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  • 99
    Thermo Fisher tris glycine sds running buffer
    Kinase assays demonstrate that p38 MAPK directly phosphorylates NHE1 at a site(s) between amino acids 703 and 793. The numbering system used corresponds to the rabbit NHE1 sequence. (A) Protein lysates from FL5.12A cells cultured with or without IL-3 for 2 h were immunoprecipitated with either an antibody specific for phospho-p38 MAPK (p38) or an antibody specific for the motif, p-TP, and the ability of these kinases to phosphorylated the WT NHE-GST fusion protein was evaluated by an in-gel kinase assay as described in Materials and Methods. Only p38 phosphorylated the WT NHE-GST fusion protein, an activity which increased 1.8-fold during IL-3 withdrawal (left panel). Protein lysates from FL5.12A cells were also immunoprecipitated with c-Jun fusion beads to capture JNK, which was tested in an in vitro kinase assay for its ability to phosphorylate the WT NHEGST fusion protein. Unlike p38 MAPK, JNK did not phosphorylate NHE (right panel). (B) Protein lysates extracted from anisomycin-activated FL5.12A cells were immunoprecipitated with antibody to phospho-p38 MAPK and a kinase assay performed as described in Materials and Methods using the WT NHE-GST fusion protein and seven mutant NHE-GST fusion proteins as substrates. GST alone was included as a negative control. (C) The results of the kinase assay from panel B are summarized in table form. In addition, phospho-p38 MAPK did not phosphorylate NHE-GST fusion proteins containing amino acids 635 to 663. Possible phosphorylation sites by p38 MAPK on NHE are located in the region from amino acids 703 to 743, which excludes the proposed p90RSK phosphorylation site (Serine 703, numbering from human NHE1 sequence). (D) FL5.12A cells, cultured without IL-3 for 0, 1, 2, and 3 h or treated with anisomycin (+), were lysed, and the precleared protein extracts were immunoprecipitated with the A7 antibody specific for NHE1 and then run on an 8% <t>Tris-glycine</t> <t>SDS</t> gel and analyzed by Western blotting with the p-TP-specific antibody (left panel). The same blots were then reprobed with a commercially available anti-NHE1 antibody (right panel). NHE1 did not contain an activated p-TP motif. (E) Mass spectrometry analysis, as described in Materials and Methods, identified four sites at Thr 717, Ser 722, Ser 725, and Ser 728 as in vitro targets for p38 MAPK activity. Phosphorylation of one or more of these sites by p38 MAPK modulates alkalinization mediated by NHE during trophic factor withdrawal.
    Tris Glycine Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: 99/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad tris glycine sds buffer
    Experimental strategy for comparing peptide- vs protein-level enrichments. (A) Experimental design applying on-resin iTRAQ-labeling. (B) Silver-staining image of <t>SDS–PAGE</t> of enriched Cys-peptides. For each lane of the 4–20% <t>Tris–HCl</t> precast gel, 5 of 30 μl of DTT-eluted Cys-peptides was loaded. The higher protein bands in protein-level enrichment samples indicate incomplete on-resin digestion. (C) An MS/MS spectrum of the Cys-peptide GVLECQVSR from obscurin protein and its zoom-in spectrum of reporter-ion region showing the SNO abundance increased in response to GSNO treatment.
    Tris Glycine Sds Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher novex tris glycine sds
    Recombinant Fs(1)h interacts with the recombinant PRC1 complex. ( A ) The purification scheme using M2 anti-Flag antibody. Nuclear extracts were generated from Sf9 cells that were infected with baculovirus expressing recombinant Fs(1)h alone or coinfected with PRC1 (Psc-Flag, Pc, Ph, and Sce) or PRC2 [Esc-Flag, E(z), Su(z)12, and Caf1-55] complex components. Entire elution fractions were boiled in <t>SDS-PAGE</t> loading buffer and resolved on an 8% <t>Tris-glycine</t> gel. ( B ) The presence of the Fs(1)h along with the intact PRC1 or PRC2 complexes was verified by silver staining. ( C ) The specificity of the Fs(1)h–PRC1 interaction was confirmed by Western blotting using anti-Fs(1)h antibodies. Nuclear extract lanes show Fs(1)h expression levels in input samples (0.5% input loaded).
    Novex Tris Glycine Sds, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 520 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Bio-Rad tris glycine sds electrophoresis buffer
    Recombinant Fs(1)h interacts with the recombinant PRC1 complex. ( A ) The purification scheme using M2 anti-Flag antibody. Nuclear extracts were generated from Sf9 cells that were infected with baculovirus expressing recombinant Fs(1)h alone or coinfected with PRC1 (Psc-Flag, Pc, Ph, and Sce) or PRC2 [Esc-Flag, E(z), Su(z)12, and Caf1-55] complex components. Entire elution fractions were boiled in <t>SDS-PAGE</t> loading buffer and resolved on an 8% <t>Tris-glycine</t> gel. ( B ) The presence of the Fs(1)h along with the intact PRC1 or PRC2 complexes was verified by silver staining. ( C ) The specificity of the Fs(1)h–PRC1 interaction was confirmed by Western blotting using anti-Fs(1)h antibodies. Nuclear extract lanes show Fs(1)h expression levels in input samples (0.5% input loaded).
    Tris Glycine Sds Electrophoresis Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 98/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad 10x tris glycine sds
    Recombinant Fs(1)h interacts with the recombinant PRC1 complex. ( A ) The purification scheme using M2 anti-Flag antibody. Nuclear extracts were generated from Sf9 cells that were infected with baculovirus expressing recombinant Fs(1)h alone or coinfected with PRC1 (Psc-Flag, Pc, Ph, and Sce) or PRC2 [Esc-Flag, E(z), Su(z)12, and Caf1-55] complex components. Entire elution fractions were boiled in <t>SDS-PAGE</t> loading buffer and resolved on an 8% <t>Tris-glycine</t> gel. ( B ) The presence of the Fs(1)h along with the intact PRC1 or PRC2 complexes was verified by silver staining. ( C ) The specificity of the Fs(1)h–PRC1 interaction was confirmed by Western blotting using anti-Fs(1)h antibodies. Nuclear extract lanes show Fs(1)h expression levels in input samples (0.5% input loaded).
    10x Tris Glycine Sds, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad 1x tris glycine sds buffer
    Recombinant Fs(1)h interacts with the recombinant PRC1 complex. ( A ) The purification scheme using M2 anti-Flag antibody. Nuclear extracts were generated from Sf9 cells that were infected with baculovirus expressing recombinant Fs(1)h alone or coinfected with PRC1 (Psc-Flag, Pc, Ph, and Sce) or PRC2 [Esc-Flag, E(z), Su(z)12, and Caf1-55] complex components. Entire elution fractions were boiled in <t>SDS-PAGE</t> loading buffer and resolved on an 8% <t>Tris-glycine</t> gel. ( B ) The presence of the Fs(1)h along with the intact PRC1 or PRC2 complexes was verified by silver staining. ( C ) The specificity of the Fs(1)h–PRC1 interaction was confirmed by Western blotting using anti-Fs(1)h antibodies. Nuclear extract lanes show Fs(1)h expression levels in input samples (0.5% input loaded).
    1x Tris Glycine Sds Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tris glycine sds buffer
    Recombinant Fs(1)h interacts with the recombinant PRC1 complex. ( A ) The purification scheme using M2 anti-Flag antibody. Nuclear extracts were generated from Sf9 cells that were infected with baculovirus expressing recombinant Fs(1)h alone or coinfected with PRC1 (Psc-Flag, Pc, Ph, and Sce) or PRC2 [Esc-Flag, E(z), Su(z)12, and Caf1-55] complex components. Entire elution fractions were boiled in <t>SDS-PAGE</t> loading buffer and resolved on an 8% <t>Tris-glycine</t> gel. ( B ) The presence of the Fs(1)h along with the intact PRC1 or PRC2 complexes was verified by silver staining. ( C ) The specificity of the Fs(1)h–PRC1 interaction was confirmed by Western blotting using anti-Fs(1)h antibodies. Nuclear extract lanes show Fs(1)h expression levels in input samples (0.5% input loaded).
    Tris Glycine Sds Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tris glycine sds running buffer
    Recombinant Fs(1)h interacts with the recombinant PRC1 complex. ( A ) The purification scheme using M2 anti-Flag antibody. Nuclear extracts were generated from Sf9 cells that were infected with baculovirus expressing recombinant Fs(1)h alone or coinfected with PRC1 (Psc-Flag, Pc, Ph, and Sce) or PRC2 [Esc-Flag, E(z), Su(z)12, and Caf1-55] complex components. Entire elution fractions were boiled in <t>SDS-PAGE</t> loading buffer and resolved on an 8% <t>Tris-glycine</t> gel. ( B ) The presence of the Fs(1)h along with the intact PRC1 or PRC2 complexes was verified by silver staining. ( C ) The specificity of the Fs(1)h–PRC1 interaction was confirmed by Western blotting using anti-Fs(1)h antibodies. Nuclear extract lanes show Fs(1)h expression levels in input samples (0.5% input loaded).
    Tris Glycine Sds Running Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kinase assays demonstrate that p38 MAPK directly phosphorylates NHE1 at a site(s) between amino acids 703 and 793. The numbering system used corresponds to the rabbit NHE1 sequence. (A) Protein lysates from FL5.12A cells cultured with or without IL-3 for 2 h were immunoprecipitated with either an antibody specific for phospho-p38 MAPK (p38) or an antibody specific for the motif, p-TP, and the ability of these kinases to phosphorylated the WT NHE-GST fusion protein was evaluated by an in-gel kinase assay as described in Materials and Methods. Only p38 phosphorylated the WT NHE-GST fusion protein, an activity which increased 1.8-fold during IL-3 withdrawal (left panel). Protein lysates from FL5.12A cells were also immunoprecipitated with c-Jun fusion beads to capture JNK, which was tested in an in vitro kinase assay for its ability to phosphorylate the WT NHEGST fusion protein. Unlike p38 MAPK, JNK did not phosphorylate NHE (right panel). (B) Protein lysates extracted from anisomycin-activated FL5.12A cells were immunoprecipitated with antibody to phospho-p38 MAPK and a kinase assay performed as described in Materials and Methods using the WT NHE-GST fusion protein and seven mutant NHE-GST fusion proteins as substrates. GST alone was included as a negative control. (C) The results of the kinase assay from panel B are summarized in table form. In addition, phospho-p38 MAPK did not phosphorylate NHE-GST fusion proteins containing amino acids 635 to 663. Possible phosphorylation sites by p38 MAPK on NHE are located in the region from amino acids 703 to 743, which excludes the proposed p90RSK phosphorylation site (Serine 703, numbering from human NHE1 sequence). (D) FL5.12A cells, cultured without IL-3 for 0, 1, 2, and 3 h or treated with anisomycin (+), were lysed, and the precleared protein extracts were immunoprecipitated with the A7 antibody specific for NHE1 and then run on an 8% Tris-glycine SDS gel and analyzed by Western blotting with the p-TP-specific antibody (left panel). The same blots were then reprobed with a commercially available anti-NHE1 antibody (right panel). NHE1 did not contain an activated p-TP motif. (E) Mass spectrometry analysis, as described in Materials and Methods, identified four sites at Thr 717, Ser 722, Ser 725, and Ser 728 as in vitro targets for p38 MAPK activity. Phosphorylation of one or more of these sites by p38 MAPK modulates alkalinization mediated by NHE during trophic factor withdrawal.

    Journal: Molecular and Cellular Biology

    Article Title: Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

    doi: 10.1128/MCB.21.22.7545-7557.2001

    Figure Lengend Snippet: Kinase assays demonstrate that p38 MAPK directly phosphorylates NHE1 at a site(s) between amino acids 703 and 793. The numbering system used corresponds to the rabbit NHE1 sequence. (A) Protein lysates from FL5.12A cells cultured with or without IL-3 for 2 h were immunoprecipitated with either an antibody specific for phospho-p38 MAPK (p38) or an antibody specific for the motif, p-TP, and the ability of these kinases to phosphorylated the WT NHE-GST fusion protein was evaluated by an in-gel kinase assay as described in Materials and Methods. Only p38 phosphorylated the WT NHE-GST fusion protein, an activity which increased 1.8-fold during IL-3 withdrawal (left panel). Protein lysates from FL5.12A cells were also immunoprecipitated with c-Jun fusion beads to capture JNK, which was tested in an in vitro kinase assay for its ability to phosphorylate the WT NHEGST fusion protein. Unlike p38 MAPK, JNK did not phosphorylate NHE (right panel). (B) Protein lysates extracted from anisomycin-activated FL5.12A cells were immunoprecipitated with antibody to phospho-p38 MAPK and a kinase assay performed as described in Materials and Methods using the WT NHE-GST fusion protein and seven mutant NHE-GST fusion proteins as substrates. GST alone was included as a negative control. (C) The results of the kinase assay from panel B are summarized in table form. In addition, phospho-p38 MAPK did not phosphorylate NHE-GST fusion proteins containing amino acids 635 to 663. Possible phosphorylation sites by p38 MAPK on NHE are located in the region from amino acids 703 to 743, which excludes the proposed p90RSK phosphorylation site (Serine 703, numbering from human NHE1 sequence). (D) FL5.12A cells, cultured without IL-3 for 0, 1, 2, and 3 h or treated with anisomycin (+), were lysed, and the precleared protein extracts were immunoprecipitated with the A7 antibody specific for NHE1 and then run on an 8% Tris-glycine SDS gel and analyzed by Western blotting with the p-TP-specific antibody (left panel). The same blots were then reprobed with a commercially available anti-NHE1 antibody (right panel). NHE1 did not contain an activated p-TP motif. (E) Mass spectrometry analysis, as described in Materials and Methods, identified four sites at Thr 717, Ser 722, Ser 725, and Ser 728 as in vitro targets for p38 MAPK activity. Phosphorylation of one or more of these sites by p38 MAPK modulates alkalinization mediated by NHE during trophic factor withdrawal.

    Article Snippet: Cell equivalent samples (20 μl containing approximately 40 μg of protein) were then separated by SDS-polyacrylamide gel electrophoresis on either 8 or 12% Tris-glycine gels (Invitrogen) run under denaturing conditions using Tris-glycine SDS running buffer (Invitrogen).

    Techniques: Sequencing, Cell Culture, Immunoprecipitation, Kinase Assay, Activity Assay, In Vitro, Mutagenesis, Negative Control, SDS-Gel, Western Blot, Mass Spectrometry

    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

    Coomassie stained tris-glycine 8×10 cm SDS-PAGE of proteins purified from eight species of synanthropic acaridid mites. A) Reducing 10% SDS-PAGE, 10 µg protein per lane; B) Reducing 20% SDS-PAGE, 10 µg protein per lane (figure has been shortened). Legend: A – actin; T1 – tropomyosin monomer; T4 – tropomyosin tetramer; P1 – paramyosin monomer; P2 – paramyosin dimer; AO – Aleuroglyphus ovatus ; GD – Glycyphagus domesticus ; BT – Blomia tropicalis ; TP – Tyrophagus putrescentiae ; LD – Lepidoglyphus destructor ; TL – Tyroborus lini ; AS – Acarus siro ; DF – Dermatophagoides farinae ; M – marker.

    Journal: PLoS ONE

    Article Title: Purification of Tropomyosin, Paramyosin, Actin, Tubulin, Troponin and Kinases for Chemiproteomics and Its Application to Different Scientific Fields

    doi: 10.1371/journal.pone.0022860

    Figure Lengend Snippet: Coomassie stained tris-glycine 8×10 cm SDS-PAGE of proteins purified from eight species of synanthropic acaridid mites. A) Reducing 10% SDS-PAGE, 10 µg protein per lane; B) Reducing 20% SDS-PAGE, 10 µg protein per lane (figure has been shortened). Legend: A – actin; T1 – tropomyosin monomer; T4 – tropomyosin tetramer; P1 – paramyosin monomer; P2 – paramyosin dimer; AO – Aleuroglyphus ovatus ; GD – Glycyphagus domesticus ; BT – Blomia tropicalis ; TP – Tyrophagus putrescentiae ; LD – Lepidoglyphus destructor ; TL – Tyroborus lini ; AS – Acarus siro ; DF – Dermatophagoides farinae ; M – marker.

    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.

    Techniques: Staining, SDS Page, Purification, Marker

    Experimental strategy for comparing peptide- vs protein-level enrichments. (A) Experimental design applying on-resin iTRAQ-labeling. (B) Silver-staining image of SDS–PAGE of enriched Cys-peptides. For each lane of the 4–20% Tris–HCl precast gel, 5 of 30 μl of DTT-eluted Cys-peptides was loaded. The higher protein bands in protein-level enrichment samples indicate incomplete on-resin digestion. (C) An MS/MS spectrum of the Cys-peptide GVLECQVSR from obscurin protein and its zoom-in spectrum of reporter-ion region showing the SNO abundance increased in response to GSNO treatment.

    Journal: Free radical biology & medicine

    Article Title: Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry

    doi: 10.1016/j.freeradbiomed.2012.12.010

    Figure Lengend Snippet: Experimental strategy for comparing peptide- vs protein-level enrichments. (A) Experimental design applying on-resin iTRAQ-labeling. (B) Silver-staining image of SDS–PAGE of enriched Cys-peptides. For each lane of the 4–20% Tris–HCl precast gel, 5 of 30 μl of DTT-eluted Cys-peptides was loaded. The higher protein bands in protein-level enrichment samples indicate incomplete on-resin digestion. (C) An MS/MS spectrum of the Cys-peptide GVLECQVSR from obscurin protein and its zoom-in spectrum of reporter-ion region showing the SNO abundance increased in response to GSNO treatment.

    Article Snippet: Gel electrophoresis was run at 170 V for 25 min in Tris/glycine/SDS buffer (Bio-Rad).

    Techniques: Labeling, Silver Staining, SDS Page, Mass Spectrometry

    SDS-PAGE of pot ale proteins separated on a 4–20% Tris-glycine gel. Samples were either run directly without treatment (lanes 1 and 6) or after concentration using either 3 (lanes 2, 3, and 4) or 10 K (lanes 7, 8, 9, and 10) nominal molecular weight tubes. The protein concentration (μl) in each sample is shown, and a broad range prestained molecular weight ladder was in lane 5. The arrows indicate the main bands that were visible on the gel.

    Journal: ACS Omega

    Article Title: Characterization of Pot Ale from a Scottish Malt Whisky Distillery and Potential Applications

    doi: 10.1021/acsomega.9b04023

    Figure Lengend Snippet: SDS-PAGE of pot ale proteins separated on a 4–20% Tris-glycine gel. Samples were either run directly without treatment (lanes 1 and 6) or after concentration using either 3 (lanes 2, 3, and 4) or 10 K (lanes 7, 8, 9, and 10) nominal molecular weight tubes. The protein concentration (μl) in each sample is shown, and a broad range prestained molecular weight ladder was in lane 5. The arrows indicate the main bands that were visible on the gel.

    Article Snippet: Gels were run using a Bio-Rad Mini-PROTEAN Tetra cell system for mini precast gels with Tris/glycine/SDS running buffer (10× Tris/glycine/SDS running buffer, Bio-Rad Laboratories).

    Techniques: SDS Page, Concentration Assay, Molecular Weight, Protein Concentration

    Recombinant Fs(1)h interacts with the recombinant PRC1 complex. ( A ) The purification scheme using M2 anti-Flag antibody. Nuclear extracts were generated from Sf9 cells that were infected with baculovirus expressing recombinant Fs(1)h alone or coinfected with PRC1 (Psc-Flag, Pc, Ph, and Sce) or PRC2 [Esc-Flag, E(z), Su(z)12, and Caf1-55] complex components. Entire elution fractions were boiled in SDS-PAGE loading buffer and resolved on an 8% Tris-glycine gel. ( B ) The presence of the Fs(1)h along with the intact PRC1 or PRC2 complexes was verified by silver staining. ( C ) The specificity of the Fs(1)h–PRC1 interaction was confirmed by Western blotting using anti-Fs(1)h antibodies. Nuclear extract lanes show Fs(1)h expression levels in input samples (0.5% input loaded).

    Journal: Genes & Development

    Article Title: Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila

    doi: 10.1101/gad.305987.117

    Figure Lengend Snippet: Recombinant Fs(1)h interacts with the recombinant PRC1 complex. ( A ) The purification scheme using M2 anti-Flag antibody. Nuclear extracts were generated from Sf9 cells that were infected with baculovirus expressing recombinant Fs(1)h alone or coinfected with PRC1 (Psc-Flag, Pc, Ph, and Sce) or PRC2 [Esc-Flag, E(z), Su(z)12, and Caf1-55] complex components. Entire elution fractions were boiled in SDS-PAGE loading buffer and resolved on an 8% Tris-glycine gel. ( B ) The presence of the Fs(1)h along with the intact PRC1 or PRC2 complexes was verified by silver staining. ( C ) The specificity of the Fs(1)h–PRC1 interaction was confirmed by Western blotting using anti-Fs(1)h antibodies. Nuclear extract lanes show Fs(1)h expression levels in input samples (0.5% input loaded).

    Article Snippet: The final crude nuclear pellet was resuspended in 600 µL of Novex Tris-glycine SDS sample buffer (Invitrogen), including NuPAGE sample-reducing agent (Invitrogen), and then boiled for 10 min. For S2 cells, ∼5 × 106 cells expressing BioTAP transgenes were harvested; washed with PBS; lysed with 200 μL of Novex Tris-glycine SDS sample buffer (Invitrogen), including NuPAGE sample-reducing agent (Invitrogen); and then boiled for 10 min. Twenty microliters was used for each Western blot.

    Techniques: Recombinant, Purification, Generated, Infection, Expressing, SDS Page, Silver Staining, Western Blot

    Solubilized MSC and SB623-derived ECM analyzed by SDS-PAGE. SDS/urea-soluble SB623-derived ECM (SB) and corresponding MSC. M: molecular weight markers. SDS/urea samples were precipitated, re-suspended in 2X loading buffer, loaded on a 1.5-mm 4–20% Tris-acetate gel, electrophoresed and stained with Coomassie Blue R-250.

    Journal: PLoS ONE

    Article Title: Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism

    doi: 10.1371/journal.pone.0079283

    Figure Lengend Snippet: Solubilized MSC and SB623-derived ECM analyzed by SDS-PAGE. SDS/urea-soluble SB623-derived ECM (SB) and corresponding MSC. M: molecular weight markers. SDS/urea samples were precipitated, re-suspended in 2X loading buffer, loaded on a 1.5-mm 4–20% Tris-acetate gel, electrophoresed and stained with Coomassie Blue R-250.

    Article Snippet: The pellet was resuspended in 40 µl Tris-glycine SDS Sample Buffer (Novex Invitrogen) supplemented with 5% 1, 4-Dithiothreitol (DTT; Sigma-Aldrich), heated to 95°C for 10 min and centrifuged at 16,000×g for 1 min. After centrifugation, each sample was loaded into a 1.5 mm 4–20% gradient Tris-Glycine gel (Invitrogen) along with Precision Plus molecular weight markers (Bio-Rad).

    Techniques: Derivative Assay, SDS Page, Molecular Weight, Staining

    IKK-β2 derived from MP-12-infected cells retained kinetic function and was bound by curcumin. A , fractions 33–36 from MP-12-infected and UV-MP-12-infected cell extracts were pooled and immunoprecipitated with anti-IKK-β antibody. Phosphorylation reactions were carried out with the immunoprecipitated material and a GST-IκBα substrate. After incubation, samples were separated on a 4–20% Tris-glycine gel, dried, and analyzed by a PhosphorImager (Amersham Biosciences). In lane 2 ( C81 ), total C81 cell extract was used as a positive control for IKK-β activity. In lane 1 , GST alone without the IκBα substrate was used as a negative control. In lanes 7 and 8 , increasing concentrations of curcumin were added to the phosphorylation reaction. In lanes 9 and 10 , fractions 18–21 were used as the source of IKK-β, and the effect of curcumin on its kinase activity was determined. B , fractions 18–21 and 33–36 were pooled independently and subjected to a pulldown assay by incubation with immobilized curcumin beads. The washed beads were resuspended in Laemmli buffer, separated by SDS-PAGE, and analyzed by Western blot using an anti-IKK-β antibody.

    Journal: The Journal of Biological Chemistry

    Article Title: Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells *

    doi: 10.1074/jbc.M112.356535

    Figure Lengend Snippet: IKK-β2 derived from MP-12-infected cells retained kinetic function and was bound by curcumin. A , fractions 33–36 from MP-12-infected and UV-MP-12-infected cell extracts were pooled and immunoprecipitated with anti-IKK-β antibody. Phosphorylation reactions were carried out with the immunoprecipitated material and a GST-IκBα substrate. After incubation, samples were separated on a 4–20% Tris-glycine gel, dried, and analyzed by a PhosphorImager (Amersham Biosciences). In lane 2 ( C81 ), total C81 cell extract was used as a positive control for IKK-β activity. In lane 1 , GST alone without the IκBα substrate was used as a negative control. In lanes 7 and 8 , increasing concentrations of curcumin were added to the phosphorylation reaction. In lanes 9 and 10 , fractions 18–21 were used as the source of IKK-β, and the effect of curcumin on its kinase activity was determined. B , fractions 18–21 and 33–36 were pooled independently and subjected to a pulldown assay by incubation with immobilized curcumin beads. The washed beads were resuspended in Laemmli buffer, separated by SDS-PAGE, and analyzed by Western blot using an anti-IKK-β antibody.

    Article Snippet: To prepare whole cell extracts, the supernatant was removed from the wells, and cells were lysed in lysis buffer (1:1 mixture of T-PER reagent (Pierce), 2× Tris-glycine SDS sample buffer (Novex, Invitrogen), 2.5% β-mercaptoethanol, and a protease and phosphatase inhibitor mixture (1× Halt mixture, Pierce)) and boiled for 10 min prior to electrophoresis.

    Techniques: Derivative Assay, Infection, Immunoprecipitation, Incubation, Positive Control, Activity Assay, Negative Control, SDS Page, Western Blot