shrimp alkaline phosphatase sap  (TaKaRa)

 
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    Name:
    Alkaline Phosphatase
    Description:
    Calf intestinal alkaline phosphatase CIAP catalyzes the hydrolysis of 5 phosphate termini from DNA RNA dNTPs and rNTPs This enzyme can be used to reduce linear vector self ligation and vector background during cloning CIAP is supplied in a buffer of 10 mM Tris HCl pH 8 0 50 mM KCl 1 mM MgCl2 0 1 mM ZnCl2 and 50 glycerol
    Catalog Number:
    2250b
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    5 000 Units
    Category:
    Alkaline phosphatase calf intestinal Phosphatases and kinases Modifying enzymes Cloning
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    Structured Review

    TaKaRa shrimp alkaline phosphatase sap
    Identification of phosphorylation sites of mouse HP1α. (A) The phosphorylation patterns of mouse HP1α analyzed by phos-tag Western blotting (phos-tag WB). Whole-cell lysates (WCLs) of NIH 3T3 cells were resolved on an SDS-PAGE gel containing phos-tag acrylamide (phos-tag) or on a standard SDS-PAGE gel (mock). As an unphosphorylated control, <t>SAP-treated</t> <t>WCL</t> was loaded on the same gel. HP1α was detected using an anti-HP1α antibody. (B) Phosphorylation patterns of mouse HP1α in cells arrested at different cell cycle stages. WCLs were prepared from NIH 3T3 cells grown under the indicated conditions and analyzed by phos-tag WB. The arrowhead indicates an HP1α with basal band shift. The asterisk represents an HP1α species with metaphase-associated phosphorylation. The same membrane was immunoblotted with anti-α-tubulin and anti-TIF1β antibodies for loading controls. (C) Amino acid sequence of mouse HP1α. Light- and dark-shaded boxes indicate the CD and CSD, respectively. The sites of serine (S)-to-alanine (A) substitutions introduced in the following experiments are shown in boldface. (D) Phos-tag and standard WB analysis of wild-type and mutant HP1αs. WCLs were prepared from NIH 3T3 cells transiently expressing FLAG-tagged wild-type HP1α or HP1α with N-terminal (N-term: S11-14A) mutations, hinge (Hinge: S92, 93, 95, 97A) mutations, or both (N-term + hinge). Each FLAG-HP1α with an SAP-treated control was analyzed by phos-tag WB using an anti-FLAG antibody. The phosphorylation state of HP1α represented by each protein band was deduced from the distance of migration level of each band (Rf) and is indicated to the right of the phos-tag WB panel. (E) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the N-terminal region. The phosphorylation patterns of the indicated FLAG-HP1α species were analyzed by phos-tag WB using a higher concentration (80 μM) of phos-tag acrylamide. (F) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the hinge region. (G) The numbers of phosphorylated peptides identified by liquid chromatography (LC)-MS/MS analysis of affinity-purified HP1α. (H) Summary of the phosphorylation sites of mouse HP1α deduced from the experiments described above.
    Calf intestinal alkaline phosphatase CIAP catalyzes the hydrolysis of 5 phosphate termini from DNA RNA dNTPs and rNTPs This enzyme can be used to reduce linear vector self ligation and vector background during cloning CIAP is supplied in a buffer of 10 mM Tris HCl pH 8 0 50 mM KCl 1 mM MgCl2 0 1 mM ZnCl2 and 50 glycerol
    https://www.bioz.com/result/shrimp alkaline phosphatase sap/product/TaKaRa
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    shrimp alkaline phosphatase sap - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "N-Terminal Phosphorylation of HP1? Promotes Its Chromatin Binding ▿"

    Article Title: N-Terminal Phosphorylation of HP1? Promotes Its Chromatin Binding ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01012-10

    Identification of phosphorylation sites of mouse HP1α. (A) The phosphorylation patterns of mouse HP1α analyzed by phos-tag Western blotting (phos-tag WB). Whole-cell lysates (WCLs) of NIH 3T3 cells were resolved on an SDS-PAGE gel containing phos-tag acrylamide (phos-tag) or on a standard SDS-PAGE gel (mock). As an unphosphorylated control, SAP-treated WCL was loaded on the same gel. HP1α was detected using an anti-HP1α antibody. (B) Phosphorylation patterns of mouse HP1α in cells arrested at different cell cycle stages. WCLs were prepared from NIH 3T3 cells grown under the indicated conditions and analyzed by phos-tag WB. The arrowhead indicates an HP1α with basal band shift. The asterisk represents an HP1α species with metaphase-associated phosphorylation. The same membrane was immunoblotted with anti-α-tubulin and anti-TIF1β antibodies for loading controls. (C) Amino acid sequence of mouse HP1α. Light- and dark-shaded boxes indicate the CD and CSD, respectively. The sites of serine (S)-to-alanine (A) substitutions introduced in the following experiments are shown in boldface. (D) Phos-tag and standard WB analysis of wild-type and mutant HP1αs. WCLs were prepared from NIH 3T3 cells transiently expressing FLAG-tagged wild-type HP1α or HP1α with N-terminal (N-term: S11-14A) mutations, hinge (Hinge: S92, 93, 95, 97A) mutations, or both (N-term + hinge). Each FLAG-HP1α with an SAP-treated control was analyzed by phos-tag WB using an anti-FLAG antibody. The phosphorylation state of HP1α represented by each protein band was deduced from the distance of migration level of each band (Rf) and is indicated to the right of the phos-tag WB panel. (E) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the N-terminal region. The phosphorylation patterns of the indicated FLAG-HP1α species were analyzed by phos-tag WB using a higher concentration (80 μM) of phos-tag acrylamide. (F) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the hinge region. (G) The numbers of phosphorylated peptides identified by liquid chromatography (LC)-MS/MS analysis of affinity-purified HP1α. (H) Summary of the phosphorylation sites of mouse HP1α deduced from the experiments described above.
    Figure Legend Snippet: Identification of phosphorylation sites of mouse HP1α. (A) The phosphorylation patterns of mouse HP1α analyzed by phos-tag Western blotting (phos-tag WB). Whole-cell lysates (WCLs) of NIH 3T3 cells were resolved on an SDS-PAGE gel containing phos-tag acrylamide (phos-tag) or on a standard SDS-PAGE gel (mock). As an unphosphorylated control, SAP-treated WCL was loaded on the same gel. HP1α was detected using an anti-HP1α antibody. (B) Phosphorylation patterns of mouse HP1α in cells arrested at different cell cycle stages. WCLs were prepared from NIH 3T3 cells grown under the indicated conditions and analyzed by phos-tag WB. The arrowhead indicates an HP1α with basal band shift. The asterisk represents an HP1α species with metaphase-associated phosphorylation. The same membrane was immunoblotted with anti-α-tubulin and anti-TIF1β antibodies for loading controls. (C) Amino acid sequence of mouse HP1α. Light- and dark-shaded boxes indicate the CD and CSD, respectively. The sites of serine (S)-to-alanine (A) substitutions introduced in the following experiments are shown in boldface. (D) Phos-tag and standard WB analysis of wild-type and mutant HP1αs. WCLs were prepared from NIH 3T3 cells transiently expressing FLAG-tagged wild-type HP1α or HP1α with N-terminal (N-term: S11-14A) mutations, hinge (Hinge: S92, 93, 95, 97A) mutations, or both (N-term + hinge). Each FLAG-HP1α with an SAP-treated control was analyzed by phos-tag WB using an anti-FLAG antibody. The phosphorylation state of HP1α represented by each protein band was deduced from the distance of migration level of each band (Rf) and is indicated to the right of the phos-tag WB panel. (E) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the N-terminal region. The phosphorylation patterns of the indicated FLAG-HP1α species were analyzed by phos-tag WB using a higher concentration (80 μM) of phos-tag acrylamide. (F) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the hinge region. (G) The numbers of phosphorylated peptides identified by liquid chromatography (LC)-MS/MS analysis of affinity-purified HP1α. (H) Summary of the phosphorylation sites of mouse HP1α deduced from the experiments described above.

    Techniques Used: Western Blot, SDS Page, Electrophoretic Mobility Shift Assay, Sequencing, Mutagenesis, Expressing, Migration, Concentration Assay, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Affinity Purification

    Related Articles

    Clone Assay:

    Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress
    Article Snippet: .. Briefly, two TonEBP responsive elements were cloned upstream of the secreted human placental alkaline phosphatase (SEAP) coding region, in the pSEAP2-Basic vector (GenBank Accession # U89937 ) from Clontech (Mountain View, CA). ..

    Transfection:

    Article Title: Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
    Article Snippet: .. NF-κB-dependent secretary alkaline phosphatase expression (SEAP) reporter Assay Approximately 1×105 SAEC per well were plated in 24-well plates, serum-starved in SABM for 24 h with or without AR inhibitor, zopolrestat (20 µM), and transiently transfected with pNF-κB-secretory alkaline phosphatase (SEAP) construct or control plasmid pTAL-SEAP DNA (Clontech, Palo Alto, CA) using the LipofectAMINE Plus reagent. .. After 6 h of transfection, medium was replaced with fresh medium and cells were treated with RWE (50 µg/mL) for 48 h. The cell culture medium was harvested, centrifuged and supernatant was analyzed for SEAP activity, essentially as described by the manufacturer (Clontech, Palo Alto, CA), using a 96-well chemiluminescence plate reader.

    Article Title: Potential Role of Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP2 in Apoptosis Inhibition
    Article Snippet: .. Since these transfected plasmids contain the secreted alkaline phosphatase (SEAP) gene as a reporter, culture supernatants were collected 72 h after transfection, and SEAP activity was detected in by the Great Escape SEAP Chemiluminescence Assay kit (Clontech) using a GloMax 20/20 Luminometer (Promega). ..

    Reporter Assay:

    Article Title: Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
    Article Snippet: .. NF-κB-dependent secretary alkaline phosphatase expression (SEAP) reporter Assay Approximately 1×105 SAEC per well were plated in 24-well plates, serum-starved in SABM for 24 h with or without AR inhibitor, zopolrestat (20 µM), and transiently transfected with pNF-κB-secretory alkaline phosphatase (SEAP) construct or control plasmid pTAL-SEAP DNA (Clontech, Palo Alto, CA) using the LipofectAMINE Plus reagent. .. After 6 h of transfection, medium was replaced with fresh medium and cells were treated with RWE (50 µg/mL) for 48 h. The cell culture medium was harvested, centrifuged and supernatant was analyzed for SEAP activity, essentially as described by the manufacturer (Clontech, Palo Alto, CA), using a 96-well chemiluminescence plate reader.

    Construct:

    Article Title: Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
    Article Snippet: .. NF-κB-dependent secretary alkaline phosphatase expression (SEAP) reporter Assay Approximately 1×105 SAEC per well were plated in 24-well plates, serum-starved in SABM for 24 h with or without AR inhibitor, zopolrestat (20 µM), and transiently transfected with pNF-κB-secretory alkaline phosphatase (SEAP) construct or control plasmid pTAL-SEAP DNA (Clontech, Palo Alto, CA) using the LipofectAMINE Plus reagent. .. After 6 h of transfection, medium was replaced with fresh medium and cells were treated with RWE (50 µg/mL) for 48 h. The cell culture medium was harvested, centrifuged and supernatant was analyzed for SEAP activity, essentially as described by the manufacturer (Clontech, Palo Alto, CA), using a 96-well chemiluminescence plate reader.

    ALP Assay:

    Article Title: Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors
    Article Snippet: .. Alkaline Phosphatase Staining Alkaline phosphatase staining was performed using a TRACP and ALP Double-Stain Kit according to the manufacturer’s instructions (Takara Bio). .. HLA Genotyping According to HLA Laboratory instructions, alleles at the human leukocyte antigen HLA-A, -B, -Cw, and -DRB1 loci were identified by the Luminex microbeads method and platform (100 System: Luminex) as follows: DNA sampling was carried out by amplifying the target genes by PCR using biotin labeled primers.

    Chemiluminescence Immunoassay:

    Article Title: Potential Role of Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP2 in Apoptosis Inhibition
    Article Snippet: .. Since these transfected plasmids contain the secreted alkaline phosphatase (SEAP) gene as a reporter, culture supernatants were collected 72 h after transfection, and SEAP activity was detected in by the Great Escape SEAP Chemiluminescence Assay kit (Clontech) using a GloMax 20/20 Luminometer (Promega). ..

    Expressing:

    Article Title: Determinants involved in subtype-specific functions of rat trace amine-associated receptors 1 and 4
    Article Snippet: .. For the CRE-SEAP (secreted alkaline phosphatase) reporter gene assay cells were co-transfected (3 μg of each) with the rat Taar1, rat Taar4 or chimeric Taar expression plasmid and the CRE-SEAP reporter plasmid (Clontech, Saint-Germain-en-Laye, France). .. SEAP reporter gene assays, analysing function in correlation to increasing amounts of Taar expression plasmid, were performed in 96-well plates (4 × 104 HEK-293 cells per well), and cells were transfected with 0.2 μg DNA per well (0.1 μg SEAP reporter plasmid and 0.025 μg Taar/0.075 μg pcDps, 0.05 μg/0.05 μg pcDps or 0.1 μg Taar).

    Article Title: Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
    Article Snippet: .. NF-κB-dependent secretary alkaline phosphatase expression (SEAP) reporter Assay Approximately 1×105 SAEC per well were plated in 24-well plates, serum-starved in SABM for 24 h with or without AR inhibitor, zopolrestat (20 µM), and transiently transfected with pNF-κB-secretory alkaline phosphatase (SEAP) construct or control plasmid pTAL-SEAP DNA (Clontech, Palo Alto, CA) using the LipofectAMINE Plus reagent. .. After 6 h of transfection, medium was replaced with fresh medium and cells were treated with RWE (50 µg/mL) for 48 h. The cell culture medium was harvested, centrifuged and supernatant was analyzed for SEAP activity, essentially as described by the manufacturer (Clontech, Palo Alto, CA), using a 96-well chemiluminescence plate reader.

    Staining:

    Article Title: Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors
    Article Snippet: .. Alkaline Phosphatase Staining Alkaline phosphatase staining was performed using a TRACP and ALP Double-Stain Kit according to the manufacturer’s instructions (Takara Bio). .. HLA Genotyping According to HLA Laboratory instructions, alleles at the human leukocyte antigen HLA-A, -B, -Cw, and -DRB1 loci were identified by the Luminex microbeads method and platform (100 System: Luminex) as follows: DNA sampling was carried out by amplifying the target genes by PCR using biotin labeled primers.

    Activity Assay:

    Article Title: Nontranscriptional modulation of intracellular Ca2+ signaling by ligand stimulated thyroid hormone receptor
    Article Snippet: .. To investigate the transcriptional activity of xTRβ A1, we coinjected oocytes with xTRβ A1 mRNA and a plasmid reporting vector containing a TRE system with two direct repeats (DR4) upstream of the secreted placental alkaline phosphatase (SEAP) gene (p-TRE-SEAP; CLONTECH Laboratories, Inc.). .. If the hormone receptor dimerizes and binds to the TRE enhancer, the oocyte expresses SEAP, which is secreted into the medium. mRNA-injected oocytes were continuously bathed in T3 (100 nM) for 3 d and the presence of SEAP was subsequently quantified by Western blot analysis and used as a marker for transcriptional activity.

    Article Title: Potential Role of Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP2 in Apoptosis Inhibition
    Article Snippet: .. Since these transfected plasmids contain the secreted alkaline phosphatase (SEAP) gene as a reporter, culture supernatants were collected 72 h after transfection, and SEAP activity was detected in by the Great Escape SEAP Chemiluminescence Assay kit (Clontech) using a GloMax 20/20 Luminometer (Promega). ..

    Reporter Gene Assay:

    Article Title: Determinants involved in subtype-specific functions of rat trace amine-associated receptors 1 and 4
    Article Snippet: .. For the CRE-SEAP (secreted alkaline phosphatase) reporter gene assay cells were co-transfected (3 μg of each) with the rat Taar1, rat Taar4 or chimeric Taar expression plasmid and the CRE-SEAP reporter plasmid (Clontech, Saint-Germain-en-Laye, France). .. SEAP reporter gene assays, analysing function in correlation to increasing amounts of Taar expression plasmid, were performed in 96-well plates (4 × 104 HEK-293 cells per well), and cells were transfected with 0.2 μg DNA per well (0.1 μg SEAP reporter plasmid and 0.025 μg Taar/0.075 μg pcDps, 0.05 μg/0.05 μg pcDps or 0.1 μg Taar).

    Plasmid Preparation:

    Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress
    Article Snippet: .. Briefly, two TonEBP responsive elements were cloned upstream of the secreted human placental alkaline phosphatase (SEAP) coding region, in the pSEAP2-Basic vector (GenBank Accession # U89937 ) from Clontech (Mountain View, CA). ..

    Article Title: Nontranscriptional modulation of intracellular Ca2+ signaling by ligand stimulated thyroid hormone receptor
    Article Snippet: .. To investigate the transcriptional activity of xTRβ A1, we coinjected oocytes with xTRβ A1 mRNA and a plasmid reporting vector containing a TRE system with two direct repeats (DR4) upstream of the secreted placental alkaline phosphatase (SEAP) gene (p-TRE-SEAP; CLONTECH Laboratories, Inc.). .. If the hormone receptor dimerizes and binds to the TRE enhancer, the oocyte expresses SEAP, which is secreted into the medium. mRNA-injected oocytes were continuously bathed in T3 (100 nM) for 3 d and the presence of SEAP was subsequently quantified by Western blot analysis and used as a marker for transcriptional activity.

    Article Title: Determinants involved in subtype-specific functions of rat trace amine-associated receptors 1 and 4
    Article Snippet: .. For the CRE-SEAP (secreted alkaline phosphatase) reporter gene assay cells were co-transfected (3 μg of each) with the rat Taar1, rat Taar4 or chimeric Taar expression plasmid and the CRE-SEAP reporter plasmid (Clontech, Saint-Germain-en-Laye, France). .. SEAP reporter gene assays, analysing function in correlation to increasing amounts of Taar expression plasmid, were performed in 96-well plates (4 × 104 HEK-293 cells per well), and cells were transfected with 0.2 μg DNA per well (0.1 μg SEAP reporter plasmid and 0.025 μg Taar/0.075 μg pcDps, 0.05 μg/0.05 μg pcDps or 0.1 μg Taar).

    Article Title: Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice
    Article Snippet: .. NF-κB-dependent secretary alkaline phosphatase expression (SEAP) reporter Assay Approximately 1×105 SAEC per well were plated in 24-well plates, serum-starved in SABM for 24 h with or without AR inhibitor, zopolrestat (20 µM), and transiently transfected with pNF-κB-secretory alkaline phosphatase (SEAP) construct or control plasmid pTAL-SEAP DNA (Clontech, Palo Alto, CA) using the LipofectAMINE Plus reagent. .. After 6 h of transfection, medium was replaced with fresh medium and cells were treated with RWE (50 µg/mL) for 48 h. The cell culture medium was harvested, centrifuged and supernatant was analyzed for SEAP activity, essentially as described by the manufacturer (Clontech, Palo Alto, CA), using a 96-well chemiluminescence plate reader.

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    TaKaRa shrimp alkaline phosphatase sap
    Identification of phosphorylation sites of mouse HP1α. (A) The phosphorylation patterns of mouse HP1α analyzed by phos-tag Western blotting (phos-tag WB). Whole-cell lysates (WCLs) of NIH 3T3 cells were resolved on an SDS-PAGE gel containing phos-tag acrylamide (phos-tag) or on a standard SDS-PAGE gel (mock). As an unphosphorylated control, <t>SAP-treated</t> <t>WCL</t> was loaded on the same gel. HP1α was detected using an anti-HP1α antibody. (B) Phosphorylation patterns of mouse HP1α in cells arrested at different cell cycle stages. WCLs were prepared from NIH 3T3 cells grown under the indicated conditions and analyzed by phos-tag WB. The arrowhead indicates an HP1α with basal band shift. The asterisk represents an HP1α species with metaphase-associated phosphorylation. The same membrane was immunoblotted with anti-α-tubulin and anti-TIF1β antibodies for loading controls. (C) Amino acid sequence of mouse HP1α. Light- and dark-shaded boxes indicate the CD and CSD, respectively. The sites of serine (S)-to-alanine (A) substitutions introduced in the following experiments are shown in boldface. (D) Phos-tag and standard WB analysis of wild-type and mutant HP1αs. WCLs were prepared from NIH 3T3 cells transiently expressing FLAG-tagged wild-type HP1α or HP1α with N-terminal (N-term: S11-14A) mutations, hinge (Hinge: S92, 93, 95, 97A) mutations, or both (N-term + hinge). Each FLAG-HP1α with an SAP-treated control was analyzed by phos-tag WB using an anti-FLAG antibody. The phosphorylation state of HP1α represented by each protein band was deduced from the distance of migration level of each band (Rf) and is indicated to the right of the phos-tag WB panel. (E) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the N-terminal region. The phosphorylation patterns of the indicated FLAG-HP1α species were analyzed by phos-tag WB using a higher concentration (80 μM) of phos-tag acrylamide. (F) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the hinge region. (G) The numbers of phosphorylated peptides identified by liquid chromatography (LC)-MS/MS analysis of affinity-purified HP1α. (H) Summary of the phosphorylation sites of mouse HP1α deduced from the experiments described above.
    Shrimp Alkaline Phosphatase Sap, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shrimp alkaline phosphatase sap/product/TaKaRa
    Average 99 stars, based on 48 article reviews
    Price from $9.99 to $1999.99
    shrimp alkaline phosphatase sap - by Bioz Stars, 2020-09
    99/100 stars
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    Identification of phosphorylation sites of mouse HP1α. (A) The phosphorylation patterns of mouse HP1α analyzed by phos-tag Western blotting (phos-tag WB). Whole-cell lysates (WCLs) of NIH 3T3 cells were resolved on an SDS-PAGE gel containing phos-tag acrylamide (phos-tag) or on a standard SDS-PAGE gel (mock). As an unphosphorylated control, SAP-treated WCL was loaded on the same gel. HP1α was detected using an anti-HP1α antibody. (B) Phosphorylation patterns of mouse HP1α in cells arrested at different cell cycle stages. WCLs were prepared from NIH 3T3 cells grown under the indicated conditions and analyzed by phos-tag WB. The arrowhead indicates an HP1α with basal band shift. The asterisk represents an HP1α species with metaphase-associated phosphorylation. The same membrane was immunoblotted with anti-α-tubulin and anti-TIF1β antibodies for loading controls. (C) Amino acid sequence of mouse HP1α. Light- and dark-shaded boxes indicate the CD and CSD, respectively. The sites of serine (S)-to-alanine (A) substitutions introduced in the following experiments are shown in boldface. (D) Phos-tag and standard WB analysis of wild-type and mutant HP1αs. WCLs were prepared from NIH 3T3 cells transiently expressing FLAG-tagged wild-type HP1α or HP1α with N-terminal (N-term: S11-14A) mutations, hinge (Hinge: S92, 93, 95, 97A) mutations, or both (N-term + hinge). Each FLAG-HP1α with an SAP-treated control was analyzed by phos-tag WB using an anti-FLAG antibody. The phosphorylation state of HP1α represented by each protein band was deduced from the distance of migration level of each band (Rf) and is indicated to the right of the phos-tag WB panel. (E) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the N-terminal region. The phosphorylation patterns of the indicated FLAG-HP1α species were analyzed by phos-tag WB using a higher concentration (80 μM) of phos-tag acrylamide. (F) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the hinge region. (G) The numbers of phosphorylated peptides identified by liquid chromatography (LC)-MS/MS analysis of affinity-purified HP1α. (H) Summary of the phosphorylation sites of mouse HP1α deduced from the experiments described above.

    Journal: Molecular and Cellular Biology

    Article Title: N-Terminal Phosphorylation of HP1? Promotes Its Chromatin Binding ▿

    doi: 10.1128/MCB.01012-10

    Figure Lengend Snippet: Identification of phosphorylation sites of mouse HP1α. (A) The phosphorylation patterns of mouse HP1α analyzed by phos-tag Western blotting (phos-tag WB). Whole-cell lysates (WCLs) of NIH 3T3 cells were resolved on an SDS-PAGE gel containing phos-tag acrylamide (phos-tag) or on a standard SDS-PAGE gel (mock). As an unphosphorylated control, SAP-treated WCL was loaded on the same gel. HP1α was detected using an anti-HP1α antibody. (B) Phosphorylation patterns of mouse HP1α in cells arrested at different cell cycle stages. WCLs were prepared from NIH 3T3 cells grown under the indicated conditions and analyzed by phos-tag WB. The arrowhead indicates an HP1α with basal band shift. The asterisk represents an HP1α species with metaphase-associated phosphorylation. The same membrane was immunoblotted with anti-α-tubulin and anti-TIF1β antibodies for loading controls. (C) Amino acid sequence of mouse HP1α. Light- and dark-shaded boxes indicate the CD and CSD, respectively. The sites of serine (S)-to-alanine (A) substitutions introduced in the following experiments are shown in boldface. (D) Phos-tag and standard WB analysis of wild-type and mutant HP1αs. WCLs were prepared from NIH 3T3 cells transiently expressing FLAG-tagged wild-type HP1α or HP1α with N-terminal (N-term: S11-14A) mutations, hinge (Hinge: S92, 93, 95, 97A) mutations, or both (N-term + hinge). Each FLAG-HP1α with an SAP-treated control was analyzed by phos-tag WB using an anti-FLAG antibody. The phosphorylation state of HP1α represented by each protein band was deduced from the distance of migration level of each band (Rf) and is indicated to the right of the phos-tag WB panel. (E) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the N-terminal region. The phosphorylation patterns of the indicated FLAG-HP1α species were analyzed by phos-tag WB using a higher concentration (80 μM) of phos-tag acrylamide. (F) Phosphorylation state of FLAG-HP1α with each single amino acid substitution in the hinge region. (G) The numbers of phosphorylated peptides identified by liquid chromatography (LC)-MS/MS analysis of affinity-purified HP1α. (H) Summary of the phosphorylation sites of mouse HP1α deduced from the experiments described above.

    Article Snippet: For dephosphorylation, an aliquot of whole-cell lysate (WCL) was incubated with 0.08 U/ml shrimp alkaline phosphatase (SAP) (TaKaRa) for 3 to 6 h at 37°C.

    Techniques: Western Blot, SDS Page, Electrophoretic Mobility Shift Assay, Sequencing, Mutagenesis, Expressing, Migration, Concentration Assay, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Affinity Purification