293t 17 hek 293t 17  (Millipore)

 
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    Name:
    DAPI dilactate
    Description:

    Catalog Number:
    D9564
    Price:
    None
    Applications:
    DAPI, dilactate has been used for the nuclear staining in cells.
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    Structured Review

    Millipore 293t 17 hek 293t 17
    DAPI dilactate

    https://www.bioz.com/result/293t 17 hek 293t 17/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    293t 17 hek 293t 17 - by Bioz Stars, 2021-07
    97/100 stars

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    Related Articles

    Incubation:

    Article Title: Autophagic and lysosomal defects in human tauopathies: analysis of post-mortem brain from patients with familial Alzheimer disease, corticobasal degeneration and progressive supranuclear palsy
    Article Snippet: After washing in TBS-T, sections were incubated (1 h at RT) with appropriate secondary antibodies [anti-mouse and anti-rabbit IgG (H + L)] conjugated to Alexa Fluor 546 (A-11003, Invitrogen) or Alexa Fluor 488 (A-11008, Invitrogen) at a concentration of 1:500 in TBS-T. After washing in TBS-T (3 × 10 min) slides were incubated with (or without) Sudan Black B (199664, Sigma-Aldrich), 5 min at RT to reduce autofluorescence. .. Sections were then washed in TBS-T and incubated with DAPI for 5 min (D9564, Sigma-Aldrich). .. After washing in TBS-T, coverslips were mounted with Vectashield Hard Set (H-1200, Vector Laboratories) and the slides were stored at 4 °C.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes
    Article Snippet: Therefore, in this study, we used rapamycin, an autophagy activator, to enhance autophagy in astrocytes and to investigate if the expression of GLT-1 could be returned to its initial level. .. Reagents Huntingtin antibody (1:2000, Cat.MAB2166, Chemicon, Billerica, MA, USA); β-actin antibody (1:5000, Cat.A5441, Sigma, St Louis, MO, USA); LC3 antibody (1:1000, Cat.Ab62721, Abcam, University of Cambridge, UK); p62 antibody (1:2000, Cat.PW9860, Enzo Life Science, Lausen, CH, USA); GFAP (1:2000, Cat.c9205, Sigma, St Louis, MO, USA); DAPI (1:10000, Cat. D9564, Sigma, St Louis, MO, USA); GLT-1 (1:3000, Cat. ab58571, Abcam, University of Cambridge, UK); GLAST (1:5000, Cat.ab416, Abcam, University of Cambridge, UK); Cy3-conjugated anti-mouse IgG and HRP-conjugated anti-mouse IgG (1:5000, Cat 715-165-150 and Cat 715-035-1500, Jackson ImmunoResearch, West Grove, PA, USA); rapamycin (Cat. R0395, Sigma, St Louis, MO, USA); 3-MA (3-methyladenine, Cat. M9281, Sigma, St Louis, MO, USA); DHK (dihydrokainate, Cat.D1064, Sigma, St Louis, MO, USA); and 3 H]glutamate (Cat.NET1082250UC, Perkin Elmer, Waltham, MA, USA) were used in this study. ..

    Article Title: Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes
    Article Snippet: Therefore, in this study, we used rapamycin, an autophagy activator, to enhance autophagy in astrocytes and to investigate if the expression of GLT-1 could be returned to its initial level. .. Huntingtin antibody (1:2000, Cat.MAB2166, Chemicon, Billerica, MA, USA); β-actin antibody (1:5000, Cat.A5441, Sigma, St Louis, MO, USA); LC3 antibody (1:1000, Cat.Ab62721, Abcam, University of Cambridge, UK); p62 antibody (1:2000, Cat.PW9860, Enzo Life Science, Lausen, CH, USA); GFAP (1:2000, Cat.c9205, Sigma, St Louis, MO, USA); DAPI (1:10000, Cat. D9564, Sigma, St Louis, MO, USA); GLT-1 (1:3000, Cat. ab58571, Abcam, University of Cambridge, UK); GLAST (1:5000, Cat.ab416, Abcam, University of Cambridge, UK); Cy3-conjugated anti-mouse IgG and HRP-conjugated anti-mouse IgG (1:5000, Cat 715-165-150 and Cat 715-035-1500, Jackson ImmunoResearch, West Grove, PA, USA); rapamycin (Cat. R0395, Sigma, St Louis, MO, USA); 3-MA (3-methyladenine, Cat. M9281, Sigma, St Louis, MO, USA); DHK (dihydrokainate, Cat.D1064, Sigma, St Louis, MO, USA); and 3 H]glutamate (Cat.NET1082250UC, Perkin Elmer, Waltham, MA, USA) were used in this study. ..

    Staining:

    Article Title: Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and ?-catenin signaling
    Article Snippet: .. Cells were washed three times with PBS, treated with 0.1% Triton X-100, washed again with PBS and treated with TRITC-phalloidin (Sigma, cat #P1951) to stain F-actin and DAPI (Sigma, cat #D9564) to stain nuclei before washing and visualizing by fluorescence microscopy. .. Images were taken using an Olympus IX-70 inverted fluorescence microscope equipped with a digital camera (Olympus DP70).

    Article Title: Identification, visualization and clonal analysis of intestinal stem cells in fish
    Article Snippet: Immunohistochemistry and H & E staining Immunostaining and Haematoxylin and Eosin (H & E) staining were performed as described previously , using the following primary antibodies (all at 1:500): chicken anti-EGFP (Life Technologies, A10262), rabbit anti-phospho H3 (Upstate Biotechnology, 06-570) and rabbit anti-caspase 3 (Abcam, ab13847). .. Nuclear DNA was stained with DAPI (Sigma, D9564) or DRAQ5 (Thermo Fisher Scientific, 62251; ). ..

    Article Title: Endothelial cell colony forming units derived from malignant breast diseases are resistant to tumor necrosis factor-α-induced apoptosis
    Article Snippet: Immunofluorescence staining After 5–7 days of culture, fibronectin-adherent cells were washed and then incubated with 2.4 mg/ml of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)-labelled acetylated low-density lipoprotein for 4 h (DiI-Ac-LDL, L-3484, Thermo Fisher Scientific, Waltham, MA, USA). .. The cells were fixed for 10 min with 1% para-formaldehyde and then stained with anti-lectin antibody (ab133629, Abcam, Cambridge, UK), goat anti-rabbit fluorescein isothiocyanate (FITC, ab6717, Abcam) and 4′, 6-diamidino-2-phenylindole (DAPI, D9564, Sigma-Aldrich, St. Louis, MO, USA). .. RNA preparation Briefly, total RNA was extracted from the cultured EC-CFUs of 10 malignant breast diseases in patients with a mean age of 54.6 ± 3.56 years and 6 healthy controls with a mean age of 46.67 ± 2.14 years using TRIzol reagent (Invitrogen, Carlsband, CA, USA).

    Article Title: Substituting Threonine 187 with Alanine in p27Kip1 Prevents Pituitary Tumorigenesis by Two-Hit Loss of Rb1 and Enhances Humoral Immunity in Old Age *
    Article Snippet: .. For germinal center B cell analysis, isolated splenocytes were stained with DAPI (Sigma, D9564), Alexa 700-anti-B220 (BioLegend, 103231), FITC-PNA (Vector Labs, FL-1071), and PE anti-CD95 (Fas) (eBioscience, 12–0951-81). .. For T cell and B cell ratios analyses and CD4 and CD8 T cell ratios analyses, isolated splenocytes were stained with PE-anti-CD3 (BioLegend, 100205), Alexa 700-anti-B220 (BioLegend, 103231), Alexa 488-anti-CD4 (BioLegend,100425), and Alexa 647-anti-CD8a (BioLegend, 100727).

    Fluorescence:

    Article Title: Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and ?-catenin signaling
    Article Snippet: .. Cells were washed three times with PBS, treated with 0.1% Triton X-100, washed again with PBS and treated with TRITC-phalloidin (Sigma, cat #P1951) to stain F-actin and DAPI (Sigma, cat #D9564) to stain nuclei before washing and visualizing by fluorescence microscopy. .. Images were taken using an Olympus IX-70 inverted fluorescence microscope equipped with a digital camera (Olympus DP70).

    Microscopy:

    Article Title: Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and ?-catenin signaling
    Article Snippet: .. Cells were washed three times with PBS, treated with 0.1% Triton X-100, washed again with PBS and treated with TRITC-phalloidin (Sigma, cat #P1951) to stain F-actin and DAPI (Sigma, cat #D9564) to stain nuclei before washing and visualizing by fluorescence microscopy. .. Images were taken using an Olympus IX-70 inverted fluorescence microscope equipped with a digital camera (Olympus DP70).

    Molecular Weight:

    Article Title: Cross-linked polyethylenimine–tripolyphosphate nanoparticles for gene delivery
    Article Snippet: .. Reagents PEI (average molecular weight 25 kDa), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), agarose, ethidium bromide, bromophenol blue, and 4′,6-diamidino-2-phenylindole (DAPI) dilactate were purchased from Sigma-Aldrich (St Louis, MO, USA). ..

    MTT Assay:

    Article Title: Cross-linked polyethylenimine–tripolyphosphate nanoparticles for gene delivery
    Article Snippet: .. Reagents PEI (average molecular weight 25 kDa), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), agarose, ethidium bromide, bromophenol blue, and 4′,6-diamidino-2-phenylindole (DAPI) dilactate were purchased from Sigma-Aldrich (St Louis, MO, USA). ..

    Isolation:

    Article Title: Substituting Threonine 187 with Alanine in p27Kip1 Prevents Pituitary Tumorigenesis by Two-Hit Loss of Rb1 and Enhances Humoral Immunity in Old Age *
    Article Snippet: .. For germinal center B cell analysis, isolated splenocytes were stained with DAPI (Sigma, D9564), Alexa 700-anti-B220 (BioLegend, 103231), FITC-PNA (Vector Labs, FL-1071), and PE anti-CD95 (Fas) (eBioscience, 12–0951-81). .. For T cell and B cell ratios analyses and CD4 and CD8 T cell ratios analyses, isolated splenocytes were stained with PE-anti-CD3 (BioLegend, 100205), Alexa 700-anti-B220 (BioLegend, 103231), Alexa 488-anti-CD4 (BioLegend,100425), and Alexa 647-anti-CD8a (BioLegend, 100727).

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  • 97
    Millipore anti ezh2
    Meg3 facilitates the non-stoichiometric interaction of the PRC2 complex and Jarid2 ( A ) <t>Ezh2,</t> Jarid2, and Suz12 immunoprecipitation specifically retrieves Meg3 RNA isoforms ( v1 and v5 ). Rnu1 RNA and Malat1 lncRNA are negative controls. 10% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments). Immunoblotting reflects the recovery of Ezh2, Jarid2 and Suz12 proteins using the corresponding antibodies. ( B ) In vitro -transcribed, biotinylated Meg3 RNA isoforms retrieved Ezh2. ( C ) Ezh2 interacts with Jarid2 in ESC~MNs, but knockdown of Meg3 impairs this interaction. The abundance of Jarid2 is shown on the right (N.S.: not significant; error bars represent SD, n = 3 independent experiments; ** p-value
    Anti Ezh2, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ezh2/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ezh2 - by Bioz Stars, 2021-07
    97/100 stars
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    97
    Millipore 293t 17
    p27 is SUMOylated on K134 in vitro and in vivo . ( A ) Western blot analysis of FLAG-p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from <t>293T/17</t> cells transfected with FLAG-p27 WT or Flag-p27 1–170 , in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. Asterisk indicates the IgG heavy chain band. ( B ) Western blot analysis of p27 WT recombinant protein in an in vitro SUMOylation assay. His-tagged p27 was incubated with SAE1/SAE2, SUMO, and increasing doses of Ubc9. p27 SUMOylation, expressed as a SUMOylated p27/p27 ratio, was calculated by densitometric analysis of the blots. ( C ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence or absence of HA-SUMO1 and untagged Ubc9. ( D ) Western blot analysis of p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. ( E ) Western blot analysis of p27 and SUMO1 expression in SUMO1 (IP SUMO1) or control (IP IgG) immunoprecipitates from HeLa cells transfected with FLAG-p27 WT or FLAG-p27 K134R in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate SUMO-modified p27 proteins detected using anti-p27 antibody (upper panel) or the immunoprecipitated SUMO1 (lower panel). Asterisk indicates the IgG heavy chain band. ( F ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) and immunoprecipitates (IP) derived from 293T/17 cells transfected with untagged p27 WT in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-p27 (lower and middle panels) or anti-HA (upper panel) antibodies.
    293t 17, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/293t 17/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    293t 17 - by Bioz Stars, 2021-07
    97/100 stars
      Buy from Supplier

    99
    Millipore mouse monoclonal anti ha antibody
    p27 is SUMOylated on K134 in vitro and in vivo . ( A ) Western blot analysis of FLAG-p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from <t>293T/17</t> cells transfected with FLAG-p27 WT or Flag-p27 1–170 , in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. Asterisk indicates the IgG heavy chain band. ( B ) Western blot analysis of p27 WT recombinant protein in an in vitro SUMOylation assay. His-tagged p27 was incubated with SAE1/SAE2, SUMO, and increasing doses of Ubc9. p27 SUMOylation, expressed as a SUMOylated p27/p27 ratio, was calculated by densitometric analysis of the blots. ( C ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence or absence of HA-SUMO1 and untagged Ubc9. ( D ) Western blot analysis of p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. ( E ) Western blot analysis of p27 and SUMO1 expression in SUMO1 (IP SUMO1) or control (IP IgG) immunoprecipitates from HeLa cells transfected with FLAG-p27 WT or FLAG-p27 K134R in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate SUMO-modified p27 proteins detected using anti-p27 antibody (upper panel) or the immunoprecipitated SUMO1 (lower panel). Asterisk indicates the IgG heavy chain band. ( F ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) and immunoprecipitates (IP) derived from 293T/17 cells transfected with untagged p27 WT in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-p27 (lower and middle panels) or anti-HA (upper panel) antibodies.
    Mouse Monoclonal Anti Ha Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti ha antibody/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti ha antibody - by Bioz Stars, 2021-07
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    85
    Millipore sophion qpatchtm hnav 1 7 alpha hek 293 cell line
    p27 is SUMOylated on K134 in vitro and in vivo . ( A ) Western blot analysis of FLAG-p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from <t>293T/17</t> cells transfected with FLAG-p27 WT or Flag-p27 1–170 , in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. Asterisk indicates the IgG heavy chain band. ( B ) Western blot analysis of p27 WT recombinant protein in an in vitro SUMOylation assay. His-tagged p27 was incubated with SAE1/SAE2, SUMO, and increasing doses of Ubc9. p27 SUMOylation, expressed as a SUMOylated p27/p27 ratio, was calculated by densitometric analysis of the blots. ( C ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence or absence of HA-SUMO1 and untagged Ubc9. ( D ) Western blot analysis of p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. ( E ) Western blot analysis of p27 and SUMO1 expression in SUMO1 (IP SUMO1) or control (IP IgG) immunoprecipitates from HeLa cells transfected with FLAG-p27 WT or FLAG-p27 K134R in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate SUMO-modified p27 proteins detected using anti-p27 antibody (upper panel) or the immunoprecipitated SUMO1 (lower panel). Asterisk indicates the IgG heavy chain band. ( F ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) and immunoprecipitates (IP) derived from 293T/17 cells transfected with untagged p27 WT in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-p27 (lower and middle panels) or anti-HA (upper panel) antibodies.
    Sophion Qpatchtm Hnav 1 7 Alpha Hek 293 Cell Line, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sophion qpatchtm hnav 1 7 alpha hek 293 cell line/product/Millipore
    Average 85 stars, based on 1 article reviews
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    sophion qpatchtm hnav 1 7 alpha hek 293 cell line - by Bioz Stars, 2021-07
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    Image Search Results


    Meg3 facilitates the non-stoichiometric interaction of the PRC2 complex and Jarid2 ( A ) Ezh2, Jarid2, and Suz12 immunoprecipitation specifically retrieves Meg3 RNA isoforms ( v1 and v5 ). Rnu1 RNA and Malat1 lncRNA are negative controls. 10% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments). Immunoblotting reflects the recovery of Ezh2, Jarid2 and Suz12 proteins using the corresponding antibodies. ( B ) In vitro -transcribed, biotinylated Meg3 RNA isoforms retrieved Ezh2. ( C ) Ezh2 interacts with Jarid2 in ESC~MNs, but knockdown of Meg3 impairs this interaction. The abundance of Jarid2 is shown on the right (N.S.: not significant; error bars represent SD, n = 3 independent experiments; ** p-value

    Journal: eLife

    Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

    doi: 10.7554/eLife.38080

    Figure Lengend Snippet: Meg3 facilitates the non-stoichiometric interaction of the PRC2 complex and Jarid2 ( A ) Ezh2, Jarid2, and Suz12 immunoprecipitation specifically retrieves Meg3 RNA isoforms ( v1 and v5 ). Rnu1 RNA and Malat1 lncRNA are negative controls. 10% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments). Immunoblotting reflects the recovery of Ezh2, Jarid2 and Suz12 proteins using the corresponding antibodies. ( B ) In vitro -transcribed, biotinylated Meg3 RNA isoforms retrieved Ezh2. ( C ) Ezh2 interacts with Jarid2 in ESC~MNs, but knockdown of Meg3 impairs this interaction. The abundance of Jarid2 is shown on the right (N.S.: not significant; error bars represent SD, n = 3 independent experiments; ** p-value

    Article Snippet: Standard Western blot procedures were applied using anti-Jarid2 (Novus Biologicals, NB100-2214) or anti-Ezh2 (Millipore, 17–662) antibodies.

    Techniques: Immunoprecipitation, In Vitro

    p27 is SUMOylated on K134 in vitro and in vivo . ( A ) Western blot analysis of FLAG-p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or Flag-p27 1–170 , in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. Asterisk indicates the IgG heavy chain band. ( B ) Western blot analysis of p27 WT recombinant protein in an in vitro SUMOylation assay. His-tagged p27 was incubated with SAE1/SAE2, SUMO, and increasing doses of Ubc9. p27 SUMOylation, expressed as a SUMOylated p27/p27 ratio, was calculated by densitometric analysis of the blots. ( C ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence or absence of HA-SUMO1 and untagged Ubc9. ( D ) Western blot analysis of p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. ( E ) Western blot analysis of p27 and SUMO1 expression in SUMO1 (IP SUMO1) or control (IP IgG) immunoprecipitates from HeLa cells transfected with FLAG-p27 WT or FLAG-p27 K134R in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate SUMO-modified p27 proteins detected using anti-p27 antibody (upper panel) or the immunoprecipitated SUMO1 (lower panel). Asterisk indicates the IgG heavy chain band. ( F ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) and immunoprecipitates (IP) derived from 293T/17 cells transfected with untagged p27 WT in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-p27 (lower and middle panels) or anti-HA (upper panel) antibodies.

    Journal: Journal of Molecular Cell Biology

    Article Title: SUMOylation regulates p27Kip1 stability and localization in response to TGFβ

    doi: 10.1093/jmcb/mjv056

    Figure Lengend Snippet: p27 is SUMOylated on K134 in vitro and in vivo . ( A ) Western blot analysis of FLAG-p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or Flag-p27 1–170 , in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. Asterisk indicates the IgG heavy chain band. ( B ) Western blot analysis of p27 WT recombinant protein in an in vitro SUMOylation assay. His-tagged p27 was incubated with SAE1/SAE2, SUMO, and increasing doses of Ubc9. p27 SUMOylation, expressed as a SUMOylated p27/p27 ratio, was calculated by densitometric analysis of the blots. ( C ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence or absence of HA-SUMO1 and untagged Ubc9. ( D ) Western blot analysis of p27 and HA-SUMO1 expression in immunoprecipitates (IP) and lysates (Input) derived from 293T/17 cells transfected with FLAG-p27 WT or indicated FLAG-p27 KR mutants in the presence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-HA (upper panel) or anti-FLAG (middle and lower panels) antibodies. ( E ) Western blot analysis of p27 and SUMO1 expression in SUMO1 (IP SUMO1) or control (IP IgG) immunoprecipitates from HeLa cells transfected with FLAG-p27 WT or FLAG-p27 K134R in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate SUMO-modified p27 proteins detected using anti-p27 antibody (upper panel) or the immunoprecipitated SUMO1 (lower panel). Asterisk indicates the IgG heavy chain band. ( F ) Western blot analysis of p27 and HA-SUMO1 expression in lysates (Input) and immunoprecipitates (IP) derived from 293T/17 cells transfected with untagged p27 WT in the presence or absence of HA-SUMO1 and untagged Ubc9. Arrowheads indicate the unmodified and SUMO-modified p27 proteins detected using anti-p27 (lower and middle panels) or anti-HA (upper panel) antibodies.

    Article Snippet: HeLa (human cervical adenocarcinoma cell line, ATCC CCL-2), MCF7 (human breast adenocarcinoma cell line, ATCC HTB-22), and 293T/17 (human embryonic kidney cell line) cells were grown in DMEM supplemented with 10% heat-inactivated FBS (Sigma).

    Techniques: In Vitro, In Vivo, Western Blot, Expressing, Derivative Assay, Transfection, Modification, Recombinant, Incubation, Immunoprecipitation

    TGFβ-induced SUMOylation decreases p27 affinity for CDK2. ( A ) In vivo ubiquitination assay in 293T/17 cells transfected with the indicated FLAG-tagged vectors and with HA-tagged ubiquitin. Cells were then treated or not with TGFβ (20 ng/ml). Lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-HA and anti-p27 antibodies. Cell lysates were probed with anti-HA antibody for the equivalent level of ubiquitin transfection in different samples (lower panel). ( B ) In vivo ubiquitination assay in 293T/17 cells transfected with the indicated FLAG-tagged vectors along with HA-tagged ubiquitin. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) antibody and probed with anti-HA and anti-p27 antibodies. Cell lysates were probed with anti-HA antibody for the equivalent level of ubiquitin transfection in different samples (lower panel). ( C ) Co-immunoprecipitation analysis (IP: FLAG) of protein lysates derived from MCF7 cells transfected with FLAG-p27 WT , FLAG-p27 K134R , or FLAG empty vector in the presence of HA-SUMO1 and untagged Ubc9, and treated or not with TGFβ (20 ng/ml) for 3 h. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) resin and probed with anti-FLAG and anti-CDK2 antibodies. Lysates (Input) were analyzed for the expression of CDK2, FLAG-p27, and Smad2 phosphorylated on Ser 465/467 (pSmad2) to confirm the activation of the TGFβ pathway. Vinculin was used as loading control. The FLAG-p27/CDK2 interaction is reported as the ratio of FLAG-p27 WT bound to CDK2 based on densitometric analysis of the blots. ( D ) Co-immunoprecipitation analysis (IP: FLAG) of protein lysates derived from HeLa cells transfected with FLAG-p27 WT , FLAG-p27 K134R , or FLAG empty vector in the presence of HA-SUMO1 and untagged Ubc9, and treated or not with TGFβ (20 ng/ml) for 3 h. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) resin and probed with anti-FLAG, anti-Skp2, and anti-CDK2 antibodies.

    Journal: Journal of Molecular Cell Biology

    Article Title: SUMOylation regulates p27Kip1 stability and localization in response to TGFβ

    doi: 10.1093/jmcb/mjv056

    Figure Lengend Snippet: TGFβ-induced SUMOylation decreases p27 affinity for CDK2. ( A ) In vivo ubiquitination assay in 293T/17 cells transfected with the indicated FLAG-tagged vectors and with HA-tagged ubiquitin. Cells were then treated or not with TGFβ (20 ng/ml). Lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-HA and anti-p27 antibodies. Cell lysates were probed with anti-HA antibody for the equivalent level of ubiquitin transfection in different samples (lower panel). ( B ) In vivo ubiquitination assay in 293T/17 cells transfected with the indicated FLAG-tagged vectors along with HA-tagged ubiquitin. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) antibody and probed with anti-HA and anti-p27 antibodies. Cell lysates were probed with anti-HA antibody for the equivalent level of ubiquitin transfection in different samples (lower panel). ( C ) Co-immunoprecipitation analysis (IP: FLAG) of protein lysates derived from MCF7 cells transfected with FLAG-p27 WT , FLAG-p27 K134R , or FLAG empty vector in the presence of HA-SUMO1 and untagged Ubc9, and treated or not with TGFβ (20 ng/ml) for 3 h. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) resin and probed with anti-FLAG and anti-CDK2 antibodies. Lysates (Input) were analyzed for the expression of CDK2, FLAG-p27, and Smad2 phosphorylated on Ser 465/467 (pSmad2) to confirm the activation of the TGFβ pathway. Vinculin was used as loading control. The FLAG-p27/CDK2 interaction is reported as the ratio of FLAG-p27 WT bound to CDK2 based on densitometric analysis of the blots. ( D ) Co-immunoprecipitation analysis (IP: FLAG) of protein lysates derived from HeLa cells transfected with FLAG-p27 WT , FLAG-p27 K134R , or FLAG empty vector in the presence of HA-SUMO1 and untagged Ubc9, and treated or not with TGFβ (20 ng/ml) for 3 h. Lysates were immunoprecipitated with anti-FLAG or control (IgG = Anti-V5) resin and probed with anti-FLAG, anti-Skp2, and anti-CDK2 antibodies.

    Article Snippet: HeLa (human cervical adenocarcinoma cell line, ATCC CCL-2), MCF7 (human breast adenocarcinoma cell line, ATCC HTB-22), and 293T/17 (human embryonic kidney cell line) cells were grown in DMEM supplemented with 10% heat-inactivated FBS (Sigma).

    Techniques: In Vivo, Ubiquitin Assay, Transfection, Immunoprecipitation, Derivative Assay, Plasmid Preparation, Expressing, Activation Assay