tris glycine sds page  (Millipore)


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  • 96
    Name:
    TRIS glycine SDS buffer solution
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    Catalog Number:
    93311
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    Structured Review

    Millipore tris glycine sds page
    Iron chelators inhibit phosphorylation of RNAPII ( a ) Chelators do not have an effect on expression of CDK9 . 293 cells were treated for 24 h with 10 μM 311 or 100 μM of ICL670. The cells were lysed were resolved on 10% <t>SDS-PAGE,</t> and immunoblotted anti-CDK9 antibodies. Lane 1 : control untreated cells; lanes 2, 3 and 4 : cells treated with DFO, 311 or ICL670. ( b ) Chelators inhibit association of CDK9 with cyclin T1 . The cell lysates prepared as in ( a ) were subjected to immunoprecipitation with anti-cyclin T1 antibody (lanes 3–6). The immunoprecipitated material was resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : input control untreated cells; lane 2: precipitation of untreated cells with non-specific preimmune IgG, lanes 3–6: immunoprecipitation with anti-cyclin T1 antibodies of lysates from untreated, DFO, 311 and ICL670 treated cells. The precipitated samples were resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK9. ( c ) Chelators inhibit phosphorylation of RNAPII . The cell lysates prepared as in ( a ) were resolved on 7.5% SDS-PAGE, and immunoblotted with the RNAPII CTD phospho-Serine 2 specific antibodies. Lane 1 : control untreated cells; lanes 2,3 and 4 : cells treated with DFO, 311 or ICL670.

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    1) Product Images from "Iron Chelators ICL670 and 311 Inhibit HIV-1 Transcription"

    Article Title: Iron Chelators ICL670 and 311 Inhibit HIV-1 Transcription

    Journal: Virology

    doi: 10.1016/j.virol.2007.06.011

    Iron chelators inhibit phosphorylation of RNAPII ( a ) Chelators do not have an effect on expression of CDK9 . 293 cells were treated for 24 h with 10 μM 311 or 100 μM of ICL670. The cells were lysed were resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : control untreated cells; lanes 2, 3 and 4 : cells treated with DFO, 311 or ICL670. ( b ) Chelators inhibit association of CDK9 with cyclin T1 . The cell lysates prepared as in ( a ) were subjected to immunoprecipitation with anti-cyclin T1 antibody (lanes 3–6). The immunoprecipitated material was resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : input control untreated cells; lane 2: precipitation of untreated cells with non-specific preimmune IgG, lanes 3–6: immunoprecipitation with anti-cyclin T1 antibodies of lysates from untreated, DFO, 311 and ICL670 treated cells. The precipitated samples were resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK9. ( c ) Chelators inhibit phosphorylation of RNAPII . The cell lysates prepared as in ( a ) were resolved on 7.5% SDS-PAGE, and immunoblotted with the RNAPII CTD phospho-Serine 2 specific antibodies. Lane 1 : control untreated cells; lanes 2,3 and 4 : cells treated with DFO, 311 or ICL670.
    Figure Legend Snippet: Iron chelators inhibit phosphorylation of RNAPII ( a ) Chelators do not have an effect on expression of CDK9 . 293 cells were treated for 24 h with 10 μM 311 or 100 μM of ICL670. The cells were lysed were resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : control untreated cells; lanes 2, 3 and 4 : cells treated with DFO, 311 or ICL670. ( b ) Chelators inhibit association of CDK9 with cyclin T1 . The cell lysates prepared as in ( a ) were subjected to immunoprecipitation with anti-cyclin T1 antibody (lanes 3–6). The immunoprecipitated material was resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : input control untreated cells; lane 2: precipitation of untreated cells with non-specific preimmune IgG, lanes 3–6: immunoprecipitation with anti-cyclin T1 antibodies of lysates from untreated, DFO, 311 and ICL670 treated cells. The precipitated samples were resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK9. ( c ) Chelators inhibit phosphorylation of RNAPII . The cell lysates prepared as in ( a ) were resolved on 7.5% SDS-PAGE, and immunoblotted with the RNAPII CTD phospho-Serine 2 specific antibodies. Lane 1 : control untreated cells; lanes 2,3 and 4 : cells treated with DFO, 311 or ICL670.

    Techniques Used: Expressing, SDS Page, Immunoprecipitation

    Iron chelators reduce HIV-1 Tat phosphorylated in cultured cells HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 2–5). Lane 1, control uninfected cells. HeLa cells were transfected with siRNAs targeting CDK2 (lane 3) or treated with 10 μM 311 or 100 μM ICL670. At 48 hours post infection cells were labeled with ( 32 P)-orthophosphate for 2 hours with the addition of 1 μM okadaic acid. Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Flag monoclonal antibodies, resolved on 15% Tris-Tricine SDS-PAGE and detected by Phosphor Imager. Phosphor Imager quantification is shown in the lower panel.
    Figure Legend Snippet: Iron chelators reduce HIV-1 Tat phosphorylated in cultured cells HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 2–5). Lane 1, control uninfected cells. HeLa cells were transfected with siRNAs targeting CDK2 (lane 3) or treated with 10 μM 311 or 100 μM ICL670. At 48 hours post infection cells were labeled with ( 32 P)-orthophosphate for 2 hours with the addition of 1 μM okadaic acid. Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Flag monoclonal antibodies, resolved on 15% Tris-Tricine SDS-PAGE and detected by Phosphor Imager. Phosphor Imager quantification is shown in the lower panel.

    Techniques Used: Cell Culture, Infection, Recombinant, Expressing, Transfection, Labeling, Immunoprecipitation, SDS Page

    Iron chelators reduce cellular activity of CDK2 (a) CDK2 expression determined by Western blotting . 293 cells were grown in 100 mm plates and treated for the indicated time with 100 μM DFO, 10 μM 311 or 100 μM ICL670. The cells were lysed and CDK2 was immunoprecipitated as described in Methods. Lane 1, input control. Lane, preimmune IgG was used for the immunoprecipitation. The precipitated CDK2 was resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK2. ( b ) CDK2 was precipitated as in (a) and the immunoprecipitated material was subsequently incubated with histone H1 in the presence of γ-( 32 P) ATP. Kinase reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager. Lane 1 : non-specific preimmune IgG. Lane 10, histone H1 phosphorylation with recombinant CDK2/Cyclin E.
    Figure Legend Snippet: Iron chelators reduce cellular activity of CDK2 (a) CDK2 expression determined by Western blotting . 293 cells were grown in 100 mm plates and treated for the indicated time with 100 μM DFO, 10 μM 311 or 100 μM ICL670. The cells were lysed and CDK2 was immunoprecipitated as described in Methods. Lane 1, input control. Lane, preimmune IgG was used for the immunoprecipitation. The precipitated CDK2 was resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK2. ( b ) CDK2 was precipitated as in (a) and the immunoprecipitated material was subsequently incubated with histone H1 in the presence of γ-( 32 P) ATP. Kinase reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager. Lane 1 : non-specific preimmune IgG. Lane 10, histone H1 phosphorylation with recombinant CDK2/Cyclin E.

    Techniques Used: Activity Assay, Expressing, Western Blot, Immunoprecipitation, SDS Page, Incubation, Recombinant

    2) Product Images from "Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response"

    Article Title: Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw959

    Initial purification of the E3 ubiquitin ligases for NEIL1. ( A ) Scheme for the purification of the E3 ubiquitin ligases for NEIL1 from HeLa cell extracts. ( B ) In vitro ubiquitylation of His-tagged NEIL1 by fractions obtained from the first ion exchange (Mono Q) chromatography. ( C and D ) In vitro ubiquitylation of His-tagged NEIL1 by fractions obtained from size exclusion (Superdex 200) chromatography containing ( C ) NEIL1-E3 1 or ( D ) NEIL1-E3 2 . Shown above the figures are the positions of elution of known molecular weight standards. In all experiments, in vitro ubiquitylation of His-tagged NEIL1 (4.6 pmol) was performed in the presence of E1 activating enzyme (0.7 pmol), ubiquitin (0.6 nmol; Ub) and all E2 conjugating enzymes (2.5 pmol) and analysed by 10% SDS-PAGE and immunoblotting using His-tag antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of unmodified and ubiquitylated NEIL1 (NEIL1 ub ) are shown.
    Figure Legend Snippet: Initial purification of the E3 ubiquitin ligases for NEIL1. ( A ) Scheme for the purification of the E3 ubiquitin ligases for NEIL1 from HeLa cell extracts. ( B ) In vitro ubiquitylation of His-tagged NEIL1 by fractions obtained from the first ion exchange (Mono Q) chromatography. ( C and D ) In vitro ubiquitylation of His-tagged NEIL1 by fractions obtained from size exclusion (Superdex 200) chromatography containing ( C ) NEIL1-E3 1 or ( D ) NEIL1-E3 2 . Shown above the figures are the positions of elution of known molecular weight standards. In all experiments, in vitro ubiquitylation of His-tagged NEIL1 (4.6 pmol) was performed in the presence of E1 activating enzyme (0.7 pmol), ubiquitin (0.6 nmol; Ub) and all E2 conjugating enzymes (2.5 pmol) and analysed by 10% SDS-PAGE and immunoblotting using His-tag antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of unmodified and ubiquitylated NEIL1 (NEIL1 ub ) are shown.

    Techniques Used: Purification, In Vitro, Chromatography, Molecular Weight, SDS Page

    Identification of sites of ubiquitylation within the C-terminus of NEIL1 by recombinant truncated Mule and TRIM26. ( A ) Schematic showing the protein domains within the full length NEIL1 protein. In vitro ubiquitylation of His-tagged NEIL1 mutants in the absence and presence of either ( B ) GST-tagged truncated Mule or ( C ) His-tagged TRIM26. In all experiments, in vitro ubiquitylation of His-tagged NEIL1 (4.6 pmol) was performed in the presence of E1 activating enzyme (0.7 pmol) and ubiquitin (0.6 nmol; Ub). Additionally in (B) the H5c E2 conjugating enzyme (2.5 pmol) was used in the presence of GST-tagged truncated Mule protein (4.7 pmol) and in (C) the H5a E2 conjugating enzyme (2.5 pmol) was used in the presence of His-tagged TRIM26 protein (12.5 pmol). All reactions were analysed by 10% SDS-PAGE and immunoblotting using His-tag antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of unmodified and ubiquitylated NEIL1 (NEIL1 ub ) are shown. For the NEIL1 mutants: 3KR = K333/356/357R; 6KR = K333/356/357/361/374/376R; 2KR = K319/333R; 4KR = K319/333/356/357R; 7KR = K319/333/356/357/361/374/376R.
    Figure Legend Snippet: Identification of sites of ubiquitylation within the C-terminus of NEIL1 by recombinant truncated Mule and TRIM26. ( A ) Schematic showing the protein domains within the full length NEIL1 protein. In vitro ubiquitylation of His-tagged NEIL1 mutants in the absence and presence of either ( B ) GST-tagged truncated Mule or ( C ) His-tagged TRIM26. In all experiments, in vitro ubiquitylation of His-tagged NEIL1 (4.6 pmol) was performed in the presence of E1 activating enzyme (0.7 pmol) and ubiquitin (0.6 nmol; Ub). Additionally in (B) the H5c E2 conjugating enzyme (2.5 pmol) was used in the presence of GST-tagged truncated Mule protein (4.7 pmol) and in (C) the H5a E2 conjugating enzyme (2.5 pmol) was used in the presence of His-tagged TRIM26 protein (12.5 pmol). All reactions were analysed by 10% SDS-PAGE and immunoblotting using His-tag antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of unmodified and ubiquitylated NEIL1 (NEIL1 ub ) are shown. For the NEIL1 mutants: 3KR = K333/356/357R; 6KR = K333/356/357/361/374/376R; 2KR = K319/333R; 4KR = K319/333/356/357R; 7KR = K319/333/356/357/361/374/376R.

    Techniques Used: Recombinant, In Vitro, SDS Page, Molecular Weight

    Cellular steady state NEIL1 protein levels are regulated by Mule and TRIM26. ( A–C ) U2OS cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine RNAiMAX transfection reagent (10 μl) in the presence of 200 pmol non-targeting (NT), Mule and/or TRIM26 siRNA for 72 h. (A) Whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Proteins were separated by biochemical fractionation, and the soluble (S) and chromatin bound (CB) fractions analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (C) Levels of NEIL1 protein relative to tubulin in the soluble fraction were quantified from at least three independent experiments. Shown is the mean NEIL1/tubulin ratio with standard errors normalised to the non-targeting (NT) siRNA-treated control which was set to 1.0. ( D ) U2OS cells were grown in 10 cm dishes for 24 h to ∼90% confluency and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 150 ng mammalian expression plasmids for Flag-tagged wild type (WT) or NEIL1 mutants (6KR = K333/356/357/361/374/376R; 7KR = K319/333/356/357/361/374/ 376R) for 24 h. Whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. ( E ) Levels of Flag-tagged NEIL1 proteins relative to tubulin were quantified from at least three independent experiments. Shown is the mean Flag-NEIL1/tubulin ratio with standard errors normalised to the WT-NEIL1 transfected cells which was set to 1.0. * P
    Figure Legend Snippet: Cellular steady state NEIL1 protein levels are regulated by Mule and TRIM26. ( A–C ) U2OS cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine RNAiMAX transfection reagent (10 μl) in the presence of 200 pmol non-targeting (NT), Mule and/or TRIM26 siRNA for 72 h. (A) Whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Proteins were separated by biochemical fractionation, and the soluble (S) and chromatin bound (CB) fractions analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. (C) Levels of NEIL1 protein relative to tubulin in the soluble fraction were quantified from at least three independent experiments. Shown is the mean NEIL1/tubulin ratio with standard errors normalised to the non-targeting (NT) siRNA-treated control which was set to 1.0. ( D ) U2OS cells were grown in 10 cm dishes for 24 h to ∼90% confluency and then treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 150 ng mammalian expression plasmids for Flag-tagged wild type (WT) or NEIL1 mutants (6KR = K333/356/357/361/374/376R; 7KR = K319/333/356/357/361/374/ 376R) for 24 h. Whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. ( E ) Levels of Flag-tagged NEIL1 proteins relative to tubulin were quantified from at least three independent experiments. Shown is the mean Flag-NEIL1/tubulin ratio with standard errors normalised to the WT-NEIL1 transfected cells which was set to 1.0. * P

    Techniques Used: Transfection, SDS Page, Fractionation, Expressing

    Recombinant truncated Mule and TRIM26 ubiquitylate NEIL1 in vitro . ( A–D ) In vitro ubiquitylation of His-tagged NEIL1 in the absence and presence of either ( A and C ) GST-tagged truncated Mule or ( B and D ) His-tagged TRIM26. In all experiments, in vitro ubiquitylation of His-tagged NEIL1 (4.6 pmol) was performed in the presence of E1 activating enzyme (0.7 pmol) and ubiquitin (0.6 nmol; Ub). Additionally in (A) the H5c E2 conjugating enzyme (2.5 pmol) was used in the presence of increasing amounts of truncated Mule protein (1.6, 3.1 and 6.3 pmol, respectively). In (B), the H5a E2 conjugating enzyme (2.5 pmol) was used in the presence of increasing amounts of TRIM26 protein (3.1, 6.3 and 12.5 pmol, respectively). In (C and D) individual E2 enzymes (2.5 pmol) were used with either truncated Mule (4.7 pmol) or TRIM26 (12.5 pmol). All reactions were analysed by 10% SDS-PAGE and immunoblotting using His-tag antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of unmodified and ubiquitylated NEIL1 (NEIL1 ub ) are shown.
    Figure Legend Snippet: Recombinant truncated Mule and TRIM26 ubiquitylate NEIL1 in vitro . ( A–D ) In vitro ubiquitylation of His-tagged NEIL1 in the absence and presence of either ( A and C ) GST-tagged truncated Mule or ( B and D ) His-tagged TRIM26. In all experiments, in vitro ubiquitylation of His-tagged NEIL1 (4.6 pmol) was performed in the presence of E1 activating enzyme (0.7 pmol) and ubiquitin (0.6 nmol; Ub). Additionally in (A) the H5c E2 conjugating enzyme (2.5 pmol) was used in the presence of increasing amounts of truncated Mule protein (1.6, 3.1 and 6.3 pmol, respectively). In (B), the H5a E2 conjugating enzyme (2.5 pmol) was used in the presence of increasing amounts of TRIM26 protein (3.1, 6.3 and 12.5 pmol, respectively). In (C and D) individual E2 enzymes (2.5 pmol) were used with either truncated Mule (4.7 pmol) or TRIM26 (12.5 pmol). All reactions were analysed by 10% SDS-PAGE and immunoblotting using His-tag antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of unmodified and ubiquitylated NEIL1 (NEIL1 ub ) are shown.

    Techniques Used: Recombinant, In Vitro, SDS Page, Molecular Weight

    Identification of Mule and TRIM26 as E3 ubiquitin ligases for NEIL1. ( A and B ) In vitro ubiquitylation of His-tagged NEIL1 by fractions obtained from the final 1 ml Mono Q chromatography containing (A) NEIL1-E3 1 or (B) NEIL1-E3 2 . Below each figure is the alignment of active fractions with either Mule or TRIM26, respectively as detected by Western blotting. ( C and D ) In vitro ubiquitylation of His-tagged NEIL1 by an active fraction purified from HeLa whole cell extracts containing either (C) NEIL1-E3 1 or (D) NEIL1-E3 2 in the presence of individual E2 conjugating enzymes. In all experiments, in vitro ubiquitylation of His-tagged NEIL1 (4.6 pmol) was performed in the presence of E1 activating enzyme (0.7 pmol), ubiquitin (0.6 nmol; Ub) and E2 conjugating enzymes (2.5 pmol) and analysed by 10% SDS-PAGE and immunoblotting using His-tag antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of unmodified and ubiquitylated NEIL1 (NEIL1 ub ) are shown.
    Figure Legend Snippet: Identification of Mule and TRIM26 as E3 ubiquitin ligases for NEIL1. ( A and B ) In vitro ubiquitylation of His-tagged NEIL1 by fractions obtained from the final 1 ml Mono Q chromatography containing (A) NEIL1-E3 1 or (B) NEIL1-E3 2 . Below each figure is the alignment of active fractions with either Mule or TRIM26, respectively as detected by Western blotting. ( C and D ) In vitro ubiquitylation of His-tagged NEIL1 by an active fraction purified from HeLa whole cell extracts containing either (C) NEIL1-E3 1 or (D) NEIL1-E3 2 in the presence of individual E2 conjugating enzymes. In all experiments, in vitro ubiquitylation of His-tagged NEIL1 (4.6 pmol) was performed in the presence of E1 activating enzyme (0.7 pmol), ubiquitin (0.6 nmol; Ub) and E2 conjugating enzymes (2.5 pmol) and analysed by 10% SDS-PAGE and immunoblotting using His-tag antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of unmodified and ubiquitylated NEIL1 (NEIL1 ub ) are shown.

    Techniques Used: In Vitro, Chromatography, Western Blot, Purification, SDS Page, Molecular Weight

    TRIM26 controls cellular sensitivity to IR. ( A–C ) U2OS cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine RNAiMAX transfection reagent (10 μl) in the presence of 200 pmol non-targeting (NT) siRNA, TRIM26 siRNA or Mule siRNA for 72 h. U2OS cells were also treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 500 ng mammalian expression plasmid for NEIL1 (NEIL1 overexpression) for 24 h. (A and C) Clonogenic survival of cells was analysed following treatment with increasing doses of ionising radiation (0–4 Gy). Shown is the surviving fraction with standard error of the mean from at least three independent experiments. ( B ) Whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. ( D ) Cells were irradiated (1.5 Gy) and DNA single strand breaks and alkali labile sites measured at various time points post-irradiation by the alkaline single cell gel electrophoresis (comet) assay. Shown is the % tail DNA with standard deviations from at least three independent experiments.
    Figure Legend Snippet: TRIM26 controls cellular sensitivity to IR. ( A–C ) U2OS cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine RNAiMAX transfection reagent (10 μl) in the presence of 200 pmol non-targeting (NT) siRNA, TRIM26 siRNA or Mule siRNA for 72 h. U2OS cells were also treated with Lipofectamine 2000 transfection reagent (10 μl) in the presence of 500 ng mammalian expression plasmid for NEIL1 (NEIL1 overexpression) for 24 h. (A and C) Clonogenic survival of cells was analysed following treatment with increasing doses of ionising radiation (0–4 Gy). Shown is the surviving fraction with standard error of the mean from at least three independent experiments. ( B ) Whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. ( D ) Cells were irradiated (1.5 Gy) and DNA single strand breaks and alkali labile sites measured at various time points post-irradiation by the alkaline single cell gel electrophoresis (comet) assay. Shown is the % tail DNA with standard deviations from at least three independent experiments.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Over Expression, SDS Page, Irradiation, Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis

    Cellular NEIL protein levels are induced in response to ionising radiation in a Mule-dependent manner. ( A–C ) U2OS cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine RNAiMAX transfection reagent (10 μl) in (A) presence of non-targeting (NT) siRNA, ( B ) 200 pmol TRIM26 siRNA or (C) 200 pmol Mule siRNA for 72 h. Cells were subsequently irradiated (10 Gy) and harvested at the various time points post-irradiation. Whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. C refers to unirradiated control in either the presence of non-targeting (NT), Mule or TRIM26 siRNA. ( D ) Levels of NEIL1 protein relative to tubulin were quantified from at least three independent experiments. Shown is the mean NEIL1/tubulin ratio with standard errors normalised to the unirradiated control which was set to 1.0. * P
    Figure Legend Snippet: Cellular NEIL protein levels are induced in response to ionising radiation in a Mule-dependent manner. ( A–C ) U2OS cells were grown in 10 cm dishes for 24 h to 30–50% confluency and then treated with Lipofectamine RNAiMAX transfection reagent (10 μl) in (A) presence of non-targeting (NT) siRNA, ( B ) 200 pmol TRIM26 siRNA or (C) 200 pmol Mule siRNA for 72 h. Cells were subsequently irradiated (10 Gy) and harvested at the various time points post-irradiation. Whole cell extracts were prepared and analysed by 10% SDS-PAGE and immunoblotting with the indicated antibodies. C refers to unirradiated control in either the presence of non-targeting (NT), Mule or TRIM26 siRNA. ( D ) Levels of NEIL1 protein relative to tubulin were quantified from at least three independent experiments. Shown is the mean NEIL1/tubulin ratio with standard errors normalised to the unirradiated control which was set to 1.0. * P

    Techniques Used: Transfection, Irradiation, SDS Page

    3) Product Images from "Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus"

    Article Title: Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02576

    DsbA is required for cbb 3 -Cox activity and maturation/assembly of its subunits. (A) Detection of active cbb 3 -Cox by BN-PAGE stained with NADI in chromatophore membranes prepared from the wild type (wt), Δ cbb 3 -Cox (Δ cox ), Δ cbb 3 -Cox overproducing cbb 3 -Cox (Δ cox /pCox) and Δ dsbA . The active 230 kDa band corresponding to cbb 3 -Cox complex is present in the wild type and Δ cox /pCox strains. In the absence of DsbA, activity of the cbb 3 -Cox is severely affected. (B) Chromatophore membranes were separated by SDS-PAGE and stained by TMBZ for detection of the c -type cyts. The wild type and Δ cox /pCox showed cyt c 1 of cyt bc 1 complex, the electron carrier c y , and the cyts c p and c o subunits of cbb 3 -Cox, which are absent in Δ cbb 3 -Cox. The levels of all c -type cyts c are decreased in Δ dsbA due to its effect on the Ccm process, and the cyts c p and c o subunits of cbb 3 -Cox were present at ~10 and 8% of the wild type levels, respectively. (C) Immunoblot analysis of chromatophore membranes separated by SDS-PAGE using polyclonal antibodies against the CcoN subunit of cbb 3 -Cox. The amount of CcoN is severely reduced in the Δ dsbA mutant to ~15% of wild type levels, but it increases by roughly three folds when CcoA is overproduced upon induction with L-arabinose.
    Figure Legend Snippet: DsbA is required for cbb 3 -Cox activity and maturation/assembly of its subunits. (A) Detection of active cbb 3 -Cox by BN-PAGE stained with NADI in chromatophore membranes prepared from the wild type (wt), Δ cbb 3 -Cox (Δ cox ), Δ cbb 3 -Cox overproducing cbb 3 -Cox (Δ cox /pCox) and Δ dsbA . The active 230 kDa band corresponding to cbb 3 -Cox complex is present in the wild type and Δ cox /pCox strains. In the absence of DsbA, activity of the cbb 3 -Cox is severely affected. (B) Chromatophore membranes were separated by SDS-PAGE and stained by TMBZ for detection of the c -type cyts. The wild type and Δ cox /pCox showed cyt c 1 of cyt bc 1 complex, the electron carrier c y , and the cyts c p and c o subunits of cbb 3 -Cox, which are absent in Δ cbb 3 -Cox. The levels of all c -type cyts c are decreased in Δ dsbA due to its effect on the Ccm process, and the cyts c p and c o subunits of cbb 3 -Cox were present at ~10 and 8% of the wild type levels, respectively. (C) Immunoblot analysis of chromatophore membranes separated by SDS-PAGE using polyclonal antibodies against the CcoN subunit of cbb 3 -Cox. The amount of CcoN is severely reduced in the Δ dsbA mutant to ~15% of wild type levels, but it increases by roughly three folds when CcoA is overproduced upon induction with L-arabinose.

    Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, SDS Page, Mutagenesis

    4) Product Images from "Type 2 diabetes-associated single nucleotide polymorphism in Sorcs1 gene results in alternative processing of the Sorcs1 protein in INS1 β-cells"

    Article Title: Type 2 diabetes-associated single nucleotide polymorphism in Sorcs1 gene results in alternative processing of the Sorcs1 protein in INS1 β-cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55873-6

    The alternative 90 kDa protein product of mutSorcs1 is unaffected by pro-protein convertases. ( A ) Representative western blot of lysates from wtSorcs1 and mutSorcs1 -expressing INS1 cells treated with DMSO or 10 µM prohormone convertase inhibitor (PCI). ( B ) Immunoprecipitation of Sorcs1 from wtSorcs1 and mutSorcs1 -expressing INS1 whole cell lysates (WCL) using anti-myc tag antibody-conjugated agarose beads. ( C ) Representative peptide coverage of 130 kDa and 90 kDa bands excised from SDS-PAGE of wtSorcs1 and mutSorcs1- expressing INS1 cells and subject to LC-MS/MS data analysis. ( D ) Representative western blot of wtSorcs1 and mutSorcs1 -expressing INS1 cells in reducing and non-reducing conditions. ( E ) Densitometry analysis of mutSorcs1 bands immunoblotted in the presence or absence of 100 mM dithiothreitol (DTT). ( F ) Representative western blot of wtSorcs1 and mutSorcs1 -expressing INS1 cells in reducing conditions in the presence of 50 mM N-Ethylmaleimide (NEM) or iodoacetamide (IAA).
    Figure Legend Snippet: The alternative 90 kDa protein product of mutSorcs1 is unaffected by pro-protein convertases. ( A ) Representative western blot of lysates from wtSorcs1 and mutSorcs1 -expressing INS1 cells treated with DMSO or 10 µM prohormone convertase inhibitor (PCI). ( B ) Immunoprecipitation of Sorcs1 from wtSorcs1 and mutSorcs1 -expressing INS1 whole cell lysates (WCL) using anti-myc tag antibody-conjugated agarose beads. ( C ) Representative peptide coverage of 130 kDa and 90 kDa bands excised from SDS-PAGE of wtSorcs1 and mutSorcs1- expressing INS1 cells and subject to LC-MS/MS data analysis. ( D ) Representative western blot of wtSorcs1 and mutSorcs1 -expressing INS1 cells in reducing and non-reducing conditions. ( E ) Densitometry analysis of mutSorcs1 bands immunoblotted in the presence or absence of 100 mM dithiothreitol (DTT). ( F ) Representative western blot of wtSorcs1 and mutSorcs1 -expressing INS1 cells in reducing conditions in the presence of 50 mM N-Ethylmaleimide (NEM) or iodoacetamide (IAA).

    Techniques Used: Western Blot, Expressing, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    5) Product Images from "Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity"

    Article Title: Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16265

    Analysis of MMS sensitivity and PARP inhibition on the radiosensitivity of HPV-negative and HPV-positive OPSCC cells ( A ) Clonogenic survival of OPSCC cells was analysed following treatment with increasing doses of MMS (0–2 mM). Shown is the surviving fraction with standard errors from at least three independent experiments. ( B ) Whole cell extracts from OPSCC cells were prepared and analysed by 10 % SDS-PAGE and immunoblotting with either MPG or actin antibodies. Clonogenic survival of ( C ) HPV-positive OPSCC cells or ( D ) HPV-negative OPSCC cells was analysed following treatment with increasing doses of IR (0–4 Gy) in the absence and presence of the PARP inhibitor olaparib (0.1 μM). Shown is the surviving fraction with standard errors from at least three independent experiments.
    Figure Legend Snippet: Analysis of MMS sensitivity and PARP inhibition on the radiosensitivity of HPV-negative and HPV-positive OPSCC cells ( A ) Clonogenic survival of OPSCC cells was analysed following treatment with increasing doses of MMS (0–2 mM). Shown is the surviving fraction with standard errors from at least three independent experiments. ( B ) Whole cell extracts from OPSCC cells were prepared and analysed by 10 % SDS-PAGE and immunoblotting with either MPG or actin antibodies. Clonogenic survival of ( C ) HPV-positive OPSCC cells or ( D ) HPV-negative OPSCC cells was analysed following treatment with increasing doses of IR (0–4 Gy) in the absence and presence of the PARP inhibitor olaparib (0.1 μM). Shown is the surviving fraction with standard errors from at least three independent experiments.

    Techniques Used: Inhibition, SDS Page

    Analysis of BER and SSB repair protein levels in HPV-negative and HPV-positive OPSCC cells ( A ) Whole cell extracts from OPSCC cells were prepared and analysed by 10 % SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Levels of BER and SSB repair proteins relative to actin were quantified from at least three independent experiments. Shown is the mean protein level relative to actin with standard errors from at least three independent experiments, normalised to those calculated in the HPV-negative UMSCC6 cell extracts which was set to 100 %. * p
    Figure Legend Snippet: Analysis of BER and SSB repair protein levels in HPV-negative and HPV-positive OPSCC cells ( A ) Whole cell extracts from OPSCC cells were prepared and analysed by 10 % SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Levels of BER and SSB repair proteins relative to actin were quantified from at least three independent experiments. Shown is the mean protein level relative to actin with standard errors from at least three independent experiments, normalised to those calculated in the HPV-negative UMSCC6 cell extracts which was set to 100 %. * p

    Techniques Used: SDS Page

    6) Product Images from "Enhanced Accumulation of Phosphorylated α-Synuclein and Elevated β-Amyloid 42/40 Ratio Caused by Expression of the Presenilin-1 ΔT440 Mutant Associated with Familial Lewy Body Disease and Variant Alzheimer's Disease"

    Article Title: Enhanced Accumulation of Phosphorylated α-Synuclein and Elevated β-Amyloid 42/40 Ratio Caused by Expression of the Presenilin-1 ΔT440 Mutant Associated with Familial Lewy Body Disease and Variant Alzheimer's Disease

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4244-07.2007

    α-Synuclein expression in cells expressing PS1 ΔT440 mutant. A , N2a neuroblastoma or C6 glioblastoma cells stably expressing PS1 WT, the FAD-linked variants (H163R, E280A, and ΔE9), or ΔT440 mutant were transiently transfected with cDNA encoding human α-synuclein. Samples sequentially extracted with Tris-HCl- and 2% SDS-containing buffers were separated on a 10/20% gradient Tris-glycine SDS-PAGE and probed with the anti-α-synuclein antibody Syn-1. Representative results of Western blots analysis using C6 cell lines were shown (top). The band corresponding to the ∼15 kDa α-synuclein monomer is indicated (arrow). The semiquantitative analysis of the expression level of α-synuclein was performed by densitometry, and the ratio of α-synuclein intensity in the Tris/SDS-extracted fraction was determined. Results from three independent experiments are shown as mean ± SE. * p
    Figure Legend Snippet: α-Synuclein expression in cells expressing PS1 ΔT440 mutant. A , N2a neuroblastoma or C6 glioblastoma cells stably expressing PS1 WT, the FAD-linked variants (H163R, E280A, and ΔE9), or ΔT440 mutant were transiently transfected with cDNA encoding human α-synuclein. Samples sequentially extracted with Tris-HCl- and 2% SDS-containing buffers were separated on a 10/20% gradient Tris-glycine SDS-PAGE and probed with the anti-α-synuclein antibody Syn-1. Representative results of Western blots analysis using C6 cell lines were shown (top). The band corresponding to the ∼15 kDa α-synuclein monomer is indicated (arrow). The semiquantitative analysis of the expression level of α-synuclein was performed by densitometry, and the ratio of α-synuclein intensity in the Tris/SDS-extracted fraction was determined. Results from three independent experiments are shown as mean ± SE. * p

    Techniques Used: Expressing, Mutagenesis, Stable Transfection, Transfection, SDS Page, Western Blot

    Aberrant accumulation of α-synuclein in autopsied brain. A , Samples extracted from brain (frontal cortex) homogenates using different detergents were run on a 10/20% gradient Tris-glycine SDS-PAGE system and subjected to immunoblotting using the α-synuclein antibodies Syn-1, LB509, and pSyn#64. In the urea-extracted fraction, the monomer α-synuclein migrating at ∼15 kDa (arrow), which was reactive to the anti-phosphorylated α-synuclein antibody pSyn#64 (arrowhead), substantially accumulated only in the brain of the patient with the PS1 ΔT440 mutation. B , Samples were extracted from various regions of the patient's brain with PS1 ΔT440 mutation, including the frontal cortex (#1), parahippocampal gyrus (#2), thalamus (#3), and cerebellum (#4). The level of α-synuclein accumulation was highest in the urea-extracted fraction prepared from the parahippocampal gyrus (#2), followed by the thalamus (#3) and the frontal cortex (#1). The accumulation of α-synuclein was not observed in the urea-extracted fraction prepared from the cerebellum.
    Figure Legend Snippet: Aberrant accumulation of α-synuclein in autopsied brain. A , Samples extracted from brain (frontal cortex) homogenates using different detergents were run on a 10/20% gradient Tris-glycine SDS-PAGE system and subjected to immunoblotting using the α-synuclein antibodies Syn-1, LB509, and pSyn#64. In the urea-extracted fraction, the monomer α-synuclein migrating at ∼15 kDa (arrow), which was reactive to the anti-phosphorylated α-synuclein antibody pSyn#64 (arrowhead), substantially accumulated only in the brain of the patient with the PS1 ΔT440 mutation. B , Samples were extracted from various regions of the patient's brain with PS1 ΔT440 mutation, including the frontal cortex (#1), parahippocampal gyrus (#2), thalamus (#3), and cerebellum (#4). The level of α-synuclein accumulation was highest in the urea-extracted fraction prepared from the parahippocampal gyrus (#2), followed by the thalamus (#3) and the frontal cortex (#1). The accumulation of α-synuclein was not observed in the urea-extracted fraction prepared from the cerebellum.

    Techniques Used: SDS Page, Mutagenesis

    7) Product Images from "Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations"

    Article Title: Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.772335

    Effect of S1359A on full-length CFTR. A, detection of S1359A and wild-type CFTR proteins in microsomal membranes of transfected HEK293 cells. 10 μg of total membrane protein were resolved by SDS-PAGE (7.5% acrylamide) and electroblotted onto a nitrocellulose membrane that was probed with primary mAb 596. B, single-channel recording after fusion of S1359A membrane vesicles with a planar lipid bilayer. The unitary conductance and open probability are indicated above the recording, and the amplitude histogram is shown to the left . Dwell time histograms below the recording were used to estimate mean closed and open times.
    Figure Legend Snippet: Effect of S1359A on full-length CFTR. A, detection of S1359A and wild-type CFTR proteins in microsomal membranes of transfected HEK293 cells. 10 μg of total membrane protein were resolved by SDS-PAGE (7.5% acrylamide) and electroblotted onto a nitrocellulose membrane that was probed with primary mAb 596. B, single-channel recording after fusion of S1359A membrane vesicles with a planar lipid bilayer. The unitary conductance and open probability are indicated above the recording, and the amplitude histogram is shown to the left . Dwell time histograms below the recording were used to estimate mean closed and open times.

    Techniques Used: Transfection, SDS Page

    Related Articles

    Electrophoresis:

    Article Title: Purification of Tropomyosin, Paramyosin, Actin, Tubulin, Troponin and Kinases for Chemiproteomics and Its Application to Different Scientific Fields
    Article Snippet: .. A 37.5∶1 Acrylamide/bisAcrylamide solution (Cat. No. A3699, Sigma-Aldrich) and tris-glycine-SDS Buffer 10× Concentrate, both from Sigma-Aldrich, were diluted in distilled water for use in SDS-PAGE tris-glycine electrophoresis. ..

    Immunoprecipitation:

    Article Title: Iron Chelators ICL670 and 311 Inhibit HIV-1 Transcription
    Article Snippet: .. In parallel, the immunoprecipitated CDK2 was resolved on 10% Tris-Glycine SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Allen, TX) and immunoblotted with anti-CDK2 antibodies. .. Lactate dehydrogenase (LDH) release, used as indicator of cell membrane damage was measured in the culture medium using an LDH assay kit (LDH-Cytotoxicity Assay Kit, BioVision, Mountain View, CA).

    Incubation:

    Article Title: Enhanced Accumulation of Phosphorylated α-Synuclein and Elevated β-Amyloid 42/40 Ratio Caused by Expression of the Presenilin-1 ΔT440 Mutant Associated with Familial Lewy Body Disease and Variant Alzheimer's Disease
    Article Snippet: .. Cell lysates were subjected to Tris-glycine SDS-PAGE, and separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) before incubation with appropriate antibodies. .. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Millipore) and semiquantified by densitometry.

    Staining:

    Article Title: Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label *
    Article Snippet: .. Supernatant and pellet samples were each run on 12% Tris-glycine SDS-PAGE gels that were stained with Coomassie G (Sigma-Aldrich); and band intensity was analyzed by densitometry using Image J ( ). .. The equilibrium constant for dissociation of S1dC from actin ( Kd ) was determined by fitting with a quadratic binding function with maximal fraction bound constrained to 1.

    Western Blot:

    Article Title: Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity
    Article Snippet: .. Western Blotting Protein extracts (40 μg) were separated by 10 % or 6 % Tris-glycine SDS-PAGE, for medium and high molecular weight proteins, respectively and proteins transferred onto an Immobilon FL PVDF membrane (Millipore, Watford, UK). .. Membranes were blocked using Odyssey blocking buffer (Li-cor Biosciences, Cambridge, UK) and incubated with the primary antibody overnight at 4°C.

    Article Title: Carbapenem Resistance in a Clinical Isolate of Enterobacter aerogenes Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs
    Article Snippet: .. Western blotting of porins was performed as follows: proteins from 4 to 12% Tris-glycine-SDS gels or 10% NuPAGE gels were transferred to Immobilon-P filters (Millipore) ( , ). ..

    SDS Page:

    Article Title: Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations
    Article Snippet: .. The lysates were separated using Tris-glycine SDS-PAGE and subsequently transferred to PVDF membrane (Millipore). .. The CFTR protein was either detected using the monoclonal 660 α-CFTR antibody (University of North Carolina/Cystic Fibrosis Foundation antibody resource) and visualized using HRP-conjugated secondary antibody (Thermo Fisher Scientific) and GE Healthcare ( ) or detected with mAb596 and visualized with secondary goat anti-mouse IgG-IR800 using Odyssey infrared fluorescent imager (Licor Corp.) ( ).

    Article Title: Iron Chelators ICL670 and 311 Inhibit HIV-1 Transcription
    Article Snippet: .. In parallel, the immunoprecipitated CDK2 was resolved on 10% Tris-Glycine SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Allen, TX) and immunoblotted with anti-CDK2 antibodies. .. Lactate dehydrogenase (LDH) release, used as indicator of cell membrane damage was measured in the culture medium using an LDH assay kit (LDH-Cytotoxicity Assay Kit, BioVision, Mountain View, CA).

    Article Title: Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label *
    Article Snippet: .. Supernatant and pellet samples were each run on 12% Tris-glycine SDS-PAGE gels that were stained with Coomassie G (Sigma-Aldrich); and band intensity was analyzed by densitometry using Image J ( ). .. The equilibrium constant for dissociation of S1dC from actin ( Kd ) was determined by fitting with a quadratic binding function with maximal fraction bound constrained to 1.

    Article Title: Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity
    Article Snippet: .. Western Blotting Protein extracts (40 μg) were separated by 10 % or 6 % Tris-glycine SDS-PAGE, for medium and high molecular weight proteins, respectively and proteins transferred onto an Immobilon FL PVDF membrane (Millipore, Watford, UK). .. Membranes were blocked using Odyssey blocking buffer (Li-cor Biosciences, Cambridge, UK) and incubated with the primary antibody overnight at 4°C.

    Article Title: Purification of Tropomyosin, Paramyosin, Actin, Tubulin, Troponin and Kinases for Chemiproteomics and Its Application to Different Scientific Fields
    Article Snippet: .. A 37.5∶1 Acrylamide/bisAcrylamide solution (Cat. No. A3699, Sigma-Aldrich) and tris-glycine-SDS Buffer 10× Concentrate, both from Sigma-Aldrich, were diluted in distilled water for use in SDS-PAGE tris-glycine electrophoresis. ..

    Article Title: Enhanced Accumulation of Phosphorylated α-Synuclein and Elevated β-Amyloid 42/40 Ratio Caused by Expression of the Presenilin-1 ΔT440 Mutant Associated with Familial Lewy Body Disease and Variant Alzheimer's Disease
    Article Snippet: .. Cell lysates were subjected to Tris-glycine SDS-PAGE, and separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) before incubation with appropriate antibodies. .. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Millipore) and semiquantified by densitometry.

    Molecular Weight:

    Article Title: Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity
    Article Snippet: .. Western Blotting Protein extracts (40 μg) were separated by 10 % or 6 % Tris-glycine SDS-PAGE, for medium and high molecular weight proteins, respectively and proteins transferred onto an Immobilon FL PVDF membrane (Millipore, Watford, UK). .. Membranes were blocked using Odyssey blocking buffer (Li-cor Biosciences, Cambridge, UK) and incubated with the primary antibody overnight at 4°C.

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    Millipore tris glycine sds page
    Iron chelators inhibit phosphorylation of RNAPII ( a ) Chelators do not have an effect on expression of CDK9 . 293 cells were treated for 24 h with 10 μM 311 or 100 μM of ICL670. The cells were lysed were resolved on 10% <t>SDS-PAGE,</t> and immunoblotted anti-CDK9 antibodies. Lane 1 : control untreated cells; lanes 2, 3 and 4 : cells treated with DFO, 311 or ICL670. ( b ) Chelators inhibit association of CDK9 with cyclin T1 . The cell lysates prepared as in ( a ) were subjected to immunoprecipitation with anti-cyclin T1 antibody (lanes 3–6). The immunoprecipitated material was resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : input control untreated cells; lane 2: precipitation of untreated cells with non-specific preimmune IgG, lanes 3–6: immunoprecipitation with anti-cyclin T1 antibodies of lysates from untreated, DFO, 311 and ICL670 treated cells. The precipitated samples were resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK9. ( c ) Chelators inhibit phosphorylation of RNAPII . The cell lysates prepared as in ( a ) were resolved on 7.5% SDS-PAGE, and immunoblotted with the RNAPII CTD phospho-Serine 2 specific antibodies. Lane 1 : control untreated cells; lanes 2,3 and 4 : cells treated with DFO, 311 or ICL670.
    Tris Glycine Sds Page, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Iron chelators reduce HIV-1 Tat phosphorylated in cultured cells HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 2–5). Lane 1, control uninfected cells. HeLa cells were transfected with siRNAs targeting CDK2 (lane 3) or treated with 10 μM 311 or 100 μM ICL670. At 48 hours post infection cells were labeled with ( 32 P)-orthophosphate for 2 hours with the addition of 1 μM okadaic acid. Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Flag monoclonal antibodies, resolved on 15% <t>Tris-Tricine</t> <t>SDS-PAGE</t> and detected by Phosphor Imager. Phosphor Imager quantification is shown in the lower panel.
    Tris Tricine Sds Page, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phosphorylated Ser8 IRAK-4 death domain interferes with the Myddosome formation. ( A–D ) Absorbance Elution spectrum of a TSKgel SuperSW3000 analytical size exclusion column at 280 nm. ( A ) 0.5 mg.ml −1 of IRAK-4 death domain only. ( B ) 0.5 mg.ml −1 of MyD88 DD only. ( C ) 1:1 mix of MyD88 and non-phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior to loading. ( D ) 1:1 mix of MyD88 and Ser8 phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior loading. ( E ) Calibration of gel filtration using protein standard markers ( F ) 4–20% reducing <t>Tris-Glycine</t> <t>SDS</t> <t>PAGE</t> of fractions corresponding to peak 1, 2 and 3 of figure 15a. Fractions corresponding to peak 1 were concentrated using a 3 kDa MW cut-off Amicon Spin Filters for better visualisation of samples. One of three repeats is shown.
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    Iron chelators inhibit phosphorylation of RNAPII ( a ) Chelators do not have an effect on expression of CDK9 . 293 cells were treated for 24 h with 10 μM 311 or 100 μM of ICL670. The cells were lysed were resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : control untreated cells; lanes 2, 3 and 4 : cells treated with DFO, 311 or ICL670. ( b ) Chelators inhibit association of CDK9 with cyclin T1 . The cell lysates prepared as in ( a ) were subjected to immunoprecipitation with anti-cyclin T1 antibody (lanes 3–6). The immunoprecipitated material was resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : input control untreated cells; lane 2: precipitation of untreated cells with non-specific preimmune IgG, lanes 3–6: immunoprecipitation with anti-cyclin T1 antibodies of lysates from untreated, DFO, 311 and ICL670 treated cells. The precipitated samples were resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK9. ( c ) Chelators inhibit phosphorylation of RNAPII . The cell lysates prepared as in ( a ) were resolved on 7.5% SDS-PAGE, and immunoblotted with the RNAPII CTD phospho-Serine 2 specific antibodies. Lane 1 : control untreated cells; lanes 2,3 and 4 : cells treated with DFO, 311 or ICL670.

    Journal: Virology

    Article Title: Iron Chelators ICL670 and 311 Inhibit HIV-1 Transcription

    doi: 10.1016/j.virol.2007.06.011

    Figure Lengend Snippet: Iron chelators inhibit phosphorylation of RNAPII ( a ) Chelators do not have an effect on expression of CDK9 . 293 cells were treated for 24 h with 10 μM 311 or 100 μM of ICL670. The cells were lysed were resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : control untreated cells; lanes 2, 3 and 4 : cells treated with DFO, 311 or ICL670. ( b ) Chelators inhibit association of CDK9 with cyclin T1 . The cell lysates prepared as in ( a ) were subjected to immunoprecipitation with anti-cyclin T1 antibody (lanes 3–6). The immunoprecipitated material was resolved on 10% SDS-PAGE, and immunoblotted anti-CDK9 antibodies. Lane 1 : input control untreated cells; lane 2: precipitation of untreated cells with non-specific preimmune IgG, lanes 3–6: immunoprecipitation with anti-cyclin T1 antibodies of lysates from untreated, DFO, 311 and ICL670 treated cells. The precipitated samples were resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK9. ( c ) Chelators inhibit phosphorylation of RNAPII . The cell lysates prepared as in ( a ) were resolved on 7.5% SDS-PAGE, and immunoblotted with the RNAPII CTD phospho-Serine 2 specific antibodies. Lane 1 : control untreated cells; lanes 2,3 and 4 : cells treated with DFO, 311 or ICL670.

    Article Snippet: In parallel, the immunoprecipitated CDK2 was resolved on 10% Tris-Glycine SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Allen, TX) and immunoblotted with anti-CDK2 antibodies.

    Techniques: Expressing, SDS Page, Immunoprecipitation

    Iron chelators reduce HIV-1 Tat phosphorylated in cultured cells HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 2–5). Lane 1, control uninfected cells. HeLa cells were transfected with siRNAs targeting CDK2 (lane 3) or treated with 10 μM 311 or 100 μM ICL670. At 48 hours post infection cells were labeled with ( 32 P)-orthophosphate for 2 hours with the addition of 1 μM okadaic acid. Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Flag monoclonal antibodies, resolved on 15% Tris-Tricine SDS-PAGE and detected by Phosphor Imager. Phosphor Imager quantification is shown in the lower panel.

    Journal: Virology

    Article Title: Iron Chelators ICL670 and 311 Inhibit HIV-1 Transcription

    doi: 10.1016/j.virol.2007.06.011

    Figure Lengend Snippet: Iron chelators reduce HIV-1 Tat phosphorylated in cultured cells HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 2–5). Lane 1, control uninfected cells. HeLa cells were transfected with siRNAs targeting CDK2 (lane 3) or treated with 10 μM 311 or 100 μM ICL670. At 48 hours post infection cells were labeled with ( 32 P)-orthophosphate for 2 hours with the addition of 1 μM okadaic acid. Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Flag monoclonal antibodies, resolved on 15% Tris-Tricine SDS-PAGE and detected by Phosphor Imager. Phosphor Imager quantification is shown in the lower panel.

    Article Snippet: In parallel, the immunoprecipitated CDK2 was resolved on 10% Tris-Glycine SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Allen, TX) and immunoblotted with anti-CDK2 antibodies.

    Techniques: Cell Culture, Infection, Recombinant, Expressing, Transfection, Labeling, Immunoprecipitation, SDS Page

    Iron chelators reduce cellular activity of CDK2 (a) CDK2 expression determined by Western blotting . 293 cells were grown in 100 mm plates and treated for the indicated time with 100 μM DFO, 10 μM 311 or 100 μM ICL670. The cells were lysed and CDK2 was immunoprecipitated as described in Methods. Lane 1, input control. Lane, preimmune IgG was used for the immunoprecipitation. The precipitated CDK2 was resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK2. ( b ) CDK2 was precipitated as in (a) and the immunoprecipitated material was subsequently incubated with histone H1 in the presence of γ-( 32 P) ATP. Kinase reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager. Lane 1 : non-specific preimmune IgG. Lane 10, histone H1 phosphorylation with recombinant CDK2/Cyclin E.

    Journal: Virology

    Article Title: Iron Chelators ICL670 and 311 Inhibit HIV-1 Transcription

    doi: 10.1016/j.virol.2007.06.011

    Figure Lengend Snippet: Iron chelators reduce cellular activity of CDK2 (a) CDK2 expression determined by Western blotting . 293 cells were grown in 100 mm plates and treated for the indicated time with 100 μM DFO, 10 μM 311 or 100 μM ICL670. The cells were lysed and CDK2 was immunoprecipitated as described in Methods. Lane 1, input control. Lane, preimmune IgG was used for the immunoprecipitation. The precipitated CDK2 was resolved on 10% SDS-PAGE and immunoblotted with antibodies against CDK2. ( b ) CDK2 was precipitated as in (a) and the immunoprecipitated material was subsequently incubated with histone H1 in the presence of γ-( 32 P) ATP. Kinase reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager. Lane 1 : non-specific preimmune IgG. Lane 10, histone H1 phosphorylation with recombinant CDK2/Cyclin E.

    Article Snippet: In parallel, the immunoprecipitated CDK2 was resolved on 10% Tris-Glycine SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Allen, TX) and immunoblotted with anti-CDK2 antibodies.

    Techniques: Activity Assay, Expressing, Western Blot, Immunoprecipitation, SDS Page, Incubation, Recombinant

    Iron chelators reduce HIV-1 Tat phosphorylated in cultured cells HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 2–5). Lane 1, control uninfected cells. HeLa cells were transfected with siRNAs targeting CDK2 (lane 3) or treated with 10 μM 311 or 100 μM ICL670. At 48 hours post infection cells were labeled with ( 32 P)-orthophosphate for 2 hours with the addition of 1 μM okadaic acid. Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Flag monoclonal antibodies, resolved on 15% Tris-Tricine SDS-PAGE and detected by Phosphor Imager. Phosphor Imager quantification is shown in the lower panel.

    Journal: Virology

    Article Title: Iron Chelators ICL670 and 311 Inhibit HIV-1 Transcription

    doi: 10.1016/j.virol.2007.06.011

    Figure Lengend Snippet: Iron chelators reduce HIV-1 Tat phosphorylated in cultured cells HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 2–5). Lane 1, control uninfected cells. HeLa cells were transfected with siRNAs targeting CDK2 (lane 3) or treated with 10 μM 311 or 100 μM ICL670. At 48 hours post infection cells were labeled with ( 32 P)-orthophosphate for 2 hours with the addition of 1 μM okadaic acid. Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Flag monoclonal antibodies, resolved on 15% Tris-Tricine SDS-PAGE and detected by Phosphor Imager. Phosphor Imager quantification is shown in the lower panel.

    Article Snippet: The immunoprecipitated Tat was recovered by heating for 2 min at 100°C in Tricine SDS-loading buffer, resolved on 15% Tris-Tricine SDS-PAGE ( ) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Allen, TX).

    Techniques: Cell Culture, Infection, Recombinant, Expressing, Transfection, Labeling, Immunoprecipitation, SDS Page

    Differences between Bcl-2 and Bcl-xL behaviour during autophagy. (A) Co-immunoprecipitation of endogenous Beclin 1 with endogenous Bcl-2 and Bcl-xL in HCT116-Bax KO cells grown in complete medium or starved for 6 hours. (B) Subcellular fractionation of HCT116-Bax KO cells grown in complete medium or starved for 6 hours. Cells were broken with a Dounce homogeniser, post-nuclear supernatants were loaded on top of a continuous 10–55% sucrose gradient for ultracentrifugation overnight. Fractions of 500 µl were collected, and total protein precipitated. The same volume of each fraction was separated on a 14% tris-tricine SDS-PAGE. Western-blots were as described in the methods section. (C) Immunocytochemistry on endogenous Bcl-xL and ATP1 in HCT116-BaxKO cells grown in complete medium or starved for 6 hours.

    Journal: PLoS ONE

    Article Title: Differential Dependence on Beclin 1 for the Regulation of Pro-Survival Autophagy by Bcl-2 and Bcl-xL in HCT116 Colorectal Cancer Cells

    doi: 10.1371/journal.pone.0008755

    Figure Lengend Snippet: Differences between Bcl-2 and Bcl-xL behaviour during autophagy. (A) Co-immunoprecipitation of endogenous Beclin 1 with endogenous Bcl-2 and Bcl-xL in HCT116-Bax KO cells grown in complete medium or starved for 6 hours. (B) Subcellular fractionation of HCT116-Bax KO cells grown in complete medium or starved for 6 hours. Cells were broken with a Dounce homogeniser, post-nuclear supernatants were loaded on top of a continuous 10–55% sucrose gradient for ultracentrifugation overnight. Fractions of 500 µl were collected, and total protein precipitated. The same volume of each fraction was separated on a 14% tris-tricine SDS-PAGE. Western-blots were as described in the methods section. (C) Immunocytochemistry on endogenous Bcl-xL and ATP1 in HCT116-BaxKO cells grown in complete medium or starved for 6 hours.

    Article Snippet: Protein extracts were separated on SDS-PAGE or tris-tricine SDS-PAGE , transferred onto PVDF membrane (Millipore) and revealed with ECL (Roche Diagnostics).

    Techniques: Immunoprecipitation, Fractionation, SDS Page, Western Blot, Immunocytochemistry

    Phosphorylated Ser8 IRAK-4 death domain interferes with the Myddosome formation. ( A–D ) Absorbance Elution spectrum of a TSKgel SuperSW3000 analytical size exclusion column at 280 nm. ( A ) 0.5 mg.ml −1 of IRAK-4 death domain only. ( B ) 0.5 mg.ml −1 of MyD88 DD only. ( C ) 1:1 mix of MyD88 and non-phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior to loading. ( D ) 1:1 mix of MyD88 and Ser8 phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior loading. ( E ) Calibration of gel filtration using protein standard markers ( F ) 4–20% reducing Tris-Glycine SDS PAGE of fractions corresponding to peak 1, 2 and 3 of figure 15a. Fractions corresponding to peak 1 were concentrated using a 3 kDa MW cut-off Amicon Spin Filters for better visualisation of samples. One of three repeats is shown.

    Journal: Scientific Reports

    Article Title: The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold

    doi: 10.1038/srep37267

    Figure Lengend Snippet: Phosphorylated Ser8 IRAK-4 death domain interferes with the Myddosome formation. ( A–D ) Absorbance Elution spectrum of a TSKgel SuperSW3000 analytical size exclusion column at 280 nm. ( A ) 0.5 mg.ml −1 of IRAK-4 death domain only. ( B ) 0.5 mg.ml −1 of MyD88 DD only. ( C ) 1:1 mix of MyD88 and non-phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior to loading. ( D ) 1:1 mix of MyD88 and Ser8 phosphorylated IRAK-4 death domains concentrated to 0.5 mg.ml −1 prior loading. ( E ) Calibration of gel filtration using protein standard markers ( F ) 4–20% reducing Tris-Glycine SDS PAGE of fractions corresponding to peak 1, 2 and 3 of figure 15a. Fractions corresponding to peak 1 were concentrated using a 3 kDa MW cut-off Amicon Spin Filters for better visualisation of samples. One of three repeats is shown.

    Article Snippet: The elution fractions corresponding to IRAK-4 DD, identified on a 4–20% Tris-Glycine reducing SDS PAGE, were pooled together, concentrated to 1 mg.ml−1 using an Amicon Ultra 3 kDa MW cut-off filter from Millipore.

    Techniques: Filtration, SDS Page

    Reciprocal pull down assays confirm inability of Ser8-P death domain to interact with MyD88. ( A ) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit polyclonal anti-human MyD88 death domain antibody. Lane 1 to 3 are control lanes each loaded with 3 μg of the protein samples: lane 1-MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 6 were loaded with the pull down experiment samples as indicated. ( B ) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit monoclonal anti-human IRAK-4 death domain antibody. Lane 1 to 3 were control lanes each loaded with 3 μg of the protein samples: lane 1 corresponded to MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 7 were loaded with the pull down experiment samples as indicated. One repeat of three biological replicates is shown.

    Journal: Scientific Reports

    Article Title: The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold

    doi: 10.1038/srep37267

    Figure Lengend Snippet: Reciprocal pull down assays confirm inability of Ser8-P death domain to interact with MyD88. ( A ) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit polyclonal anti-human MyD88 death domain antibody. Lane 1 to 3 are control lanes each loaded with 3 μg of the protein samples: lane 1-MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 6 were loaded with the pull down experiment samples as indicated. ( B ) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit monoclonal anti-human IRAK-4 death domain antibody. Lane 1 to 3 were control lanes each loaded with 3 μg of the protein samples: lane 1 corresponded to MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 7 were loaded with the pull down experiment samples as indicated. One repeat of three biological replicates is shown.

    Article Snippet: The elution fractions corresponding to IRAK-4 DD, identified on a 4–20% Tris-Glycine reducing SDS PAGE, were pooled together, concentrated to 1 mg.ml−1 using an Amicon Ultra 3 kDa MW cut-off filter from Millipore.

    Techniques: SDS Page

    Purification of IRAK-4 death domain. ( A ) IRAK-4 full length was cleaved with thrombin and TEV proteases and analysed by XX% SDS-PAGE ( B ) Cleaved IRAK-4 was fractionated by gel filtration on 16/60 Superdex™ 75 pg ( C ) SDS-PAGE analysis of gel filtration peaks 1–3. ( D ) LC/MS spectrum showing deconvoluted masses of IRAK-4 DD purified in peak 3. The mass of 12782 Da corresponded to IRAK-4 death domain residues (−7) to 107 (see Methods). The mass of 12862 Da represents IRAK-4 death domain with a phosphorylated residue (+80 Da) ( C ).

    Journal: Scientific Reports

    Article Title: The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold

    doi: 10.1038/srep37267

    Figure Lengend Snippet: Purification of IRAK-4 death domain. ( A ) IRAK-4 full length was cleaved with thrombin and TEV proteases and analysed by XX% SDS-PAGE ( B ) Cleaved IRAK-4 was fractionated by gel filtration on 16/60 Superdex™ 75 pg ( C ) SDS-PAGE analysis of gel filtration peaks 1–3. ( D ) LC/MS spectrum showing deconvoluted masses of IRAK-4 DD purified in peak 3. The mass of 12782 Da corresponded to IRAK-4 death domain residues (−7) to 107 (see Methods). The mass of 12862 Da represents IRAK-4 death domain with a phosphorylated residue (+80 Da) ( C ).

    Article Snippet: The elution fractions corresponding to IRAK-4 DD, identified on a 4–20% Tris-Glycine reducing SDS PAGE, were pooled together, concentrated to 1 mg.ml−1 using an Amicon Ultra 3 kDa MW cut-off filter from Millipore.

    Techniques: Purification, SDS Page, Filtration, Liquid Chromatography with Mass Spectroscopy