied to streptavidin magnetic beads. After rotation for 1 h at 4°C, the unbound fraction (fraction 1) was removed, the beads were washed three times, then washed with 25% formamide, and RNA was eluted with 60% formamide (fraction 2). The fractions were dialyzed overnight in EMSA buffer, and aliquots were treated with RNase A and subjected to EMSA. ( Right ) Fraction 1 from thrombin-treated EC lysates was used for EMSA without RNase treatment to detect activated dbpB.
    Figure Legend Snippet: ( A ) Latent dbpB is associated with RNA in nonstimulated EC. Cytosolic extracts were prepared from either control bovine aortic EC or EC treated with bovine α-thrombin (10 units/ml, 2 h), as described in Experimental Procedures . Extracts from control EC were incubated with RNase A (1 mg/ml) or micrococcal nuclease (10,000 units/ml) for 30 min at 37°C before EMSA with the thrombin-response element (CCACCCACC) oligonucleotide as a probe. Arrow shows the position of dbpB activated by thrombin treatment of intact cells or ribonuclease treatment of extracts from untreated EC; McNase, micrococcal nuclease. ( B ) Latent dbpB coimmunoprecipitates with RNA. Cytosolic extracts prepared from control EC were incubated with preimmune serum or anti-dbpB antiserum raised against a C-terminal peptide of dbpB, followed by precipitation with protein A/G Sepharose. Protein was eluted by heat treatment. Aliquots of the eluants were treated with RNase A to release dbpB coimmunoprecipitated in association with RNA, and EMSA was performed. −Ab, preimmune serum used as a control; +Ab, immune serum used for precipitation of dbpB; RNase A, sample treated with ribonuclease A. ( C ) Oligo(dT) affinity chromatography of dbpB. ( Left ) EC cytosolic extracts were prepared from control and thrombin-treated EC as described in Experimental Procedures and dialyzed overnight in 0.2 M NaCl/0.01 M Tris⋅HCl, pH 7.5/0.002 M EDTA/0.2% Nonidet P-40/0.005 M β-mercaptoethanol. Oligo(dT)–biotin complex was added to the extract, and the mixture was applied to streptavidin magnetic beads. After rotation for 1 h at 4°C, the unbound fraction (fraction 1) was removed, the beads were washed three times, then washed with 25% formamide, and RNA was eluted with 60% formamide (fraction 2). The fractions were dialyzed overnight in EMSA buffer, and aliquots were treated with RNase A and subjected to EMSA. ( Right ) Fraction 1 from thrombin-treated EC lysates was used for EMSA without RNase treatment to detect activated dbpB.

    Techniques Used: Incubation, Affinity Chromatography, Magnetic Beads

    Related Articles

    Labeling:

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes
    Article Snippet: Thin-section EM was performed as described ( ). .. Immunogold labeling of whole-mount cytoskeletons was performed by settling washed cells onto charged, formvar-coated grids, treating with 1% Nonidet P-40 in PBS for 5 min, block (1% BSA in PBS) for 20 min, staining with α-GFP (Invitrogen ; 1/500) in block for 45 min, washing three times in PBS, staining with 10 nm gold particle conjugated secondary antibody (Sigma G7402; 1/50) in block for 45 min, washing two times in PBS, followed by fixation for 10 min in 2.5% glutaraldehyde. .. Samples were then washed twice in PEME, stained for 1 s in 1% aurothioglucose, dried on filter paper, and imaged by TEM.

    Blocking Assay:

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes
    Article Snippet: Thin-section EM was performed as described ( ). .. Immunogold labeling of whole-mount cytoskeletons was performed by settling washed cells onto charged, formvar-coated grids, treating with 1% Nonidet P-40 in PBS for 5 min, block (1% BSA in PBS) for 20 min, staining with α-GFP (Invitrogen ; 1/500) in block for 45 min, washing three times in PBS, staining with 10 nm gold particle conjugated secondary antibody (Sigma G7402; 1/50) in block for 45 min, washing two times in PBS, followed by fixation for 10 min in 2.5% glutaraldehyde. .. Samples were then washed twice in PEME, stained for 1 s in 1% aurothioglucose, dried on filter paper, and imaged by TEM.

    Staining:

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes
    Article Snippet: Thin-section EM was performed as described ( ). .. Immunogold labeling of whole-mount cytoskeletons was performed by settling washed cells onto charged, formvar-coated grids, treating with 1% Nonidet P-40 in PBS for 5 min, block (1% BSA in PBS) for 20 min, staining with α-GFP (Invitrogen ; 1/500) in block for 45 min, washing three times in PBS, staining with 10 nm gold particle conjugated secondary antibody (Sigma G7402; 1/50) in block for 45 min, washing two times in PBS, followed by fixation for 10 min in 2.5% glutaraldehyde. .. Samples were then washed twice in PEME, stained for 1 s in 1% aurothioglucose, dried on filter paper, and imaged by TEM.

    Article Title: Influenza A Virus Proteins NS1 and Hemagglutinin Along with M2 Are Involved in Stimulation of Autophagy in Infected Cells
    Article Snippet: After incubation, cells were subjected to polypeptide analysis by polyacrylamide gel electrophoresis (PAGE). .. Uninfected cells and cells infected with either delNS1 or WT virus were fixed with 4% paraformaldehyde (Merck, Germany) overnight at 4°C, permeabilized with 0.1% detergent NP-40 (Pierce) for 3 min, and stained sequentially with rabbit antibody specific to LC3 (Cell Signaling) and secondary anti-rabbit–tetramethyl rhodamine isocyanate (TRITC) conjugate (Jackson) dissolved in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). .. Nuclei were stained with 0.1 mg/ml of DAPI (4′,6-diamidino-2-phenylindole) for 30 min in the dark.

    Infection:

    Article Title: Influenza A Virus Proteins NS1 and Hemagglutinin Along with M2 Are Involved in Stimulation of Autophagy in Infected Cells
    Article Snippet: After incubation, cells were subjected to polypeptide analysis by polyacrylamide gel electrophoresis (PAGE). .. Uninfected cells and cells infected with either delNS1 or WT virus were fixed with 4% paraformaldehyde (Merck, Germany) overnight at 4°C, permeabilized with 0.1% detergent NP-40 (Pierce) for 3 min, and stained sequentially with rabbit antibody specific to LC3 (Cell Signaling) and secondary anti-rabbit–tetramethyl rhodamine isocyanate (TRITC) conjugate (Jackson) dissolved in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). .. Nuclei were stained with 0.1 mg/ml of DAPI (4′,6-diamidino-2-phenylindole) for 30 min in the dark.

    Incubation:

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM
    Article Snippet: Western blots were probed with NC1 and R7 to detect virion proteins VP5 and gD, respectively, and R140 was used to detect HVEM. .. Fifty-microliter reactions containing 3 μg of gD-1(Δ290–299t) and 16 μg of HVEMt per ml were incubated in binding buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.1% Nonidet P-40, 0.05% bovine serum albumin [BSA], 0.05% chicken egg albumin) on ice for 1 h. MAb ascites (0.1 μl) was added for 1 h, followed by 50 μl of protein A-agarose (GIBCO BRL) (50 mg/ml) for 1 h. Bound material was collected by centrifugation at 13,000 × g for 3 min. Pellets were washed four times with high-salt buffer (10 mM Tris [pH 8.0], 500 mM NaCl, 0.1% Nonidet P-40, 0.05% BSA, 0.05% chicken egg albumin) and then boiled in SDS sample buffer for 3 min. .. Following SDS-PAGE (12% polyacrylamide), Western blots were probed with R7 and R140.

    Article Title: Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *
    Article Snippet: .. After incubation for 4 h, cells were collected and lysed with the Nonidet P-40 containing buffer, and cellular protein was concentration measured by the BCA method (Pierce/Thermo Scientific kit). ..

    Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination
    Article Snippet: The membrane was incubated with 1 mL of coelenterazine h solution (20 μg/mL) in TBS containing 100 μM CaCl2, and the luminescence signal was detected with a LAS-3000mini lumino-image analyzer (FUJIFILM, Tokyo, Japan). .. For pulldown-binding assay, aliquots of the suspensions of beads were incubated with 0.2 mL of NlucALG-2 solution (300 ng/mL) in lysis buffer HKM containing 0.2% Nonidet P-40, 0.1% gelatin, protease inhibitors, and 100 μM CaCl2 for 2 h at 4 °C. .. After the beads had been collected with a magnetic stand and washed three times with 0.5 mL of lysis buffer HKM containing 0.2% Nonidet P-40 and 100 μM CaCl2 , the beads were suspended with 50 μL of passive lysis buffer (PLB) (Promega, Madison, WI, USA).

    Binding Assay:

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM
    Article Snippet: Western blots were probed with NC1 and R7 to detect virion proteins VP5 and gD, respectively, and R140 was used to detect HVEM. .. Fifty-microliter reactions containing 3 μg of gD-1(Δ290–299t) and 16 μg of HVEMt per ml were incubated in binding buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.1% Nonidet P-40, 0.05% bovine serum albumin [BSA], 0.05% chicken egg albumin) on ice for 1 h. MAb ascites (0.1 μl) was added for 1 h, followed by 50 μl of protein A-agarose (GIBCO BRL) (50 mg/ml) for 1 h. Bound material was collected by centrifugation at 13,000 × g for 3 min. Pellets were washed four times with high-salt buffer (10 mM Tris [pH 8.0], 500 mM NaCl, 0.1% Nonidet P-40, 0.05% BSA, 0.05% chicken egg albumin) and then boiled in SDS sample buffer for 3 min. .. Following SDS-PAGE (12% polyacrylamide), Western blots were probed with R7 and R140.

    Centrifugation:

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM
    Article Snippet: Western blots were probed with NC1 and R7 to detect virion proteins VP5 and gD, respectively, and R140 was used to detect HVEM. .. Fifty-microliter reactions containing 3 μg of gD-1(Δ290–299t) and 16 μg of HVEMt per ml were incubated in binding buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.1% Nonidet P-40, 0.05% bovine serum albumin [BSA], 0.05% chicken egg albumin) on ice for 1 h. MAb ascites (0.1 μl) was added for 1 h, followed by 50 μl of protein A-agarose (GIBCO BRL) (50 mg/ml) for 1 h. Bound material was collected by centrifugation at 13,000 × g for 3 min. Pellets were washed four times with high-salt buffer (10 mM Tris [pH 8.0], 500 mM NaCl, 0.1% Nonidet P-40, 0.05% BSA, 0.05% chicken egg albumin) and then boiled in SDS sample buffer for 3 min. .. Following SDS-PAGE (12% polyacrylamide), Western blots were probed with R7 and R140.

    Concentration Assay:

    Article Title: Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *
    Article Snippet: .. After incubation for 4 h, cells were collected and lysed with the Nonidet P-40 containing buffer, and cellular protein was concentration measured by the BCA method (Pierce/Thermo Scientific kit). ..

    BIA-KA:

    Article Title: Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *
    Article Snippet: .. After incubation for 4 h, cells were collected and lysed with the Nonidet P-40 containing buffer, and cellular protein was concentration measured by the BCA method (Pierce/Thermo Scientific kit). ..

    Lysis:

    Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination
    Article Snippet: The membrane was incubated with 1 mL of coelenterazine h solution (20 μg/mL) in TBS containing 100 μM CaCl2, and the luminescence signal was detected with a LAS-3000mini lumino-image analyzer (FUJIFILM, Tokyo, Japan). .. For pulldown-binding assay, aliquots of the suspensions of beads were incubated with 0.2 mL of NlucALG-2 solution (300 ng/mL) in lysis buffer HKM containing 0.2% Nonidet P-40, 0.1% gelatin, protease inhibitors, and 100 μM CaCl2 for 2 h at 4 °C. .. After the beads had been collected with a magnetic stand and washed three times with 0.5 mL of lysis buffer HKM containing 0.2% Nonidet P-40 and 100 μM CaCl2 , the beads were suspended with 50 μL of passive lysis buffer (PLB) (Promega, Madison, WI, USA).

    other:

    Article Title: Divergent roles of BECN1 in LC3 lipidation and autophagosomal function
    Article Snippet: The NP40 detergent solution (85124) was purchased from Thermo, and protein A beads (17-0963-02) were from GE Health Care.

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    Thermo Fisher triton x 114
    Blocking TgMLC1 palmitoylation causes the protein to shift into the aqueous phase in Triton X-114. WT, C8S, C11S, C(  8 ,  11 )S and C(  8 ,  11 )A parasites were extracted at 4°C in Triton X-114 and the extracted proteins phase partitioned by shifting the temperature to 20°C. The amounts of  (A)  TgMLC1and  (B)  TgGRA8 recovered in the detergent (grey) and aqueous (black) phases from each sample were determined by quantitative western blotting (see Fig. S2 for a representative western blot), and are displayed here as the percentage of the total TgMLC1 recovered in the two phases combined. The data shown are the means and standard error of the mean (SEM) from 2 (C8S, C11S) or 4 (WT, C[8,11]S) independent replicates; C(  8 ,  11 )A parasites were analyzed once.
    Triton X 114, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nonidet p 40
    Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4  PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.
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    BAG3_Pro209 mutants also aggregate in muscle (C2C12) and motoneuron-like cells (NSC-34). We transiently transfected GFP-tagged BAG3 wild type or mutant constructs in C2C12 and NSC-34 cells. We then verified protein aggregation by separating the soluble fraction (western blot) and insoluble fraction (filter retardation assay (FRA)) ( a , c ) or verified protein aggregation by immunofluorescence ( b , d ). The FRA analysis is displayed for the <t>NP-40</t> insoluble fraction. Relative optical densities are reported in the graphs as means ± SD of normalized values. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis (n = 3). Scale bar = 10 µm.
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    Image Search Results


    Blocking TgMLC1 palmitoylation causes the protein to shift into the aqueous phase in Triton X-114. WT, C8S, C11S, C(  8 ,  11 )S and C(  8 ,  11 )A parasites were extracted at 4°C in Triton X-114 and the extracted proteins phase partitioned by shifting the temperature to 20°C. The amounts of  (A)  TgMLC1and  (B)  TgGRA8 recovered in the detergent (grey) and aqueous (black) phases from each sample were determined by quantitative western blotting (see Fig. S2 for a representative western blot), and are displayed here as the percentage of the total TgMLC1 recovered in the two phases combined. The data shown are the means and standard error of the mean (SEM) from 2 (C8S, C11S) or 4 (WT, C[8,11]S) independent replicates; C(  8 ,  11 )A parasites were analyzed once.

    Journal: bioRxiv

    Article Title: Blocking palmitoylation of Toxoplasma gondii myosin light chain 1 disrupts glideosome composition but has little impact on parasite motility

    doi: 10.1101/2020.08.13.250399

    Figure Lengend Snippet: Blocking TgMLC1 palmitoylation causes the protein to shift into the aqueous phase in Triton X-114. WT, C8S, C11S, C( 8 , 11 )S and C( 8 , 11 )A parasites were extracted at 4°C in Triton X-114 and the extracted proteins phase partitioned by shifting the temperature to 20°C. The amounts of (A) TgMLC1and (B) TgGRA8 recovered in the detergent (grey) and aqueous (black) phases from each sample were determined by quantitative western blotting (see Fig. S2 for a representative western blot), and are displayed here as the percentage of the total TgMLC1 recovered in the two phases combined. The data shown are the means and standard error of the mean (SEM) from 2 (C8S, C11S) or 4 (WT, C[8,11]S) independent replicates; C( 8 , 11 )A parasites were analyzed once.

    Article Snippet: Phase separation of parasite proteins in Triton X-114 The phase separation was performed as previously described ( - ).

    Techniques: Blocking Assay, Western Blot

    Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4  PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.

    Journal: Journal of Virology

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

    doi:

    Figure Lengend Snippet: Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4 PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.

    Article Snippet: Fifty-microliter reactions containing 3 μg of gD-1(Δ290–299t) and 16 μg of HVEMt per ml were incubated in binding buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.1% Nonidet P-40, 0.05% bovine serum albumin [BSA], 0.05% chicken egg albumin) on ice for 1 h. MAb ascites (0.1 μl) was added for 1 h, followed by 50 μl of protein A-agarose (GIBCO BRL) (50 mg/ml) for 1 h. Bound material was collected by centrifugation at 13,000 × g for 3 min. Pellets were washed four times with high-salt buffer (10 mM Tris [pH 8.0], 500 mM NaCl, 0.1% Nonidet P-40, 0.05% BSA, 0.05% chicken egg albumin) and then boiled in SDS sample buffer for 3 min.

    Techniques: Neutralization, Purification, Incubation, IA, Infection, Activity Assay

    BAG3_Pro209 mutants also aggregate in muscle (C2C12) and motoneuron-like cells (NSC-34). We transiently transfected GFP-tagged BAG3 wild type or mutant constructs in C2C12 and NSC-34 cells. We then verified protein aggregation by separating the soluble fraction (western blot) and insoluble fraction (filter retardation assay (FRA)) ( a , c ) or verified protein aggregation by immunofluorescence ( b , d ). The FRA analysis is displayed for the NP-40 insoluble fraction. Relative optical densities are reported in the graphs as means ± SD of normalized values. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis (n = 3). Scale bar = 10 µm.

    Journal: Scientific Reports

    Article Title: BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes

    doi: 10.1038/s41598-020-65664-z

    Figure Lengend Snippet: BAG3_Pro209 mutants also aggregate in muscle (C2C12) and motoneuron-like cells (NSC-34). We transiently transfected GFP-tagged BAG3 wild type or mutant constructs in C2C12 and NSC-34 cells. We then verified protein aggregation by separating the soluble fraction (western blot) and insoluble fraction (filter retardation assay (FRA)) ( a , c ) or verified protein aggregation by immunofluorescence ( b , d ). The FRA analysis is displayed for the NP-40 insoluble fraction. Relative optical densities are reported in the graphs as means ± SD of normalized values. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis (n = 3). Scale bar = 10 µm.

    Article Snippet: Lysed cells were centrifuged at 16,100 g for 15 min. Supernatants were collected and pellets resuspended in the same volume of NP-40 buffer without protease inhibitors and DTT, and finally sonicated.

    Techniques: Transfection, Mutagenesis, Construct, Western Blot, Immunofluorescence

    BAG3_Pro209 mutations cause cytoplasmic aggregation. ( a ) Schematic representation of the structure of BAG3, including the WW-domain, the two IPV-motifs, the PxxP-domain and BAG-domain. The known interactors of each motif are shown at the top and the missense mutations that were studied in this manuscript are shown at the bottom in red. ( b ) HEK293T cells stably expressing HSPB8-V5 were transiently transfected with BAG3-GFP constructs. Six random fields were selected for analysis. The mean number of cells counted per field was 95 and thus over 400 cells per genotype were counted. (scale bar = 10 μm) ( c ) Quantification of BAG3-GFP inclusions using Flow cytometric analysis of inclusions (FloIT). Transiently transfected HEK293T cells were collected and stained with DAPI prior to 0.1% Triton X-100 treatment. The intracellular BAG3-GFP inclusions and Hoechst-positive nuclei are subsequently quantified using flow cytometry. Bar graph represents the means of BAG3-GFP cytoplasmic inclusions per 100 transfected cells. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis. ( d,e ) Bio-informatic analysis of ( d ) the solubility of wild type or mutant BAG3 with CamSol and ( e ) of the aggregation propensity with Tango software. ( f ) Western blot analysis of the NP-40 soluble fraction from HEK293T cells stably expressing HSPB8-V5 and transiently transfected with BAG3-GFP constructs. The constructs were abbreviated as followed: wild type (WT), Pro209Ser (PS), Pro209Leu (PL), Pro209Gln (PQ), Glu455Lys (EK). One of three representative western blots is shown. ( g ) Filter retardation assay (FRA) analysis of the NP-40 insoluble fraction. Anti-GFP and anti-HSPB8 antibodies were used to detect insoluble levels of BAG3 (wild type or mutants) and HSPB8. Relative optical densities are reported in the graphs as means ± SD of normalized values. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis (n = 3). The constructs were abbreviated as followed: non-transfected (NT), empty vector (EV), wild type (WT), Pro209Ser (PS), Pro209Leu (PL), Pro209Gln (PQ), Glu455Lys (EK).

    Journal: Scientific Reports

    Article Title: BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes

    doi: 10.1038/s41598-020-65664-z

    Figure Lengend Snippet: BAG3_Pro209 mutations cause cytoplasmic aggregation. ( a ) Schematic representation of the structure of BAG3, including the WW-domain, the two IPV-motifs, the PxxP-domain and BAG-domain. The known interactors of each motif are shown at the top and the missense mutations that were studied in this manuscript are shown at the bottom in red. ( b ) HEK293T cells stably expressing HSPB8-V5 were transiently transfected with BAG3-GFP constructs. Six random fields were selected for analysis. The mean number of cells counted per field was 95 and thus over 400 cells per genotype were counted. (scale bar = 10 μm) ( c ) Quantification of BAG3-GFP inclusions using Flow cytometric analysis of inclusions (FloIT). Transiently transfected HEK293T cells were collected and stained with DAPI prior to 0.1% Triton X-100 treatment. The intracellular BAG3-GFP inclusions and Hoechst-positive nuclei are subsequently quantified using flow cytometry. Bar graph represents the means of BAG3-GFP cytoplasmic inclusions per 100 transfected cells. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis. ( d,e ) Bio-informatic analysis of ( d ) the solubility of wild type or mutant BAG3 with CamSol and ( e ) of the aggregation propensity with Tango software. ( f ) Western blot analysis of the NP-40 soluble fraction from HEK293T cells stably expressing HSPB8-V5 and transiently transfected with BAG3-GFP constructs. The constructs were abbreviated as followed: wild type (WT), Pro209Ser (PS), Pro209Leu (PL), Pro209Gln (PQ), Glu455Lys (EK). One of three representative western blots is shown. ( g ) Filter retardation assay (FRA) analysis of the NP-40 insoluble fraction. Anti-GFP and anti-HSPB8 antibodies were used to detect insoluble levels of BAG3 (wild type or mutants) and HSPB8. Relative optical densities are reported in the graphs as means ± SD of normalized values. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis (n = 3). The constructs were abbreviated as followed: non-transfected (NT), empty vector (EV), wild type (WT), Pro209Ser (PS), Pro209Leu (PL), Pro209Gln (PQ), Glu455Lys (EK).

    Article Snippet: Lysed cells were centrifuged at 16,100 g for 15 min. Supernatants were collected and pellets resuspended in the same volume of NP-40 buffer without protease inhibitors and DTT, and finally sonicated.

    Techniques: Stable Transfection, Expressing, Transfection, Construct, Staining, Flow Cytometry, Solubility, Mutagenesis, Software, Western Blot, Plasmid Preparation

    The TTK-LKS region of C-terminus of DNAJB8 is required to suppress the aggregation of Fluc DM into inclusions. (A) Schematic overview of DNAJB6 and DNAJB8 C-terminal mutational variants used in this work. M1, M2 and M3 are mutations in the S/T-rich region of DNAJB6 in which underlined amino acids represent 6, 13 and 18 S/T-to-A substitutions, respectively. Regions identified between sets of arrows indicate deletion mutations. HEK293 cells were co-transfected with Fluc DM -EGFP and DNAJB6 or DNAJB8 C-terminal mutational variants (or mRFP as a negative control) and analysed 48 h post-transfection by quantitative flow cytometry (B and D) or NP-40 fractionation and subsequent immunoblotting (C and E) . Data in (B and D) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA (P

    Journal: bioRxiv

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation

    doi: 10.1101/2020.10.08.326280

    Figure Lengend Snippet: The TTK-LKS region of C-terminus of DNAJB8 is required to suppress the aggregation of Fluc DM into inclusions. (A) Schematic overview of DNAJB6 and DNAJB8 C-terminal mutational variants used in this work. M1, M2 and M3 are mutations in the S/T-rich region of DNAJB6 in which underlined amino acids represent 6, 13 and 18 S/T-to-A substitutions, respectively. Regions identified between sets of arrows indicate deletion mutations. HEK293 cells were co-transfected with Fluc DM -EGFP and DNAJB6 or DNAJB8 C-terminal mutational variants (or mRFP as a negative control) and analysed 48 h post-transfection by quantitative flow cytometry (B and D) or NP-40 fractionation and subsequent immunoblotting (C and E) . Data in (B and D) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA (P

    Article Snippet: Cellular protein extraction, quantification and fractionation Transfected cells were trypsinised, harvested, washed twice in PBS (300 × g for 5 min at RT) and total cellular protein was extracted by lysis with Nonidet™ P-40 (NP-40; Thermo Fisher Scientific) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40 supplemented with 0.5% (v/v) Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), pH 8.0).

    Techniques: Transfection, Negative Control, Flow Cytometry, Fractionation

    DNAJBs rely upon interaction with Hsp70 to deliver Fluc DM for the degradation via the proteasome. HEK293 cells co-transfected with Fluc DM -EGFP and mRFP (as a negative control), DNAJB1, DNAJB6 or DNAJB8 H/Q variants. Cells were treated with a proteasome inhibitor MG132 (10µM) or a DMSO vehicle control 24 h post-transfection. Cells were incubated for a further 18 h and analysed 42 h post-transfection by (A) quantitative flow cytometry or (B) NP-40 fractionation and subsequent Western blotting. Data in (A) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA ( P

    Journal: bioRxiv

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation

    doi: 10.1101/2020.10.08.326280

    Figure Lengend Snippet: DNAJBs rely upon interaction with Hsp70 to deliver Fluc DM for the degradation via the proteasome. HEK293 cells co-transfected with Fluc DM -EGFP and mRFP (as a negative control), DNAJB1, DNAJB6 or DNAJB8 H/Q variants. Cells were treated with a proteasome inhibitor MG132 (10µM) or a DMSO vehicle control 24 h post-transfection. Cells were incubated for a further 18 h and analysed 42 h post-transfection by (A) quantitative flow cytometry or (B) NP-40 fractionation and subsequent Western blotting. Data in (A) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA ( P

    Article Snippet: Cellular protein extraction, quantification and fractionation Transfected cells were trypsinised, harvested, washed twice in PBS (300 × g for 5 min at RT) and total cellular protein was extracted by lysis with Nonidet™ P-40 (NP-40; Thermo Fisher Scientific) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40 supplemented with 0.5% (v/v) Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), pH 8.0).

    Techniques: Transfection, Negative Control, Incubation, Flow Cytometry, Fractionation, Western Blot

    DNAJBs require an active proteasome to facilitate the degradation of Fluc DM . HEK293 cells were co-transfected to express Fluc DM -EGFP and mRFP (as a negative control), DNAJB1 or DNAJB6 and 24 h post-transfection, cells were treated with the proteasome inhibitor MG132 (10µM) or autophagy inhibitors 3-methyladenine (5mM) and bafilomycin A1 (1µM), or a DMSO vehicle control. Cells were incubated for a further 24 h and then analysed by (A) quantitative flow cytometry or (B) NP-40 fractionation and subsequent immunoblotting. Data in (A) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA ( P

    Journal: bioRxiv

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation

    doi: 10.1101/2020.10.08.326280

    Figure Lengend Snippet: DNAJBs require an active proteasome to facilitate the degradation of Fluc DM . HEK293 cells were co-transfected to express Fluc DM -EGFP and mRFP (as a negative control), DNAJB1 or DNAJB6 and 24 h post-transfection, cells were treated with the proteasome inhibitor MG132 (10µM) or autophagy inhibitors 3-methyladenine (5mM) and bafilomycin A1 (1µM), or a DMSO vehicle control. Cells were incubated for a further 24 h and then analysed by (A) quantitative flow cytometry or (B) NP-40 fractionation and subsequent immunoblotting. Data in (A) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA ( P

    Article Snippet: Cellular protein extraction, quantification and fractionation Transfected cells were trypsinised, harvested, washed twice in PBS (300 × g for 5 min at RT) and total cellular protein was extracted by lysis with Nonidet™ P-40 (NP-40; Thermo Fisher Scientific) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40 supplemented with 0.5% (v/v) Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), pH 8.0).

    Techniques: Transfection, Negative Control, Incubation, Flow Cytometry, Fractionation

    Interaction with Hsp70 is required for DNAJBs to suppress Fluc DM aggregation. (A) Schematic overview of DNAJB proteins identifying location of mutation within the J-domain, in which the histidine residue has been substituted for a glutamine (termed H/Q) at amino acid position 31 within the HPD (Hsp70-interacting) motif. HEK293 cells were co-transfected to express Fluc DM -EGFP and mRFP (as a negative control), DNAJB1, DNAJB6, DNAJB8 or their H/Q variants. Cells were analysed 48 h post-transfection by (B) quantitative flow cytometry or (C) NP-40 cell fractionation followed by immunoblotting. Data in (B) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA (P

    Journal: bioRxiv

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation

    doi: 10.1101/2020.10.08.326280

    Figure Lengend Snippet: Interaction with Hsp70 is required for DNAJBs to suppress Fluc DM aggregation. (A) Schematic overview of DNAJB proteins identifying location of mutation within the J-domain, in which the histidine residue has been substituted for a glutamine (termed H/Q) at amino acid position 31 within the HPD (Hsp70-interacting) motif. HEK293 cells were co-transfected to express Fluc DM -EGFP and mRFP (as a negative control), DNAJB1, DNAJB6, DNAJB8 or their H/Q variants. Cells were analysed 48 h post-transfection by (B) quantitative flow cytometry or (C) NP-40 cell fractionation followed by immunoblotting. Data in (B) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA (P

    Article Snippet: Cellular protein extraction, quantification and fractionation Transfected cells were trypsinised, harvested, washed twice in PBS (300 × g for 5 min at RT) and total cellular protein was extracted by lysis with Nonidet™ P-40 (NP-40; Thermo Fisher Scientific) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40 supplemented with 0.5% (v/v) Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), pH 8.0).

    Techniques: Mutagenesis, Transfection, Negative Control, Flow Cytometry, Cell Fractionation

    Fluc DM readily aggregates to form inclusions in cells, which can be assessed using FloIT. HEK293 cells were transfected with Fluc WT -EGFP or Fluc DM -EGFP and analysed 48 h post-transfection by (A) epifluorescence microscopy, (B) NP-40 cell fractionation followed by immunoblotting or (C) quantitative flow cytometry. In (A) green fluorescence was detected by excitation at 488 nm. Examples of cells containing inclusions are denoted by the arrows. All images were taken at 20X magnification using a Leica DMi8 fluorescence microscope. Scale bars = 60 μm. In (B) an anti-GFP antibody was used to detect Fluc WT or Fluc DM in the insoluble pellet fraction and total protein was used as a loading control. Data in (C) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a student’s t-test (** = P

    Journal: bioRxiv

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation

    doi: 10.1101/2020.10.08.326280

    Figure Lengend Snippet: Fluc DM readily aggregates to form inclusions in cells, which can be assessed using FloIT. HEK293 cells were transfected with Fluc WT -EGFP or Fluc DM -EGFP and analysed 48 h post-transfection by (A) epifluorescence microscopy, (B) NP-40 cell fractionation followed by immunoblotting or (C) quantitative flow cytometry. In (A) green fluorescence was detected by excitation at 488 nm. Examples of cells containing inclusions are denoted by the arrows. All images were taken at 20X magnification using a Leica DMi8 fluorescence microscope. Scale bars = 60 μm. In (B) an anti-GFP antibody was used to detect Fluc WT or Fluc DM in the insoluble pellet fraction and total protein was used as a loading control. Data in (C) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a student’s t-test (** = P

    Article Snippet: Cellular protein extraction, quantification and fractionation Transfected cells were trypsinised, harvested, washed twice in PBS (300 × g for 5 min at RT) and total cellular protein was extracted by lysis with Nonidet™ P-40 (NP-40; Thermo Fisher Scientific) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40 supplemented with 0.5% (v/v) Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), pH 8.0).

    Techniques: Transfection, Epifluorescence Microscopy, Cell Fractionation, Flow Cytometry, Fluorescence, Microscopy

    Disease-related mutations in the G/F-rich domain of DNAJB6 do not affect its capacity to inhibit Fluc DM inclusions formation. (A) Schematic overview of DNAJB6 disease-related missense mutations at amino acid positions 93 and 96 in the G/F-rich region. HEK293 cells were co-transfected with Fluc DM -EGFP and mRFP (as a negative control) or DNAJB6 G/F-domain disease-related mutational variants. Cells were analysed 48 h post-transfection by (B) quantitative flow cytometry or (C) NP-40 fractionation and immunoblotting. Data in (B) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA ( P

    Journal: bioRxiv

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation

    doi: 10.1101/2020.10.08.326280

    Figure Lengend Snippet: Disease-related mutations in the G/F-rich domain of DNAJB6 do not affect its capacity to inhibit Fluc DM inclusions formation. (A) Schematic overview of DNAJB6 disease-related missense mutations at amino acid positions 93 and 96 in the G/F-rich region. HEK293 cells were co-transfected with Fluc DM -EGFP and mRFP (as a negative control) or DNAJB6 G/F-domain disease-related mutational variants. Cells were analysed 48 h post-transfection by (B) quantitative flow cytometry or (C) NP-40 fractionation and immunoblotting. Data in (B) is presented as the mean ± S.E.M (n=3) of the number of inclusions per 100 cells. Significant differences between group means in the data were determined using a one-way ANOVA ( P

    Article Snippet: Cellular protein extraction, quantification and fractionation Transfected cells were trypsinised, harvested, washed twice in PBS (300 × g for 5 min at RT) and total cellular protein was extracted by lysis with Nonidet™ P-40 (NP-40; Thermo Fisher Scientific) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40 supplemented with 0.5% (v/v) Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), pH 8.0).

    Techniques: Transfection, Negative Control, Flow Cytometry, Fractionation