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Roche tris np40 buffer
Tris Np40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Protease Inhibitor:

Article Title: SIRT3, a Mitochondrial NAD+-Dependent Deacetylase, Is Involved in the Regulation of Myoblast Differentiation
Article Snippet: .. Immunoblotting Cells were lysed in Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). ..

Article Title: Protective Activity of Total Polyphenols from Genista quadriflora Munby and Teucrium polium geyrii Maire in Acetaminophen-Induced Hepatotoxicity in Rats
Article Snippet: .. Western Blot Analysis Livers were homogenized using a Polytron homogenizer in a Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). .. The homogenates were incubated on ice for 10 min and centrifuged at 10000 g for 10 min to remove tissue debris.

Pyrolysis Gas Chromatography:

Article Title: SIRT3, a Mitochondrial NAD+-Dependent Deacetylase, Is Involved in the Regulation of Myoblast Differentiation
Article Snippet: Immunoblotting Cells were lysed in Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). .. Membranes were blocked (1 h at room temperature) with a 5% skim milk in 1×TBST (Tris-buffered saline Tween-20: 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.2% Tween-20) solution and probed with an antibody raised against SIRT3, SIRT1 (Cell Signaling, 1∶1000), Myogenin, MyoD (Santa Cruz Biotechnology, 1∶50), voltage-dependent anion-selective channel (VDAC) (Abcam 1∶3000), Citrate synthase (Genetex, 1∶1000), PGC-1α (Calbiochem,1∶500) and Tubulin (Sigma, 1∶10000) overnight at 4°C.

Blocking Assay:

Article Title: MET overexpression and activation favors invasiveness in a model of anaplastic thyroid cancer
Article Snippet: Western blotting Total cell protein lysates were extracted using Tris-NP40 buffer (20 mM TrisHCl (pH 7.5) 1 mM EDTA, 150 mM NaCl, NP40 1%) containing protease and phosphatase inhibitors (Roche). .. After blocking with 5% non-fat milk or 5% bovine serum albumin, proteins were immunoblotted overnight with primary antibodies followed by incubation with HRP-conjugated secondary antibodies.

Article Title: Iron-Responsive Olfactory Uptake of Manganese Improves Motor Function Deficits Associated with Iron Deficiency
Article Snippet: Western blot analysis of dopamine transporters and receptors Microdissected striatal regions were homogenized in Tris-NP40 buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP40, pH 7.5) containing protease inhibitors (Complete Mini, Roche). .. After blocking, the membrane was incubated in goat anti-dopamine transporter (DAT) antibody (1∶100, Santa Cruz), mouse anti-dopamine receptor D2 (D2R) antibody (1∶100, Santa Cruz) or rat anti-dopamine receptor D1 (D1R) antibody (1∶100, Sigma).

Concentration Assay:

Article Title: SIRT3, a Mitochondrial NAD+-Dependent Deacetylase, Is Involved in the Regulation of Myoblast Differentiation
Article Snippet: Immunoblotting Cells were lysed in Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). .. Fifty µg of proteins were run on SDS-PAGE mini-gels at the appropriate concentration of acrylamide and transferred onto nitrocellulose membrane.

Article Title: Protective Activity of Total Polyphenols from Genista quadriflora Munby and Teucrium polium geyrii Maire in Acetaminophen-Induced Hepatotoxicity in Rats
Article Snippet: Western Blot Analysis Livers were homogenized using a Polytron homogenizer in a Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). .. Fifty µg of proteins were run on SDS-PAGE mini-gels at the appropriate concentration of acrylamide and transferred onto a polyvinylidene difluoride membrane.

Incubation:

Article Title: MET overexpression and activation favors invasiveness in a model of anaplastic thyroid cancer
Article Snippet: Western blotting Total cell protein lysates were extracted using Tris-NP40 buffer (20 mM TrisHCl (pH 7.5) 1 mM EDTA, 150 mM NaCl, NP40 1%) containing protease and phosphatase inhibitors (Roche). .. After blocking with 5% non-fat milk or 5% bovine serum albumin, proteins were immunoblotted overnight with primary antibodies followed by incubation with HRP-conjugated secondary antibodies.

Article Title: Iron-Responsive Olfactory Uptake of Manganese Improves Motor Function Deficits Associated with Iron Deficiency
Article Snippet: Western blot analysis of dopamine transporters and receptors Microdissected striatal regions were homogenized in Tris-NP40 buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP40, pH 7.5) containing protease inhibitors (Complete Mini, Roche). .. After blocking, the membrane was incubated in goat anti-dopamine transporter (DAT) antibody (1∶100, Santa Cruz), mouse anti-dopamine receptor D2 (D2R) antibody (1∶100, Santa Cruz) or rat anti-dopamine receptor D1 (D1R) antibody (1∶100, Sigma).

Article Title: Protective Activity of Total Polyphenols from Genista quadriflora Munby and Teucrium polium geyrii Maire in Acetaminophen-Induced Hepatotoxicity in Rats
Article Snippet: Western Blot Analysis Livers were homogenized using a Polytron homogenizer in a Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). .. The homogenates were incubated on ice for 10 min and centrifuged at 10000 g for 10 min to remove tissue debris.

Protein Concentration:

Article Title: MET overexpression and activation favors invasiveness in a model of anaplastic thyroid cancer
Article Snippet: Western blotting Total cell protein lysates were extracted using Tris-NP40 buffer (20 mM TrisHCl (pH 7.5) 1 mM EDTA, 150 mM NaCl, NP40 1%) containing protease and phosphatase inhibitors (Roche). .. Protein concentration was determined using the Bradford reagent (BioRad).

Western Blot:

Article Title: MET overexpression and activation favors invasiveness in a model of anaplastic thyroid cancer
Article Snippet: .. Western blotting Total cell protein lysates were extracted using Tris-NP40 buffer (20 mM TrisHCl (pH 7.5) 1 mM EDTA, 150 mM NaCl, NP40 1%) containing protease and phosphatase inhibitors (Roche). .. Protein concentration was determined using the Bradford reagent (BioRad).

Article Title: Iron-Responsive Olfactory Uptake of Manganese Improves Motor Function Deficits Associated with Iron Deficiency
Article Snippet: .. Western blot analysis of dopamine transporters and receptors Microdissected striatal regions were homogenized in Tris-NP40 buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP40, pH 7.5) containing protease inhibitors (Complete Mini, Roche). .. Samples (50–100 µg proteins) were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Millipore).

Article Title: Protective Activity of Total Polyphenols from Genista quadriflora Munby and Teucrium polium geyrii Maire in Acetaminophen-Induced Hepatotoxicity in Rats
Article Snippet: .. Western Blot Analysis Livers were homogenized using a Polytron homogenizer in a Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). .. The homogenates were incubated on ice for 10 min and centrifuged at 10000 g for 10 min to remove tissue debris.

SDS Page:

Article Title: MET overexpression and activation favors invasiveness in a model of anaplastic thyroid cancer
Article Snippet: Western blotting Total cell protein lysates were extracted using Tris-NP40 buffer (20 mM TrisHCl (pH 7.5) 1 mM EDTA, 150 mM NaCl, NP40 1%) containing protease and phosphatase inhibitors (Roche). .. Proteins were separated by 7–12% SDS-PAGE and transferred on nitrocellulose membranes (GE Healthcare).

Article Title: SIRT3, a Mitochondrial NAD+-Dependent Deacetylase, Is Involved in the Regulation of Myoblast Differentiation
Article Snippet: Immunoblotting Cells were lysed in Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). .. Fifty µg of proteins were run on SDS-PAGE mini-gels at the appropriate concentration of acrylamide and transferred onto nitrocellulose membrane.

Article Title: Protective Activity of Total Polyphenols from Genista quadriflora Munby and Teucrium polium geyrii Maire in Acetaminophen-Induced Hepatotoxicity in Rats
Article Snippet: Western Blot Analysis Livers were homogenized using a Polytron homogenizer in a Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). .. Fifty µg of proteins were run on SDS-PAGE mini-gels at the appropriate concentration of acrylamide and transferred onto a polyvinylidene difluoride membrane.

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  • 99
    Roche ripa buffer
    Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P <t>exosomes</t> collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of <t>RIPA</t> buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P
    Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 4719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ripa buffer/product/Roche
    Average 99 stars, based on 4719 article reviews
    Price from $9.99 to $1999.99
    ripa buffer - by Bioz Stars, 2020-04
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    96
    Roche immunoprecipitation buffer
    The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) <t>Immunoprecipitation</t> of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.
    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
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    93
    Roche iph buffer
    Irreparable DSBs induced by a 16 hour etoposide treatment lead to an increased interaction of MTA3 with HIC1 and favor its recruitment to the HIC1-response elements in the SIRT1 promoter ( A ) Etoposide-induced non-repairable DSBs lead to an increase of MTA3 interaction with HIC1. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, and FLAG-MTA3 expression vectors. 32 hours after <t>transfection</t> cells were incubated for 16 hours with 20 μM etoposide (+) or with DMSO (–) as control. After lysis in <t>IPH</t> buffer, cell extracts were co-immunoprecipitated with anti-MTA3 antibodies. The immunoprecipitates as well as 1% of the whole cell extracts were analyzed by SDS/PAGE and transferred to membranes. Relevant pieces of the membranes were cut and analyzed by Western blot with anti-FLAG antibodies to detect MTA3 and HIC1. ΔH2AX and actin levels were used as controls for DSB induction and equal loading, respectively. ( B ) Etoposide-induced irreparable DSB lead to an increase of MTA3 recruitment on the HiRE in the SIRT1 promoter. Chromatin was prepared from BJ-hTERT fibroblasts mock-treated with DMSO or treated with 80 uM etoposide for 16 hours to induce irreparable DSB and ChIP experiments were performed with antibodies against MTA3 or rabbit IgG. The bound material was eluted and analysed by quantitative PCR using primers flanking the HIC1-responsive elements (HiRE) in the SIRT1 promoter [ 6 ], as previously described [ 46 ]. GAPDH was used as a nonbinding control. Values that are statistically significantly different are indicated by bars and asterisks as follows: *P
    Iph Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/iph buffer/product/Roche
    Average 93 stars, based on 8 article reviews
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    Image Search Results


    Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P

    Journal: PLoS ONE

    Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0138849

    Figure Lengend Snippet: Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P

    Article Snippet: Western blot analysis Cells and exosomes were lysed in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate in Tris-buffered saline) with complete protease inhibitors (Roche Diagnostics).

    Techniques: Electron Microscopy, Isolation, Western Blot, Cell Culture, Activity Assay

    Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.

    Journal: PLoS ONE

    Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0138849

    Figure Lengend Snippet: Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.

    Article Snippet: Western blot analysis Cells and exosomes were lysed in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate in Tris-buffered saline) with complete protease inhibitors (Roche Diagnostics).

    Techniques: Derivative Assay, Electron Microscopy, Isolation, Western Blot, Cell Culture, SDS Page, Electrophoresis, Staining, Software

    SUMOylation of KHSRP is involved in tumorigenesis. a KHSRP-K87R downregulates the anchorage-independent growth in DU145 stable cell lines. In soft agar colony forming assays, stable cell lines DU145 shRNA control, shKHSRP, shKHSRP-KHSRP-WT or shKHSRP-KHSRP-K87R were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 2 × 10 3 cells/well and layered onto the base. The photographs were taken 21 days later and the number of colonies was scored. b KHSRP-K87R downregulates the migration ability in DU145 stable cell lines. The RTCA migration assay was performed to detect the migration ability in above stable DU145 cell lines with xCELLigene RTCA-DP instrument. The kinetic cell index of their migration was recorded every 15 min for 24 h (left panel) and the relative slope value was calculated (right panel). c KHSRP-K87R downregulates the invasive ability in above stable DU145 cell lines. The 3D–culture assay was performed to detect the invasive ability of DU145 stable cell lines. The photos were taken at day 7. The first image was taken under the white light, and the green signals indicates the expression of GFP (Green Fluorescent Protein) in the plasmid CD513B-HA-KHSRP. d KHSRP-K87R downregulates the aggressive ability in DU145 stable cell lines in vasculogenic mimicry (VM) assay. VM assay was performed to detect the aggressive ability in above stable DU145 cell lines. The photos were taken 20 h later. Scale: 500 μm. Independent experiments ( a - d ) were repeated three times. e KHSRP-K87R suppresses xenograft tumor growth in vivo. 5 male BALB/c nude mice were injected subcutaneously with stable DU145 cell lines (2.5 × 10 6 cells/each) expressing the shRNA control in the left back and shKHSRP in the right back, respectively. Another 5 male BALB/c nude mice were injected subcutaneously with stable DU145 cell line expressing shKHSRP-KHSRP-WT in the left back and shKHSRP-KHSRP-K87R in the right back, respectively. Mice were sacrificed 5 weeks later, and tumors were dissected (upper panel) and assessed by weight (low panel). ( a - d ) f KHSRP SUMOylation could be detected in tumors of nude mice. The tumors of nude mice, which was chosen from the groups of shKHSRP, shKHSRP-KHSRP-WT or -K87R, were lysed in NEM-RIPA as described in the Methods. The proteins was immunoprecipitated by anti-SUMO1 antibody, Western blotting was detected with anti-HA antibody

    Journal: Molecular Cancer

    Article Title: SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis

    doi: 10.1186/s12943-017-0724-6

    Figure Lengend Snippet: SUMOylation of KHSRP is involved in tumorigenesis. a KHSRP-K87R downregulates the anchorage-independent growth in DU145 stable cell lines. In soft agar colony forming assays, stable cell lines DU145 shRNA control, shKHSRP, shKHSRP-KHSRP-WT or shKHSRP-KHSRP-K87R were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 2 × 10 3 cells/well and layered onto the base. The photographs were taken 21 days later and the number of colonies was scored. b KHSRP-K87R downregulates the migration ability in DU145 stable cell lines. The RTCA migration assay was performed to detect the migration ability in above stable DU145 cell lines with xCELLigene RTCA-DP instrument. The kinetic cell index of their migration was recorded every 15 min for 24 h (left panel) and the relative slope value was calculated (right panel). c KHSRP-K87R downregulates the invasive ability in above stable DU145 cell lines. The 3D–culture assay was performed to detect the invasive ability of DU145 stable cell lines. The photos were taken at day 7. The first image was taken under the white light, and the green signals indicates the expression of GFP (Green Fluorescent Protein) in the plasmid CD513B-HA-KHSRP. d KHSRP-K87R downregulates the aggressive ability in DU145 stable cell lines in vasculogenic mimicry (VM) assay. VM assay was performed to detect the aggressive ability in above stable DU145 cell lines. The photos were taken 20 h later. Scale: 500 μm. Independent experiments ( a - d ) were repeated three times. e KHSRP-K87R suppresses xenograft tumor growth in vivo. 5 male BALB/c nude mice were injected subcutaneously with stable DU145 cell lines (2.5 × 10 6 cells/each) expressing the shRNA control in the left back and shKHSRP in the right back, respectively. Another 5 male BALB/c nude mice were injected subcutaneously with stable DU145 cell line expressing shKHSRP-KHSRP-WT in the left back and shKHSRP-KHSRP-K87R in the right back, respectively. Mice were sacrificed 5 weeks later, and tumors were dissected (upper panel) and assessed by weight (low panel). ( a - d ) f KHSRP SUMOylation could be detected in tumors of nude mice. The tumors of nude mice, which was chosen from the groups of shKHSRP, shKHSRP-KHSRP-WT or -K87R, were lysed in NEM-RIPA as described in the Methods. The proteins was immunoprecipitated by anti-SUMO1 antibody, Western blotting was detected with anti-HA antibody

    Article Snippet: Co-immunoprecipitation (co-IP) 293T cells transfected with HA-Drosha and Flag-KHSRP-WT or -K87R, or cells transfected with Flag-DGCR8 and HA-KHSRP-WT or -K87R respectively were lysed in RIPA buffer (50 mM Tris-HCl pH = 7.4, 150 mM NaCl, 1% NP40, 10% glycerol and a complete protease inhibitor cocktail (Roche)).

    Techniques: Stable Transfection, shRNA, Migration, Expressing, Plasmid Preparation, VM Assay, In Vivo, Mouse Assay, Injection, Immunoprecipitation, Western Blot

    The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) Immunoprecipitation of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.

    Journal: Nucleic Acids Research

    Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

    doi: 10.1093/nar/gku692

    Figure Lengend Snippet: The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) Immunoprecipitation of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.

    Article Snippet: For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively.

    Techniques: Mutagenesis, Binding Assay, Proximity Ligation Assay, Labeling, Confocal Microscopy, Immunoprecipitation, Nuclear Magnetic Resonance, Concentration Assay, SYBR Green Assay, Staining

    RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. ( A ) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. ( B ) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( D ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.

    Journal: Nucleic Acids Research

    Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

    doi: 10.1093/nar/gku692

    Figure Lengend Snippet: RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. ( A ) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. ( B ) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( D ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.

    Article Snippet: For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively.

    Techniques: In Vitro, In Vivo, Clone Assay, Isolation, Immunoprecipitation, Incubation, Ligation, Proximity Ligation Assay, Labeling, Confocal Microscopy

    Irreparable DSBs induced by a 16 hour etoposide treatment lead to an increased interaction of MTA3 with HIC1 and favor its recruitment to the HIC1-response elements in the SIRT1 promoter ( A ) Etoposide-induced non-repairable DSBs lead to an increase of MTA3 interaction with HIC1. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, and FLAG-MTA3 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or with DMSO (–) as control. After lysis in IPH buffer, cell extracts were co-immunoprecipitated with anti-MTA3 antibodies. The immunoprecipitates as well as 1% of the whole cell extracts were analyzed by SDS/PAGE and transferred to membranes. Relevant pieces of the membranes were cut and analyzed by Western blot with anti-FLAG antibodies to detect MTA3 and HIC1. ΔH2AX and actin levels were used as controls for DSB induction and equal loading, respectively. ( B ) Etoposide-induced irreparable DSB lead to an increase of MTA3 recruitment on the HiRE in the SIRT1 promoter. Chromatin was prepared from BJ-hTERT fibroblasts mock-treated with DMSO or treated with 80 uM etoposide for 16 hours to induce irreparable DSB and ChIP experiments were performed with antibodies against MTA3 or rabbit IgG. The bound material was eluted and analysed by quantitative PCR using primers flanking the HIC1-responsive elements (HiRE) in the SIRT1 promoter [ 6 ], as previously described [ 46 ]. GAPDH was used as a nonbinding control. Values that are statistically significantly different are indicated by bars and asterisks as follows: *P

    Journal: Oncotarget

    Article Title: HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs)

    doi: 10.18632/oncotarget.13807

    Figure Lengend Snippet: Irreparable DSBs induced by a 16 hour etoposide treatment lead to an increased interaction of MTA3 with HIC1 and favor its recruitment to the HIC1-response elements in the SIRT1 promoter ( A ) Etoposide-induced non-repairable DSBs lead to an increase of MTA3 interaction with HIC1. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, and FLAG-MTA3 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or with DMSO (–) as control. After lysis in IPH buffer, cell extracts were co-immunoprecipitated with anti-MTA3 antibodies. The immunoprecipitates as well as 1% of the whole cell extracts were analyzed by SDS/PAGE and transferred to membranes. Relevant pieces of the membranes were cut and analyzed by Western blot with anti-FLAG antibodies to detect MTA3 and HIC1. ΔH2AX and actin levels were used as controls for DSB induction and equal loading, respectively. ( B ) Etoposide-induced irreparable DSB lead to an increase of MTA3 recruitment on the HiRE in the SIRT1 promoter. Chromatin was prepared from BJ-hTERT fibroblasts mock-treated with DMSO or treated with 80 uM etoposide for 16 hours to induce irreparable DSB and ChIP experiments were performed with antibodies against MTA3 or rabbit IgG. The bound material was eluted and analysed by quantitative PCR using primers flanking the HIC1-responsive elements (HiRE) in the SIRT1 promoter [ 6 ], as previously described [ 46 ]. GAPDH was used as a nonbinding control. Values that are statistically significantly different are indicated by bars and asterisks as follows: *P

    Article Snippet: For co-immunoprecipitation analyses (Co-IPs), 48 h after transfection, cells were rinsed with cold PBS and lysed in cold IPH buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, protease inhibitor cocktail [Roche]).

    Techniques: Transfection, Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Repairable DNA double-strand breaks (DSBs) induced by a 1 hour etoposide treatment do not lead to an ATM-dependent increase of HIC1 SUMOylation ( A ) Etoposide-induced non-repairable DSBs lead to an increase of HIC1 SUMOylation. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, SENP2 and SUMO2 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or DMSO (–) as control before direct lysis in denaturing conditions. Total cell extracts were analyzed by Western Blotting (WB) using the indicated antibodies. ( B ) HEK 293T cells were transfected with FLAG-HIC1 and treated with etoposide or DMSO for 1 hour or 16 hours. Cell extracts were prepared as described in panel A) and analyzed by immunoblotting using the indicated antibodies. Quantification of SUMO-HIC1 to total HIC1 (FLAG) was performed with the Fujifilm MultiGauge software (Bottom Panel) ( C ) HEK 293T cells were transfected with FLAG-HIC1 and treated with etoposide or DMSO for 1 hour. Transfected cells were pre-treated or not with the following inhibitors (Wortmannin; ATMi, ATM inhibitor and DNAPKcsi, DNA-PKcs inhibitor) 1 hour before etoposide treatment, as indicated. Cell extracts were prepared as described in panel B) and analyzed by immunoblotting using the indicated antibodies. ( D ) HEK293T cells were co-transfected with the indicated combinations of expression vectors for FLAG-HIC1 and MTA1 and then incubated for 1 hour in etoposide or with DMSO as control. After lysis in IPH buffer, cells extracts were co-immunoprecipitated with anti-HIC1 antibodies. The immunoprecipitates as well as 2% of the whole cell extract (Input) were analyzed by Western blotting with the anti FLAG and anti MTA1 antibody. Note that the MTA1 antibodies detect a doublet of endogenous proteins in non transfected cells whereas the ectopically expressed MTA1 protein co-migrates with the upper band of the doublet (arrowheads).

    Journal: Oncotarget

    Article Title: HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs)

    doi: 10.18632/oncotarget.13807

    Figure Lengend Snippet: Repairable DNA double-strand breaks (DSBs) induced by a 1 hour etoposide treatment do not lead to an ATM-dependent increase of HIC1 SUMOylation ( A ) Etoposide-induced non-repairable DSBs lead to an increase of HIC1 SUMOylation. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, SENP2 and SUMO2 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or DMSO (–) as control before direct lysis in denaturing conditions. Total cell extracts were analyzed by Western Blotting (WB) using the indicated antibodies. ( B ) HEK 293T cells were transfected with FLAG-HIC1 and treated with etoposide or DMSO for 1 hour or 16 hours. Cell extracts were prepared as described in panel A) and analyzed by immunoblotting using the indicated antibodies. Quantification of SUMO-HIC1 to total HIC1 (FLAG) was performed with the Fujifilm MultiGauge software (Bottom Panel) ( C ) HEK 293T cells were transfected with FLAG-HIC1 and treated with etoposide or DMSO for 1 hour. Transfected cells were pre-treated or not with the following inhibitors (Wortmannin; ATMi, ATM inhibitor and DNAPKcsi, DNA-PKcs inhibitor) 1 hour before etoposide treatment, as indicated. Cell extracts were prepared as described in panel B) and analyzed by immunoblotting using the indicated antibodies. ( D ) HEK293T cells were co-transfected with the indicated combinations of expression vectors for FLAG-HIC1 and MTA1 and then incubated for 1 hour in etoposide or with DMSO as control. After lysis in IPH buffer, cells extracts were co-immunoprecipitated with anti-HIC1 antibodies. The immunoprecipitates as well as 2% of the whole cell extract (Input) were analyzed by Western blotting with the anti FLAG and anti MTA1 antibody. Note that the MTA1 antibodies detect a doublet of endogenous proteins in non transfected cells whereas the ectopically expressed MTA1 protein co-migrates with the upper band of the doublet (arrowheads).

    Article Snippet: For co-immunoprecipitation analyses (Co-IPs), 48 h after transfection, cells were rinsed with cold PBS and lysed in cold IPH buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, protease inhibitor cocktail [Roche]).

    Techniques: Transfection, Expressing, Incubation, Lysis, Western Blot, Software, Immunoprecipitation