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Roche nacl protease inhibitors
Nacl Protease Inhibitors, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 5 article reviews
Price from $9.99 to $1999.99
nacl protease inhibitors - by Bioz Stars, 2020-04
96/100 stars

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Centrifugation:

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: .. Powder was thawed in buffer A / 0.3 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and insoluble material removed by centrifugation (235,000 x g, 4°C, 1 h). .. At this point, 2 mM CaCl2 was added to the soluble extract, followed by 2 mL calmodulin affinity resin, and the mixture incubated for 90 min at 4°C.

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Powder was thawed in buffer C / 0.2 M NaCl / protease inhibitors (Roche tablets + Sigma protease inhibitors + homemade cocktail). .. Insoluble material was removed by centrifugation (235,000 x g, 4°C, 1 h) and the supernatant was mixed with 2.5 mL anti-FLAG M2 affinity gel (Sigma-Aldrich) at 4°C for 90 min.

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: .. Powder was thawed in buffer A / 0.5 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and insoluble material was removed by centrifugation (235,000 x g, 4°C, 1 h). .. The soluble extract was mixed with 4 mL anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220) and the mixture incubated with rotation at 4°C for 90 min.

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: .. Powder was thawed in buffer A / 0.2 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and the insoluble material removed by centrifugation (235,000 x g, 4°C, 1 h). .. The soluble extract was supplemented with 2 mM CaCl2 , 2 mL calmodulin affinity resin was added, and the mixture incubated with rotation at 4°C for 90 min.

Affinity Chromatography:

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Buffer A / 0.4 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) was added to cell powder and the resuspension was centrifuged (235,000 x g, 4°C, 1 h). .. The soluble extract was supplemented with 2 mM CaCl2 and Pol α / primase was purified by calmodulin affinity chromatography using 2 mL calmodulin affinity resin (GE Healthcare, 17052901).

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Powder was thawed in buffer A / 0.15 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and then centrifuged (235,000 x g, 4°C, 1 h). .. The soluble extract was recovered and supplemented with 2 mM CaCl2 and RFC purified by camodulin affinity chromatography using 1 mL resin.

Homogenization:

Article Title: Airway Inflammation in Chronic Rhinosinusitis with Nasal Polyps and Asthma: The United Airways Concept Further Supported
Article Snippet: .. One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue (Compete # 1 697 498; Roche Diagnostics) was added after disruption and homogenization. .. From tissue homogenates, the protein concentration was determined using a standard BCA assay and the measured values were normalized to a concentration of 100 μg total protein per sample (in 25 μl pr well), based on parallel analysis of protein concentrations.

Protease Inhibitor:

Article Title: Airway Inflammation in Chronic Rhinosinusitis with Nasal Polyps and Asthma: The United Airways Concept Further Supported
Article Snippet: .. One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue (Compete # 1 697 498; Roche Diagnostics) was added after disruption and homogenization. .. From tissue homogenates, the protein concentration was determined using a standard BCA assay and the measured values were normalized to a concentration of 100 μg total protein per sample (in 25 μl pr well), based on parallel analysis of protein concentrations.

Protein Concentration:

Article Title: Airway Inflammation in Chronic Rhinosinusitis with Nasal Polyps and Asthma: The United Airways Concept Further Supported
Article Snippet: One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue (Compete # 1 697 498; Roche Diagnostics) was added after disruption and homogenization. .. From tissue homogenates, the protein concentration was determined using a standard BCA assay and the measured values were normalized to a concentration of 100 μg total protein per sample (in 25 μl pr well), based on parallel analysis of protein concentrations.

Purification:

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Buffer A / 0.4 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) was added to cell powder and the resuspension was centrifuged (235,000 x g, 4°C, 1 h). .. The soluble extract was supplemented with 2 mM CaCl2 and Pol α / primase was purified by calmodulin affinity chromatography using 2 mL calmodulin affinity resin (GE Healthcare, 17052901).

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Powder was thawed in buffer A / 0.15 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and then centrifuged (235,000 x g, 4°C, 1 h). .. The soluble extract was recovered and supplemented with 2 mM CaCl2 and RFC purified by camodulin affinity chromatography using 1 mL resin.

Activation Assay:

Article Title: Airway Inflammation in Chronic Rhinosinusitis with Nasal Polyps and Asthma: The United Airways Concept Further Supported
Article Snippet: One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue (Compete # 1 697 498; Roche Diagnostics) was added after disruption and homogenization. .. Included biomarkers were: eotaxin, interleukin (IL)-8, interferon-inducable protein (IP)-10, monocyte chemotactic protein (MCP)-1, MCP-4, macrophage inflammatory protein (MIP)-1β, thymus- and activation-regulated chemokine (TARC), interferon (INF)-γ, IL-10, IL-12p70, IL-13, IL-2, IL-4, IL-5 (for BAL, only the chemokine assay was performed).

Incubation:

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Powder was thawed in buffer A / 0.3 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and insoluble material removed by centrifugation (235,000 x g, 4°C, 1 h). .. At this point, 2 mM CaCl2 was added to the soluble extract, followed by 2 mL calmodulin affinity resin, and the mixture incubated for 90 min at 4°C.

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Powder was thawed in buffer A / 0.5 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and insoluble material was removed by centrifugation (235,000 x g, 4°C, 1 h). .. The soluble extract was mixed with 4 mL anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220) and the mixture incubated with rotation at 4°C for 90 min.

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Buffer A / 0.2 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) was added to thawed powder and the sample was centrifuged (235,000 x g, 4°C, 1 h). .. At this point, 1.5 mL calmodulin affinity resin was added and the mixture incubated at 4°C for 90 min with rotation.

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: Powder was thawed in buffer A / 0.2 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and the insoluble material removed by centrifugation (235,000 x g, 4°C, 1 h). .. The soluble extract was supplemented with 2 mM CaCl2 , 2 mL calmodulin affinity resin was added, and the mixture incubated with rotation at 4°C for 90 min.

Cytokine Assay:

Article Title: Airway Inflammation in Chronic Rhinosinusitis with Nasal Polyps and Asthma: The United Airways Concept Further Supported
Article Snippet: One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue (Compete # 1 697 498; Roche Diagnostics) was added after disruption and homogenization. .. Homogenates were analyzed using an ultra-sensitive human chemokine 7-plex assay (cat# K15031C-2) and an ultra-sensitive human Th1/Th2 cytokine assay (cat# K15011-C2, Mesoscale Diagnostics, LCC, Rockwill, US), according to the protocol from the manufacturer.

BIA-KA:

Article Title: Airway Inflammation in Chronic Rhinosinusitis with Nasal Polyps and Asthma: The United Airways Concept Further Supported
Article Snippet: One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue (Compete # 1 697 498; Roche Diagnostics) was added after disruption and homogenization. .. From tissue homogenates, the protein concentration was determined using a standard BCA assay and the measured values were normalized to a concentration of 100 μg total protein per sample (in 25 μl pr well), based on parallel analysis of protein concentrations.

Concentration Assay:

Article Title: Airway Inflammation in Chronic Rhinosinusitis with Nasal Polyps and Asthma: The United Airways Concept Further Supported
Article Snippet: One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue (Compete # 1 697 498; Roche Diagnostics) was added after disruption and homogenization. .. From tissue homogenates, the protein concentration was determined using a standard BCA assay and the measured values were normalized to a concentration of 100 μg total protein per sample (in 25 μl pr well), based on parallel analysis of protein concentrations.

Lysis:

Article Title: Airway Inflammation in Chronic Rhinosinusitis with Nasal Polyps and Asthma: The United Airways Concept Further Supported
Article Snippet: Samples were precooled in liquid nitrogen for at least 30 minutes and then disrupted and homogenized without lysis buffer. .. One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue (Compete # 1 697 498; Roche Diagnostics) was added after disruption and homogenization.

other:

Article Title: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes
Article Snippet: The resin was collected and washed extensively with 120 mL buffer A / 0.5 M NaCl / protease inhibitors, then 20 mL buffer A / 0.5 M NaCl, then 40 mL buffer A / 0.5 M NaCl / 10 mM MgOAc / 1 mM ATP, then 20 mL buffer A / 0.2 M NaCl.

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  • 96
    Roche immunoprecipitation buffer
    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of <t>co-immunoprecipitation</t> experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
    Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoprecipitation buffer/product/Roche
    Average 96 stars, based on 228 article reviews
    Price from $9.99 to $1999.99
    immunoprecipitation buffer - by Bioz Stars, 2020-04
    96/100 stars
      Buy from Supplier

    93
    Roche tnte buffer
    Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in <t>TNTE</t> buffer (20 mM <t>Tris,</t> pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .
    Tnte Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tnte buffer/product/Roche
    Average 93 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    tnte buffer - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    93
    Roche gfp trap lysis buffer
    HeV V undergoes <t>importin</t> α1-dependent nuclear import. HeLa cells were transfected with siRNAs targeting KPNA2 (encoding importin α1), KPNA4 (encoding importin α3) siRNA, or control (scrambled - Scr) siRNA or mock-transfected (48 h), followed by transfection to express <t>GFP-HeV</t> V or GFP-HeV V L174A/L177A (24 h), then imaged by live-cell CLSM. ( a ) Importin α1, ( b ) importin α3 and actin expression levels were determined by western analysis. ( c ) Representative images from live-cell CLSM. Scale bars represent 10 μM. ( d ) Images such as those shown in ( c ) were used to calculate the Fn/c; results represent the mean ± SEM (n ≥ 100 cells) from a single assay representative of two independent assays. ****p
    Gfp Trap Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp trap lysis buffer/product/Roche
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    gfp trap lysis buffer - by Bioz Stars, 2020-04
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    Image Search Results


    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Journal: Molecular psychiatry

    Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

    doi: 10.1038/s41380-018-0027-3

    Figure Lengend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C.

    Techniques: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

    Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: Hedgehog signaling leads to reduced nuclear Sufu protein levels and Sufu dissociation from Gli2 and Gli3 primarily in the nucleus. ( A ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions derived from MEFs treated with Shh-conditioned medium. Cytoplasmic tubulin and nuclear lamin A were used to assess the purity of cytoplasmic and nuclear fractions. Nuclear Sufu protein levels were reduced by 60% upon Hh pathway activation, while cytoplasmic Sufu levels were largely unaltered. ( B ) Western blot analysis and quantification of Sufu protein levels in the nuclear and cytoplasmic fractions in the presence of Shh-conditioned medium and proteasome inhibitor MG132. Nuclear Sufu protein levels were notably restored upon MG132 addition. ( C ) Western blot analysis and quantification of immunoprecipitated Sufu, Gli2, and Gli3 using lysates from the nuclear and cytoplasmic fractions derived from MEFs expressing Flag-tagged Sufu. The amount of coimmunoprecipitated Gli2 and Gli3 by Sufu was significantly reduced in the nuclear fraction but only marginally decreased in the cytoplasmic fraction at indicated time points after Hh stimulation. (In) Input; (IP) immunoprecipitation. (*) P

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Expressing

    A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Journal: Genes & Development

    Article Title: Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling

    doi: 10.1101/gad.249425.114

    Figure Lengend Snippet: A proteomic approach to identify Sufu-interacting proteins uncovers p66β and Mycbp. ( A ) Coomassie Blue-stained gel of control and Sufu immunoprecipitates treated with mock- or Shh-conditioned medium. Distinct bands were detected and were candidates for new Sufu-interacting proteins. Numbers at the right indicate locations of protein size standards. Large-scale immunoprecipitation (IP) and mass spectrometry were performed to identify new Sufu-interacting proteins and Sufu phosphorylation sites. Mass spectrometric analysis was performed directly on immunoprecipitates or specific bands cut out from SDS-PAGE gels. Immunoprecipitation and mass spectrometric analysis were repeated multiple times to eliminate nonspecific Sufu-binding proteins. ( B ) Western blot analysis of Sufu immunoprecipitates probed with anti-Sufu, anti-Gli2, and Gli3 antibodies. Endogenous Gli2 and Gli3 were detected in Sufu immunoprecipitates (but not in the control), suggesting that a physiologically relevant protein complex was pulled down. ( C ). ( D ) Western blot analysis of proteins pulled down by Sufu from lysates expressing Sufu and the indicated proteins, which were epitope-tagged. Both p66β and Mycbp physically interacted with Sufu by coimmunoprecipitation. Fu and Prc1 served as negative controls. ( E , top panels) Western blot analysis of Sufu immunoprecipitates using lysates derived from MEFs expressing Flag-tagged Sufu. Endogenous p66β and HDAC1 were coimmunoprecipitated. In contrast, HDAC2 and RBBP7/4 could not be detected in Sufu immunoprecipitates. ( Bottom panels) Western blot analysis of endogenous Sufu immunoprecipitated by an anti-Sufu antibody. p66β was coimmunoprecipitated by Sufu in wild-type MEFs but not in Sufu -deficient MEFs. p66β/Sufu interaction was not altered by Hh stimulation (Supplemental Fig. S6). ( F–H ) Immunofluorescence studies to assess the subcellular distribution of p66β and Mycbp. MEFs were transfected or transduced with p66β- and Mycbp-expressing constructs. p66β and Mycbp localized to the nucleus (marked by DAPI) of Hh-responsive cells. Cytoplasmic expression of Mycbp was also detected. Acetylated (Ac)-tubulin marks the primary cilium. Interestingly, Mycbp immunoreactivity can also be detected at the base of the cilium (white arrow).

    Article Snippet: Cells were collected at 48 h post-transfection and lysed in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 50mM Tris-Cl at pH 7.5, 1 mM EDTA, protease inhibitor cocktail [Roche], PhosSTOP [Roche]).

    Techniques: Staining, Immunoprecipitation, Mass Spectrometry, SDS Page, Binding Assay, Western Blot, Expressing, Derivative Assay, Immunofluorescence, Transfection, Transduction, Construct

    Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

    Journal: Current Biology

    Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy

    doi: 10.1016/j.cub.2017.06.021

    Figure Lengend Snippet: Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

    Article Snippet: To inhibit deubiquitinases cells were lysed in TNTE buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1x Complete protease inhibitor (Roche), 1x PhosSTOP (Roche)) supplemented with 20 mM N-Ethylmaleimide (NEM) prior to immunoprecipitation as described.

    Techniques: Expressing, Plasmid Preparation, Construct, Incubation, Recombinant, Immunoprecipitation, Lysis, Mass Spectrometry, Mutagenesis

    HeV V undergoes importin α1-dependent nuclear import. HeLa cells were transfected with siRNAs targeting KPNA2 (encoding importin α1), KPNA4 (encoding importin α3) siRNA, or control (scrambled - Scr) siRNA or mock-transfected (48 h), followed by transfection to express GFP-HeV V or GFP-HeV V L174A/L177A (24 h), then imaged by live-cell CLSM. ( a ) Importin α1, ( b ) importin α3 and actin expression levels were determined by western analysis. ( c ) Representative images from live-cell CLSM. Scale bars represent 10 μM. ( d ) Images such as those shown in ( c ) were used to calculate the Fn/c; results represent the mean ± SEM (n ≥ 100 cells) from a single assay representative of two independent assays. ****p

    Journal: Scientific Reports

    Article Title: Recognition by host nuclear transport proteins drives disorder-to-order transition in Hendra virus V

    doi: 10.1038/s41598-017-18742-8

    Figure Lengend Snippet: HeV V undergoes importin α1-dependent nuclear import. HeLa cells were transfected with siRNAs targeting KPNA2 (encoding importin α1), KPNA4 (encoding importin α3) siRNA, or control (scrambled - Scr) siRNA or mock-transfected (48 h), followed by transfection to express GFP-HeV V or GFP-HeV V L174A/L177A (24 h), then imaged by live-cell CLSM. ( a ) Importin α1, ( b ) importin α3 and actin expression levels were determined by western analysis. ( c ) Representative images from live-cell CLSM. Scale bars represent 10 μM. ( d ) Images such as those shown in ( c ) were used to calculate the Fn/c; results represent the mean ± SEM (n ≥ 100 cells) from a single assay representative of two independent assays. ****p

    Article Snippet: For importin α1 coimmunoprecipitation assays, transfected HEK293T cells were lysed with GFP-Trap lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) Nonidet P-40) containing 1 x cOmplete EDTA-free protease inhibitor cocktail tablet (Roche).

    Techniques: Transfection, Confocal Laser Scanning Microscopy, Expressing, Western Blot

    HeV V undergoes NES-dependent, exportin-1 mediated nuclear export. ( a ) Vero cells were transfected to express the indicated GFP-fused HeV proteins (18 h) before treatment with (+) or without (no add.) LMB (3.5 h), then imaged by live-cell CLSM. ‘GFP-W contrast ++’ represents the same cells as for GFP-W, but with enhanced brightness and contrast to allow visualization of cytoplasmic fluorescence. ( b ) Images such as those in ( a ) were analyzed to determine the nuclear/cytoplasmic fluorescence ratio, Fn/c; results represent the mean ± SEM (n ≥ 30 cells) from a single assay representative of three independent assays. ****p

    Journal: Scientific Reports

    Article Title: Recognition by host nuclear transport proteins drives disorder-to-order transition in Hendra virus V

    doi: 10.1038/s41598-017-18742-8

    Figure Lengend Snippet: HeV V undergoes NES-dependent, exportin-1 mediated nuclear export. ( a ) Vero cells were transfected to express the indicated GFP-fused HeV proteins (18 h) before treatment with (+) or without (no add.) LMB (3.5 h), then imaged by live-cell CLSM. ‘GFP-W contrast ++’ represents the same cells as for GFP-W, but with enhanced brightness and contrast to allow visualization of cytoplasmic fluorescence. ( b ) Images such as those in ( a ) were analyzed to determine the nuclear/cytoplasmic fluorescence ratio, Fn/c; results represent the mean ± SEM (n ≥ 30 cells) from a single assay representative of three independent assays. ****p

    Article Snippet: For importin α1 coimmunoprecipitation assays, transfected HEK293T cells were lysed with GFP-Trap lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) Nonidet P-40) containing 1 x cOmplete EDTA-free protease inhibitor cocktail tablet (Roche).

    Techniques: Transfection, Confocal Laser Scanning Microscopy, Fluorescence