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Agilent technologies quikchange polymerase chain reaction pcr
Quikchange Polymerase Chain Reaction Pcr, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/quikchange polymerase chain reaction pcr/product/Agilent technologies
Average 96 stars, based on 1 article reviews
Price from $9.99 to $1999.99
quikchange polymerase chain reaction pcr - by Bioz Stars, 2020-04
96/100 stars

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Related Articles

Transfection:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: Paragraph title: cDNA Expression Plasmids and Transfection of Human Embryonic Kidney-293 and COS7 Cells. ... The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara).

Fluorescence:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Polymerase Chain Reaction:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: .. The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: .. The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Mutagenesis:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: .. The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: .. The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Construct:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: N-terminally tagged YFP and CFP versions of MKK7 α 1, β 1, and γ 1 were constructed by transfer of Flag-MKK7 encoding HindIII-XbaI fragments into pEC/YFPC1 (Takara Bio USA, Inc., Mountain View, CA). .. The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara).

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: N-terminally tagged YFP and CFP versions of MKK7 α 1, β 1, and γ 1 were constructed by transfer of Flag-MKK7 encoding HindIII-XbaI fragments into pEC/YFPC1 (Takara Bio USA, Inc., Mountain View, CA). .. The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara).

Immunoprecipitation:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Förster Resonance Energy Transfer:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Expressing:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: Paragraph title: cDNA Expression Plasmids and Transfection of Human Embryonic Kidney-293 and COS7 Cells. ... The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara).

Modification:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Cells were then washed with phosphate-buffered saline (PBS), fed with normal growth medium (Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 1% penicillin/streptomycin; Invitrogen, Carlsbad, CA) and grown for 24–48 hours prior to fluorescence imaging or preparation of cell extracts for IP.

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Cells were then washed with phosphate-buffered saline (PBS), fed with normal growth medium (Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 1% penicillin/streptomycin; Invitrogen, Carlsbad, CA) and grown for 24–48 hours prior to fluorescence imaging or preparation of cell extracts for IP.

Plasmid Preparation:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Imaging:

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

Article Title: Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin
Article Snippet: The ΔPIX mutation deleting residues 41PIIVIT46 in MKK7 γ 1 was introduced by Stratagene QuikChange polymerase chain reaction (PCR) (Agilent Technologies Inc., Santa Clara). .. Human embryonic kidney (HEK)-293 and COS7 cells at 20%–50% confluency (24–48 hours after plating) were transfected by calcium phosphate precipitation with the various plasmid cDNA expression constructs [1–2 μ g each plasmid per 25-mm glass coverslip in six-well plates for fluorescence resonance energy transfer (FRET) imaging or 2–10 μ g each plasmid for 10-cm plates for immunoprecipitation (IP)] for 4–6 hours at 5% CO2 and 37°C.

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    Agilent technologies mouse cblb promoter
    <t>HLXB9</t> suppresses the expression of <t>Cblb</t> mRNA by suppressing Cblb promoter activity. A and B , among targets identified by anti-HB9-PO4 ChIP-Seq, the expression of only Arid1b and Cblb was affected by HLXB9. Quantitative RT-PCR is shown of the indicated genes using RNA prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ), empty vector, or mh-HB9-WT. A , Western blot confirming knockdown or overexpression of HLXB9 with anti-HLXB9 or anti-myc-tag, respectively, and β-actin as the loading control. B , HLXB9 knockdown or overexpression regulated the relative mRNA level of only two genes (Arid1b and Cblb). Both were reduced upon HLXB9 overexpression, but only Cblb was increased upon HLXB9 knockdown. Error bar = mean and S.D. from three experiments. * = p
    Mouse Cblb Promoter, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse cblb promoter/product/Agilent technologies
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    99
    Agilent technologies quikchange ii site directed mutagenesis kit
    <t>HLXB9</t> suppresses the expression of <t>Cblb</t> mRNA by suppressing Cblb promoter activity. A and B , among targets identified by anti-HB9-PO4 ChIP-Seq, the expression of only Arid1b and Cblb was affected by HLXB9. Quantitative RT-PCR is shown of the indicated genes using RNA prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ), empty vector, or mh-HB9-WT. A , Western blot confirming knockdown or overexpression of HLXB9 with anti-HLXB9 or anti-myc-tag, respectively, and β-actin as the loading control. B , HLXB9 knockdown or overexpression regulated the relative mRNA level of only two genes (Arid1b and Cblb). Both were reduced upon HLXB9 overexpression, but only Cblb was increased upon HLXB9 knockdown. Error bar = mean and S.D. from three experiments. * = p
    Quikchange Ii Site Directed Mutagenesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 1317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quikchange ii site directed mutagenesis kit/product/Agilent technologies
    Average 99 stars, based on 1317 article reviews
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    Agilent technologies quikchange lightning mutagenic pcr kit
    <t>HLXB9</t> suppresses the expression of <t>Cblb</t> mRNA by suppressing Cblb promoter activity. A and B , among targets identified by anti-HB9-PO4 ChIP-Seq, the expression of only Arid1b and Cblb was affected by HLXB9. Quantitative RT-PCR is shown of the indicated genes using RNA prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ), empty vector, or mh-HB9-WT. A , Western blot confirming knockdown or overexpression of HLXB9 with anti-HLXB9 or anti-myc-tag, respectively, and β-actin as the loading control. B , HLXB9 knockdown or overexpression regulated the relative mRNA level of only two genes (Arid1b and Cblb). Both were reduced upon HLXB9 overexpression, but only Cblb was increased upon HLXB9 knockdown. Error bar = mean and S.D. from three experiments. * = p
    Quikchange Lightning Mutagenic Pcr Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies pfu quikchange pcr
    <t>HLXB9</t> suppresses the expression of <t>Cblb</t> mRNA by suppressing Cblb promoter activity. A and B , among targets identified by anti-HB9-PO4 ChIP-Seq, the expression of only Arid1b and Cblb was affected by HLXB9. Quantitative RT-PCR is shown of the indicated genes using RNA prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ), empty vector, or mh-HB9-WT. A , Western blot confirming knockdown or overexpression of HLXB9 with anti-HLXB9 or anti-myc-tag, respectively, and β-actin as the loading control. B , HLXB9 knockdown or overexpression regulated the relative mRNA level of only two genes (Arid1b and Cblb). Both were reduced upon HLXB9 overexpression, but only Cblb was increased upon HLXB9 knockdown. Error bar = mean and S.D. from three experiments. * = p
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    Image Search Results


    HLXB9 suppresses the expression of Cblb mRNA by suppressing Cblb promoter activity. A and B , among targets identified by anti-HB9-PO4 ChIP-Seq, the expression of only Arid1b and Cblb was affected by HLXB9. Quantitative RT-PCR is shown of the indicated genes using RNA prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ), empty vector, or mh-HB9-WT. A , Western blot confirming knockdown or overexpression of HLXB9 with anti-HLXB9 or anti-myc-tag, respectively, and β-actin as the loading control. B , HLXB9 knockdown or overexpression regulated the relative mRNA level of only two genes (Arid1b and Cblb). Both were reduced upon HLXB9 overexpression, but only Cblb was increased upon HLXB9 knockdown. Error bar = mean and S.D. from three experiments. * = p

    Journal: The Journal of Biological Chemistry

    Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

    doi: 10.1074/jbc.M115.661413

    Figure Lengend Snippet: HLXB9 suppresses the expression of Cblb mRNA by suppressing Cblb promoter activity. A and B , among targets identified by anti-HB9-PO4 ChIP-Seq, the expression of only Arid1b and Cblb was affected by HLXB9. Quantitative RT-PCR is shown of the indicated genes using RNA prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ), empty vector, or mh-HB9-WT. A , Western blot confirming knockdown or overexpression of HLXB9 with anti-HLXB9 or anti-myc-tag, respectively, and β-actin as the loading control. B , HLXB9 knockdown or overexpression regulated the relative mRNA level of only two genes (Arid1b and Cblb). Both were reduced upon HLXB9 overexpression, but only Cblb was increased upon HLXB9 knockdown. Error bar = mean and S.D. from three experiments. * = p

    Article Snippet: For luciferase reporter assays the promoter-less pEZX-PG02 vector and the promoter constructs pEZX-PG02-Arid1b and pEZX-PG02-Cblb were purchased and confirmed by sequencing (GeneCopoeia). pEZX-PG02-Cblb-SDM2 was constructed by site-directed mutagenesis of the two HLXB9 binding motifs at −735 and −722 in the mouse Cblb promoter (Agilent, QuikChange site-directed mutagenesis kit).

    Techniques: Expressing, Activity Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Over Expression

    HLXB9 binding motif in the Cblb promoter. A , consensus motif in anti-HB9-PO4 ChIP-Seq tags located at promoter regions. De novo ). B , sequence of the HLXB9 binding motifs in the Cblb promoter. The top line shows the putative HLXB9 binding motifs ( red ) in the DNA sequence of the Cblb promoter (−741 to −710 region from the transcriptional start site is shown). The bottom line shows nucleotide substitutions ( blue ) to mutate the motifs by site-directed mutagenesis in the Cblb-promoter construct used in C. C and D , HLXB9 did not suppress the activity of the Cblb promoter containing mutations at the HLXB9 binding motifs. The putative HLXB9 binding motifs shown in B were mutated by site-directed mutagenesis of the PG02-Cblb promoter construct and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb-SDM2 plasmid showed significantly high RLU and co-expression of increasing amounts of HLXB9 did not suppress the Cblb promoter activity. Error bar = Mean and S.D. from 3 experiments, * = p

    Journal: The Journal of Biological Chemistry

    Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

    doi: 10.1074/jbc.M115.661413

    Figure Lengend Snippet: HLXB9 binding motif in the Cblb promoter. A , consensus motif in anti-HB9-PO4 ChIP-Seq tags located at promoter regions. De novo ). B , sequence of the HLXB9 binding motifs in the Cblb promoter. The top line shows the putative HLXB9 binding motifs ( red ) in the DNA sequence of the Cblb promoter (−741 to −710 region from the transcriptional start site is shown). The bottom line shows nucleotide substitutions ( blue ) to mutate the motifs by site-directed mutagenesis in the Cblb-promoter construct used in C. C and D , HLXB9 did not suppress the activity of the Cblb promoter containing mutations at the HLXB9 binding motifs. The putative HLXB9 binding motifs shown in B were mutated by site-directed mutagenesis of the PG02-Cblb promoter construct and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb-SDM2 plasmid showed significantly high RLU and co-expression of increasing amounts of HLXB9 did not suppress the Cblb promoter activity. Error bar = Mean and S.D. from 3 experiments, * = p

    Article Snippet: For luciferase reporter assays the promoter-less pEZX-PG02 vector and the promoter constructs pEZX-PG02-Arid1b and pEZX-PG02-Cblb were purchased and confirmed by sequencing (GeneCopoeia). pEZX-PG02-Cblb-SDM2 was constructed by site-directed mutagenesis of the two HLXB9 binding motifs at −735 and −722 in the mouse Cblb promoter (Agilent, QuikChange site-directed mutagenesis kit).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Sequencing, Mutagenesis, Construct, Activity Assay, Transfection, Plasmid Preparation, Expressing

    Increased phospho-HLXB9, decreased Cblb, and increased c-Met in insulinoma from the conventional mouse model of menin loss ( Men1 +/− ). Shown are images of immunofluorescence for insulin and IHC for the indicated proteins in the pancreas section of an 18-month-old Men1 +/− mouse. Insulin staining shows the location of the normal-looking islet ( panels on the left ) and the large islet tumor that covers the entire viewing field ( panels on the right ). Compared with a normal-looking insulin-positive islet in the same section, the insulin-positive islet tumor shows increased nuclear staining for HB9 and HB9-PO4 and increased nuclear and cytoplasmic staining for c-Met but almost no staining for Cblb (cytoplasm).

    Journal: The Journal of Biological Chemistry

    Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

    doi: 10.1074/jbc.M115.661413

    Figure Lengend Snippet: Increased phospho-HLXB9, decreased Cblb, and increased c-Met in insulinoma from the conventional mouse model of menin loss ( Men1 +/− ). Shown are images of immunofluorescence for insulin and IHC for the indicated proteins in the pancreas section of an 18-month-old Men1 +/− mouse. Insulin staining shows the location of the normal-looking islet ( panels on the left ) and the large islet tumor that covers the entire viewing field ( panels on the right ). Compared with a normal-looking insulin-positive islet in the same section, the insulin-positive islet tumor shows increased nuclear staining for HB9 and HB9-PO4 and increased nuclear and cytoplasmic staining for c-Met but almost no staining for Cblb (cytoplasm).

    Article Snippet: For luciferase reporter assays the promoter-less pEZX-PG02 vector and the promoter constructs pEZX-PG02-Arid1b and pEZX-PG02-Cblb were purchased and confirmed by sequencing (GeneCopoeia). pEZX-PG02-Cblb-SDM2 was constructed by site-directed mutagenesis of the two HLXB9 binding motifs at −735 and −722 in the mouse Cblb promoter (Agilent, QuikChange site-directed mutagenesis kit).

    Techniques: Immunofluorescence, Immunohistochemistry, Staining

    Increased phospho-HLXB9, decreased Cblb, and increased c-Met in insulinoma from the conditional mouse model of menin loss (RIP-Cre- Men1 f/f ). Images of immunofluorescence for insulin and IHC for the indicated proteins in pancreas sections from a 12-month-old Men1 f/f mouse and RIP-Cre- Men1 f/f mouse. Compared with the insulin-positive normal islet ( panels on the left ), the large insulin-positive islet tumor that covers the entire viewing field ( panels on the right ) shows increased nuclear staining for HB9 and HB9-PO4, increased nuclear and cytoplasmic staining for c-Met, and decreased staining for Cblb (cytoplasm).

    Journal: The Journal of Biological Chemistry

    Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

    doi: 10.1074/jbc.M115.661413

    Figure Lengend Snippet: Increased phospho-HLXB9, decreased Cblb, and increased c-Met in insulinoma from the conditional mouse model of menin loss (RIP-Cre- Men1 f/f ). Images of immunofluorescence for insulin and IHC for the indicated proteins in pancreas sections from a 12-month-old Men1 f/f mouse and RIP-Cre- Men1 f/f mouse. Compared with the insulin-positive normal islet ( panels on the left ), the large insulin-positive islet tumor that covers the entire viewing field ( panels on the right ) shows increased nuclear staining for HB9 and HB9-PO4, increased nuclear and cytoplasmic staining for c-Met, and decreased staining for Cblb (cytoplasm).

    Article Snippet: For luciferase reporter assays the promoter-less pEZX-PG02 vector and the promoter constructs pEZX-PG02-Arid1b and pEZX-PG02-Cblb were purchased and confirmed by sequencing (GeneCopoeia). pEZX-PG02-Cblb-SDM2 was constructed by site-directed mutagenesis of the two HLXB9 binding motifs at −735 and −722 in the mouse Cblb promoter (Agilent, QuikChange site-directed mutagenesis kit).

    Techniques: Immunofluorescence, Immunohistochemistry, Staining

    Cblb overexpression or HLXB9 knockdown inactivates the oncogenic c-Met pathway. A , overexpression of Cblb or knockdown of HLXB9 decreases c-Met levels. Western blot analysis of the indicated proteins using WCE prepared from MIN6-4N cells transfected (by nucleofection) with empty vector or Cblb expression plasmid, control siRNA ( siC ), or HLXB9 siRNA ( siHB9 ) and control shRNA ( shC ) or Nono shRNA ( shNono ). Cblb overexpression reduced the level of endogenous c-Met. HLXB9 knockdown increased the level of endogenous Cblb and reduced the level of endogenous c-Met. p84 was used as the loading control. The upper band marked pro-c-Met is the glycosylated c-Met precursor form that is cleaved and processed into mature c-Met ( lower band ). B , reduced cell proliferation from Cblb overexpression but not from HLXB9 or Nono knockdown. An MTT assay assessed cell proliferation of MIN6-4N cells transfected in A . Overexpression of Cblb caused a slight but significant reduction in cell proliferation, but cell proliferation was unaffected upon HLXB9 or Nono knockdown. Note that the Western blots in A were performed using WCE prepared at 96 h post-transfection. Error bar = mean and S.D. from 3 experiments, * = p

    Journal: The Journal of Biological Chemistry

    Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

    doi: 10.1074/jbc.M115.661413

    Figure Lengend Snippet: Cblb overexpression or HLXB9 knockdown inactivates the oncogenic c-Met pathway. A , overexpression of Cblb or knockdown of HLXB9 decreases c-Met levels. Western blot analysis of the indicated proteins using WCE prepared from MIN6-4N cells transfected (by nucleofection) with empty vector or Cblb expression plasmid, control siRNA ( siC ), or HLXB9 siRNA ( siHB9 ) and control shRNA ( shC ) or Nono shRNA ( shNono ). Cblb overexpression reduced the level of endogenous c-Met. HLXB9 knockdown increased the level of endogenous Cblb and reduced the level of endogenous c-Met. p84 was used as the loading control. The upper band marked pro-c-Met is the glycosylated c-Met precursor form that is cleaved and processed into mature c-Met ( lower band ). B , reduced cell proliferation from Cblb overexpression but not from HLXB9 or Nono knockdown. An MTT assay assessed cell proliferation of MIN6-4N cells transfected in A . Overexpression of Cblb caused a slight but significant reduction in cell proliferation, but cell proliferation was unaffected upon HLXB9 or Nono knockdown. Note that the Western blots in A were performed using WCE prepared at 96 h post-transfection. Error bar = mean and S.D. from 3 experiments, * = p

    Article Snippet: For luciferase reporter assays the promoter-less pEZX-PG02 vector and the promoter constructs pEZX-PG02-Arid1b and pEZX-PG02-Cblb were purchased and confirmed by sequencing (GeneCopoeia). pEZX-PG02-Cblb-SDM2 was constructed by site-directed mutagenesis of the two HLXB9 binding motifs at −735 and −722 in the mouse Cblb promoter (Agilent, QuikChange site-directed mutagenesis kit).

    Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, shRNA, MTT Assay

    Identification of Cblb as a phospho-HLXB9 target gene. A , the anti-HB9-PO4 antibody specifically recognizes the phosphorylated isoform of HLXB9. WCE and chromatin were prepared from MIN6-4N cells transfected with a plasmid expressing myc-his-tagged HLXB9 ( mh-HB9-WT ). WCE was used IP with rabbit antibodies anti-myc-tag or anti-HB9-PO4 and detected by Western blot ( WB ) with mouse anti-myc-tag. Rabbit anti-HA-tag was used as the negative control. The input WCE and anti-myc-tag IP display a doublet corresponding to phospho-HLXB9 and unphosphorylated HLXB9. Anti-HB9-PO4 could specifically immunoprecipitate phospho-HLXB9 corresponding to the top band of the doublet. B , significant enrichment of promoter regions among the anti-HB9-PO4 ChIP-Seq tags. Chromatin prepared from MIN6-4N cells transfected in A was used for ChIP with anti-HB9-PO4. DNA obtained before and after ChIP was used for preparing libraries followed by deep sequencing (ChIP-Seq) and mapping of the anti-HB9-PO4-specific ChIP-Seq tags to the mouse genome. The pie chart shows the percent distribution of tags in the mouse genome (a typical input library, Genomatix) and in the anti-HB9-PO4 ChIP-Seq at the indicated regions; 20% of the anti-HB9-PO4 ChIP-Seq tags were located near promoter regions and selected for further analysis. C , phospho-HLXB9 occupancy is highest at the Arid1b and Cblb gene in cells overexpressing HLXB9. ChIP-quantitative PCR assay for validating the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells expressing mh-HB9-WT was used for anti-HB9-PO4 ChIP. Also shown is a Western blot confirming overexpression of HLXB9 (myc-tag antibody) and β-actin as the loading control. D , endogenous phospho-HLXB9 occupancy is highest at the Cblb gene. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells was used for endogenous anti-HB9-PO4 ChIP. Endogenous phospho-HLXB9 occupancy was only detected at Cblb. E and F , H3K4me3 at Cblb unaffected but reduced H3K27me3 upon HLXB9 knockdown. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) was used for endogenous anti-H3K4me3 ChIP ( E ) or H3K27me3 ChIP ( F ). Also shown is a Western blot confirming knockdown of HLXB9 (HLXB9 antibody) and β-actin as the loading control. In siC versus siHB9, reciprocal H3K4me3 or H3K27me3 at only Cblb was HLXB9 binding-dependent because endogenous phospho-HLXB9 was only found to occupy Cblb ( D ).

    Journal: The Journal of Biological Chemistry

    Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

    doi: 10.1074/jbc.M115.661413

    Figure Lengend Snippet: Identification of Cblb as a phospho-HLXB9 target gene. A , the anti-HB9-PO4 antibody specifically recognizes the phosphorylated isoform of HLXB9. WCE and chromatin were prepared from MIN6-4N cells transfected with a plasmid expressing myc-his-tagged HLXB9 ( mh-HB9-WT ). WCE was used IP with rabbit antibodies anti-myc-tag or anti-HB9-PO4 and detected by Western blot ( WB ) with mouse anti-myc-tag. Rabbit anti-HA-tag was used as the negative control. The input WCE and anti-myc-tag IP display a doublet corresponding to phospho-HLXB9 and unphosphorylated HLXB9. Anti-HB9-PO4 could specifically immunoprecipitate phospho-HLXB9 corresponding to the top band of the doublet. B , significant enrichment of promoter regions among the anti-HB9-PO4 ChIP-Seq tags. Chromatin prepared from MIN6-4N cells transfected in A was used for ChIP with anti-HB9-PO4. DNA obtained before and after ChIP was used for preparing libraries followed by deep sequencing (ChIP-Seq) and mapping of the anti-HB9-PO4-specific ChIP-Seq tags to the mouse genome. The pie chart shows the percent distribution of tags in the mouse genome (a typical input library, Genomatix) and in the anti-HB9-PO4 ChIP-Seq at the indicated regions; 20% of the anti-HB9-PO4 ChIP-Seq tags were located near promoter regions and selected for further analysis. C , phospho-HLXB9 occupancy is highest at the Arid1b and Cblb gene in cells overexpressing HLXB9. ChIP-quantitative PCR assay for validating the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells expressing mh-HB9-WT was used for anti-HB9-PO4 ChIP. Also shown is a Western blot confirming overexpression of HLXB9 (myc-tag antibody) and β-actin as the loading control. D , endogenous phospho-HLXB9 occupancy is highest at the Cblb gene. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells was used for endogenous anti-HB9-PO4 ChIP. Endogenous phospho-HLXB9 occupancy was only detected at Cblb. E and F , H3K4me3 at Cblb unaffected but reduced H3K27me3 upon HLXB9 knockdown. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells transfected with control siRNA ( siC ) or HLXB9 siRNA ( siHB9 ) was used for endogenous anti-H3K4me3 ChIP ( E ) or H3K27me3 ChIP ( F ). Also shown is a Western blot confirming knockdown of HLXB9 (HLXB9 antibody) and β-actin as the loading control. In siC versus siHB9, reciprocal H3K4me3 or H3K27me3 at only Cblb was HLXB9 binding-dependent because endogenous phospho-HLXB9 was only found to occupy Cblb ( D ).

    Article Snippet: For luciferase reporter assays the promoter-less pEZX-PG02 vector and the promoter constructs pEZX-PG02-Arid1b and pEZX-PG02-Cblb were purchased and confirmed by sequencing (GeneCopoeia). pEZX-PG02-Cblb-SDM2 was constructed by site-directed mutagenesis of the two HLXB9 binding motifs at −735 and −722 in the mouse Cblb promoter (Agilent, QuikChange site-directed mutagenesis kit).

    Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control, Chromatin Immunoprecipitation, Sequencing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Over Expression, Binding Assay