nonidet p 40 buffer  (Thermo Fisher)


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    Name:
    NP40 Cell Lysis Buffer
    Description:
    NP40 Cell Lysis Buffer is suitable for the preparation of cell extracts to be analyzed by Antibody Bead Immunoassay Luminex ELISA and Western blotting
    Catalog Number:
    FNN0021
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    Build Your Own Immunoassay|Cell Analysis|Cell Lysis & Fractionation|ELISA|Luminex® Assays|Protein Assays and Analysis|Protein Biology|Protein Purification & Isolation|Ready-To-Use Immunoassay
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    Structured Review

    Thermo Fisher nonidet p 40 buffer
    NP40 Cell Lysis Buffer is suitable for the preparation of cell extracts to be analyzed by Antibody Bead Immunoassay Luminex ELISA and Western blotting
    https://www.bioz.com/result/nonidet p 40 buffer/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nonidet p 40 buffer - by Bioz Stars, 2021-05
    97/100 stars

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    Lysis:

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation
    Article Snippet: .. The pellet was washed in ice-cold TNE buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) and centrifuged again at 20,000 × g for 30 mins at 4° C. The supernatant was carefully removed and discarded and the pellet resuspended in 50 µL NP-40 lysis buffer. .. The insoluble pellet was sonicated at 50% amplitude for 5 sec (NP-40 insoluble fraction).

    Article Title: The E3 ubiquitin ligase MARCH2 regulates ERGIC3-dependent trafficking of secretory proteins
    Article Snippet: To immunoprecipitate the endogenous ERGIC3, cell lysates were incubated with 500 ng of anti-ERGIC3 antibody and protein G–agarose beads (Invitrogen) overnight at 4 °C. .. Samples were washed with 1% NP-40 lysis buffer three times, eluted in 2× SDS sample buffer, and analyzed by Western blotting using antibodies as indicated in the figure legends. .. Analysis of secreted proteins Cells were transiently transfected with the secreted protein expression plasmids.

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation
    Article Snippet: Data were analysed by one-way analysis of variance (ANOVA) and Tukey’s post-hoc test or, where appropriate, assessed assuming unequal variance using the two-tailed student t-test. .. 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). .. Cell lysates were then sonicated using the Sonifer® 250 Digital cell disruptor and a double step micro-tip (Branson Ultrasonics, Brookfield, CT, USA) at 50% amplitude for 5 sec.

    Pull Down Assay:

    Article Title: Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis
    Article Snippet: Protein detection on the blotting membranes was performed using anti-FLAG mAb M2 (Sigma-Aldrich, St. Louis, MO, USA), followed by peroxidase-conjugated affinity-purified F(ab′)2 fragment of goat anti-mouse IgG, F(ab′)2 fragment-specific (Jackson ImmunoResearch, West Grove, PA, USA) for FLAG-tagged proteins, or HRP-conjugated streptavidin (Nacalai, Kyoto, Japan) for biotinylated proteins. .. Pull-Down Assay One-microgram biotinylated-Nod2-WT (Nod2-WT-Btn) and 1 μ g FLAG-tagged RICK-WT (FLAG-RICK-WT) lysed in 300 μ L NP-40 buffer [1% Nonidet P-40, 142.5 mmol/L KCl, 5 mmol/L MgCl2 , 10 mmol/L HEPES (pH 7.6), 0.2 mmol/L phenylmethylsulfonylfluoride (PMSF), and 1 mmol/L EDTA] were precipitated with 20 μ L streptavidin-conjugated agarose beads (Invitrogen) with or without 5.33 mg/mL MDP (Sigma-Aldrich) and incubated for 3 hours at 4°C. .. The precipitations were subjected to SDS-PAGE and immunoblotting.

    Incubation:

    Article Title: Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis
    Article Snippet: Protein detection on the blotting membranes was performed using anti-FLAG mAb M2 (Sigma-Aldrich, St. Louis, MO, USA), followed by peroxidase-conjugated affinity-purified F(ab′)2 fragment of goat anti-mouse IgG, F(ab′)2 fragment-specific (Jackson ImmunoResearch, West Grove, PA, USA) for FLAG-tagged proteins, or HRP-conjugated streptavidin (Nacalai, Kyoto, Japan) for biotinylated proteins. .. Pull-Down Assay One-microgram biotinylated-Nod2-WT (Nod2-WT-Btn) and 1 μ g FLAG-tagged RICK-WT (FLAG-RICK-WT) lysed in 300 μ L NP-40 buffer [1% Nonidet P-40, 142.5 mmol/L KCl, 5 mmol/L MgCl2 , 10 mmol/L HEPES (pH 7.6), 0.2 mmol/L phenylmethylsulfonylfluoride (PMSF), and 1 mmol/L EDTA] were precipitated with 20 μ L streptavidin-conjugated agarose beads (Invitrogen) with or without 5.33 mg/mL MDP (Sigma-Aldrich) and incubated for 3 hours at 4°C. .. The precipitations were subjected to SDS-PAGE and immunoblotting.

    Western Blot:

    Article Title: The E3 ubiquitin ligase MARCH2 regulates ERGIC3-dependent trafficking of secretory proteins
    Article Snippet: To immunoprecipitate the endogenous ERGIC3, cell lysates were incubated with 500 ng of anti-ERGIC3 antibody and protein G–agarose beads (Invitrogen) overnight at 4 °C. .. Samples were washed with 1% NP-40 lysis buffer three times, eluted in 2× SDS sample buffer, and analyzed by Western blotting using antibodies as indicated in the figure legends. .. Analysis of secreted proteins Cells were transiently transfected with the secreted protein expression plasmids.

    Article Title: A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking
    Article Snippet: .. GST-GGA3VHS+GAT (3 μg) was preincubated with 3 μg Trx-PACS-1FBR in GST-binding buffer containing 4% NP40 for 2 h at RT, followed by the addition of 3 μg Trx-CK2β for 2 h at RT, then glutathione agarose for 30 min. Glutathione beads were pelleted by centrifugation, washed with GST-binding buffer containing 4% NP40, and analyzed by Western blotting with mAb anti-Thioredoxin (Trx; IgG1, Invitrogen no. R920–25). .. A7 cells or HeLa:CD8-CIMPR cells grown to 80% confluency were infected with VV expressing PACS-1, PACS-1GGAmut , PACS-1CKmut , PACS-1Admut or PACS-1S278D/CKmut (m.o.i.=10) or transfected with pcDNA FLAG-furin.

    other:

    Article Title: The Sec63/BiP complex suppresses higher-order oligomerization and RNase activity of IRE1α during ER stress
    Article Snippet: The beads were washed 3 times with either 1ml of Buffer A including 0.1% digitonin or 1 ml of NP40 buffer and eluted by directly boiling in 50 μl of 2x SDS sample buffer for 5 min and analyzed by immunoblotting.

    Sonication:

    Article Title: BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes
    Article Snippet: Cells were resuspended in NP-40 lysis buffer (150 mM NaCl, 20 mM TrisBase, NP-40 0.05%, 1.5 mM MgCl2 , Glycerol 3%, pH 7.4) added DTT and Complete Protease inhibitor (Roche Applied Science, Indianapolis, IN, USA), and passed through a syringe 10 times. .. 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. .. For the evaluation of the effects of BAG3 mutations on its chaperone-activity towards aggregation-prone proteins (SOD1_G93A) (Fig. ), HEK293T cells were co-transfected with BAG3-GFP constructs and SOD1_G93A encoding plasmid, as described above.

    Centrifugation:

    Article Title: A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking
    Article Snippet: .. GST-GGA3VHS+GAT (3 μg) was preincubated with 3 μg Trx-PACS-1FBR in GST-binding buffer containing 4% NP40 for 2 h at RT, followed by the addition of 3 μg Trx-CK2β for 2 h at RT, then glutathione agarose for 30 min. Glutathione beads were pelleted by centrifugation, washed with GST-binding buffer containing 4% NP40, and analyzed by Western blotting with mAb anti-Thioredoxin (Trx; IgG1, Invitrogen no. R920–25). .. A7 cells or HeLa:CD8-CIMPR cells grown to 80% confluency were infected with VV expressing PACS-1, PACS-1GGAmut , PACS-1CKmut , PACS-1Admut or PACS-1S278D/CKmut (m.o.i.=10) or transfected with pcDNA FLAG-furin.

    Concentration Assay:

    Article Title: Alzheimer’s disease pathogenesis is dependent on neuronal receptor PTPσ
    Article Snippet: Protein extraction, immunoprecipitation, and western blot analysisFor the co-immunoprecipitation of APP and PTPσ, RIPA buffer was used (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate). .. For the co-immunoprecipitation of APP and BACE1, NP40 buffer was used (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% NP40) without or with SDS at concentration of 0.1%, 0.3%, and 0.4%. .. For total protein extraction and immunopurification of CTFβ, SDS concentration in RIPA buffer was adjusted to 1% to ensure protein extraction from the lipid rafts.

    Protein Extraction:

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation
    Article Snippet: Data were analysed by one-way analysis of variance (ANOVA) and Tukey’s post-hoc test or, where appropriate, assessed assuming unequal variance using the two-tailed student t-test. .. 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). .. Cell lysates were then sonicated using the Sonifer® 250 Digital cell disruptor and a double step micro-tip (Branson Ultrasonics, Brookfield, CT, USA) at 50% amplitude for 5 sec.

    Fractionation:

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation
    Article Snippet: Data were analysed by one-way analysis of variance (ANOVA) and Tukey’s post-hoc test or, where appropriate, assessed assuming unequal variance using the two-tailed student t-test. .. 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). .. Cell lysates were then sonicated using the Sonifer® 250 Digital cell disruptor and a double step micro-tip (Branson Ultrasonics, Brookfield, CT, USA) at 50% amplitude for 5 sec.

    Transfection:

    Article Title: DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation
    Article Snippet: Data were analysed by one-way analysis of variance (ANOVA) and Tukey’s post-hoc test or, where appropriate, assessed assuming unequal variance using the two-tailed student t-test. .. 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). .. Cell lysates were then sonicated using the Sonifer® 250 Digital cell disruptor and a double step micro-tip (Branson Ultrasonics, Brookfield, CT, USA) at 50% amplitude for 5 sec.

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    Thermo Fisher np 40 buffer
    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.
    Np 40 Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher immunoprecipitation ip buffer
    HERC2 is an E3 ligase important for the degradation of USP33. A , <t>co-immunoprecipitation</t> ( IP ) of endogenous USP33 and USP20 with endogenous HERC2. Cell lysates were prepared from HeLa cells and immunoprecipitation experiments were performed using control
    Immunoprecipitation Ip Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 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

    Construction of Nod2-nodosome containing the BS/EOS-associated mutation in a cell-free system. Synthetic protein-protein interactions were detected by pull-down assay and amplified luminescent proximity homogeneous assay (ALPHA). (a) Biotinylated-Nod2-WT (Nod2-WT-Btn) and 1 μ g FLAG-tagged RICK-WT (FLAG-RICK-WT) lysed in 300 μ L NP-40 buffer were precipitated with 20 μ L streptavidin-conjugated agarose beads with or without MDP. The precipitations were subjected to SDS-PAGE and immunoblotting. Detection on the blotting membranes was performed using anti-FLAG mAb M2 or anti-Nod2 mAb. (b) A total of 100 ng of each protein indicated was incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL MDP or 5 mg/mL N-Acetylmuramyl-D-Alanyl-D-Isoglutamine (MDP-D-isomer). Responses (counts) were measured using the EnSpire ™ Multimode Plate Reader. (c) Activation of Nod2-nodosome in a cell-free system by MDP degradation components. Interactions between Nod2-WT-Btn and FLAG-RICK-WT were detected by ALPHA. A total of 100 ng of Nod2-WT-Btn and FLAG-RICK-WT were incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, without or with 5.33 mg/mL MDP, 5.33 mg/mL MDP (D-isomer), 5.33 mg/mL N-acetylglucosamine (GlcNAc), 5.33 mg/mL L-alanine (L-Ala), or 5.33 mg/mL D-isoglutamine (D-isoGlu). Responses (counts) were measured using the EnSpire Multimode Plate Reader. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. (d) A total of 100 ng of Nod2-WT-Btn, or Nod2-R334W-Btn, or Nod2-N670K-Btn with FLAG-RICK-WT was incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL or 13.33 mg/mL MDP or 13.33 mg/mL MDP-D-isomer. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. CARD, caspase recruitment domain; MDP, muramyl dipeptide; WT, wild-type; P, precipitation; WB, western blot. ∗ p value

    Journal: The Scientific World Journal

    Article Title: Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

    doi: 10.1155/2016/2597376

    Figure Lengend Snippet: Construction of Nod2-nodosome containing the BS/EOS-associated mutation in a cell-free system. Synthetic protein-protein interactions were detected by pull-down assay and amplified luminescent proximity homogeneous assay (ALPHA). (a) Biotinylated-Nod2-WT (Nod2-WT-Btn) and 1 μ g FLAG-tagged RICK-WT (FLAG-RICK-WT) lysed in 300 μ L NP-40 buffer were precipitated with 20 μ L streptavidin-conjugated agarose beads with or without MDP. The precipitations were subjected to SDS-PAGE and immunoblotting. Detection on the blotting membranes was performed using anti-FLAG mAb M2 or anti-Nod2 mAb. (b) A total of 100 ng of each protein indicated was incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL MDP or 5 mg/mL N-Acetylmuramyl-D-Alanyl-D-Isoglutamine (MDP-D-isomer). Responses (counts) were measured using the EnSpire ™ Multimode Plate Reader. (c) Activation of Nod2-nodosome in a cell-free system by MDP degradation components. Interactions between Nod2-WT-Btn and FLAG-RICK-WT were detected by ALPHA. A total of 100 ng of Nod2-WT-Btn and FLAG-RICK-WT were incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, without or with 5.33 mg/mL MDP, 5.33 mg/mL MDP (D-isomer), 5.33 mg/mL N-acetylglucosamine (GlcNAc), 5.33 mg/mL L-alanine (L-Ala), or 5.33 mg/mL D-isoglutamine (D-isoGlu). Responses (counts) were measured using the EnSpire Multimode Plate Reader. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. (d) A total of 100 ng of Nod2-WT-Btn, or Nod2-R334W-Btn, or Nod2-N670K-Btn with FLAG-RICK-WT was incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL or 13.33 mg/mL MDP or 13.33 mg/mL MDP-D-isomer. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. CARD, caspase recruitment domain; MDP, muramyl dipeptide; WT, wild-type; P, precipitation; WB, western blot. ∗ p value

    Article Snippet: Pull-Down Assay One-microgram biotinylated-Nod2-WT (Nod2-WT-Btn) and 1 μ g FLAG-tagged RICK-WT (FLAG-RICK-WT) lysed in 300 μ L NP-40 buffer [1% Nonidet P-40, 142.5 mmol/L KCl, 5 mmol/L MgCl2 , 10 mmol/L HEPES (pH 7.6), 0.2 mmol/L phenylmethylsulfonylfluoride (PMSF), and 1 mmol/L EDTA] were precipitated with 20 μ L streptavidin-conjugated agarose beads (Invitrogen) with or without 5.33 mg/mL MDP (Sigma-Aldrich) and incubated for 3 hours at 4°C.

    Techniques: Mutagenesis, Pull Down Assay, Amplification, SDS Page, Incubation, Activation Assay, Standard Deviation, Western Blot

    HERC2 is an E3 ligase important for the degradation of USP33. A , co-immunoprecipitation ( IP ) of endogenous USP33 and USP20 with endogenous HERC2. Cell lysates were prepared from HeLa cells and immunoprecipitation experiments were performed using control

    Journal: The Journal of Biological Chemistry

    Article Title: Degradation of the Deubiquitinating Enzyme USP33 Is Mediated by p97 and the Ubiquitin Ligase HERC2 *

    doi: 10.1074/jbc.M114.569392

    Figure Lengend Snippet: HERC2 is an E3 ligase important for the degradation of USP33. A , co-immunoprecipitation ( IP ) of endogenous USP33 and USP20 with endogenous HERC2. Cell lysates were prepared from HeLa cells and immunoprecipitation experiments were performed using control

    Article Snippet: Freshly harvested cells were washed once in ice-cold phosphate-buffered saline (PBS) followed by lysis in immunoprecipitation (IP) buffer (50 m m HEPES-KOH, pH 7.0, 250 m m NaCl, 5 m m EDTA, 10% glycerol, 0.1% Igepal CA-630) supplemented with 1× Halt protease inhibitors mixture (Pierce) and 10 μ m MG132.

    Techniques: Immunoprecipitation

    p97 regulates the interaction between HERC2 and USP33. A , co-immunoprecipitation of HERC2 and USP33 upon p97 inhibition. HeLa cells were treated with control ( DMSO ) or 10 μ m NMS-873 for 12 h, and total lysates were harvested for immunoprecipitation

    Journal: The Journal of Biological Chemistry

    Article Title: Degradation of the Deubiquitinating Enzyme USP33 Is Mediated by p97 and the Ubiquitin Ligase HERC2 *

    doi: 10.1074/jbc.M114.569392

    Figure Lengend Snippet: p97 regulates the interaction between HERC2 and USP33. A , co-immunoprecipitation of HERC2 and USP33 upon p97 inhibition. HeLa cells were treated with control ( DMSO ) or 10 μ m NMS-873 for 12 h, and total lysates were harvested for immunoprecipitation

    Article Snippet: Freshly harvested cells were washed once in ice-cold phosphate-buffered saline (PBS) followed by lysis in immunoprecipitation (IP) buffer (50 m m HEPES-KOH, pH 7.0, 250 m m NaCl, 5 m m EDTA, 10% glycerol, 0.1% Igepal CA-630) supplemented with 1× Halt protease inhibitors mixture (Pierce) and 10 μ m MG132.

    Techniques: Immunoprecipitation, Inhibition