np 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
    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
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher np 40 buffer
    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 <t>NP-40</t> 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
    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/np 40 buffer/product/Thermo Fisher
    Average 99 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    np 40 buffer - by Bioz Stars, 2021-01
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    Images

    1) Product Images from "Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis"

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

    Journal: The Scientific World Journal

    doi: 10.1155/2016/2597376

    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
    Figure Legend 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

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

    2) Product Images from "Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation"

    Article Title: Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201102142

    DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.
    Figure Legend Snippet: DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.

    Techniques Used: Mouse Assay, Expressing, Transfection, Cell Culture, Over Expression, Isolation, Staining, Immunostaining

    3) Product Images from "A Duplicated ESCRT-III Factor Blocks Enveloped Virus Budding Without Inhibiting Cellular ESCRT Functions"

    Article Title: A Duplicated ESCRT-III Factor Blocks Enveloped Virus Budding Without Inhibiting Cellular ESCRT Functions

    Journal: bioRxiv

    doi: 10.1101/2020.08.30.273656

    RetroCHMP3 SS is detoxified. (A) Viability of cells transiently transfected with HA-tagged CHMP3 SS variants, as determined in a luminescent ATP cell viability assay. Mean ± SD from 5 experimental replicates with n≥6 each. (B) Immunofluorescence showing the dispersed (CHMP3 SS and retroCHMP3 SS ) and punctate (CHMP3 SS (155)) subcellular localization phenotypes. Scale bar 10 μm. Insets show percentages of cells with the two depicted phenotypes (diffuse or punctate: n? 450 cells/condition). White arrowhead points to a midbody. (C) Number of midbody-linked cells in cultures transiently transfected with the designated HA-tagged CHMP3 SS variants. Mean ± SD from 3 experimental replicates, each with 150 cells/condition. (D) Pulldown of myc-tagged human CHMP4B with STrEP/FLAG-tagged human CHMP3 (CHMP3 HS ), C-terminally truncated human CHMP3 (CHMP3 HS (150)), or CHMP3 SS variants. Cell lysis and pulldowns were performed in RIPA or NP-40 buffer, as indicated. Representative Western blot from 3 experimental repeats. **** p
    Figure Legend Snippet: RetroCHMP3 SS is detoxified. (A) Viability of cells transiently transfected with HA-tagged CHMP3 SS variants, as determined in a luminescent ATP cell viability assay. Mean ± SD from 5 experimental replicates with n≥6 each. (B) Immunofluorescence showing the dispersed (CHMP3 SS and retroCHMP3 SS ) and punctate (CHMP3 SS (155)) subcellular localization phenotypes. Scale bar 10 μm. Insets show percentages of cells with the two depicted phenotypes (diffuse or punctate: n? 450 cells/condition). White arrowhead points to a midbody. (C) Number of midbody-linked cells in cultures transiently transfected with the designated HA-tagged CHMP3 SS variants. Mean ± SD from 3 experimental replicates, each with 150 cells/condition. (D) Pulldown of myc-tagged human CHMP4B with STrEP/FLAG-tagged human CHMP3 (CHMP3 HS ), C-terminally truncated human CHMP3 (CHMP3 HS (150)), or CHMP3 SS variants. Cell lysis and pulldowns were performed in RIPA or NP-40 buffer, as indicated. Representative Western blot from 3 experimental repeats. **** p

    Techniques Used: Transfection, Viability Assay, Immunofluorescence, Lysis, Western Blot

    4) Product Images from "Interaction and Interdependent Packaging of Tegument Protein UL11 and Glycoprotein E of Herpes Simplex Virus ▿"

    Article Title: Interaction and Interdependent Packaging of Tegument Protein UL11 and Glycoprotein E of Herpes Simplex Virus ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.05207-11

    Direct binding of UL11 to the cytoplasmic tail of gE. UL11 was expressed as a His 6 -tagged fusion protein. Purified GST-gE.CT or GST-only were incubated with 2-fold increasing amounts of purified His 6 -UL11 in NP-40 lysis buffer at room temperature for
    Figure Legend Snippet: Direct binding of UL11 to the cytoplasmic tail of gE. UL11 was expressed as a His 6 -tagged fusion protein. Purified GST-gE.CT or GST-only were incubated with 2-fold increasing amounts of purified His 6 -UL11 in NP-40 lysis buffer at room temperature for

    Techniques Used: Binding Assay, Purification, Incubation, Lysis

    5) Product Images from "BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes"

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

    Journal: bioRxiv

    doi: 10.1101/853804

    Quantification of total amount of BAG3 wild type or mutants. HEK293T cells that stably overexpress HSPB8-V5 were transiently transfected with wild type or mutant BAG3-GFP constructs. Cells were collected in NP-40 containing buffer and the soluble fraction was separated from the NP-40 insoluble fraction. Both fractions were analysed by western blot and quantified using densitometric analysis. Samples were quantified relative to the wild type (WT). (n=3)
    Figure Legend Snippet: Quantification of total amount of BAG3 wild type or mutants. HEK293T cells that stably overexpress HSPB8-V5 were transiently transfected with wild type or mutant BAG3-GFP constructs. Cells were collected in NP-40 containing buffer and the soluble fraction was separated from the NP-40 insoluble fraction. Both fractions were analysed by western blot and quantified using densitometric analysis. Samples were quantified relative to the wild type (WT). (n=3)

    Techniques Used: Stable Transfection, Transfection, Mutagenesis, Construct, Western Blot

    Clearance assay of wild type and mutant BAG3 is prohibited by inhibition of autophagy. HEK293T cells that stably overexpress HSPB8-V5 were transiently transfected with mutant BAG3-GFP constructs. Cells were left untreated or treated with 3-MA for 36 hours after which protein lysates were collected and analysed by western blot and filter retardation assay (FRA). The FRA analysis is displayed for the NP-40 insoluble fraction. Relative optical densities are reported in the graph as means ± SD of normalized values. Student T tests were used for statistical analysis to compare the treated with untreated condition for each respective BAG3 variant (n=3).
    Figure Legend Snippet: Clearance assay of wild type and mutant BAG3 is prohibited by inhibition of autophagy. HEK293T cells that stably overexpress HSPB8-V5 were transiently transfected with mutant BAG3-GFP constructs. Cells were left untreated or treated with 3-MA for 36 hours after which protein lysates were collected and analysed by western blot and filter retardation assay (FRA). The FRA analysis is displayed for the NP-40 insoluble fraction. Relative optical densities are reported in the graph as means ± SD of normalized values. Student T tests were used for statistical analysis to compare the treated with untreated condition for each respective BAG3 variant (n=3).

    Techniques Used: Mutagenesis, Inhibition, Stable Transfection, Transfection, Construct, Western Blot, Variant 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 and c ) or verified protein aggregation by immunofluorescence ( b and 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.
    Figure Legend 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 and c ) or verified protein aggregation by immunofluorescence ( b and 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.

    Techniques Used: 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).
    Figure Legend 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).

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

    6) Product Images from "BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-65664-z

    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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

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

    7) Product Images from "Trafficking of an Acylated Cytosolic Protein: Newly Synthesized p56lck Travels to the Plasma Membrane via the Exocytic Pathway "

    Article Title: Trafficking of an Acylated Cytosolic Protein: Newly Synthesized p56lck Travels to the Plasma Membrane via the Exocytic Pathway

    Journal: The Journal of Cell Biology

    doi:

    Kinetics of the association of Lck with CD4. (A) SupT1 cells (1.25 × 10 8 ) were labeled for 5 min with [ 35 S]methionine/cysteine (1.5 mCi) and chased as indicated. The cells were lysed in NP-40 buffer and subjected to sequential immunoprecipitations, first with two rounds of anti-CD4 antibodies (CD4.1 and CD4.2) and next with anti-Lck antibodies (Lck). (B) SupT1 cells (10 8 ) were labeled with [ 35 S]methionine/cysteine (1 mCi) for 2 min and chased for 0, 3, or 6 min. Lck was immunoprecipitated from membrane (M) and soluble (S) fractions with LckN and analyzed by SDS-PAGE and autoradiography. (C) SupT1 cells (7.5 × 10 7 ) were labeled with [ 35 S]methionine/cysteine (1 mCi) for 2 min and chased for 0, 3, or 6 min. Cell lysates were subjected to sequential immunoprecipitations, first with anti-CD4 antibodies (CD4) and next with anti-Lck antibodies (Lck). The last of three rounds of preclears with normal rabbit serum (NRS) and protein A–Sepharose is shown for the 6-min chase (NRS). Lck, CD4, and the background band (★) are indicated.
    Figure Legend Snippet: Kinetics of the association of Lck with CD4. (A) SupT1 cells (1.25 × 10 8 ) were labeled for 5 min with [ 35 S]methionine/cysteine (1.5 mCi) and chased as indicated. The cells were lysed in NP-40 buffer and subjected to sequential immunoprecipitations, first with two rounds of anti-CD4 antibodies (CD4.1 and CD4.2) and next with anti-Lck antibodies (Lck). (B) SupT1 cells (10 8 ) were labeled with [ 35 S]methionine/cysteine (1 mCi) for 2 min and chased for 0, 3, or 6 min. Lck was immunoprecipitated from membrane (M) and soluble (S) fractions with LckN and analyzed by SDS-PAGE and autoradiography. (C) SupT1 cells (7.5 × 10 7 ) were labeled with [ 35 S]methionine/cysteine (1 mCi) for 2 min and chased for 0, 3, or 6 min. Cell lysates were subjected to sequential immunoprecipitations, first with anti-CD4 antibodies (CD4) and next with anti-Lck antibodies (Lck). The last of three rounds of preclears with normal rabbit serum (NRS) and protein A–Sepharose is shown for the 6-min chase (NRS). Lck, CD4, and the background band (★) are indicated.

    Techniques Used: Labeling, Immunoprecipitation, SDS Page, Autoradiography

    Binding of Lck to newly synthesized CD4 and membranes in the presence of BFA. (A) SupT1 cells were pulse-labeled for 5 min and chased for 40 min in the absence or presence of BFA (10 μg/ml during pulse, 2 μg/ml during chase). Pretreatment with BFA was 2 min before the pulse. NP-40 cell lysates were subjected to immunoprecipitation, first with anti-Lck (αLck lanes), next with anti-CD4 antibodies (αCD4). Lck, CD4, and a background band (★) are indicated. (B) SupT1 cells were pretreated and labeled for 5 min in the presence of BFA (10 μg/ml) and chased for indicated times in the presence of 2 μg/ml BFA. Lck was immunoprecipitated at indicated chase times from membrane (M) and soluble (S) fractions.
    Figure Legend Snippet: Binding of Lck to newly synthesized CD4 and membranes in the presence of BFA. (A) SupT1 cells were pulse-labeled for 5 min and chased for 40 min in the absence or presence of BFA (10 μg/ml during pulse, 2 μg/ml during chase). Pretreatment with BFA was 2 min before the pulse. NP-40 cell lysates were subjected to immunoprecipitation, first with anti-Lck (αLck lanes), next with anti-CD4 antibodies (αCD4). Lck, CD4, and a background band (★) are indicated. (B) SupT1 cells were pretreated and labeled for 5 min in the presence of BFA (10 μg/ml) and chased for indicated times in the presence of 2 μg/ml BFA. Lck was immunoprecipitated at indicated chase times from membrane (M) and soluble (S) fractions.

    Techniques Used: Binding Assay, Synthesized, Labeling, Immunoprecipitation

    Kinetics of the association of CD4 with Lck. (A) SupT1 cells (5 × 10 7 ) were labeled for 5 min with [ 35 S]methionine/cysteine (0.5 mCi) and chased for the indicated times. The cells were lysed in NP-40 buffer and subjected to immunoprecipitation with anti-LckN serum. Association of CD4 with Lck was detected by coimmunoprecipitation with Lck. Lck, CD4, and the background band (★) are indicated. (B) SupT1 cells were labeled as described in A, lysed, and CD4 was immunoprecipitated from the cell lysates. Immunoprecipitates were incubated with Endo H (1 mU) for 1 h at 37°C before separation by SDS-PAGE. Endo H–sensitive (S) forms of CD4 from which two carbohydrate chains were removed and resistant (R) forms from which only one carbohydrate chain was removed are indicated. Nondigested CD4, containing two carbohydrate chains, migrates slower than Endo H–resistant CD4 (not shown). Coimmunoprecipitated Lck is indicated and is unaffected by Endo H treatment.
    Figure Legend Snippet: Kinetics of the association of CD4 with Lck. (A) SupT1 cells (5 × 10 7 ) were labeled for 5 min with [ 35 S]methionine/cysteine (0.5 mCi) and chased for the indicated times. The cells were lysed in NP-40 buffer and subjected to immunoprecipitation with anti-LckN serum. Association of CD4 with Lck was detected by coimmunoprecipitation with Lck. Lck, CD4, and the background band (★) are indicated. (B) SupT1 cells were labeled as described in A, lysed, and CD4 was immunoprecipitated from the cell lysates. Immunoprecipitates were incubated with Endo H (1 mU) for 1 h at 37°C before separation by SDS-PAGE. Endo H–sensitive (S) forms of CD4 from which two carbohydrate chains were removed and resistant (R) forms from which only one carbohydrate chain was removed are indicated. Nondigested CD4, containing two carbohydrate chains, migrates slower than Endo H–resistant CD4 (not shown). Coimmunoprecipitated Lck is indicated and is unaffected by Endo H treatment.

    Techniques Used: Labeling, Immunoprecipitation, Incubation, SDS Page

    8) Product Images from "HRS–WASH axis governs actin-mediated endosomal recycling and cell invasion"

    Article Title: HRS–WASH axis governs actin-mediated endosomal recycling and cell invasion

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201710051

    HRS is required for the endosomal recruitment of WASH. (A) HeLa cells were treated with the indicated siRNA over 120 h before fixation in 4% PFA/PBS and labeling with antibodies targeting HRS and WASH. Maximum-projection images. (B) Airyscan single confocal slice images of HeLa cells fixed and labeled for WASH and HRS. (C and D) PLA of HeLa cells probed for both HRS and WASH or technical single WASH or HRS antibody controls. Data represented as mean number of signals per cell. Maximum-projection images (nuclei stained with DAPI). Bars, 20 µm. (E and F) HeLa S3 Flp-In cells stably transfected with GFP-HRS (mouse) were treated with siRNA targeting endogenous HRS over 120 h before fixation in 4% PFA/PBS or lysis in NP-40 buffer. Single confocal slice images. Molecular masses are given in kilodaltons. IB, immunoblot. (G) Quantification of Pearson’s R correlation between EEA1 and WASH ( > 150 cells total). (H and I) HeLa cells were treated with siRNA over 120 h before fixation in 4% PFA/PBS and labeling with antibodies targeting EEA1 and 647-phalloidin. Quantification of sum intensity of actin on endosome. Maximum-projection images ( > 30 cells total). n = 3; error bars indicate SEM. **, P
    Figure Legend Snippet: HRS is required for the endosomal recruitment of WASH. (A) HeLa cells were treated with the indicated siRNA over 120 h before fixation in 4% PFA/PBS and labeling with antibodies targeting HRS and WASH. Maximum-projection images. (B) Airyscan single confocal slice images of HeLa cells fixed and labeled for WASH and HRS. (C and D) PLA of HeLa cells probed for both HRS and WASH or technical single WASH or HRS antibody controls. Data represented as mean number of signals per cell. Maximum-projection images (nuclei stained with DAPI). Bars, 20 µm. (E and F) HeLa S3 Flp-In cells stably transfected with GFP-HRS (mouse) were treated with siRNA targeting endogenous HRS over 120 h before fixation in 4% PFA/PBS or lysis in NP-40 buffer. Single confocal slice images. Molecular masses are given in kilodaltons. IB, immunoblot. (G) Quantification of Pearson’s R correlation between EEA1 and WASH ( > 150 cells total). (H and I) HeLa cells were treated with siRNA over 120 h before fixation in 4% PFA/PBS and labeling with antibodies targeting EEA1 and 647-phalloidin. Quantification of sum intensity of actin on endosome. Maximum-projection images ( > 30 cells total). n = 3; error bars indicate SEM. **, P

    Techniques Used: Labeling, Proximity Ligation Assay, Staining, Stable Transfection, Transfection, Lysis

    9) Product Images from "Glycoprotein D of HSV-1 is dependent on tegument protein UL16 for packaging and contains a motif that is differentially required for syncytia formation"

    Article Title: Glycoprotein D of HSV-1 is dependent on tegument protein UL16 for packaging and contains a motif that is differentially required for syncytia formation

    Journal: Virology

    doi: 10.1016/j.virol.2018.09.018

    A regulated interaction between UL16 and the gD cytoplasmic tail. ( A ) Vero cells were singly transfected (top row) with plasmids that express full-length UL16-GFP, UL16 NTD-GFP, or UL16 CTD-GFP. Additionally, each of these constructs were co-expressed with gD-HA (bottom three rows). Cells were fixed, permeabilized, and stained with a monoclonal antibody against the HA tag at one day post-transfection. ( B ) Purified His 6 -UL16(1–155) protein was incubated with the indicated GST-fusion proteins either in the presence or absence of NEM bound to glutathione-sepharose beads in 0.5% NP-40 buffer at 37°C. The beads were then washed, boiled in sample buffer, and the proteins were analyzed by western blotting.
    Figure Legend Snippet: A regulated interaction between UL16 and the gD cytoplasmic tail. ( A ) Vero cells were singly transfected (top row) with plasmids that express full-length UL16-GFP, UL16 NTD-GFP, or UL16 CTD-GFP. Additionally, each of these constructs were co-expressed with gD-HA (bottom three rows). Cells were fixed, permeabilized, and stained with a monoclonal antibody against the HA tag at one day post-transfection. ( B ) Purified His 6 -UL16(1–155) protein was incubated with the indicated GST-fusion proteins either in the presence or absence of NEM bound to glutathione-sepharose beads in 0.5% NP-40 buffer at 37°C. The beads were then washed, boiled in sample buffer, and the proteins were analyzed by western blotting.

    Techniques Used: Transfection, Construct, Staining, Purification, Incubation, Western Blot

    The UL16-gD interaction is not critical for the gBsyn phenotype. ( A ) Cells were infected with gBsyn, M6/syn, ΔCT/syn, or ΔCT.R/syn at a low MOI. Images were taken with an inverted light microscope at 2–3 days post infection. ( B ) Vero cells were infected with the indicated viruses at an MOI of 1. At 24 hr post infection, extracellular virions in the media were pelleted through a 30% sucrose cushion and the cells were collected. Samples were analyzed by western blotting. (C) Duplicate cultures of Vero cells were infected with the indicated viruses at an MOI of 0.1. The media containing extracellular virions was collected, and the infected cells were harvested and processed separately at 6-hour time points. Infectious virus was titered by plaque assay. ( D ) Purified His 6 -UL16 was incubated in 0.5% NP-40 buffer at 37°C for 5 hr with glutathione-sepharose beads bearing GST fusion proteins with the entire tail of gD or truncated sections of the tail. GST-UL11 and GST alone served as respective positive and negative controls. After incubation, the beads were washed, boiled in sample buffer, and the presence of bound His 6 -UL16 was analyzed by western blotting.
    Figure Legend Snippet: The UL16-gD interaction is not critical for the gBsyn phenotype. ( A ) Cells were infected with gBsyn, M6/syn, ΔCT/syn, or ΔCT.R/syn at a low MOI. Images were taken with an inverted light microscope at 2–3 days post infection. ( B ) Vero cells were infected with the indicated viruses at an MOI of 1. At 24 hr post infection, extracellular virions in the media were pelleted through a 30% sucrose cushion and the cells were collected. Samples were analyzed by western blotting. (C) Duplicate cultures of Vero cells were infected with the indicated viruses at an MOI of 0.1. The media containing extracellular virions was collected, and the infected cells were harvested and processed separately at 6-hour time points. Infectious virus was titered by plaque assay. ( D ) Purified His 6 -UL16 was incubated in 0.5% NP-40 buffer at 37°C for 5 hr with glutathione-sepharose beads bearing GST fusion proteins with the entire tail of gD or truncated sections of the tail. GST-UL11 and GST alone served as respective positive and negative controls. After incubation, the beads were washed, boiled in sample buffer, and the presence of bound His 6 -UL16 was analyzed by western blotting.

    Techniques Used: Infection, Light Microscopy, Western Blot, Plaque Assay, Purification, Incubation

    UL16 interacts with the cytoplasmic tail of gD. ( A ) Vero cells were infected with WT, ΔUL11, or ΔUL16 viruses. At 24 hr post infection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After 5 hr of incubation at room temperature or at 37°C, the beads were washed, boiled in sample buffer, and the proteins were analyzed by western blotting with an antibody specific for UL16. ( B ) Vero cells were transfected with expression plasmids for UL16-GFP or GFP-only. At 18–24 hr post transfection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gE.CT, GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After incubation, the beads were processed as in (A) and an antibody specific for GFP was used. ( C ) Increasing amounts of purified His 6 -UL16 produced in E. coli was incubated with GST-gD.CT, GST-gE.CT, or GST-only bound to glutathionesepharose beads in 0.5% NP-40 buffer at 37°C. After incubation, the beads were processed as in (A), a Ponceau stain for total protein was performed, and an antibody specific for the His tag was used to probe the western blot.
    Figure Legend Snippet: UL16 interacts with the cytoplasmic tail of gD. ( A ) Vero cells were infected with WT, ΔUL11, or ΔUL16 viruses. At 24 hr post infection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After 5 hr of incubation at room temperature or at 37°C, the beads were washed, boiled in sample buffer, and the proteins were analyzed by western blotting with an antibody specific for UL16. ( B ) Vero cells were transfected with expression plasmids for UL16-GFP or GFP-only. At 18–24 hr post transfection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gE.CT, GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After incubation, the beads were processed as in (A) and an antibody specific for GFP was used. ( C ) Increasing amounts of purified His 6 -UL16 produced in E. coli was incubated with GST-gD.CT, GST-gE.CT, or GST-only bound to glutathionesepharose beads in 0.5% NP-40 buffer at 37°C. After incubation, the beads were processed as in (A), a Ponceau stain for total protein was performed, and an antibody specific for the His tag was used to probe the western blot.

    Techniques Used: Infection, Incubation, Purification, Staining, Western Blot, Transfection, Expressing, Produced

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    Pull Down Assay:

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    Isolation:

    Article Title: Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation
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    Sonication:

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    Western Blot:

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    Thermo Fisher rna processing buffer
    Dependence of <t>RNA</t> targeting on crRNA variants, temperature and point mutations a, <t>LbuC2c2</t> ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.
    Rna Processing Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Interactions of FIGLA, LHX8 and SOHLH1. ( A ) Representative protein domains (bHLH, LIM, homeobox) of FIGLA, SOHLH1 and LHX8. ( B ) FIGLA HA and LHX8 FLAG expression vectors were co-transfected into HEK-293T cells. Cell lysates were probed with HA and FLAG antibodies to detect input protein FIGLA and LHX8, respectively. After <t>immunoprecipitation</t> with HA and FLAG antibody, immunoblots were performed to detect FIGLA and associated LHX8 protein or LHX8 and associated FIGLA protein, respectively. ( C ) Same as (B) except that FIGLA HA and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to HA and MYC were used to detect input proteins and immunoprecipitate FIGLA and SOHLH1, respectively, and their associated proteins. ( D ) Same as (B) except that LHX8 FLAG and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to FLAG and MYC were used to detect input proteins and immunoprecipitate LHX8 and SOHLH1, respectively, and their associated proteins. ( E ) Co-expression of FIGLA, LHX8 and SOHLH1 in P0 ovaries from Figla FLAG mice. The dashed circles indicate co-expression of FIGLA, LHX8 and SOHLH1 in the same oocytes. Scale bar, 20 μm. ( F ) FIGLA, LHX8 and SOHLH1 appear to form a nuclear complex in oocytes. Representative of n = 3 (B–E) independent biological replicates with similar results per condition.
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    DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts <t>(NP-40</t> lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.
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    Dependence of RNA targeting on crRNA variants, temperature and point mutations a, LbuC2c2 ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: Dependence of RNA targeting on crRNA variants, temperature and point mutations a, LbuC2c2 ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: Cleavage Assay, Activity Assay

    Binding data for LbuC2c2 to mature crRNA and target ssRNA a, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GG to LbuC2c2-WT, LbuC2c2-dHEPN1, LbuC2c2-dHEPN2, or LbuC2c2-dHEPN1/dHEPN2. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 27.1 ± 7.5 nM (LbuC2c2-WT), 15.2 ± 3.2 nM (LbuC2c2-dHEPN1), 11.5 ± 2.5 nM (LbuC2c2-dHEPN2), and 43.3 ± 11.5 nM (LbuC2c2- dHEPN1/dHEPN2). b, Representative electrophoretic mobility shift assay for binding reactions between LbuC2c2-dHEPN1/dHEPN2: crRNA-A_GG and either ‘on-target’ A ssRNA or ‘off-target’ B ssRNA, as indicated. Three independent experiments were conducted as described in the Methods. The gel was cropped for clarity. c, Quantified binding data from (b) were fitted to standard binding isoforms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 1.62 ± 0.43 nM for ssRNA A and N.D (≫10 nM) for ssRNA B. d, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GA to LbuC2c2-WT and LbuC2c2-R1079A. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 4.65 ± 0.6 nM (LbuC2c2-WT) and 2.52 ± 0.5 nM (LbuC2c2-R1079A). It is of note that these binding affinities differ from panel a. This difference is accounted for in a slight difference in the 5′ sequence of the guide with panel a guides beginning with a 5′-G G CCA… and panel d 5′-G A CCA. While the native sequence guide (5′-G A CCA) binds tighter to LbuC2c2, no difference is seen in the RNA targeting efficiencies of these guide variants (). Extended Data Fig. 6c

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: Binding data for LbuC2c2 to mature crRNA and target ssRNA a, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GG to LbuC2c2-WT, LbuC2c2-dHEPN1, LbuC2c2-dHEPN2, or LbuC2c2-dHEPN1/dHEPN2. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 27.1 ± 7.5 nM (LbuC2c2-WT), 15.2 ± 3.2 nM (LbuC2c2-dHEPN1), 11.5 ± 2.5 nM (LbuC2c2-dHEPN2), and 43.3 ± 11.5 nM (LbuC2c2- dHEPN1/dHEPN2). b, Representative electrophoretic mobility shift assay for binding reactions between LbuC2c2-dHEPN1/dHEPN2: crRNA-A_GG and either ‘on-target’ A ssRNA or ‘off-target’ B ssRNA, as indicated. Three independent experiments were conducted as described in the Methods. The gel was cropped for clarity. c, Quantified binding data from (b) were fitted to standard binding isoforms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 1.62 ± 0.43 nM for ssRNA A and N.D (≫10 nM) for ssRNA B. d, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GA to LbuC2c2-WT and LbuC2c2-R1079A. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 4.65 ± 0.6 nM (LbuC2c2-WT) and 2.52 ± 0.5 nM (LbuC2c2-R1079A). It is of note that these binding affinities differ from panel a. This difference is accounted for in a slight difference in the 5′ sequence of the guide with panel a guides beginning with a 5′-G G CCA… and panel d 5′-G A CCA. While the native sequence guide (5′-G A CCA) binds tighter to LbuC2c2, no difference is seen in the RNA targeting efficiencies of these guide variants (). Extended Data Fig. 6c

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: Binding Assay, Standard Deviation, Electrophoretic Mobility Shift Assay, Sequencing

    C2c2 provides sensitive detection of transcripts in complex mixtures a , Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b , Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c ,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β -actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β -actin null negative control). (mean ± s.d., n = 3) d , Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e , Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: C2c2 provides sensitive detection of transcripts in complex mixtures a , Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b , Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c ,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β -actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β -actin null negative control). (mean ± s.d., n = 3) d , Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e , Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: RNA Detection, Fluorescence, Generated, Negative Control, CRISPR

    Interactions of FIGLA, LHX8 and SOHLH1. ( A ) Representative protein domains (bHLH, LIM, homeobox) of FIGLA, SOHLH1 and LHX8. ( B ) FIGLA HA and LHX8 FLAG expression vectors were co-transfected into HEK-293T cells. Cell lysates were probed with HA and FLAG antibodies to detect input protein FIGLA and LHX8, respectively. After immunoprecipitation with HA and FLAG antibody, immunoblots were performed to detect FIGLA and associated LHX8 protein or LHX8 and associated FIGLA protein, respectively. ( C ) Same as (B) except that FIGLA HA and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to HA and MYC were used to detect input proteins and immunoprecipitate FIGLA and SOHLH1, respectively, and their associated proteins. ( D ) Same as (B) except that LHX8 FLAG and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to FLAG and MYC were used to detect input proteins and immunoprecipitate LHX8 and SOHLH1, respectively, and their associated proteins. ( E ) Co-expression of FIGLA, LHX8 and SOHLH1 in P0 ovaries from Figla FLAG mice. The dashed circles indicate co-expression of FIGLA, LHX8 and SOHLH1 in the same oocytes. Scale bar, 20 μm. ( F ) FIGLA, LHX8 and SOHLH1 appear to form a nuclear complex in oocytes. Representative of n = 3 (B–E) independent biological replicates with similar results per condition.

    Journal: Nucleic Acids Research

    Article Title: FIGLA, LHX8 and SOHLH1 transcription factor networks regulate mouse oocyte growth and differentiation

    doi: 10.1093/nar/gkaa101

    Figure Lengend Snippet: Interactions of FIGLA, LHX8 and SOHLH1. ( A ) Representative protein domains (bHLH, LIM, homeobox) of FIGLA, SOHLH1 and LHX8. ( B ) FIGLA HA and LHX8 FLAG expression vectors were co-transfected into HEK-293T cells. Cell lysates were probed with HA and FLAG antibodies to detect input protein FIGLA and LHX8, respectively. After immunoprecipitation with HA and FLAG antibody, immunoblots were performed to detect FIGLA and associated LHX8 protein or LHX8 and associated FIGLA protein, respectively. ( C ) Same as (B) except that FIGLA HA and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to HA and MYC were used to detect input proteins and immunoprecipitate FIGLA and SOHLH1, respectively, and their associated proteins. ( D ) Same as (B) except that LHX8 FLAG and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to FLAG and MYC were used to detect input proteins and immunoprecipitate LHX8 and SOHLH1, respectively, and their associated proteins. ( E ) Co-expression of FIGLA, LHX8 and SOHLH1 in P0 ovaries from Figla FLAG mice. The dashed circles indicate co-expression of FIGLA, LHX8 and SOHLH1 in the same oocytes. Scale bar, 20 μm. ( F ) FIGLA, LHX8 and SOHLH1 appear to form a nuclear complex in oocytes. Representative of n = 3 (B–E) independent biological replicates with similar results per condition.

    Article Snippet: Immunoprecipitation and co-IPHarvested cells were lysed in immunoprecipitation (IP) lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA) supplemented with 1× protease inhibitor cocktail (Thermo Fisher Scientific) on ice for 30 min.

    Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Mouse Assay

    DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.

    Journal: The Journal of Cell Biology

    Article Title: Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation

    doi: 10.1083/jcb.201102142

    Figure Lengend Snippet: DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.

    Article Snippet: Isolated hepatocytes were either lysed in NP-40 buffer or subjected to subcellular fractionation using the NE-PER cytoplasmic/nuclear fractionation kit (Thermo Fisher Scientific) to obtain total, cytoplasmic, and nuclear-enriched fractions.

    Techniques: Mouse Assay, Expressing, Transfection, Cell Culture, Over Expression, Isolation, Staining, Immunostaining