np 40  (Millipore)


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    Name:
    Triton X 114
    Description:
    Triton TM X 114 is a nonionic detergent It is a good detergent for solubilizing membranes having reversible phase separation property useful for the separation of intrinsic membrane proteins Solution of Triton TM X 114 is homogeneous at 0C but phase separation into aqueous phase and a detergent phase occurs above 20C Phase separation of proteins in solutions of Triton TM X 114 has been investigated This octylphenol ethoxylate is compatible with anionic cationic and other nonionic surfactants and chemically stable in most acidic alkaline solutions
    Catalog Number:
    x114
    Price:
    None
    Applications:
    Triton(TM) X-114 has been used:. To remove endotoxin from solutions of protein using phase separation technique. In the extraction and purification of DNA from endotoxin. In the biphasic extraction of lipoproteins from bacterial cell wall
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    Structured Review

    Millipore np 40
    Triton X 114
    Triton TM X 114 is a nonionic detergent It is a good detergent for solubilizing membranes having reversible phase separation property useful for the separation of intrinsic membrane proteins Solution of Triton TM X 114 is homogeneous at 0C but phase separation into aqueous phase and a detergent phase occurs above 20C Phase separation of proteins in solutions of Triton TM X 114 has been investigated This octylphenol ethoxylate is compatible with anionic cationic and other nonionic surfactants and chemically stable in most acidic alkaline solutions
    https://www.bioz.com/result/np 40/product/Millipore
    Average 99 stars, based on 517 article reviews
    Price from $9.99 to $1999.99
    np 40 - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "An Essential Role for a Small Synaptic Vesicle-Associated Phosphatidylinositol 4-Kinase in Neurotransmitter Release"

    Article Title: An Essential Role for a Small Synaptic Vesicle-Associated Phosphatidylinositol 4-Kinase in Neurotransmitter Release

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-15-05594.1998

    Localization of a membrane-associated PtdIns 4-kinase activity on SSVs. Crude SSV preparations were further purified on a continuous sucrose gradient. Six fractions (I–VI, from top to bottom of the gradient) were collected and analyzed. a , Separation of SSV from contaminating plasma membrane was demonstrated by immunoblot analysis of 30 μg of protein of each fraction. SSV and plasma membranes were detected by the use of antibodies to the marker proteins VAMP/synaptobrevin and the α-subunit of Na + /K + -ATPase, respectively. SSV accumulated in fractions IV and V, whereas most of the plasma membrane was recovered in the first fraction. The two bands at 140 and 100 kDa detected by the antibodies to Na + /K + -ATPase in fraction I represent the heterodimer of the α- and β-subunits, and the α-subunit monomer, respectively. b , Membrane-associated PtdIns 4-kinase activity within the sucrose gradient peaked in fractions III–V, enriched in SSVs and devoid of plasma membrane. Relative mPtdIns 4-kinase activity was measured by incubation of aliquots from fractions I–VI without addition of exogenous substrate. Phosphorylation of membrane-derived PtdIns was determined by separation of labeled phospholipids on thin layer chromatograms. PtdIns(4) 32 P was quantitated and expressed in arbitrary units. c , Solubilized PtdIns-kinase activity was determined in 1% NP-40 extracts of aliquots of fractions I–VI. When extracts were incubated with [γ- 32 P]ATP (10 μCi/assay) and exogenous phospholipids as substrate, PtdIns(4) 32 P ( top panel ) and PtdIns(4,5) 32 P 2 ( bottom panel ) were formed. Relative amounts were quantitated and displayed as described for b . Distribution of sPtdIns 4-kinase and sPtdIns(4)P 5-kinase activity followed the distribution of the SSV-marker VAMP/synaptobrevin and of the plasma membrane marker Na + /K + -ATPase, respectively.
    Figure Legend Snippet: Localization of a membrane-associated PtdIns 4-kinase activity on SSVs. Crude SSV preparations were further purified on a continuous sucrose gradient. Six fractions (I–VI, from top to bottom of the gradient) were collected and analyzed. a , Separation of SSV from contaminating plasma membrane was demonstrated by immunoblot analysis of 30 μg of protein of each fraction. SSV and plasma membranes were detected by the use of antibodies to the marker proteins VAMP/synaptobrevin and the α-subunit of Na + /K + -ATPase, respectively. SSV accumulated in fractions IV and V, whereas most of the plasma membrane was recovered in the first fraction. The two bands at 140 and 100 kDa detected by the antibodies to Na + /K + -ATPase in fraction I represent the heterodimer of the α- and β-subunits, and the α-subunit monomer, respectively. b , Membrane-associated PtdIns 4-kinase activity within the sucrose gradient peaked in fractions III–V, enriched in SSVs and devoid of plasma membrane. Relative mPtdIns 4-kinase activity was measured by incubation of aliquots from fractions I–VI without addition of exogenous substrate. Phosphorylation of membrane-derived PtdIns was determined by separation of labeled phospholipids on thin layer chromatograms. PtdIns(4) 32 P was quantitated and expressed in arbitrary units. c , Solubilized PtdIns-kinase activity was determined in 1% NP-40 extracts of aliquots of fractions I–VI. When extracts were incubated with [γ- 32 P]ATP (10 μCi/assay) and exogenous phospholipids as substrate, PtdIns(4) 32 P ( top panel ) and PtdIns(4,5) 32 P 2 ( bottom panel ) were formed. Relative amounts were quantitated and displayed as described for b . Distribution of sPtdIns 4-kinase and sPtdIns(4)P 5-kinase activity followed the distribution of the SSV-marker VAMP/synaptobrevin and of the plasma membrane marker Na + /K + -ATPase, respectively.

    Techniques Used: Activity Assay, Purification, Marker, Incubation, Derivative Assay, Labeling

    2) Product Images from "An Essential Role for a Small Synaptic Vesicle-Associated Phosphatidylinositol 4-Kinase in Neurotransmitter Release"

    Article Title: An Essential Role for a Small Synaptic Vesicle-Associated Phosphatidylinositol 4-Kinase in Neurotransmitter Release

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-15-05594.1998

    Localization of a membrane-associated PtdIns 4-kinase activity on SSVs. Crude SSV preparations were further purified on a continuous sucrose gradient. Six fractions (I–VI, from top to bottom of the gradient) were collected and analyzed. a , Separation of SSV from contaminating plasma membrane was demonstrated by immunoblot analysis of 30 μg of protein of each fraction. SSV and plasma membranes were detected by the use of antibodies to the marker proteins VAMP/synaptobrevin and the α-subunit of Na + /K + -ATPase, respectively. SSV accumulated in fractions IV and V, whereas most of the plasma membrane was recovered in the first fraction. The two bands at 140 and 100 kDa detected by the antibodies to Na + /K + -ATPase in fraction I represent the heterodimer of the α- and β-subunits, and the α-subunit monomer, respectively. b , Membrane-associated PtdIns 4-kinase activity within the sucrose gradient peaked in fractions III–V, enriched in SSVs and devoid of plasma membrane. Relative mPtdIns 4-kinase activity was measured by incubation of aliquots from fractions I–VI without addition of exogenous substrate. Phosphorylation of membrane-derived PtdIns was determined by separation of labeled phospholipids on thin layer chromatograms. PtdIns(4) 32 P was quantitated and expressed in arbitrary units. c , Solubilized PtdIns-kinase activity was determined in 1% NP-40 extracts of aliquots of fractions I–VI. When extracts were incubated with [γ- 32 P]ATP (10 μCi/assay) and exogenous phospholipids as substrate, PtdIns(4) 32 P ( top panel ) and PtdIns(4,5) 32 P 2 ( bottom panel ) were formed. Relative amounts were quantitated and displayed as described for b . Distribution of sPtdIns 4-kinase and sPtdIns(4)P 5-kinase activity followed the distribution of the SSV-marker VAMP/synaptobrevin and of the plasma membrane marker Na + /K + -ATPase, respectively.
    Figure Legend Snippet: Localization of a membrane-associated PtdIns 4-kinase activity on SSVs. Crude SSV preparations were further purified on a continuous sucrose gradient. Six fractions (I–VI, from top to bottom of the gradient) were collected and analyzed. a , Separation of SSV from contaminating plasma membrane was demonstrated by immunoblot analysis of 30 μg of protein of each fraction. SSV and plasma membranes were detected by the use of antibodies to the marker proteins VAMP/synaptobrevin and the α-subunit of Na + /K + -ATPase, respectively. SSV accumulated in fractions IV and V, whereas most of the plasma membrane was recovered in the first fraction. The two bands at 140 and 100 kDa detected by the antibodies to Na + /K + -ATPase in fraction I represent the heterodimer of the α- and β-subunits, and the α-subunit monomer, respectively. b , Membrane-associated PtdIns 4-kinase activity within the sucrose gradient peaked in fractions III–V, enriched in SSVs and devoid of plasma membrane. Relative mPtdIns 4-kinase activity was measured by incubation of aliquots from fractions I–VI without addition of exogenous substrate. Phosphorylation of membrane-derived PtdIns was determined by separation of labeled phospholipids on thin layer chromatograms. PtdIns(4) 32 P was quantitated and expressed in arbitrary units. c , Solubilized PtdIns-kinase activity was determined in 1% NP-40 extracts of aliquots of fractions I–VI. When extracts were incubated with [γ- 32 P]ATP (10 μCi/assay) and exogenous phospholipids as substrate, PtdIns(4) 32 P ( top panel ) and PtdIns(4,5) 32 P 2 ( bottom panel ) were formed. Relative amounts were quantitated and displayed as described for b . Distribution of sPtdIns 4-kinase and sPtdIns(4)P 5-kinase activity followed the distribution of the SSV-marker VAMP/synaptobrevin and of the plasma membrane marker Na + /K + -ATPase, respectively.

    Techniques Used: Activity Assay, Purification, Marker, Incubation, Derivative Assay, Labeling

    3) Product Images from "Ectromelia Virus Encodes a Novel Family of F-Box Proteins That Interact with the SCF Complex ▿"

    Article Title: Ectromelia Virus Encodes a Novel Family of F-Box Proteins That Interact with the SCF Complex ▿

    Journal:

    doi: 10.1128/JVI.00953-08

    EVM005 interacts with HA-cullin-1 during infection. (A) HEK293T cells were infected with VV Copenhagen (VVCop), VV-Flag-EVM005, or VV-Flag-EVM004 and mock transfected or transfected with pSC66-HA-cullin-1. At 12 h postinfection, cells were lysed in NP-40
    Figure Legend Snippet: EVM005 interacts with HA-cullin-1 during infection. (A) HEK293T cells were infected with VV Copenhagen (VVCop), VV-Flag-EVM005, or VV-Flag-EVM004 and mock transfected or transfected with pSC66-HA-cullin-1. At 12 h postinfection, cells were lysed in NP-40

    Techniques Used: Infection, Transfection

    EVM005 associates with conjugated ubiquitin. (A) HEK293T cells were mock infected or infected with VV Copenhagen (VVCop), VV-Flag-EVM005, VV-Flag-EVM004, VV-Flag-EVM150, or VV-Flag-FPV039 at an MOI of 5. At 12 h postinfection, cells were lysed in NP-40
    Figure Legend Snippet: EVM005 associates with conjugated ubiquitin. (A) HEK293T cells were mock infected or infected with VV Copenhagen (VVCop), VV-Flag-EVM005, VV-Flag-EVM004, VV-Flag-EVM150, or VV-Flag-FPV039 at an MOI of 5. At 12 h postinfection, cells were lysed in NP-40

    Techniques Used: Infection

    4) Product Images from "Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell ▿Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell ▿ †"

    Article Title: Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell ▿Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell ▿ †

    Journal:

    doi: 10.1128/JVI.01223-08

    Sensitivity to NEM and NP-40 is conserved among alphaherpesviruses. Extracellular HSV and PRV were harvested from infected Vero cells and treated with NEM or vehicle for 30 min at 37°C. Virions were then treated with NP-40 (+) or left
    Figure Legend Snippet: Sensitivity to NEM and NP-40 is conserved among alphaherpesviruses. Extracellular HSV and PRV were harvested from infected Vero cells and treated with NEM or vehicle for 30 min at 37°C. Virions were then treated with NP-40 (+) or left

    Techniques Used: Infection

    Related Articles

    Protease Inhibitor:

    Article Title: Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein
    Article Snippet: .. The outer membrane of L. kirschneri was solubilized with Triton X-114 as described previously , except that 0.5% protease inhibitor cocktail (catalog no. P8849; Sigma) was included in the lysis buffer. .. The membrane was isolated and washed as described previously , with some minor modifications.

    Solubility:

    Article Title: Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions ▿
    Article Snippet: .. For Triton X-114 extractions, soluble and integral membrane proteins were separated based on solubility in Triton X-114 (Sigma) to examine the partitioning of selected proteins during chlamydial infection. .. Cultures were harvested at 2 or 20 h postinfection in the presence of protease inhibitors (Roche), and material was extracted with 1% Triton X-114 essentially as described previously ( ).

    Concentration Assay:

    Article Title: Effect of the GPI anchor of human Thy-1 on antibody recognition and function
    Article Snippet: .. Partitioning of Conditioned Media using Triton X-114 Triton X-114 (Sigma-Aldrich 93422) was added to concentrated CM to a final concentration of 2%. ..

    other:

    Article Title: Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it
    Article Snippet: BHA, NAC, bacterial LPS, Nec-1, PI, 4-hydroxytamoxifen (4OHT), and Triton X-114 were from Sigma-Aldrich (St. Louis, MO, USA).

    Infection:

    Article Title: Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions ▿
    Article Snippet: .. For Triton X-114 extractions, soluble and integral membrane proteins were separated based on solubility in Triton X-114 (Sigma) to examine the partitioning of selected proteins during chlamydial infection. .. Cultures were harvested at 2 or 20 h postinfection in the presence of protease inhibitors (Roche), and material was extracted with 1% Triton X-114 essentially as described previously ( ).

    Lysis:

    Article Title: Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein
    Article Snippet: .. The outer membrane of L. kirschneri was solubilized with Triton X-114 as described previously , except that 0.5% protease inhibitor cocktail (catalog no. P8849; Sigma) was included in the lysis buffer. .. The membrane was isolated and washed as described previously , with some minor modifications.

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  • 99
    Millipore triton x 114
    Assembly of transgenic  HA Gγ 1  and  HA Gγ 1 C71S with the endogenous Gβ 1  in retinal photoreceptors.  (A)  Transgenic construct used for generation of  HA Gγ 1  mice and amino acid sequence of the encoded protein with HA-tag (underlined) and C71 (asterisk).  (B)  SDS polyacrylamide gel stained with Coomassie blue showing anti-HA pull downs from 10 retinas of the transgene-negative (1) and transgene-positive (2)  HA Gγ 1  mice.  (B)  Western blot analysis of anti-HA pull downs from the retinas of  HA Gγ 1  and  HA Gγ 1 C71S mice with antibodies against Gβ 1  and HA.  (C)  Representative Western blot showing co-precipitation of phosducin (Pdc) with  HA Gγ 1  or  HA Gγ 1 C71S (HA). Tg(-) indicates transgene-negative littermates. The break separates two blots that were adjusted differently. Identical amounts of phosducin co-precipitated with  HA Gγ 1  and  HA Gγ 1  C71S ( n  = 4).  (E)  Representative experiments illustrating partitioning of  HA Gγ 1 C71S,  HA Gγ 1 , and endogenous Gγ 1  between the detergent (Triton X-114) and aqueous phases. Specific bands were visualized by Western blotting with anti-HA ( HA Gγ 1 C71S,  HA Gγ 1 ) and anti-Gγ 1  (Gγ 1 ).  (D)  Average distribution of each protein in the aqueous and detergent phases ( HA Gγ 1 C71S: 87/13 ± 4%;  HA Gγ 1 : 36/64 ± 9%, Gγ 1 : 41/59 ± 8%, error bars are SEM,  n  = 4).
    Triton X 114, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 114/product/Millipore
    Average 99 stars, based on 68 article reviews
    Price from $9.99 to $1999.99
    triton x 114 - by Bioz Stars, 2020-09
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    99
    Millipore triton x 100
    14-3-3 protein expression is ubiquitous across early embryonic stages and tissues. (A) Whole embryo lysates (1% Triton X-100) were immunoblotted for 14-3-3 using a pan antibody that detects multiple isoforms. Each lane represents approximately 50 μg. (B) Colored schematic of a bisected  Xenopus  embryo at gastrula depicting major tissue divisions. The tissues include the animal cap (AC), mesendoderm (ME), marginal zone (MZ), vegetal hemisphere (VG), and whole embryo lysate (WEL). (C) Embryos were dissected into separate tissues and corresponding lysates (1% Triton X-100) were immunoblotted using pan 14-3-3 antibody to examine expression across the gastrulating embryo. Each lane represents a portion of protein equivalent to approximately 1 embryo.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 100/product/Millipore
    Average 99 stars, based on 379 article reviews
    Price from $9.99 to $1999.99
    triton x 100 - by Bioz Stars, 2020-09
    99/100 stars
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    99
    Millipore mg132
    Strigolactones (SLs) promote ubiquitination and degradation of D14 in rice. (A) Protein levels of D14-GFP in calli of the Act:D14-GFP/d14 transgenic line at different time points after 20 μM rac -GR24 treatment with DMSO as a control. D14-GFP was detected by immunoblotting with an anti-GFP monoclonal antibody. Relative protein levels were determined by densitometry and normalized to loadings determined by Ponceau staining (red) in the immunoblotting analyses. (B) Relative expression levels of D14 and D53 in 2-week-old seedlings after 5 μM rac -GR24 treatment. Values represent means ± SEM, n = 3. (C) D14-GFP protein levels in calli of the D14-GFP/d14 transgenic line at different time points after rac -GR24 treatment in the presence or absence of <t>MG132.</t> Calli are pretreated with 50 μM MG132 for 1 h and then treated with 20 μM rac -GR24 or DMSO for 3 h. D14-GFP was detected by immunoblotting with an anti-GFP monoclonal antibody. Actin1 were used as loading control in the immunoblotting analyses. (D) Ubiquitination analysis of D14-GFP in rice protoplasts. Rice (Nipponbare) protoplasts were transformed with 35S:D14-GFP plasmids and incubated for 12 h, then pretreated with 50 μM MG132 for 1 h and immediately treated with 20 μM rac -GR24 or DMSO for 3 h. Proteins were extracted for affinity purification with an agarose-immobolized anti-GFP monoclonal antibody and followed by immunoblotting analysis with an anti-ubiquitin (upper panel) or anti-GFP (lower panel) monoclonal antibody.
    Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mg132/product/Millipore
    Average 99 stars, based on 1367 article reviews
    Price from $9.99 to $1999.99
    mg132 - by Bioz Stars, 2020-09
    99/100 stars
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    Image Search Results


    Assembly of transgenic  HA Gγ 1  and  HA Gγ 1 C71S with the endogenous Gβ 1  in retinal photoreceptors.  (A)  Transgenic construct used for generation of  HA Gγ 1  mice and amino acid sequence of the encoded protein with HA-tag (underlined) and C71 (asterisk).  (B)  SDS polyacrylamide gel stained with Coomassie blue showing anti-HA pull downs from 10 retinas of the transgene-negative (1) and transgene-positive (2)  HA Gγ 1  mice.  (B)  Western blot analysis of anti-HA pull downs from the retinas of  HA Gγ 1  and  HA Gγ 1 C71S mice with antibodies against Gβ 1  and HA.  (C)  Representative Western blot showing co-precipitation of phosducin (Pdc) with  HA Gγ 1  or  HA Gγ 1 C71S (HA). Tg(-) indicates transgene-negative littermates. The break separates two blots that were adjusted differently. Identical amounts of phosducin co-precipitated with  HA Gγ 1  and  HA Gγ 1  C71S ( n  = 4).  (E)  Representative experiments illustrating partitioning of  HA Gγ 1 C71S,  HA Gγ 1 , and endogenous Gγ 1  between the detergent (Triton X-114) and aqueous phases. Specific bands were visualized by Western blotting with anti-HA ( HA Gγ 1 C71S,  HA Gγ 1 ) and anti-Gγ 1  (Gγ 1 ).  (D)  Average distribution of each protein in the aqueous and detergent phases ( HA Gγ 1 C71S: 87/13 ± 4%;  HA Gγ 1 : 36/64 ± 9%, Gγ 1 : 41/59 ± 8%, error bars are SEM,  n  = 4).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Farnesylation of the Transducin G Protein Gamma Subunit Is a Prerequisite for Its Ciliary Targeting in Rod Photoreceptors

    doi: 10.3389/fnmol.2018.00016

    Figure Lengend Snippet: Assembly of transgenic HA Gγ 1 and HA Gγ 1 C71S with the endogenous Gβ 1 in retinal photoreceptors. (A) Transgenic construct used for generation of HA Gγ 1 mice and amino acid sequence of the encoded protein with HA-tag (underlined) and C71 (asterisk). (B) SDS polyacrylamide gel stained with Coomassie blue showing anti-HA pull downs from 10 retinas of the transgene-negative (1) and transgene-positive (2) HA Gγ 1 mice. (B) Western blot analysis of anti-HA pull downs from the retinas of HA Gγ 1 and HA Gγ 1 C71S mice with antibodies against Gβ 1 and HA. (C) Representative Western blot showing co-precipitation of phosducin (Pdc) with HA Gγ 1 or HA Gγ 1 C71S (HA). Tg(-) indicates transgene-negative littermates. The break separates two blots that were adjusted differently. Identical amounts of phosducin co-precipitated with HA Gγ 1 and HA Gγ 1 C71S ( n = 4). (E) Representative experiments illustrating partitioning of HA Gγ 1 C71S, HA Gγ 1 , and endogenous Gγ 1 between the detergent (Triton X-114) and aqueous phases. Specific bands were visualized by Western blotting with anti-HA ( HA Gγ 1 C71S, HA Gγ 1 ) and anti-Gγ 1 (Gγ 1 ). (D) Average distribution of each protein in the aqueous and detergent phases ( HA Gγ 1 C71S: 87/13 ± 4%; HA Gγ 1 : 36/64 ± 9%, Gγ 1 : 41/59 ± 8%, error bars are SEM, n = 4).

    Article Snippet: 20 μl of 10% Triton X-114 (648468, Calbiochem) was added to 180 μl of the retinal extract, mixed by gentle inversion and pre-warmed to 37°C for 5 min.

    Techniques: Transgenic Assay, Construct, Mouse Assay, Sequencing, Staining, Western Blot

    14-3-3 protein expression is ubiquitous across early embryonic stages and tissues. (A) Whole embryo lysates (1% Triton X-100) were immunoblotted for 14-3-3 using a pan antibody that detects multiple isoforms. Each lane represents approximately 50 μg. (B) Colored schematic of a bisected  Xenopus  embryo at gastrula depicting major tissue divisions. The tissues include the animal cap (AC), mesendoderm (ME), marginal zone (MZ), vegetal hemisphere (VG), and whole embryo lysate (WEL). (C) Embryos were dissected into separate tissues and corresponding lysates (1% Triton X-100) were immunoblotted using pan 14-3-3 antibody to examine expression across the gastrulating embryo. Each lane represents a portion of protein equivalent to approximately 1 embryo.

    Journal: Molecular Biology of the Cell

    Article Title: 14-3-3 targets keratin intermediate filaments to mechanically sensitive cell–cell contacts

    doi: 10.1091/mbc.E18-06-0373

    Figure Lengend Snippet: 14-3-3 protein expression is ubiquitous across early embryonic stages and tissues. (A) Whole embryo lysates (1% Triton X-100) were immunoblotted for 14-3-3 using a pan antibody that detects multiple isoforms. Each lane represents approximately 50 μg. (B) Colored schematic of a bisected Xenopus embryo at gastrula depicting major tissue divisions. The tissues include the animal cap (AC), mesendoderm (ME), marginal zone (MZ), vegetal hemisphere (VG), and whole embryo lysate (WEL). (C) Embryos were dissected into separate tissues and corresponding lysates (1% Triton X-100) were immunoblotted using pan 14-3-3 antibody to examine expression across the gastrulating embryo. Each lane represents a portion of protein equivalent to approximately 1 embryo.

    Article Snippet: ImmunoprecipitationWhole embryos or dissected mesendoderm for immunoprecipitation were processed in aforementioned lysis buffer with 1% Tergitol type NP-40 or 1% Triton X-100 as indicated in figure legends.

    Techniques: Expressing

    Mesendoderm keratin filament dynamic exchange is decreased by 14-3-3 inhibition. (A) Immunoblot analyses of protein extracts (1% Triton X-100) of stage 10.5  Xenopus  embryos expressing mCh-R18 or mCh-R18M. (B) Coimmunoprecipitation (1% Tergitol type NP-40) performed using stage 10.5 lysates expressing human FLAG-14-3-3 β with either mCherry-R18 or mCherry-R18M. (C, D) Still images from photobleach and recovery time lapse movies (Supplemental Movie S1). Mesendoderm explants expressing either 14-3-3 inhibitor peptide mCherry-R18 or control peptide mCherry-R18M (red) with eGFP-K19 (green) were exposed to GFP photobleaching and fluorescence recovery at the site was measured. (C’–D’’) Enlarged view of the region of filament bleaching (white boxes) during recovery measurements. The time annotations in seconds refer to start of capture ( t  = 0 s) and end of capture ( t  = 330 s). (E) Representative analysis plotting fluorescence recovery against time of image capture. (F) Comparison of mean eGFP-K19 recovery rate in explants expressing either mCherry-R18 or mCherry-R18M. Analysis was performed using a one-tailed  t  test, * p

    Journal: Molecular Biology of the Cell

    Article Title: 14-3-3 targets keratin intermediate filaments to mechanically sensitive cell–cell contacts

    doi: 10.1091/mbc.E18-06-0373

    Figure Lengend Snippet: Mesendoderm keratin filament dynamic exchange is decreased by 14-3-3 inhibition. (A) Immunoblot analyses of protein extracts (1% Triton X-100) of stage 10.5 Xenopus embryos expressing mCh-R18 or mCh-R18M. (B) Coimmunoprecipitation (1% Tergitol type NP-40) performed using stage 10.5 lysates expressing human FLAG-14-3-3 β with either mCherry-R18 or mCherry-R18M. (C, D) Still images from photobleach and recovery time lapse movies (Supplemental Movie S1). Mesendoderm explants expressing either 14-3-3 inhibitor peptide mCherry-R18 or control peptide mCherry-R18M (red) with eGFP-K19 (green) were exposed to GFP photobleaching and fluorescence recovery at the site was measured. (C’–D’’) Enlarged view of the region of filament bleaching (white boxes) during recovery measurements. The time annotations in seconds refer to start of capture ( t = 0 s) and end of capture ( t = 330 s). (E) Representative analysis plotting fluorescence recovery against time of image capture. (F) Comparison of mean eGFP-K19 recovery rate in explants expressing either mCherry-R18 or mCherry-R18M. Analysis was performed using a one-tailed t test, * p

    Article Snippet: ImmunoprecipitationWhole embryos or dissected mesendoderm for immunoprecipitation were processed in aforementioned lysis buffer with 1% Tergitol type NP-40 or 1% Triton X-100 as indicated in figure legends.

    Techniques: Inhibition, Expressing, Fluorescence, One-tailed Test

    14-3-3 proteins target keratins to cell–cell adhesions. (A) Schematic of fusion peptides created by the insertion of R18 or R18M (R18/M) into FLAG/eGFP-K19. This construct was generated using full-length K19.L from  X. laevis.  (B) Protein lysates from stage 10.5  Xenopus  embryos expressing C-cadherin-eGFP and either FLAG-R18-K19 or FLAG-R18M-K19 were prepared in 1% Triton X-100. FLAG constructs were immunoprecipitated and analyzed by immunoblot for associated proteins. (C) Protein lysates from stage 10.5  Xenopus  embryos expressing human FLAG-14-3-3β and either eGFP-R18-K19 or eGFP-R18M-K19 were prepared in 1% Tergitol-type NP-40. FLAG constructs were immunoprecipitated and analyzed by immunoblot for associated proteins. (D, E) Explanted mesendoderm cells mosaically expressing eGFP-R18M-K19 (in D) or eGFP-R18-K19 (in E). (F, G) Explanted mesendoderm coexpressing mem-RFP and eGFP-R18M-K19 (in F) or eGFP-R18-K19 (in G). Arrows indicate areas where filament densities have localized. Images are z-stacks (maximum intensity projection). Scale bars are 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: 14-3-3 targets keratin intermediate filaments to mechanically sensitive cell–cell contacts

    doi: 10.1091/mbc.E18-06-0373

    Figure Lengend Snippet: 14-3-3 proteins target keratins to cell–cell adhesions. (A) Schematic of fusion peptides created by the insertion of R18 or R18M (R18/M) into FLAG/eGFP-K19. This construct was generated using full-length K19.L from X. laevis. (B) Protein lysates from stage 10.5 Xenopus embryos expressing C-cadherin-eGFP and either FLAG-R18-K19 or FLAG-R18M-K19 were prepared in 1% Triton X-100. FLAG constructs were immunoprecipitated and analyzed by immunoblot for associated proteins. (C) Protein lysates from stage 10.5 Xenopus embryos expressing human FLAG-14-3-3β and either eGFP-R18-K19 or eGFP-R18M-K19 were prepared in 1% Tergitol-type NP-40. FLAG constructs were immunoprecipitated and analyzed by immunoblot for associated proteins. (D, E) Explanted mesendoderm cells mosaically expressing eGFP-R18M-K19 (in D) or eGFP-R18-K19 (in E). (F, G) Explanted mesendoderm coexpressing mem-RFP and eGFP-R18M-K19 (in F) or eGFP-R18-K19 (in G). Arrows indicate areas where filament densities have localized. Images are z-stacks (maximum intensity projection). Scale bars are 10 μm.

    Article Snippet: ImmunoprecipitationWhole embryos or dissected mesendoderm for immunoprecipitation were processed in aforementioned lysis buffer with 1% Tergitol type NP-40 or 1% Triton X-100 as indicated in figure legends.

    Techniques: Construct, Generated, Expressing, Immunoprecipitation

    Comigration of ISKNV MCP with mCav-1 in subcellular fractions in the early stages of ISKNV infection. Mandarin fish fry (MFF-1) cells infected with ISKNV were lysed with cold 1% Triton X-100 (1× PBS). The caveola-rich microdomains were purified

    Journal: Journal of Virology

    Article Title: Mandarin Fish Caveolin 1 Interaction with Major Capsid Protein of Infectious Spleen and Kidney Necrosis Virus and Its Role in Early Stages of Infection

    doi: 10.1128/JVI.00552-12

    Figure Lengend Snippet: Comigration of ISKNV MCP with mCav-1 in subcellular fractions in the early stages of ISKNV infection. Mandarin fish fry (MFF-1) cells infected with ISKNV were lysed with cold 1% Triton X-100 (1× PBS). The caveola-rich microdomains were purified

    Article Snippet: The cells were lysed on ice with lysis buffer (10 mM Tris-HCl [pH 7.5], 0.4 M NaCl, 1% NP-40, 0.4% Triton X-100, 0.2% sodium deoxycholate, 1 mM EDTA, protease inhibitors [Calbiochem]) for 30 min at 48 h after transfection.

    Techniques: Infection, Fluorescence In Situ Hybridization, Purification

    Strigolactones (SLs) promote ubiquitination and degradation of D14 in rice. (A) Protein levels of D14-GFP in calli of the Act:D14-GFP/d14 transgenic line at different time points after 20 μM rac -GR24 treatment with DMSO as a control. D14-GFP was detected by immunoblotting with an anti-GFP monoclonal antibody. Relative protein levels were determined by densitometry and normalized to loadings determined by Ponceau staining (red) in the immunoblotting analyses. (B) Relative expression levels of D14 and D53 in 2-week-old seedlings after 5 μM rac -GR24 treatment. Values represent means ± SEM, n = 3. (C) D14-GFP protein levels in calli of the D14-GFP/d14 transgenic line at different time points after rac -GR24 treatment in the presence or absence of MG132. Calli are pretreated with 50 μM MG132 for 1 h and then treated with 20 μM rac -GR24 or DMSO for 3 h. D14-GFP was detected by immunoblotting with an anti-GFP monoclonal antibody. Actin1 were used as loading control in the immunoblotting analyses. (D) Ubiquitination analysis of D14-GFP in rice protoplasts. Rice (Nipponbare) protoplasts were transformed with 35S:D14-GFP plasmids and incubated for 12 h, then pretreated with 50 μM MG132 for 1 h and immediately treated with 20 μM rac -GR24 or DMSO for 3 h. Proteins were extracted for affinity purification with an agarose-immobolized anti-GFP monoclonal antibody and followed by immunoblotting analysis with an anti-ubiquitin (upper panel) or anti-GFP (lower panel) monoclonal antibody.

    Journal: Frontiers in Plant Science

    Article Title: DWARF14, A Receptor Covalently Linked with the Active Form of Strigolactones, Undergoes Strigolactone-Dependent Degradation in Rice

    doi: 10.3389/fpls.2017.01935

    Figure Lengend Snippet: Strigolactones (SLs) promote ubiquitination and degradation of D14 in rice. (A) Protein levels of D14-GFP in calli of the Act:D14-GFP/d14 transgenic line at different time points after 20 μM rac -GR24 treatment with DMSO as a control. D14-GFP was detected by immunoblotting with an anti-GFP monoclonal antibody. Relative protein levels were determined by densitometry and normalized to loadings determined by Ponceau staining (red) in the immunoblotting analyses. (B) Relative expression levels of D14 and D53 in 2-week-old seedlings after 5 μM rac -GR24 treatment. Values represent means ± SEM, n = 3. (C) D14-GFP protein levels in calli of the D14-GFP/d14 transgenic line at different time points after rac -GR24 treatment in the presence or absence of MG132. Calli are pretreated with 50 μM MG132 for 1 h and then treated with 20 μM rac -GR24 or DMSO for 3 h. D14-GFP was detected by immunoblotting with an anti-GFP monoclonal antibody. Actin1 were used as loading control in the immunoblotting analyses. (D) Ubiquitination analysis of D14-GFP in rice protoplasts. Rice (Nipponbare) protoplasts were transformed with 35S:D14-GFP plasmids and incubated for 12 h, then pretreated with 50 μM MG132 for 1 h and immediately treated with 20 μM rac -GR24 or DMSO for 3 h. Proteins were extracted for affinity purification with an agarose-immobolized anti-GFP monoclonal antibody and followed by immunoblotting analysis with an anti-ubiquitin (upper panel) or anti-GFP (lower panel) monoclonal antibody.

    Article Snippet: After incubation at 28°C for 12 h in W5 solution (154 mM NaCl, 125 mM CaCl2 , 5 mM KCl, 2 mM MES, pH 5.7), proteins were extracted from the collected protoplasts using the extraction buffer (50 mM sodium phosphate buffer, pH 7.0, 150 mM NaCl, 10% (v/v) glycerol, 0.1% NP-40, 50 μM MG132, 1× complete protease inhibitor cocktail) by centrifugation at 20,000 g for 20 min at 4°C and then the supernatant was taken out for Co-IP experiments.

    Techniques: Activated Clotting Time Assay, Transgenic Assay, Staining, Expressing, Transformation Assay, Incubation, Affinity Purification