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  • 99
    Millipore sds gel electrophoresis
    Mapping of Hsp90–WT1 interacting domains by GST pull-down assay . (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by <t>SDS-PAGE</t> and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.
    Sds Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4345 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gel electrophoresis
    Mapping of Hsp90–WT1 interacting domains by GST pull-down assay . (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by <t>SDS-PAGE</t> and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.
    Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sodium dodecyl sulfate page
    Mapping of Hsp90–WT1 interacting domains by GST pull-down assay . (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by <t>SDS-PAGE</t> and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.
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    99
    Millipore sds polyacrylamide gels
    Intracellular trafficking and oligomerization of Ib in MDCK cells. (A) MDCK cells were incubated with Ib (1 μg/ml) at 4°C for 1 h, washed, and incubated at 37°C for the period indicated. Cells were fixed, permeabilized, and stained with anti-Ib antibody and DAPI. The Ib (red) and nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 5 μm. (B) MDCK cells were incubated with Ib (500 ng/ml) at 4°C for 1 h. The washed cells were incubated at 37°C for the period indicated. The cells were treated with Ia (100 ng/ml) and incubated at 37°C for 4 h. Pictures were taken. The total number of cells and number of round cells were counted from the pictures, and the percentage of round cells was calculated. Values are given as the mean ± standard deviation (SD) ( n = 3). (C) Cells were incubated with Ib (1 μg/ml) at 4°C for 1 h. The cells were rinsed and incubated at 37°C for the period indicated. They were lysed in <t>Tricine-SDS</t> sample buffer (Invitrogen). The cell lysates were subjected to Tricine-SDS-PAGE and Western blot analysis of Ib and β-actin as a control. A typical result from three experiments is shown.
    Sds Polyacrylamide Gels, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sds polyacrylamide gel electrophoresis page gels
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gel Electrophoresis Page Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher sds polyacrylamide gel electrophoresis gels
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
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    94
    Bio-Rad sds polyacrylamide gel electrophoresis gel
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gel Electrophoresis Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Bio-Rad gel sds polyacrylamide gel electrophoresis
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Gel Sds Polyacrylamide Gel Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Jule Inc sds polyacrylamide gel electrophoresis gels
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gel Electrophoresis Gels, supplied by Jule Inc, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific sds polyacrylamide gel electrophoresis gel
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gel Electrophoresis Gel, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    iNtRON Biotechnology sds polyacrylamide gel electrophoresis gel
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gel Electrophoresis Gel, supplied by iNtRON Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher sds polyarcylamide gel electrophoresis gel
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyarcylamide Gel Electrophoresis Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare sds polyacrylamide gel electrophoresis page gels
    Western blot analysis of expression of SF2/ASF in a clonal population of U2-OS cells stably expressing HPV-16 E2 protein. (A) Cell extracts in E buffer were prepared from U2-OS cells transformed with the E2 expression vector (E2) and from cells stably transformed with the vector alone (V). The protein concentration was measured by the Bradford assay, and equal quantities (20 μg) were electrophoresed in each <t>SDS-PAGE</t> track. Panels were Western blotted with monoclonal antibodies against the proteins shown to the right. Each experiment was carried out three times with very similar results. (B) Quantification by densitometry scanning of levels of SF2/ASF in pCMV-transformed (V) and pCMVE2-transformed (E2) U2-OS cells, showing the mean and standard deviation from the mean of three separate experiments.
    Sds Polyacrylamide Gel Electrophoresis Page Gels, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sds gel electrophoresis
    Generation of a T cell-specific conditional Coro1a knock-out mouse. A , schematic presentation of genetic modification leading to T cell-specific deletion of Coro1a. B , efficiency of T cell-specific deletion of Coro1a was determined on protein level by <t>SDS</t> Page and <t>immunoblotting.</t> Protein lysate from 1 × 10 5 MACS-sorted CD4 + and CD8 + cells of the three genotypes (Cd4-Cre transgenic mice harboring wild-type or floxed Coro1a gene and germline Coro1a −/− mice) was applied per lane. One representative Western blot of two independent experiments is shown. C, intracellular Coro1A expression was analyzed in splenocytes by flow cytometry; representative dot blots depict Coro1A and CD4 staining (gated on CD3 + cells). D , for quantification and comparison between the three different genotypes the mean fluorescent intensity of Coro1A staining in wild-type (CD4-Cre) cells was set to 100%. Results for CD4 + and CD8 + T cells and non-T cells (CD3 − ) are presented as means and single values of 4 independent experiments with a total of 4 or 5 mice. n.d ., not detectable.
    Sds Gel Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Gel Electrophoresis, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Mapping of Hsp90–WT1 interacting domains by GST pull-down assay . (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by SDS-PAGE and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.

    Journal: Blood

    Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

    doi: 10.1182/blood-2009-10-247239

    Figure Lengend Snippet: Mapping of Hsp90–WT1 interacting domains by GST pull-down assay . (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by SDS-PAGE and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.

    Article Snippet: For Western blot analysis, cell extracts or immunoprecipitates were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore).

    Techniques: Pull Down Assay, Expressing, Construct, Binding Assay, Negative Control, In Vitro, Labeling, SDS Page

    Pharmacologic inhibition of Hsp90 down-regulates WT1 protein . (A) K562 and (B) KG-1 leukemia cells were treated with the Hsp90 inhibitor 17-AAG for 24 hours. The cells were lysed, and protein extracts were subjected to SDS-PAGE and analyzed by Western blotting for WT1, Hsp90, and/or the WT1-regulated protein c-Myc. β-actin was used as loading control. (C) K562, (D) KG1, (E) Kasumi-1, and (F) MV4-11 leukemia cells were treated with the Hsp90 inhibitor STA-9090 for 24 hours and analyzed for WT1 expression by Western blotting. (G) Primary myeloid leukemia blasts from 5 AML patients (PS#1-5) were isolated, treated with STA-9090 for 24 hours, and analyzed for WT1 and β-actin by Western blotting.

    Journal: Blood

    Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

    doi: 10.1182/blood-2009-10-247239

    Figure Lengend Snippet: Pharmacologic inhibition of Hsp90 down-regulates WT1 protein . (A) K562 and (B) KG-1 leukemia cells were treated with the Hsp90 inhibitor 17-AAG for 24 hours. The cells were lysed, and protein extracts were subjected to SDS-PAGE and analyzed by Western blotting for WT1, Hsp90, and/or the WT1-regulated protein c-Myc. β-actin was used as loading control. (C) K562, (D) KG1, (E) Kasumi-1, and (F) MV4-11 leukemia cells were treated with the Hsp90 inhibitor STA-9090 for 24 hours and analyzed for WT1 expression by Western blotting. (G) Primary myeloid leukemia blasts from 5 AML patients (PS#1-5) were isolated, treated with STA-9090 for 24 hours, and analyzed for WT1 and β-actin by Western blotting.

    Article Snippet: For Western blot analysis, cell extracts or immunoprecipitates were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore).

    Techniques: Inhibition, SDS Page, Western Blot, Expressing, Isolation

    Direct interaction between WT1 and Hsp90 . (A) Equal amounts of K562 protein extracts were immunoprecipitated (IP) with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp90 antibodies, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for WT1 (top panel) and Hsp90 (bottom panel). Input represents ∼5% of the total protein extract used for immunoprecipitation. (B) Subcellular colocalization of WT1 and Hsp90. K562 cells were stained with DAPI (blue, nuclear stain) and antibodies to WT1 (green) or Hsp90 (red), and confocal images were acquired at 100× magnification. (C) GST pull-down assay. In vitro–translated and 35 S-methionine–labeled full-length Hsp90 was incubated with GST or GST-WT1 protein immobilized on glutathione-sepharose beads, and bound WT1 was detected by fluorography (top panel). 20% of the in vitro–translated protein was used for pull-downs. The bottom panel shows the purity of GST-fused proteins on a Coomassie blue-stained SDS-PAGE gel. (D) Dose-dependent binding of Hsp90 to WT1. Increasing amounts of 35 S-methionine–labeled Hsp90 were added to GST-WT1, and binding was analyzed by autoradiography.

    Journal: Blood

    Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

    doi: 10.1182/blood-2009-10-247239

    Figure Lengend Snippet: Direct interaction between WT1 and Hsp90 . (A) Equal amounts of K562 protein extracts were immunoprecipitated (IP) with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp90 antibodies, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for WT1 (top panel) and Hsp90 (bottom panel). Input represents ∼5% of the total protein extract used for immunoprecipitation. (B) Subcellular colocalization of WT1 and Hsp90. K562 cells were stained with DAPI (blue, nuclear stain) and antibodies to WT1 (green) or Hsp90 (red), and confocal images were acquired at 100× magnification. (C) GST pull-down assay. In vitro–translated and 35 S-methionine–labeled full-length Hsp90 was incubated with GST or GST-WT1 protein immobilized on glutathione-sepharose beads, and bound WT1 was detected by fluorography (top panel). 20% of the in vitro–translated protein was used for pull-downs. The bottom panel shows the purity of GST-fused proteins on a Coomassie blue-stained SDS-PAGE gel. (D) Dose-dependent binding of Hsp90 to WT1. Increasing amounts of 35 S-methionine–labeled Hsp90 were added to GST-WT1, and binding was analyzed by autoradiography.

    Article Snippet: For Western blot analysis, cell extracts or immunoprecipitates were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore).

    Techniques: Immunoprecipitation, SDS Page, Staining, Pull Down Assay, In Vitro, Labeling, Incubation, Binding Assay, Autoradiography

    Swo1p and Rng3p are required for the stability of myo2-E1p. (A) Lysates of the indicated strains were arrested at the restrictive temperature of 36°C for 5 h, resolved by SDS-PAGE (12% polyacrylamide), immunoblotted, and probed with antibodies

    Journal:

    Article Title: Hsp90 Protein in Fission Yeast Swo1p and UCS Protein Rng3p Facilitate Myosin II Assembly and Function

    doi: 10.1128/EC.4.3.567-576.2005

    Figure Lengend Snippet: Swo1p and Rng3p are required for the stability of myo2-E1p. (A) Lysates of the indicated strains were arrested at the restrictive temperature of 36°C for 5 h, resolved by SDS-PAGE (12% polyacrylamide), immunoblotted, and probed with antibodies

    Article Snippet: After the final wash, the beads were resuspended in SDS-polyacrylamide gel electrophoresis (PAGE) gel loading buffer and heated at 95°C for 5 min. Proteins were separated on SDS-8% polyacrylamide gels and transferred to a polyvinylidene difluoride sequencing. membrane (Millipore Corp., Bedford, Mass.).

    Techniques: SDS Page

    Certain PRG/IRGs are significantly expressed in the presence of FVP during a mitogenic response. (A) Effects of FVP in the cell cycle were determined by FACS of Propidium Iodide(PI)stained cells. The percent of cells in S phase is indicated at the top of each panel. BJ-TERT fibroblasts were treated with either DMSO or 300 nM FVP followed by serum stimulation for 0, 14, 22 and 30 h and stained with PI. (B) Western blot analysis of cell cycle progression, transcription markers and MCL1. BJ-TERT fibroblasts were harvested and lysed. Ten microgram of protein was resolved by 8% polyacrylamide/SDS gel electrophoresis, transferred to a PVDF membrane and specific antibodies were used to detect proteins, as indicated above. (C) 300 nM FVP inhibits phosphorylation of the CTD of RNAPII on Ser-2 and Ser-5 without affecting the phosphorylation state of pocket proteins. Exponentially growing BJ-TERT fibroblasts were treated with FVP at 10 nM, 30 nM, 100 nM, 300 nM, 1 μM and 10 μM for 2 and 4 h. Whole cell lysates were resolved by 6% SDS/PAGE and immunoblotted with antibodies to the indicated proteins/phosphorylation sites. Solid and dashed arrows at the bottom indicate the concentration of FVP leading to dephosphorylation of RNAPII and pocket proteins, respectively. The asterisk indicates a crossreacting band recognized by the anti-p130 antibody. (D) mRNA levels of selected genes at the indicated time points were detected by Q-RT-PCR. The results of three different experiments are shown. Data are represented as a fold change value of levels normalized to 1 at time zero. The results of three different experiments are shown.

    Journal: Cell Division

    Article Title: Complex effects of flavopiridol on the expression of primary response genes

    doi: 10.1186/1747-1028-7-11

    Figure Lengend Snippet: Certain PRG/IRGs are significantly expressed in the presence of FVP during a mitogenic response. (A) Effects of FVP in the cell cycle were determined by FACS of Propidium Iodide(PI)stained cells. The percent of cells in S phase is indicated at the top of each panel. BJ-TERT fibroblasts were treated with either DMSO or 300 nM FVP followed by serum stimulation for 0, 14, 22 and 30 h and stained with PI. (B) Western blot analysis of cell cycle progression, transcription markers and MCL1. BJ-TERT fibroblasts were harvested and lysed. Ten microgram of protein was resolved by 8% polyacrylamide/SDS gel electrophoresis, transferred to a PVDF membrane and specific antibodies were used to detect proteins, as indicated above. (C) 300 nM FVP inhibits phosphorylation of the CTD of RNAPII on Ser-2 and Ser-5 without affecting the phosphorylation state of pocket proteins. Exponentially growing BJ-TERT fibroblasts were treated with FVP at 10 nM, 30 nM, 100 nM, 300 nM, 1 μM and 10 μM for 2 and 4 h. Whole cell lysates were resolved by 6% SDS/PAGE and immunoblotted with antibodies to the indicated proteins/phosphorylation sites. Solid and dashed arrows at the bottom indicate the concentration of FVP leading to dephosphorylation of RNAPII and pocket proteins, respectively. The asterisk indicates a crossreacting band recognized by the anti-p130 antibody. (D) mRNA levels of selected genes at the indicated time points were detected by Q-RT-PCR. The results of three different experiments are shown. Data are represented as a fold change value of levels normalized to 1 at time zero. The results of three different experiments are shown.

    Article Snippet: Proteins were resolved by 8% polyacrylamide/SDS gel electrophoresis, and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-FL, Millipore) in 10 mM CAPS/10% methanol buffer (pH 11).

    Techniques: FACS, Staining, Western Blot, SDS-Gel, Electrophoresis, SDS Page, Concentration Assay, De-Phosphorylation Assay, Reverse Transcription Polymerase Chain Reaction

    DEF-A DNA-binding activity is associated with a hypophosphorylated form of the L4 33-kDa protein. Whole-cell extracts were prepared from HeLa cells infected with Ad5 for the times indicated or mock infected (M) and examined for proteins that bind to the ML DE sequence (A) or by immunoblotting for the L4 33-kDa protein (B). The positions of the a, b, and c complexes are indicated on the right of panel A. Extracts prepared from 293FT cells transiently synthesizing the L4 33-kDa protein were loaded onto the lanes marked L4 in panel B before (−) or after (+) treatment with 400 units phosphatase (PPase) for 1 h at 30°C, followed by incubation for 1 h at 65°C. (C) Proteins present in the 24-h-infected cell extract (I) and that prepared from L4 33-kDa protein-producing 293FT cells (L4) shown in panel A were separated by electrophoresis in 15% SDS-polyacrylamide gel electrophoresis and examined by immunoblotting them with polyclonal antibodies against the L4 33-kDa protein. (D) The infected cell extract (I) was examined as described above before (−) and after (+) treatment with phosphatase. p.i., postinfection.

    Journal: Journal of Virology

    Article Title: The Adenovirus L4 33-Kilodalton Protein Binds to Intragenic Sequences of the Major Late Promoter Required for Late Phase-Specific Stimulation of Transcription ▿

    doi: 10.1128/JVI.01584-06

    Figure Lengend Snippet: DEF-A DNA-binding activity is associated with a hypophosphorylated form of the L4 33-kDa protein. Whole-cell extracts were prepared from HeLa cells infected with Ad5 for the times indicated or mock infected (M) and examined for proteins that bind to the ML DE sequence (A) or by immunoblotting for the L4 33-kDa protein (B). The positions of the a, b, and c complexes are indicated on the right of panel A. Extracts prepared from 293FT cells transiently synthesizing the L4 33-kDa protein were loaded onto the lanes marked L4 in panel B before (−) or after (+) treatment with 400 units phosphatase (PPase) for 1 h at 30°C, followed by incubation for 1 h at 65°C. (C) Proteins present in the 24-h-infected cell extract (I) and that prepared from L4 33-kDa protein-producing 293FT cells (L4) shown in panel A were separated by electrophoresis in 15% SDS-polyacrylamide gel electrophoresis and examined by immunoblotting them with polyclonal antibodies against the L4 33-kDa protein. (D) The infected cell extract (I) was examined as described above before (−) and after (+) treatment with phosphatase. p.i., postinfection.

    Article Snippet: Unless otherwise indicated, proteins were separated by electrophoresis in 10% SDS-polyacrylamide gel electrophoresis gels and examined by immunoblotting, as described previously , using the M2 anti-FLAG monoclonal antibody (Sigma) or the antibodies against the L4 33-kDa and IVa2 proteins.

    Techniques: Binding Assay, Activity Assay, Infection, Sequencing, Incubation, Electrophoresis, Polyacrylamide Gel Electrophoresis

    Effect of IFN γ and TNF α costimulation on cytochrome c , Bcl-2, and Bax levels in MC3T3-E1 cells. Cells were cultured as described in Figure 4 and treated with 10 ng/ml IFN γ and 5 ng/ml TNF α . Samples were collected at various time points and fractionated into cytosolic and mitochondrial (Mito) fractions, of which 20 μ g protein was subjected to 12% SDS-PAGE and immunoblotted with antibodies to cytochrome c . The mitochondrial fraction was also analyzed by western blot for Bcl-2 and Bax. Results are representative of three independent experiments.

    Journal: Mediators of Inflammation

    Article Title: Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria

    doi: 10.1155/2018/3979606

    Figure Lengend Snippet: Effect of IFN γ and TNF α costimulation on cytochrome c , Bcl-2, and Bax levels in MC3T3-E1 cells. Cells were cultured as described in Figure 4 and treated with 10 ng/ml IFN γ and 5 ng/ml TNF α . Samples were collected at various time points and fractionated into cytosolic and mitochondrial (Mito) fractions, of which 20 μ g protein was subjected to 12% SDS-PAGE and immunoblotted with antibodies to cytochrome c . The mitochondrial fraction was also analyzed by western blot for Bcl-2 and Bax. Results are representative of three independent experiments.

    Article Snippet: Western Blot Protein samples obtained as described were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) using a semidry transfer cell (Bio-Rad).

    Techniques: Cell Culture, SDS Page, Western Blot

    Overexpression of Bcl-2 attenuates IFN γ - and TNF α -induced cytotoxicity in MC3T3-E1 cells. (a) Western blot for Bcl-2 in cells stably expressing Bcl-2. Total cell lysates were prepared from cells stably transfected with control vector (pcDNA3) or a Bcl-2 expression vector (pCMV-Bcl-2), of which 20 μ g protein was subjected to 12% SDS-PAGE and analyzed with antibodies to Bcl-2. The blot was stripped and reprobed with anti-tubulin to confirm loading of equal amounts of total protein. (b) Effect of IFN γ and TNF α costimulation on the viability of cells stably expressing Bcl-2. Cells stably transfected with control vector or pCMV-Bcl-2 were seeded in 96-well plates, incubated for 5 days to form a confluent monolayer, and costimulated with 10 ng/ml IFN γ and 5 ng/ml TNF α . Cell viability was monitored over time. Data represent the mean ± SEM of three independent experiments. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria

    doi: 10.1155/2018/3979606

    Figure Lengend Snippet: Overexpression of Bcl-2 attenuates IFN γ - and TNF α -induced cytotoxicity in MC3T3-E1 cells. (a) Western blot for Bcl-2 in cells stably expressing Bcl-2. Total cell lysates were prepared from cells stably transfected with control vector (pcDNA3) or a Bcl-2 expression vector (pCMV-Bcl-2), of which 20 μ g protein was subjected to 12% SDS-PAGE and analyzed with antibodies to Bcl-2. The blot was stripped and reprobed with anti-tubulin to confirm loading of equal amounts of total protein. (b) Effect of IFN γ and TNF α costimulation on the viability of cells stably expressing Bcl-2. Cells stably transfected with control vector or pCMV-Bcl-2 were seeded in 96-well plates, incubated for 5 days to form a confluent monolayer, and costimulated with 10 ng/ml IFN γ and 5 ng/ml TNF α . Cell viability was monitored over time. Data represent the mean ± SEM of three independent experiments. ∗ p

    Article Snippet: Western Blot Protein samples obtained as described were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) using a semidry transfer cell (Bio-Rad).

    Techniques: Over Expression, Western Blot, Stable Transfection, Expressing, Transfection, Plasmid Preparation, SDS Page, Incubation

    IRF3-dependent enhancement of gene transduction by recombinant baculovirus. (A) Cell extracts of MEFs derived from WT or IRF3 −/− mice and FLAG-mIRF3-transduced IRF3 −/− MEFs were subjected to SDS-PAGE and immunoblotted with antibodies against FLAG, IRF3, or β-actin, respectively. (B) MEFs derived from WT or IRF3 −/− mice and FLAG-mIRF3-transduced IRF3-deficient MEFs were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent the means ± SD from 3 independent experiments. (C) MEFs were inoculated with rBV-luc at an MOI of 100. At 6 h after inoculation, cells were fixed in 50% methanol–50% acetone for 10 min. IRF3 (green) was stained with the appropriate antibodies, followed by staining with Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained by DAPI.

    Journal: Journal of Virology

    Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

    doi: 10.1128/JVI.03055-13

    Figure Lengend Snippet: IRF3-dependent enhancement of gene transduction by recombinant baculovirus. (A) Cell extracts of MEFs derived from WT or IRF3 −/− mice and FLAG-mIRF3-transduced IRF3 −/− MEFs were subjected to SDS-PAGE and immunoblotted with antibodies against FLAG, IRF3, or β-actin, respectively. (B) MEFs derived from WT or IRF3 −/− mice and FLAG-mIRF3-transduced IRF3-deficient MEFs were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent the means ± SD from 3 independent experiments. (C) MEFs were inoculated with rBV-luc at an MOI of 100. At 6 h after inoculation, cells were fixed in 50% methanol–50% acetone for 10 min. IRF3 (green) was stained with the appropriate antibodies, followed by staining with Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained by DAPI.

    Article Snippet: The proteins were boiled in 20 μl of sample buffer, subjected to sodium dodecyl sulfate–12.5% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan).

    Techniques: Transduction, Recombinant, Derivative Assay, Mouse Assay, SDS Page, Luciferase, Activity Assay, Staining

    Involvement of TBK1 in efficient transgene expression by recombinant baculovirus. (A) MEFs derived from TBK1 +/− IKKβ +/− (WT) and TBK1 −/− IKKβ +/− (TBK1 −/− ) mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, the GFP signal was determined by microscopic observation after fixation in 4% paraformaldehyde. (B) MEFs derived from WT and TBK1 −/− mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, TBK1, or β-actin, respectively. (C) MEFs derived from WT and TBK1-deficient mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNA was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA.

    Journal: Journal of Virology

    Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

    doi: 10.1128/JVI.03055-13

    Figure Lengend Snippet: Involvement of TBK1 in efficient transgene expression by recombinant baculovirus. (A) MEFs derived from TBK1 +/− IKKβ +/− (WT) and TBK1 −/− IKKβ +/− (TBK1 −/− ) mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, the GFP signal was determined by microscopic observation after fixation in 4% paraformaldehyde. (B) MEFs derived from WT and TBK1 −/− mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, TBK1, or β-actin, respectively. (C) MEFs derived from WT and TBK1-deficient mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNA was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA.

    Article Snippet: The proteins were boiled in 20 μl of sample buffer, subjected to sodium dodecyl sulfate–12.5% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan).

    Techniques: Expressing, Recombinant, Derivative Assay, Mouse Assay, SDS Page, Real-time Polymerase Chain Reaction

    Efficient gene transduction by recombinant baculovirus in HCV replicon-harboring cells. (A) Huh7OK1 cells (cured) and the HCV replicon-harboring cells derived from genotype 1a (RMT strain), 1b (con1 strain), and 2a (JFH1 strain) were inoculated with rBV-luc (MOI of 100) or VSV-luc at an MOI of 5 and (B) with rBV-luc at MOI of 5, 10, 25, and 50. At 24 h after inoculation, the luciferase activity of cell lysates was determined. (C) Huh7 cells were inoculated with rBV-GFP (MOI of 100) or VSV-GFP (NCP mutant) at an MOI of 0.05. At 24 h after inoculation, total RNA was extracted, and the expression of IP-10, ISG15, and IL-8 mRNAs was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA. (D) Huh7 cells infected with HCVcc at an MOI of 1 and incubated for 72 h were inoculated with rBV-GFP (MOI of 100) in the presence or absence of human recombinant IFN-α (rIFN-α) (100 U/ml). At 24 h after inoculation, the cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, NS5A, or β-actin, respectively. (E) Relative luciferase activity in Huh7 cells with IRF3, IPS-1, or STING knocked down. Cells were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity of cell lysates was determined (right panel). Luciferase activity is normalized to control shNC cells, and data represent the means ± SD from 2 independent experiments. Total RNA was extracted, and the expression of IRF3, IPS-1, or STING mRNA was determined by real-time PCR (left panel). Data from the real-time PCR were normalized to the amount of GAPDH mRNA.

    Journal: Journal of Virology

    Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

    doi: 10.1128/JVI.03055-13

    Figure Lengend Snippet: Efficient gene transduction by recombinant baculovirus in HCV replicon-harboring cells. (A) Huh7OK1 cells (cured) and the HCV replicon-harboring cells derived from genotype 1a (RMT strain), 1b (con1 strain), and 2a (JFH1 strain) were inoculated with rBV-luc (MOI of 100) or VSV-luc at an MOI of 5 and (B) with rBV-luc at MOI of 5, 10, 25, and 50. At 24 h after inoculation, the luciferase activity of cell lysates was determined. (C) Huh7 cells were inoculated with rBV-GFP (MOI of 100) or VSV-GFP (NCP mutant) at an MOI of 0.05. At 24 h after inoculation, total RNA was extracted, and the expression of IP-10, ISG15, and IL-8 mRNAs was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA. (D) Huh7 cells infected with HCVcc at an MOI of 1 and incubated for 72 h were inoculated with rBV-GFP (MOI of 100) in the presence or absence of human recombinant IFN-α (rIFN-α) (100 U/ml). At 24 h after inoculation, the cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, NS5A, or β-actin, respectively. (E) Relative luciferase activity in Huh7 cells with IRF3, IPS-1, or STING knocked down. Cells were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity of cell lysates was determined (right panel). Luciferase activity is normalized to control shNC cells, and data represent the means ± SD from 2 independent experiments. Total RNA was extracted, and the expression of IRF3, IPS-1, or STING mRNA was determined by real-time PCR (left panel). Data from the real-time PCR were normalized to the amount of GAPDH mRNA.

    Article Snippet: The proteins were boiled in 20 μl of sample buffer, subjected to sodium dodecyl sulfate–12.5% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan).

    Techniques: Transduction, Recombinant, Derivative Assay, Luciferase, Activity Assay, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Infection, Incubation, SDS Page

    Efficient gene transduction by recombinant baculovirus in the IRF3 −/− MEFs. (A) MEFs derived from wild-type (WT) and IRF3 −/− , IRF7 −/− , or MyD88 −/− mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, GFP signal was determined by microscopic observation after fixation in 4% paraformaldehyde. (B) MEFs derived from WT and IRF3 −/− mice were inoculated with rBV-GFP at an MOI of 100, incubated at 4°C for 30 min, and washed three times with PBS. The total cellular DNA was extracted, and the amounts of baculovirus genome were quantified by real-time PCR. Data represent means ± SD from 2 independent experiments. (C) MEFs derived from WT and IRF3 −/− mice were inoculated with 2 doses of rBV-GFP (MOI of 100 or 20). At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNAs was determined by real-time PCR. (D) MEFs derived from WT and IRF3-deficient mice were inoculated with 2 doses of rBV-GFP (MOI of 100 and 20). At 24 h after inoculation, cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, IRF3, or β-actin, respectively.

    Journal: Journal of Virology

    Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

    doi: 10.1128/JVI.03055-13

    Figure Lengend Snippet: Efficient gene transduction by recombinant baculovirus in the IRF3 −/− MEFs. (A) MEFs derived from wild-type (WT) and IRF3 −/− , IRF7 −/− , or MyD88 −/− mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, GFP signal was determined by microscopic observation after fixation in 4% paraformaldehyde. (B) MEFs derived from WT and IRF3 −/− mice were inoculated with rBV-GFP at an MOI of 100, incubated at 4°C for 30 min, and washed three times with PBS. The total cellular DNA was extracted, and the amounts of baculovirus genome were quantified by real-time PCR. Data represent means ± SD from 2 independent experiments. (C) MEFs derived from WT and IRF3 −/− mice were inoculated with 2 doses of rBV-GFP (MOI of 100 or 20). At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNAs was determined by real-time PCR. (D) MEFs derived from WT and IRF3-deficient mice were inoculated with 2 doses of rBV-GFP (MOI of 100 and 20). At 24 h after inoculation, cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, IRF3, or β-actin, respectively.

    Article Snippet: The proteins were boiled in 20 μl of sample buffer, subjected to sodium dodecyl sulfate–12.5% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan).

    Techniques: Transduction, Recombinant, Derivative Assay, Mouse Assay, Incubation, Real-time Polymerase Chain Reaction, Expressing, SDS Page

    RLR signaling pathways participate in the suppression of gene transduction in MEFs upon infection with recombinant baculovirus. (A) MEFs derived from wild-type (WT) and IPS-1-, TBK1-, ZBP1-, or IRF3-deficient mice were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent means ± SD from 3 independent experiments. (B) MEFs derived from WT and IPS-1 −/− mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, GFP expression was detected by microscopic observation after fixation in 4% paraformaldehyde. (C) MEFs derived from WT or IPS-1-deficient mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNAs was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA. (D) Cell extracts of MEFs derived from WT or IPS-1 −/− mice and FLAG-mIPS-1-transduced IPS-1 −/− MEFs were subjected to SDS-PAGE and immunoblotted with antibodies against FLAG, IPS-1, or β-actin, respectively. (E) MEFs derived from WT or IPS-1 −/− mice and FLAG-mIPS-1-transduced IPS-1-deficient MEFs were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent means ± SD from 3 independent experiments.

    Journal: Journal of Virology

    Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

    doi: 10.1128/JVI.03055-13

    Figure Lengend Snippet: RLR signaling pathways participate in the suppression of gene transduction in MEFs upon infection with recombinant baculovirus. (A) MEFs derived from wild-type (WT) and IPS-1-, TBK1-, ZBP1-, or IRF3-deficient mice were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent means ± SD from 3 independent experiments. (B) MEFs derived from WT and IPS-1 −/− mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, GFP expression was detected by microscopic observation after fixation in 4% paraformaldehyde. (C) MEFs derived from WT or IPS-1-deficient mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNAs was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA. (D) Cell extracts of MEFs derived from WT or IPS-1 −/− mice and FLAG-mIPS-1-transduced IPS-1 −/− MEFs were subjected to SDS-PAGE and immunoblotted with antibodies against FLAG, IPS-1, or β-actin, respectively. (E) MEFs derived from WT or IPS-1 −/− mice and FLAG-mIPS-1-transduced IPS-1-deficient MEFs were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent means ± SD from 3 independent experiments.

    Article Snippet: The proteins were boiled in 20 μl of sample buffer, subjected to sodium dodecyl sulfate–12.5% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan).

    Techniques: Transduction, Infection, Recombinant, Derivative Assay, Mouse Assay, Luciferase, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, SDS Page

    Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Down regulation of cIAP-1, cIAP-2, XIAP, MDM2 and activation of p53 AGS and SNU-484 were treated with indicated concentrations of scutellarein or 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of cIAP-1,-2, XIAP, MDM2, p53 and p-p53 proteins expressions were expressed as mean ± SD of three independent experiments. ( ** P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Activation Assay, SDS Page

    Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Regulatory effect of Scutellarein on cell cycle progression of AGS and SNU484 cells AGS and SNU-484cells were treated with indicated concentrations of scutellarein for 24 h. ( A – B ) Cell cycle distribution was determined by using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).The data were analyzed using CXP Software. ( C–D ) Effect of Scutellarein on cell cycle-related proteins (cyclin B1, CDK1 and CDC25C) expression level in A549 cells. Cells were treated with Scutellarein (0, 25, 50 and 100 μM) for 24 h. Cell lysates were subjected to SDS–PAGE and analyzed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of Scutellarein on expression of cell cycle-related proteins level were represented. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗ P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Software, Expressing, SDS Page, Western Blot, Standard Deviation

    Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

    Journal: Oncotarget

    Article Title: Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

    doi: 10.18632/oncotarget.23202

    Figure Lengend Snippet: Caspases activation and subsequent cleavage of PARP in scutellarein -treated AGS and SNU-484cells AGS and SNU-484 cells were treated with indicated concentrations of scutellarein for 24 h. The cell lysates were subjected to SDS–PAGE and analyzed by immune-blotting. Densitometry analyses of Bax/Bcl-xL ratio, Cl.caspase-9, Cl.caspase-3 and Cl.PARP proteins expressions were expressed as the mean ± standard deviation (SD) of at least three independent experiments. ( ∗∗ P

    Article Snippet: Proteins were separated by 8%–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare Life Sciences, Buckinghamshire, UK).

    Techniques: Activation Assay, SDS Page, Standard Deviation

    Immunostaining of SDS-PAGE-fractionated native C. parvum (Cp) or C. baileyi (Cb) oocyst protein or Ni-NTA-purified (P) and unpurified (IP) recombinant CP41 protein (rCp41) with rabbit antiserum to whole C. parvum oocyst protein (R α CpOO) or to native (R α NATIVE Cp41) or recombinant (R α RECOMBINANT Cp41) CP41 antigen or with normal control rabbit serum (NRS). The positions of the 28-, 36-, and 41-kDa proteins are noted. MrS, molecular weight standards.

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Cloning and Expression of a DNA Sequence Encoding a 41-Kilodalton Cryptosporidium parvum Oocyst Wall Protein

    doi:

    Figure Lengend Snippet: Immunostaining of SDS-PAGE-fractionated native C. parvum (Cp) or C. baileyi (Cb) oocyst protein or Ni-NTA-purified (P) and unpurified (IP) recombinant CP41 protein (rCp41) with rabbit antiserum to whole C. parvum oocyst protein (R α CpOO) or to native (R α NATIVE Cp41) or recombinant (R α RECOMBINANT Cp41) CP41 antigen or with normal control rabbit serum (NRS). The positions of the 28-, 36-, and 41-kDa proteins are noted. MrS, molecular weight standards.

    Article Snippet: Protein extracts of Cryptosporidium oocysts were treated with sample buffer containing 2-mercaptoethanol , heated for 3 min in a boiling-water bath, fractionated by 7.5 to 15% gradient SDS-polyacrylamide gel electrophoresis (PAGE), and transblotted to an Immobilon (Millipore, Bedford, Mass.) membrane as described previously ( ).

    Techniques: Immunostaining, SDS Page, Purification, Recombinant, Molecular Weight

    Purification and characterization of Chia from chicken glandular stomach. ( a ) Chia was purified from chicken glandular stomach tissues using chitin beads chromatography as described in Methods and analyzed by SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining. 1, extract; 2, flow- through; 3, purified enzyme. Purified Chia (1 μg of protein) was electrophoresed and visualized in the gel by CBB. ( b ) Zymogram of chicken Chia. After electrophoresis, the gel was analyzed by Zymography (left), followed by CBB staining (right). Zymography was performed as described in Methods. ( c ) Optimal pH and ( d ) optimal temperature for Chia activity. Values in ( c , d ) represent mean ± SD conducted in triplicate. **p

    Journal: Scientific Reports

    Article Title: Gastric and intestinal proteases resistance of chicken acidic chitinase nominates chitin-containing organisms for alternative whole edible diets for poultry

    doi: 10.1038/s41598-017-07146-3

    Figure Lengend Snippet: Purification and characterization of Chia from chicken glandular stomach. ( a ) Chia was purified from chicken glandular stomach tissues using chitin beads chromatography as described in Methods and analyzed by SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining. 1, extract; 2, flow- through; 3, purified enzyme. Purified Chia (1 μg of protein) was electrophoresed and visualized in the gel by CBB. ( b ) Zymogram of chicken Chia. After electrophoresis, the gel was analyzed by Zymography (left), followed by CBB staining (right). Zymography was performed as described in Methods. ( c ) Optimal pH and ( d ) optimal temperature for Chia activity. Values in ( c , d ) represent mean ± SD conducted in triplicate. **p

    Article Snippet: SDS-polyacrylamide gel electrophoresis and CBB staining or SYPRO Ruby staining The obtained protein fractions were performed using standard SDS-polyacrylamide gel electrophoresis (PAGE) , followed by Coomassie Brilliant Blue R-250 (Sigma-Aldrich) or SYPRO Ruby staining (Thermo Fisher Scientific) and analyzed using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare).

    Techniques: Purification, Chromatography, SDS Page, Staining, Flow Cytometry, Electrophoresis, Zymography, Activity Assay

    Functional stability of chicken Chia against gastrointestinal proteases. Purified Chia was incubated at 37 °C for 0, 10, 30, and 60 min under stomach-like ( a , b ) or intestine-like ( c , d ) environment in the presence of pepsin or trypsin and chymotrypsin at equal mass concentration of Chia. The samples were analyzed by SDS-PAGE and following ( a , c ) SYPRO Ruby staining, ( b , d ) chitinolytic activities. C, purified enzyme only; P, pepsin only; T/C, trypsin and chymotrypsin only; numbers, incubation time. Values in ( b , d ) represent mean ± SD conducted in triplicate. *p

    Journal: Scientific Reports

    Article Title: Gastric and intestinal proteases resistance of chicken acidic chitinase nominates chitin-containing organisms for alternative whole edible diets for poultry

    doi: 10.1038/s41598-017-07146-3

    Figure Lengend Snippet: Functional stability of chicken Chia against gastrointestinal proteases. Purified Chia was incubated at 37 °C for 0, 10, 30, and 60 min under stomach-like ( a , b ) or intestine-like ( c , d ) environment in the presence of pepsin or trypsin and chymotrypsin at equal mass concentration of Chia. The samples were analyzed by SDS-PAGE and following ( a , c ) SYPRO Ruby staining, ( b , d ) chitinolytic activities. C, purified enzyme only; P, pepsin only; T/C, trypsin and chymotrypsin only; numbers, incubation time. Values in ( b , d ) represent mean ± SD conducted in triplicate. *p

    Article Snippet: SDS-polyacrylamide gel electrophoresis and CBB staining or SYPRO Ruby staining The obtained protein fractions were performed using standard SDS-polyacrylamide gel electrophoresis (PAGE) , followed by Coomassie Brilliant Blue R-250 (Sigma-Aldrich) or SYPRO Ruby staining (Thermo Fisher Scientific) and analyzed using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare).

    Techniques: Functional Assay, Purification, Incubation, Concentration Assay, SDS Page, Staining

    Functional stability of chicken Chia against endogenous pepsin. Soluble proteins obtained from chicken glandular stomach were incubated at pH 2.0 and 37 °C for 0, 10, 40 and 60 min ( a – c ). After the incubation, the samples were further incubated at pH 7.6 for 1 hour with trypsin and chymotrypsin in extract to the reaction ( d – f ). The samples were analyzed as follows: ( a , d ) SDS-PAGE and following SYPRO Ruby staining, ( b , e ) zymography, ( c , f ) chitinolytic activities. Ex, extract only; T/C, trypsin and chymotrypsin only; numbers, incubation time at pH 2.0 (red) or at pH 7.6 (light blue). Values in ( c , f ) represent mean ± SD conducted in triplicate.

    Journal: Scientific Reports

    Article Title: Gastric and intestinal proteases resistance of chicken acidic chitinase nominates chitin-containing organisms for alternative whole edible diets for poultry

    doi: 10.1038/s41598-017-07146-3

    Figure Lengend Snippet: Functional stability of chicken Chia against endogenous pepsin. Soluble proteins obtained from chicken glandular stomach were incubated at pH 2.0 and 37 °C for 0, 10, 40 and 60 min ( a – c ). After the incubation, the samples were further incubated at pH 7.6 for 1 hour with trypsin and chymotrypsin in extract to the reaction ( d – f ). The samples were analyzed as follows: ( a , d ) SDS-PAGE and following SYPRO Ruby staining, ( b , e ) zymography, ( c , f ) chitinolytic activities. Ex, extract only; T/C, trypsin and chymotrypsin only; numbers, incubation time at pH 2.0 (red) or at pH 7.6 (light blue). Values in ( c , f ) represent mean ± SD conducted in triplicate.

    Article Snippet: SDS-polyacrylamide gel electrophoresis and CBB staining or SYPRO Ruby staining The obtained protein fractions were performed using standard SDS-polyacrylamide gel electrophoresis (PAGE) , followed by Coomassie Brilliant Blue R-250 (Sigma-Aldrich) or SYPRO Ruby staining (Thermo Fisher Scientific) and analyzed using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare).

    Techniques: Functional Assay, Incubation, SDS Page, Staining, Zymography

    Biochemical interaction of the GPA-12–CeRhoGEF pathway. ( A ) CeRGS-RhoGEF interacts with activated GPA-12 in the presence of . Myc-tagged gpa-12 was cotransfected with the flag-tagged RGS domain of CeRhoGEF (CeRGS-RhoGEF) in COS-7 cells. GPA-12 was immunoprecipitated (IP) with anti-myc antibody from the cell lysates in the presence or absence of . The immunoprecipitates were separated on SDS/PAGE and immunoblotted with anti-flag ( Top ) or anti-myc (second panel from Top ) antibody. Expression of gpa-12 ( Bottom , with anti-myc antibody) or CeRGS-RhoGEF (second panel from Bottom , with anti-flag antibody) in lysates is shown. ( B ) Stimulation of SRF activity by the DH–PH domain of CeRhoGEF. COS-7 cells were transfected with SRE.L-luciferase reporter (0.5 μg), pCMV-β-galactosidase (0.5 μg), and the indicated expression plasmids, myc- LARG DH–PH (0.5 μg), myc- CeRhoGEF DH–PH (6 μg), or pEGFP-C3 (0.5 μg). SRF activities of cell lysates were measured as described in Methods . The expression of LARG DH–PH or CeRhoGEF DH–PH in lysates was detected by immunoblotting with anti-myc antibody ( Bottom ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Identification and molecular characterization of the G?12-Rho guanine nucleotide exchange factor pathway in Caenorhabditis elegans

    doi: 10.1073/pnas.2533143100

    Figure Lengend Snippet: Biochemical interaction of the GPA-12–CeRhoGEF pathway. ( A ) CeRGS-RhoGEF interacts with activated GPA-12 in the presence of . Myc-tagged gpa-12 was cotransfected with the flag-tagged RGS domain of CeRhoGEF (CeRGS-RhoGEF) in COS-7 cells. GPA-12 was immunoprecipitated (IP) with anti-myc antibody from the cell lysates in the presence or absence of . The immunoprecipitates were separated on SDS/PAGE and immunoblotted with anti-flag ( Top ) or anti-myc (second panel from Top ) antibody. Expression of gpa-12 ( Bottom , with anti-myc antibody) or CeRGS-RhoGEF (second panel from Bottom , with anti-flag antibody) in lysates is shown. ( B ) Stimulation of SRF activity by the DH–PH domain of CeRhoGEF. COS-7 cells were transfected with SRE.L-luciferase reporter (0.5 μg), pCMV-β-galactosidase (0.5 μg), and the indicated expression plasmids, myc- LARG DH–PH (0.5 μg), myc- CeRhoGEF DH–PH (6 μg), or pEGFP-C3 (0.5 μg). SRF activities of cell lysates were measured as described in Methods . The expression of LARG DH–PH or CeRhoGEF DH–PH in lysates was detected by immunoblotting with anti-myc antibody ( Bottom ).

    Article Snippet: The immunoprecipitates were loaded onto 10% SDS/polyacrylamide gel for electrophoresis and immunoblotted with anti-myc antibody (Sigma) or anti-flag M2 antibody (Sigma).

    Techniques: Immunoprecipitation, SDS Page, Expressing, Activity Assay, Transfection, Luciferase

    Association of Rsu-1 and PINCH-1. (A) 293T and HA-PINCH-1-expressing 293T cells were lysed and affinity precipitated using anti-HA affinity matrix. The precipitates were separated by SDS-PAGE and silver stained. Each band was excised and identified by LC-MS/MS. (B) 293T cells were transfected with HA-tagged FL, ΔLIM1, ΔC, and LIM1 and immunoprecipitated with anti-HA antibody. Immunoprecipitates were probed for Rsu-1 and HA. (C) 293T cells were transfected with myc-tagged Rsu-1, and cell lysates were affinity precipitated with GST and fusion proteins of GST and the indicated fragments. Precipitates were subjected to immunoblotting with anti-myc antibody. The lower panel shows Coomassie blue staining of GST fusion proteins. LIM5: aa251–304, LIM5+C: aa251–325, C: aa305–325. (D) Schematic representation of Rsu-1 deletion mutants that were used in the experiments: FL, aa1–277; LRR, aa1–202; C, aa203–277. (E) 293T cells were transfected with myc-tagged full-length or mutant Rsu-1, and cell lysates were affinity precipitated with a fusion protein of GST and LIM5+C. Precipitates were subjected to immunoblotting with anti-myc antibody. (F) FL cells were seeded onto fibronectin-coated coverslips and fixed 20 min, 60 min, or 8 h later to visualize the localization of GFP-PINCH-1 and Rsu-1 during cell spreading (scale bar = 20 μm): 20 min, attached and round; 60 min, initial spread; 8 h, stable spread. (G) shCtrl and shPINCH cells were immunostained with anti-vinculin and anti–Rsu-1 antibodies (scale bar = 20 μm).

    Journal: Molecular Biology of the Cell

    Article Title: The Roles of Two Distinct Regions of PINCH-1 in the Regulation of Cell Attachment and Spreading

    doi: 10.1091/mbc.E10-05-0459

    Figure Lengend Snippet: Association of Rsu-1 and PINCH-1. (A) 293T and HA-PINCH-1-expressing 293T cells were lysed and affinity precipitated using anti-HA affinity matrix. The precipitates were separated by SDS-PAGE and silver stained. Each band was excised and identified by LC-MS/MS. (B) 293T cells were transfected with HA-tagged FL, ΔLIM1, ΔC, and LIM1 and immunoprecipitated with anti-HA antibody. Immunoprecipitates were probed for Rsu-1 and HA. (C) 293T cells were transfected with myc-tagged Rsu-1, and cell lysates were affinity precipitated with GST and fusion proteins of GST and the indicated fragments. Precipitates were subjected to immunoblotting with anti-myc antibody. The lower panel shows Coomassie blue staining of GST fusion proteins. LIM5: aa251–304, LIM5+C: aa251–325, C: aa305–325. (D) Schematic representation of Rsu-1 deletion mutants that were used in the experiments: FL, aa1–277; LRR, aa1–202; C, aa203–277. (E) 293T cells were transfected with myc-tagged full-length or mutant Rsu-1, and cell lysates were affinity precipitated with a fusion protein of GST and LIM5+C. Precipitates were subjected to immunoblotting with anti-myc antibody. (F) FL cells were seeded onto fibronectin-coated coverslips and fixed 20 min, 60 min, or 8 h later to visualize the localization of GFP-PINCH-1 and Rsu-1 during cell spreading (scale bar = 20 μm): 20 min, attached and round; 60 min, initial spread; 8 h, stable spread. (G) shCtrl and shPINCH cells were immunostained with anti-vinculin and anti–Rsu-1 antibodies (scale bar = 20 μm).

    Article Snippet: Equal protein quantities were separated on SDS-polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to PVDF membrane (Millipore, Billerica, MA).

    Techniques: Expressing, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transfection, Immunoprecipitation, Mutagenesis

    Phosphorylation of Akt by different forms of CK2. (A) Increasing amounts of CK2α (1.25, 2.5 and 5 ng), CK2α’ (40, 80 and 160 ng) or CK2α2β2 (2.5, 5 and 10 ng), corresponding to equal activity towards CK2-tide peptide, were incubated with 0.3 μg of active Akt1 or 0.3 μg of inactive Akt1, or 1 μg β-casein as a control. In the right panel, 0.3 μg of active form of Akt was incubated with CK2α (1.25 ng), CK2α2β2 (5 ng), CK2α’ (40 ng) or CK2α’2β2 (25 ng) in the presence of 150 mM NaCl. The amount of each enzyme used was equally active against the CK2 specific peptide (CK2-tide: RRRADDSDDDDD ) as determined by kinase activity assays (not shown). Upon radioactive phosphorylation, proteins were separated by SDS-PAGE. A representative digital autoradiography of the dried gel is shown. The bar graph on the right shows the relative quantitation of the bands (means ± SEM, n = 3), performed by analysis with CyclonePlus Storage Phosphor System, (PerkinElmer); the unit amounts of each isoform are indicated, and activity is reported as % of that measured with 5 units of CK2α. (B) shCV HK-2 and shCK2β HK-2 were transiently transfected with CK2β pCMV-HA vector. Representative Western blot analysis with the indicated antibodies is shown. β-actin was used as a loading control. Quantification (p-Akt(S129)/ AKT) is shown on the bar graph on the right (means ± SEM, n = 2).

    Journal: PLoS ONE

    Article Title: Effects of CK2β subunit down-regulation on Akt signalling in HK-2 renal cells

    doi: 10.1371/journal.pone.0227340

    Figure Lengend Snippet: Phosphorylation of Akt by different forms of CK2. (A) Increasing amounts of CK2α (1.25, 2.5 and 5 ng), CK2α’ (40, 80 and 160 ng) or CK2α2β2 (2.5, 5 and 10 ng), corresponding to equal activity towards CK2-tide peptide, were incubated with 0.3 μg of active Akt1 or 0.3 μg of inactive Akt1, or 1 μg β-casein as a control. In the right panel, 0.3 μg of active form of Akt was incubated with CK2α (1.25 ng), CK2α2β2 (5 ng), CK2α’ (40 ng) or CK2α’2β2 (25 ng) in the presence of 150 mM NaCl. The amount of each enzyme used was equally active against the CK2 specific peptide (CK2-tide: RRRADDSDDDDD ) as determined by kinase activity assays (not shown). Upon radioactive phosphorylation, proteins were separated by SDS-PAGE. A representative digital autoradiography of the dried gel is shown. The bar graph on the right shows the relative quantitation of the bands (means ± SEM, n = 3), performed by analysis with CyclonePlus Storage Phosphor System, (PerkinElmer); the unit amounts of each isoform are indicated, and activity is reported as % of that measured with 5 units of CK2α. (B) shCV HK-2 and shCK2β HK-2 were transiently transfected with CK2β pCMV-HA vector. Representative Western blot analysis with the indicated antibodies is shown. β-actin was used as a loading control. Quantification (p-Akt(S129)/ AKT) is shown on the bar graph on the right (means ± SEM, n = 2).

    Article Snippet: Equal amounts of proteins were loaded in 10% SDS polyacrylamide gels (SDS-PAGE), subjected to electrophoresis and subsequently electrotransfered to polyvinylidene fluoride membranes (PVDF, Immobilion P, Millipore).

    Techniques: Activity Assay, Incubation, SDS Page, Autoradiography, Quantitation Assay, Transfection, Plasmid Preparation, Western Blot

    Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.

    Journal: PLoS ONE

    Article Title: Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli

    doi: 10.1371/journal.pone.0149718

    Figure Lengend Snippet: Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.

    Article Snippet: The proteins were separated by SDS-polyacrylamide (12% or 10%) gel electrophoresis (SDS-PAGE) and were transferred onto an Immobilon membrane (Millipore).

    Techniques: Incubation, Magnetic Beads, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Intracellular trafficking and oligomerization of Ib in MDCK cells. (A) MDCK cells were incubated with Ib (1 μg/ml) at 4°C for 1 h, washed, and incubated at 37°C for the period indicated. Cells were fixed, permeabilized, and stained with anti-Ib antibody and DAPI. The Ib (red) and nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 5 μm. (B) MDCK cells were incubated with Ib (500 ng/ml) at 4°C for 1 h. The washed cells were incubated at 37°C for the period indicated. The cells were treated with Ia (100 ng/ml) and incubated at 37°C for 4 h. Pictures were taken. The total number of cells and number of round cells were counted from the pictures, and the percentage of round cells was calculated. Values are given as the mean ± standard deviation (SD) ( n = 3). (C) Cells were incubated with Ib (1 μg/ml) at 4°C for 1 h. The cells were rinsed and incubated at 37°C for the period indicated. They were lysed in Tricine-SDS sample buffer (Invitrogen). The cell lysates were subjected to Tricine-SDS-PAGE and Western blot analysis of Ib and β-actin as a control. A typical result from three experiments is shown.

    Journal: Infection and Immunity

    Article Title: Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

    doi: 10.1128/IAI.00483-12

    Figure Lengend Snippet: Intracellular trafficking and oligomerization of Ib in MDCK cells. (A) MDCK cells were incubated with Ib (1 μg/ml) at 4°C for 1 h, washed, and incubated at 37°C for the period indicated. Cells were fixed, permeabilized, and stained with anti-Ib antibody and DAPI. The Ib (red) and nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 5 μm. (B) MDCK cells were incubated with Ib (500 ng/ml) at 4°C for 1 h. The washed cells were incubated at 37°C for the period indicated. The cells were treated with Ia (100 ng/ml) and incubated at 37°C for 4 h. Pictures were taken. The total number of cells and number of round cells were counted from the pictures, and the percentage of round cells was calculated. Values are given as the mean ± standard deviation (SD) ( n = 3). (C) Cells were incubated with Ib (1 μg/ml) at 4°C for 1 h. The cells were rinsed and incubated at 37°C for the period indicated. They were lysed in Tricine-SDS sample buffer (Invitrogen). The cell lysates were subjected to Tricine-SDS-PAGE and Western blot analysis of Ib and β-actin as a control. A typical result from three experiments is shown.

    Article Snippet: For Western blot analysis, the proteins on SDS-polyacrylamide gels and Tricine-SDS-polyacrylamide gels were transferred to polyvinylidene difluoride membranes (Immobilon P; Millipore).

    Techniques: Incubation, Staining, Microscopy, IA, Standard Deviation, SDS Page, Western Blot

    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by SDS–PAGE and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).

    Journal: Immunology

    Article Title: Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes

    doi: 10.1046/j.1365-2567.2001.01249.x

    Figure Lengend Snippet: Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by SDS–PAGE and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).

    Article Snippet: Samples were run on 15%, 10% or 6% SDS–polyacrylamide gel electrophoresis (PAGE) gels for C3, factor H or C4bp, respectively, in a Mini-PROTEAN II system (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK), then electroblotted onto nitrocellulose.

    Techniques: Incubation, SDS Page, Western Blot, Produced, Southern Blot, Polymerase Chain Reaction, Amplification, Negative Control

    Western blot analysis of expression of SF2/ASF in a clonal population of U2-OS cells stably expressing HPV-16 E2 protein. (A) Cell extracts in E buffer were prepared from U2-OS cells transformed with the E2 expression vector (E2) and from cells stably transformed with the vector alone (V). The protein concentration was measured by the Bradford assay, and equal quantities (20 μg) were electrophoresed in each SDS-PAGE track. Panels were Western blotted with monoclonal antibodies against the proteins shown to the right. Each experiment was carried out three times with very similar results. (B) Quantification by densitometry scanning of levels of SF2/ASF in pCMV-transformed (V) and pCMVE2-transformed (E2) U2-OS cells, showing the mean and standard deviation from the mean of three separate experiments.

    Journal: Journal of Virology

    Article Title: SF2/ASF Binds the Human Papillomavirus Type 16 Late RNA Control Element and Is Regulated during Differentiation of Virus-Infected Epithelial Cells

    doi: 10.1128/JVI.78.19.10598-10605.2004

    Figure Lengend Snippet: Western blot analysis of expression of SF2/ASF in a clonal population of U2-OS cells stably expressing HPV-16 E2 protein. (A) Cell extracts in E buffer were prepared from U2-OS cells transformed with the E2 expression vector (E2) and from cells stably transformed with the vector alone (V). The protein concentration was measured by the Bradford assay, and equal quantities (20 μg) were electrophoresed in each SDS-PAGE track. Panels were Western blotted with monoclonal antibodies against the proteins shown to the right. Each experiment was carried out three times with very similar results. (B) Quantification by densitometry scanning of levels of SF2/ASF in pCMV-transformed (V) and pCMVE2-transformed (E2) U2-OS cells, showing the mean and standard deviation from the mean of three separate experiments.

    Article Snippet: For Western blot analysis, proteins fractionated on SDS-polyacrylamide gel electrophoresis (PAGE) gels were electroblotted onto polyvinylidene difluoride membranes (Amersham).

    Techniques: Western Blot, Expressing, Stable Transfection, Transformation Assay, Plasmid Preparation, Protein Concentration, Bradford Assay, SDS Page, Standard Deviation

    Generation of a T cell-specific conditional Coro1a knock-out mouse. A , schematic presentation of genetic modification leading to T cell-specific deletion of Coro1a. B , efficiency of T cell-specific deletion of Coro1a was determined on protein level by SDS Page and immunoblotting. Protein lysate from 1 × 10 5 MACS-sorted CD4 + and CD8 + cells of the three genotypes (Cd4-Cre transgenic mice harboring wild-type or floxed Coro1a gene and germline Coro1a −/− mice) was applied per lane. One representative Western blot of two independent experiments is shown. C, intracellular Coro1A expression was analyzed in splenocytes by flow cytometry; representative dot blots depict Coro1A and CD4 staining (gated on CD3 + cells). D , for quantification and comparison between the three different genotypes the mean fluorescent intensity of Coro1A staining in wild-type (CD4-Cre) cells was set to 100%. Results for CD4 + and CD8 + T cells and non-T cells (CD3 − ) are presented as means and single values of 4 independent experiments with a total of 4 or 5 mice. n.d ., not detectable.

    Journal: The Journal of Biological Chemistry

    Article Title: Proof of Principle for a T Lymphocyte Intrinsic Function of Coronin 1A *

    doi: 10.1074/jbc.M116.748012

    Figure Lengend Snippet: Generation of a T cell-specific conditional Coro1a knock-out mouse. A , schematic presentation of genetic modification leading to T cell-specific deletion of Coro1a. B , efficiency of T cell-specific deletion of Coro1a was determined on protein level by SDS Page and immunoblotting. Protein lysate from 1 × 10 5 MACS-sorted CD4 + and CD8 + cells of the three genotypes (Cd4-Cre transgenic mice harboring wild-type or floxed Coro1a gene and germline Coro1a −/− mice) was applied per lane. One representative Western blot of two independent experiments is shown. C, intracellular Coro1A expression was analyzed in splenocytes by flow cytometry; representative dot blots depict Coro1A and CD4 staining (gated on CD3 + cells). D , for quantification and comparison between the three different genotypes the mean fluorescent intensity of Coro1A staining in wild-type (CD4-Cre) cells was set to 100%. Results for CD4 + and CD8 + T cells and non-T cells (CD3 − ) are presented as means and single values of 4 independent experiments with a total of 4 or 5 mice. n.d ., not detectable.

    Article Snippet: Cell lysates from splenocytes were prepared and subjected together with precision plus protein standard (Bio-Rad) to SDS-gel electrophoresis and immunoblotting as described previously ( ).

    Techniques: Knock-Out, Modification, SDS Page, Magnetic Cell Separation, Transgenic Assay, Mouse Assay, Western Blot, Expressing, Flow Cytometry, Cytometry, Staining

    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. SDS-PAGE analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by SyproRed staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.

    Journal: The Journal of Cell Biology

    Article Title: ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

    doi: 10.1083/jcb.200112092

    Figure Lengend Snippet: ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. SDS-PAGE analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by SyproRed staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.

    Article Snippet: Eluted proteins were analyzed by SDS gel electrophoresis, SyproRed staining, and scanning with a Storm PhosphorImager (Amersham Pharmacia Biotech).

    Techniques: Binding Assay, SDS Page, In Vitro, Concentration Assay, Centrifugation, Staining

    Glo3p induce conformational changes on v-SNARE–GST fusion proteins. (A) Bet1p-GST and Sec22p-GST were mock treated (−Glo3p) or pretreated with ARF-GAP (+Glo3p). The ARF-GAP was removed before the addition of the proteases to the SNAREs. Samples were withdrawn at the indicated time points and analyzed by SDS-PAGE and SyproRed staining. B shows a quantification of A. The standard derivation was calculated from six and nine experiments for Bet1p-GST and Sec22p-GST, respectively. (C) Sec23/24p complex can bind to Bet1p-(1–65)–GST even in the absence of Sar1p upon activation by Glo3p. Binding reactions were performed under the same conditions as above. Lane 5 shows 20% of the Sar1p that was present in the binding reactions. Sec23/24p complex was added to all reactions. (D) Binding of Sar1p and Sec23/24p complex is enhanced in the presence of Glo3p. Binding assays to Bet1p-GST were performed under optimized conditions for COPII binding as described by Springer and Schekman (1998) . GTP-γ–S was present in all reactions in C and D.

    Journal: The Journal of Cell Biology

    Article Title: ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

    doi: 10.1083/jcb.200112092

    Figure Lengend Snippet: Glo3p induce conformational changes on v-SNARE–GST fusion proteins. (A) Bet1p-GST and Sec22p-GST were mock treated (−Glo3p) or pretreated with ARF-GAP (+Glo3p). The ARF-GAP was removed before the addition of the proteases to the SNAREs. Samples were withdrawn at the indicated time points and analyzed by SDS-PAGE and SyproRed staining. B shows a quantification of A. The standard derivation was calculated from six and nine experiments for Bet1p-GST and Sec22p-GST, respectively. (C) Sec23/24p complex can bind to Bet1p-(1–65)–GST even in the absence of Sar1p upon activation by Glo3p. Binding reactions were performed under the same conditions as above. Lane 5 shows 20% of the Sar1p that was present in the binding reactions. Sec23/24p complex was added to all reactions. (D) Binding of Sar1p and Sec23/24p complex is enhanced in the presence of Glo3p. Binding assays to Bet1p-GST were performed under optimized conditions for COPII binding as described by Springer and Schekman (1998) . GTP-γ–S was present in all reactions in C and D.

    Article Snippet: Eluted proteins were analyzed by SDS gel electrophoresis, SyproRed staining, and scanning with a Storm PhosphorImager (Amersham Pharmacia Biotech).

    Techniques: SDS Page, Staining, Activation Assay, Binding Assay