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    Thermo Fisher lipofectamine 2000
    Mdm2 interacts with Itch and regulates the stability of p73 in Mdm2-null MEFs A. Mdm2 interacts with Itch in vivo . Wild type MEFs and Mdm2-null MEFs were transfected with a plasmid expressing Mdm2. The cells were treated with the proteasome inhibitor MG132 (20 μM) for 6 hours prior to harvest. The cell extracts were immunoprecipitated with anti-Mdm2 or anti-Itch antibodies and analyzed by western blot with indicated antibodies. B. Wild type MEFs and Mdm2 null MEFs cells were treated with cycloheximide (CHX) (20 μg/ml) as indicated. Endogenous p73 levels were determined by immunoblotting with a p73-specific antibody (H-79). An antibody against β-actin was used as a loading control. C. Expression levels were determined by densitometry of the immunoblots in (B) Errors bars indicate the SEM ( n = 3). D. Mdm2 null MEFs were transfected with plasmids expressing HA-Ub and p73α or Myc-Itch. Cell extracts were immunoprecipitated with a p73-specific antibody (H-79) and analyzed by western blotting with HA-specific, Myc-specific (for Itch), and p73-specific antibodies. E. <t>HEK293</t> cells were transfected with <t>ITCH-siRNA</t> or control-siRNA. Two days later, the cells were further transfected with plasmids expressing HA-Ub and an MDM2 expression plasmid or empty vector as indicated. Cell extracts were immunoprecipitated with a p73-specific antibody (ER-15) and analyzed by western blotting with HA-specific, p73-specific, Lys63-specific and Lys48-specific antibodies (lower image) as indicated. Direct western blots for p73, ITCH, MDM2 and actin are shown in the lower panel. F. Mdm2 null MEFs were transfected with plasmids expressing His-Ub, p73α or p73β, and Mdm2 or Itch. His-ubiquitinated proteins were isolated from denatured whole extract extracts, and analyzed by western blot with a p73 specific antibody (H-79). Direct western blots for Mdm2 and Itch are shown in the lower panels.
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 519573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine 2000/product/Thermo Fisher
    Average 99 stars, based on 519573 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher lipofectamine 2000 cd transfection reagent
    Mdm2 interacts with Itch and regulates the stability of p73 in Mdm2-null MEFs A. Mdm2 interacts with Itch in vivo . Wild type MEFs and Mdm2-null MEFs were transfected with a plasmid expressing Mdm2. The cells were treated with the proteasome inhibitor MG132 (20 μM) for 6 hours prior to harvest. The cell extracts were immunoprecipitated with anti-Mdm2 or anti-Itch antibodies and analyzed by western blot with indicated antibodies. B. Wild type MEFs and Mdm2 null MEFs cells were treated with cycloheximide (CHX) (20 μg/ml) as indicated. Endogenous p73 levels were determined by immunoblotting with a p73-specific antibody (H-79). An antibody against β-actin was used as a loading control. C. Expression levels were determined by densitometry of the immunoblots in (B) Errors bars indicate the SEM ( n = 3). D. Mdm2 null MEFs were transfected with plasmids expressing HA-Ub and p73α or Myc-Itch. Cell extracts were immunoprecipitated with a p73-specific antibody (H-79) and analyzed by western blotting with HA-specific, Myc-specific (for Itch), and p73-specific antibodies. E. <t>HEK293</t> cells were transfected with <t>ITCH-siRNA</t> or control-siRNA. Two days later, the cells were further transfected with plasmids expressing HA-Ub and an MDM2 expression plasmid or empty vector as indicated. Cell extracts were immunoprecipitated with a p73-specific antibody (ER-15) and analyzed by western blotting with HA-specific, p73-specific, Lys63-specific and Lys48-specific antibodies (lower image) as indicated. Direct western blots for p73, ITCH, MDM2 and actin are shown in the lower panel. F. Mdm2 null MEFs were transfected with plasmids expressing His-Ub, p73α or p73β, and Mdm2 or Itch. His-ubiquitinated proteins were isolated from denatured whole extract extracts, and analyzed by western blot with a p73 specific antibody (H-79). Direct western blots for Mdm2 and Itch are shown in the lower panels.
    Lipofectamine 2000 Cd Transfection Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine 2000 cd transfection reagent/product/Thermo Fisher
    Average 99 stars, based on 291 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 cd transfection reagent - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

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    Mdm2 interacts with Itch and regulates the stability of p73 in Mdm2-null MEFs A. Mdm2 interacts with Itch in vivo . Wild type MEFs and Mdm2-null MEFs were transfected with a plasmid expressing Mdm2. The cells were treated with the proteasome inhibitor MG132 (20 μM) for 6 hours prior to harvest. The cell extracts were immunoprecipitated with anti-Mdm2 or anti-Itch antibodies and analyzed by western blot with indicated antibodies. B. Wild type MEFs and Mdm2 null MEFs cells were treated with cycloheximide (CHX) (20 μg/ml) as indicated. Endogenous p73 levels were determined by immunoblotting with a p73-specific antibody (H-79). An antibody against β-actin was used as a loading control. C. Expression levels were determined by densitometry of the immunoblots in (B) Errors bars indicate the SEM ( n = 3). D. Mdm2 null MEFs were transfected with plasmids expressing HA-Ub and p73α or Myc-Itch. Cell extracts were immunoprecipitated with a p73-specific antibody (H-79) and analyzed by western blotting with HA-specific, Myc-specific (for Itch), and p73-specific antibodies. E. HEK293 cells were transfected with ITCH-siRNA or control-siRNA. Two days later, the cells were further transfected with plasmids expressing HA-Ub and an MDM2 expression plasmid or empty vector as indicated. Cell extracts were immunoprecipitated with a p73-specific antibody (ER-15) and analyzed by western blotting with HA-specific, p73-specific, Lys63-specific and Lys48-specific antibodies (lower image) as indicated. Direct western blots for p73, ITCH, MDM2 and actin are shown in the lower panel. F. Mdm2 null MEFs were transfected with plasmids expressing His-Ub, p73α or p73β, and Mdm2 or Itch. His-ubiquitinated proteins were isolated from denatured whole extract extracts, and analyzed by western blot with a p73 specific antibody (H-79). Direct western blots for Mdm2 and Itch are shown in the lower panels.

    Journal: Oncotarget

    Article Title: MDM2 mediates p73 ubiquitination: a new molecular mechanism for suppression of p73 function

    doi:

    Figure Lengend Snippet: Mdm2 interacts with Itch and regulates the stability of p73 in Mdm2-null MEFs A. Mdm2 interacts with Itch in vivo . Wild type MEFs and Mdm2-null MEFs were transfected with a plasmid expressing Mdm2. The cells were treated with the proteasome inhibitor MG132 (20 μM) for 6 hours prior to harvest. The cell extracts were immunoprecipitated with anti-Mdm2 or anti-Itch antibodies and analyzed by western blot with indicated antibodies. B. Wild type MEFs and Mdm2 null MEFs cells were treated with cycloheximide (CHX) (20 μg/ml) as indicated. Endogenous p73 levels were determined by immunoblotting with a p73-specific antibody (H-79). An antibody against β-actin was used as a loading control. C. Expression levels were determined by densitometry of the immunoblots in (B) Errors bars indicate the SEM ( n = 3). D. Mdm2 null MEFs were transfected with plasmids expressing HA-Ub and p73α or Myc-Itch. Cell extracts were immunoprecipitated with a p73-specific antibody (H-79) and analyzed by western blotting with HA-specific, Myc-specific (for Itch), and p73-specific antibodies. E. HEK293 cells were transfected with ITCH-siRNA or control-siRNA. Two days later, the cells were further transfected with plasmids expressing HA-Ub and an MDM2 expression plasmid or empty vector as indicated. Cell extracts were immunoprecipitated with a p73-specific antibody (ER-15) and analyzed by western blotting with HA-specific, p73-specific, Lys63-specific and Lys48-specific antibodies (lower image) as indicated. Direct western blots for p73, ITCH, MDM2 and actin are shown in the lower panel. F. Mdm2 null MEFs were transfected with plasmids expressing His-Ub, p73α or p73β, and Mdm2 or Itch. His-ubiquitinated proteins were isolated from denatured whole extract extracts, and analyzed by western blot with a p73 specific antibody (H-79). Direct western blots for Mdm2 and Itch are shown in the lower panels.

    Article Snippet: siRNA experiments For siRNA experiments, HEK293 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen).

    Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Isolation

    MDM2 is required for p73 ubiquitination in vivo A. HEK293 cells were transfected with MDM2-siRNA or control-siRNA constructs. Forty hours later, the cells were further transfected with a plasmid expressing HA-Ub. Lysates were immunoprecipitated with a p73specific antibody (ER-15) and analyzed by western blotting with an HA-specific antibody to detect ubiquitinated p73. The western blots for p73, MDM2, and actin are shown in the lower panel. B. The same procedure as (A) was used, except that after the second transfection with the HA-Ub expression plasmid, the cells were treated with the proteasome inhibitor MG132 (20 μM) 6 hr prior to harvest. C. Similar to (A) except that the cell extracts were obtained from the wild-type mouse embryonic fibroblasts (MEFs) and Mdm2 null MEFs. In addition, Mdm2 expression plasmid was reintroduced into Mdm2 null MEFs (lane 3). D. HEK293 cells were immunoprecipitated with anti-p73 (ER-15) or anti-MDM2 (SMP14) as indicated, and immunoblotted with antip73 (upper image, ER-15) and anti-MDM2 antibodies (lower image).

    Journal: Oncotarget

    Article Title: MDM2 mediates p73 ubiquitination: a new molecular mechanism for suppression of p73 function

    doi:

    Figure Lengend Snippet: MDM2 is required for p73 ubiquitination in vivo A. HEK293 cells were transfected with MDM2-siRNA or control-siRNA constructs. Forty hours later, the cells were further transfected with a plasmid expressing HA-Ub. Lysates were immunoprecipitated with a p73specific antibody (ER-15) and analyzed by western blotting with an HA-specific antibody to detect ubiquitinated p73. The western blots for p73, MDM2, and actin are shown in the lower panel. B. The same procedure as (A) was used, except that after the second transfection with the HA-Ub expression plasmid, the cells were treated with the proteasome inhibitor MG132 (20 μM) 6 hr prior to harvest. C. Similar to (A) except that the cell extracts were obtained from the wild-type mouse embryonic fibroblasts (MEFs) and Mdm2 null MEFs. In addition, Mdm2 expression plasmid was reintroduced into Mdm2 null MEFs (lane 3). D. HEK293 cells were immunoprecipitated with anti-p73 (ER-15) or anti-MDM2 (SMP14) as indicated, and immunoblotted with antip73 (upper image, ER-15) and anti-MDM2 antibodies (lower image).

    Article Snippet: siRNA experiments For siRNA experiments, HEK293 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen).

    Techniques: In Vivo, Transfection, Construct, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot

    Overexpression of Mdm2 promotes p73 degradation in Mdm2-null MEFs A. Mdm2 null MEFs were transfected with plasmids expressing p73α or an empty vector with Myc-tagged Itch, along with a GFP expressing plasmid (pEGFP) and analyzed by western blotting with a p73-specific antibody (H-79), a Myc-specific antibody for Myc-Itch, a GFP-specific antibody (B-2), and actin as a loading control. B. The same procedure as (A) was used, except that Mdm2 null MEFs were cotransfected with plasmids expressing GFP, p73α along with Itch and Mdm2. C. and D. Mdm2-null MEFs were transfected with an Itch-specific siRNA or a control siRNA. Thirty hours later, the cells were transfected with plasmids expressing GFP, Mdm2 and analyzed by western blot using anti-p73, anti-Itch, anti-p53 (Pab421), anti-GFP (B-2), and anti-Mdm2 (MD-219) antibodies as indicated. E. Mdm2-null MEFs were transfected with plasmids expressing GFP and increased amounts of Mdm2 and analyzed by western blotting with p73-specific (H-79), GFP-specific (B-2) and Mdm2-specific (MD-219) antibodies.

    Journal: Oncotarget

    Article Title: MDM2 mediates p73 ubiquitination: a new molecular mechanism for suppression of p73 function

    doi:

    Figure Lengend Snippet: Overexpression of Mdm2 promotes p73 degradation in Mdm2-null MEFs A. Mdm2 null MEFs were transfected with plasmids expressing p73α or an empty vector with Myc-tagged Itch, along with a GFP expressing plasmid (pEGFP) and analyzed by western blotting with a p73-specific antibody (H-79), a Myc-specific antibody for Myc-Itch, a GFP-specific antibody (B-2), and actin as a loading control. B. The same procedure as (A) was used, except that Mdm2 null MEFs were cotransfected with plasmids expressing GFP, p73α along with Itch and Mdm2. C. and D. Mdm2-null MEFs were transfected with an Itch-specific siRNA or a control siRNA. Thirty hours later, the cells were transfected with plasmids expressing GFP, Mdm2 and analyzed by western blot using anti-p73, anti-Itch, anti-p53 (Pab421), anti-GFP (B-2), and anti-Mdm2 (MD-219) antibodies as indicated. E. Mdm2-null MEFs were transfected with plasmids expressing GFP and increased amounts of Mdm2 and analyzed by western blotting with p73-specific (H-79), GFP-specific (B-2) and Mdm2-specific (MD-219) antibodies.

    Article Snippet: siRNA experiments For siRNA experiments, HEK293 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen).

    Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot

    Sister chromatid separation in cells arrested with nondegradable cyclinB1. ( A ) Mitotic Onk2 mB1dm cells were harvested 24 h after induction and analysed by chromosome spreading. One metaphase cell and one cell arrested in anaphase are shown. Size bar indicates 5 μm. The bar graph shows a summary of mB1dm and mB1NΔ157 cells induced for 24 h and analysed by chromosome spreading. White bars=metaphase cells; black bars=anaphase cells ( n > 100 in both cell lines). ( B ) Chromosome spread analysis of transiently transfected mitotic HeLa cells. A cell arrested at metaphase by nocodazole treatment for 16 h is shown as a control. A representative chromosome spread of a HeLa cells transfected with mB1dm is shown (anaphase). The bar graph summarises experiments using HeLa cells that were transiently transfected with the indicated plasmids ( n > 100 for each transfection experiment). ( C ) Left panel: Total cell extracts from adherent interphase (I) or mitotic (M) Onk2-mB1NΔ157 cells that were either left untreated (lanes 1 and 4), nocodazole treated (0.5 μM, 16 h, lane 2), 4 h released from a 16 h nocodazole block (lane 3), or dox-treated (lanes 4 and 5) were analysed for Cdc27, cyclin B1 and Cdc2. G1=G1 phase. Right panel: total cell extracts from nonmitotic (lane 1) and mitotic (lane 2) nocodazole-treated as well as nonmitotic (lane 3) and mitotic dox-induced (lane 4) mB1NΔ157 cells were analysed for securin and GAPDH expression by immunoblotting.

    Journal: The EMBO Journal

    Article Title: Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells

    doi: 10.1038/sj.emboj.7601163

    Figure Lengend Snippet: Sister chromatid separation in cells arrested with nondegradable cyclinB1. ( A ) Mitotic Onk2 mB1dm cells were harvested 24 h after induction and analysed by chromosome spreading. One metaphase cell and one cell arrested in anaphase are shown. Size bar indicates 5 μm. The bar graph shows a summary of mB1dm and mB1NΔ157 cells induced for 24 h and analysed by chromosome spreading. White bars=metaphase cells; black bars=anaphase cells ( n > 100 in both cell lines). ( B ) Chromosome spread analysis of transiently transfected mitotic HeLa cells. A cell arrested at metaphase by nocodazole treatment for 16 h is shown as a control. A representative chromosome spread of a HeLa cells transfected with mB1dm is shown (anaphase). The bar graph summarises experiments using HeLa cells that were transiently transfected with the indicated plasmids ( n > 100 for each transfection experiment). ( C ) Left panel: Total cell extracts from adherent interphase (I) or mitotic (M) Onk2-mB1NΔ157 cells that were either left untreated (lanes 1 and 4), nocodazole treated (0.5 μM, 16 h, lane 2), 4 h released from a 16 h nocodazole block (lane 3), or dox-treated (lanes 4 and 5) were analysed for Cdc27, cyclin B1 and Cdc2. G1=G1 phase. Right panel: total cell extracts from nonmitotic (lane 1) and mitotic (lane 2) nocodazole-treated as well as nonmitotic (lane 3) and mitotic dox-induced (lane 4) mB1NΔ157 cells were analysed for securin and GAPDH expression by immunoblotting.

    Article Snippet: For transient transfection experiments, Lipofectamine 2000 (Invitrogen) was used according to the manufacturers' protocols; stable transfection was carried out as described ( ).

    Techniques: Transfection, Blocking Assay, Expressing