chromatography analysis  (Millipore)


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    Name:
    Liquid Chromatography
    Description:
    With the advent of new interfacing technology the benefits of LC MS liquid chromatography with mass spectrometry are being realized by the growth in applications of this technique in both the chemical and life sciences Topics covered include Limitations of the component techniques when used in isolation and how a combination of the two allows these to be overcome Descriptions of the various approaches including thermospray electrospray and atmospheric pressure chemical ionization which are used to interface the two techniques along with the advantages and disadvantages of each system Illustrative examples of the applications of LC MS including the molecular weight and structure determinations of both biopolymers and low molecular compounds
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    z702080
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    Structured Review

    Millipore chromatography analysis
    Liquid Chromatography
    With the advent of new interfacing technology the benefits of LC MS liquid chromatography with mass spectrometry are being realized by the growth in applications of this technique in both the chemical and life sciences Topics covered include Limitations of the component techniques when used in isolation and how a combination of the two allows these to be overcome Descriptions of the various approaches including thermospray electrospray and atmospheric pressure chemical ionization which are used to interface the two techniques along with the advantages and disadvantages of each system Illustrative examples of the applications of LC MS including the molecular weight and structure determinations of both biopolymers and low molecular compounds
    https://www.bioz.com/result/chromatography analysis/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    chromatography analysis - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    High Performance Liquid Chromatography:

    Article Title: Morusinol Exhibits Selective and Potent Antitumor Activity Against Human Liver Carcinoma by Inducing Autophagy, G2/M Cell Cycle Arrest, Inhibition of Cell Invasion and Migration, and Targeting of Ras/MEK/ERK Pathway
    Article Snippet: .. Chemicals and other reagents Morusinol (purity > 98%; determined by high-performance liquid chromatography), 3-(4, 5-dimethyl-2-thiazolyl)-2, and 5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from SigmaAldrich Chemical Co. (St. Louis, MO, USA). .. Propidium iodide was purchased from Wuhan Boster Biological Technology (Wuhan, China).

    Article Title: Remodeling of Retinal Fatty Acids in an Animal Model of Diabetes
    Article Snippet: .. High-performance liquid chromatography (HPLC)-grade acetonitrile, acetic acid, methanol, chloroform, streptozotocin (STZ), and commonly used chemicals and reagents were from Sigma-Aldrich Chemical (St. Louis, MO). ..

    MTT Assay:

    Article Title: Morusinol Exhibits Selective and Potent Antitumor Activity Against Human Liver Carcinoma by Inducing Autophagy, G2/M Cell Cycle Arrest, Inhibition of Cell Invasion and Migration, and Targeting of Ras/MEK/ERK Pathway
    Article Snippet: .. Chemicals and other reagents Morusinol (purity > 98%; determined by high-performance liquid chromatography), 3-(4, 5-dimethyl-2-thiazolyl)-2, and 5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from SigmaAldrich Chemical Co. (St. Louis, MO, USA). .. Propidium iodide was purchased from Wuhan Boster Biological Technology (Wuhan, China).

    Mass Spectrometry:

    Article Title: Enhanced dissolution and oral absorption of tacrolimus by supersaturable self-emulsifying drug delivery system
    Article Snippet: .. Ascomycin (purity > 98% w/w%) used as an internal standard for FK506 Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) analysis was obtained from Sigma-Aldrich Co. (St Louis, MO, USA). .. Cremophor EL and Soluplus were kindly provided by BASF (Ludwigshafen, Germany).

    Chromatography:

    Article Title: Enhanced dissolution and oral absorption of tacrolimus by supersaturable self-emulsifying drug delivery system
    Article Snippet: .. Ascomycin (purity > 98% w/w%) used as an internal standard for FK506 Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) analysis was obtained from Sigma-Aldrich Co. (St Louis, MO, USA). .. Cremophor EL and Soluplus were kindly provided by BASF (Ludwigshafen, Germany).

    Article Title: Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14
    Article Snippet: .. For liquid chromatography–mass spectrometry (LC–MS) analysis of modified nucleosides, nuclease P1 (Sigma), snake venom phosphodiesterase I (Worthington Biochemical Corporation) and Antarctic phosphatase (New England Biolabs) were used to digest Trm14-reacted transcripts and control unmethylated transcripts to nucleosides. .. The nucleosides were separated using a Hitachi D-7000 HPLC with a Hitachi L-7400 UV detector at 0.3 ml/min at room temperature on an LC-18 S 2.1 × 250 mm column from Supelco using a gradient of 5 mM ammonium acetate pH 5.3 and acetonitrile:water (40:60 v/v) as described ( ).

    Article Title: Unexpected Different Binding of Mistletoe Lectins from Plant Extracts to Immobilized Lactose and N-acetylgalactosamine
    Article Snippet: .. Fast protein liquid chromatography (FPLC) separation of eluted MLs using cation exchange chromatography The eluate from the affigel-asialofetuin column was dialysed against 15 mM citrate buffer (pH 4.2), then concentrated to 2 mg/ml using Microcon YM-30 tubes (Millipore, Switzerland) before applying to a cation exchange chromatography column (Mono S 5/50 GL; Amersham Bioscience, Switzerland). .. The column was adapted to a FPLC system and equilibrated with citrate buffer (pH 4.2).

    Isolation:

    Article Title: Attenuation of atherogenic apo B-48-dependent hyperlipidemia and high density lipoprotein remodeling induced by vitamin C and E combination and their beneficial effect on lethal ischemic heart disease in mice
    Article Snippet: .. Lipoprotein fractionation and high density lipoprotein isolation Serum was fractionated by fast-protein liquid chromatography (FPLC) and apolipoprotein A-I-containing fractions were pooled and concentrated using Amicon Ultra-4 centrifugal 10 K filters (Millipore, Merck, Darmstadt, HD, Germany). .. Pooled fractions were subjected to dynabead-based immunoprecipitation with an anti-apolipoprotein B (apo B) antibody to remove apo B-containing lipoproteins.

    Fractionation:

    Article Title: Attenuation of atherogenic apo B-48-dependent hyperlipidemia and high density lipoprotein remodeling induced by vitamin C and E combination and their beneficial effect on lethal ischemic heart disease in mice
    Article Snippet: .. Lipoprotein fractionation and high density lipoprotein isolation Serum was fractionated by fast-protein liquid chromatography (FPLC) and apolipoprotein A-I-containing fractions were pooled and concentrated using Amicon Ultra-4 centrifugal 10 K filters (Millipore, Merck, Darmstadt, HD, Germany). .. Pooled fractions were subjected to dynabead-based immunoprecipitation with an anti-apolipoprotein B (apo B) antibody to remove apo B-containing lipoproteins.

    Fast Protein Liquid Chromatography:

    Article Title: Attenuation of atherogenic apo B-48-dependent hyperlipidemia and high density lipoprotein remodeling induced by vitamin C and E combination and their beneficial effect on lethal ischemic heart disease in mice
    Article Snippet: .. Serum was fractionated by fast-protein liquid chromatography (FPLC) and apolipoprotein A-I-containing fractions were pooled and concentrated using Amicon Ultra-4 centrifugal 10 K filters (Millipore, Merck, Darmstadt, HD, Germany). .. Pooled fractions were subjected to dynabead-based immunoprecipitation with an anti-apolipoprotein B (apo B) antibody to remove apo B-containing lipoproteins.

    Article Title: Attenuation of atherogenic apo B-48-dependent hyperlipidemia and high density lipoprotein remodeling induced by vitamin C and E combination and their beneficial effect on lethal ischemic heart disease in mice
    Article Snippet: .. Lipoprotein fractionation and high density lipoprotein isolation Serum was fractionated by fast-protein liquid chromatography (FPLC) and apolipoprotein A-I-containing fractions were pooled and concentrated using Amicon Ultra-4 centrifugal 10 K filters (Millipore, Merck, Darmstadt, HD, Germany). .. Pooled fractions were subjected to dynabead-based immunoprecipitation with an anti-apolipoprotein B (apo B) antibody to remove apo B-containing lipoproteins.

    Article Title: Unexpected Different Binding of Mistletoe Lectins from Plant Extracts to Immobilized Lactose and N-acetylgalactosamine
    Article Snippet: .. Fast protein liquid chromatography (FPLC) separation of eluted MLs using cation exchange chromatography The eluate from the affigel-asialofetuin column was dialysed against 15 mM citrate buffer (pH 4.2), then concentrated to 2 mg/ml using Microcon YM-30 tubes (Millipore, Switzerland) before applying to a cation exchange chromatography column (Mono S 5/50 GL; Amersham Bioscience, Switzerland). .. The column was adapted to a FPLC system and equilibrated with citrate buffer (pH 4.2).

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14
    Article Snippet: .. For liquid chromatography–mass spectrometry (LC–MS) analysis of modified nucleosides, nuclease P1 (Sigma), snake venom phosphodiesterase I (Worthington Biochemical Corporation) and Antarctic phosphatase (New England Biolabs) were used to digest Trm14-reacted transcripts and control unmethylated transcripts to nucleosides. .. The nucleosides were separated using a Hitachi D-7000 HPLC with a Hitachi L-7400 UV detector at 0.3 ml/min at room temperature on an LC-18 S 2.1 × 250 mm column from Supelco using a gradient of 5 mM ammonium acetate pH 5.3 and acetonitrile:water (40:60 v/v) as described ( ).

    Modification:

    Article Title: Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14
    Article Snippet: .. For liquid chromatography–mass spectrometry (LC–MS) analysis of modified nucleosides, nuclease P1 (Sigma), snake venom phosphodiesterase I (Worthington Biochemical Corporation) and Antarctic phosphatase (New England Biolabs) were used to digest Trm14-reacted transcripts and control unmethylated transcripts to nucleosides. .. The nucleosides were separated using a Hitachi D-7000 HPLC with a Hitachi L-7400 UV detector at 0.3 ml/min at room temperature on an LC-18 S 2.1 × 250 mm column from Supelco using a gradient of 5 mM ammonium acetate pH 5.3 and acetonitrile:water (40:60 v/v) as described ( ).

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  • 92
    Millipore maldi ms
    Characterization of ADP-ribosyl-HNP-1 from BAL fluid by <t>RP-HPLC,</t> <t>MALDI,</t> and enzymatic digestion. ( a ) Alignment of RP-HPLC chromatograms of products of ART-1-catalyzed ADP-ribosylation of HNP-1 (continuous line) and a chromatogram of a BAL sample from a smoker (dotted line). Arrows indicate elution times of ADP-ribosyl-HNP-1 and HNP-1. ( b ) ( i ) The peak with elution time compatible with ADP-ribosyl-HNP-1 (46.5 min), analyzed by MALDI-MS, shows a mass of 3,983 Da, consistent with ADP-ribosyl-HNP-1 (calculated 3,983 Da). ( ii ) Incubation of ADP-ribosyl-HNP-1 with pyrophosphatase and alkaline phosphatase produced ribosyl-HNP-1 (calculated 3,574 Da). ( iii ) ADP-ribosylarginine hydrolase cleaved the ribose-arginine linkage to release HNP-1 (calculated 3,443 Da). m / z is on the x axis.
    Maldi Ms, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore lc ms analysis biocrust soil water samples
    Metabolite patterns detected in <t>biocrust</t> soil water. Metabolite dynamics (85 metabolites displayed as the average peak area, normalized across each row) were observed in biocrust soil water across wetting and successional stages. Unique patterns are indicated by cluster 1 (early metabolites including fatty acids), cluster 2 (early-to-mid time point metabolites) and cluster 3 (late metabolites). Putative metabolites are indicated by parentheses. n = 2–5 for each group
    Lc Ms Analysis Biocrust Soil Water Samples, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore vitro phosphorylated mbp hd fusion protein
    <t>Csx/Nkx2.5</t> phosphorylation increases DNA binding affinity. Csx/Nkx2.5 protein expressed in COS cells was dephosphorylated with CIAP, and DNA binding activity was compared to that of phosphorylated Csx/Nkx2.5. (A) Transfected-cell lysates were separated by SDS-PAGE and blotted with Csx/Nkx2.5 MAb. The majority of Csx/Nkx2.5 protein was dephosphorylated by CIAP treatment (lane 2). A 5-μl aliquot of each cell lysate (lanes 1 and 2) and 5 ng of <t>MBP-Csx/Nkx2.5</t> fusion protein contained equivalent amounts of Csx/Nkx2.5 protein. (B) EMSAs with ANF Csx/Nkx2.5 binding site (TGAAGTG). The DNA-Csx/Nkx2.5 protein complex (arrowhead) was supershifted by anti-Csx/Nkx2.5 MAb (arrow) in a dose-dependent manner. ANF probe was end-labeled in this experiment. (C) EMSA with ANF Csx/Nkx2.5 binding site. Lanes CIAP+ contain twofold serially diluted CIAP-treated Csx/Nkx2.5 protein; lanes CIAP− contain untreated Csx/Nkx2.5 protein. The shifted bands were scanned and plotted against protein concentration (lower panel). The plotted line was shifted downward by CIAP treatment, indicating phosphorylated Csx/Nkx2.5 has higher DNA binding affinity than dephosphorylated protein. (D) DNA mobility shift assay (similar to that shown in panel C) using A20 Csx/Nkx2.5 binding site (AGTTAATTG). Phosphorylated protein (lanes CIAP−) showed higher DNA binding affinity than dephosphorylated protein.
    Vitro Phosphorylated Mbp Hd Fusion Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore immunoprecipitates
    Fig. 6. Co-localization of Δγ1 with paxillin. ( A ) Co-immunoprecipitation of paxillin and PP2A B56γ isoforms by PP2A C subunit Ab. Cells were lysed 30 min after non-adherent cell cultivation and immunoprecipitated with PP2A C subunit Ab. The blot was probed with a mixture of paxillin (mAb) and PP2A C antibodies (upper panel), or B56γ Ab alone (lower panel). ( B ) Cells were lysed 30 min after being plated on FN, and immunoprecipitated with paxillin mAb. The <t>immunoprecipitates</t> and whole-cell lysate (50 μg) of 3T3 Δγ1 -1 cells were blotted and probed with anti-B56γ Ab. The positions of the B56γ isoforms, γ3, γ2, γ1 and Δγ1, are indicated on the left of the blots in (A) and (B). NC indicates a representative result of negative controls in (A) and (B). Arrows in (A) and (B) indicate the position of the IgG heavy chain. ( C ) Subcellular localization of HA-tagged B56γ3, γ2, γ1 and Δγ1 proteins. COS-7 cells were transiently transfected with HA-tagged full length B56γ3 (COS-7 HA-γ3 ), γ2 (COS-7 HA-γ2 ), γ1 (COS-7 HA-γ1 ) or Δγ1 (COS-7 HA-Δγ1 ) vector. After transfection, cells were replaced on chamber slides, reacted with anti-HA and paxillin polyclonal Ab, and stained with Cy2 (green; upper row) and Cy3 (red; center row), respectively. Cy2 and Cy3 images were merged into one (lower row).
    Immunoprecipitates, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 615 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of ADP-ribosyl-HNP-1 from BAL fluid by RP-HPLC, MALDI, and enzymatic digestion. ( a ) Alignment of RP-HPLC chromatograms of products of ART-1-catalyzed ADP-ribosylation of HNP-1 (continuous line) and a chromatogram of a BAL sample from a smoker (dotted line). Arrows indicate elution times of ADP-ribosyl-HNP-1 and HNP-1. ( b ) ( i ) The peak with elution time compatible with ADP-ribosyl-HNP-1 (46.5 min), analyzed by MALDI-MS, shows a mass of 3,983 Da, consistent with ADP-ribosyl-HNP-1 (calculated 3,983 Da). ( ii ) Incubation of ADP-ribosyl-HNP-1 with pyrophosphatase and alkaline phosphatase produced ribosyl-HNP-1 (calculated 3,574 Da). ( iii ) ADP-ribosylarginine hydrolase cleaved the ribose-arginine linkage to release HNP-1 (calculated 3,443 Da). m / z is on the x axis.

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

    Article Title: ADP ribosylation of human neutrophil peptide-1 regulates its biological properties

    doi: 10.1073/pnas.122238899

    Figure Lengend Snippet: Characterization of ADP-ribosyl-HNP-1 from BAL fluid by RP-HPLC, MALDI, and enzymatic digestion. ( a ) Alignment of RP-HPLC chromatograms of products of ART-1-catalyzed ADP-ribosylation of HNP-1 (continuous line) and a chromatogram of a BAL sample from a smoker (dotted line). Arrows indicate elution times of ADP-ribosyl-HNP-1 and HNP-1. ( b ) ( i ) The peak with elution time compatible with ADP-ribosyl-HNP-1 (46.5 min), analyzed by MALDI-MS, shows a mass of 3,983 Da, consistent with ADP-ribosyl-HNP-1 (calculated 3,983 Da). ( ii ) Incubation of ADP-ribosyl-HNP-1 with pyrophosphatase and alkaline phosphatase produced ribosyl-HNP-1 (calculated 3,574 Da). ( iii ) ADP-ribosylarginine hydrolase cleaved the ribose-arginine linkage to release HNP-1 (calculated 3,443 Da). m / z is on the x axis.

    Article Snippet: We incubated ADP-ribosyl-HNP-1 (6 pmol/0.2 μg) in 200 μl of 250 mM NaHCO3 /25 mM MgCl2 , alkaline phosphatase (5 μg), and pyrophosphatase (5 μg) (Sigma) for 30 min at 37°C before termination of the reaction with 6 M guanidine, separation of reaction products by RP-HPLC, and analysis by MALDI-MS. We also incubated ADP-ribosyl-HNP-1 (11 pmol, 0.42 μg) overnight at 37°C with recombinant human ADP-ribosylarginine hydrolase (1 μg) in 100 μl of 50 mM Tris (pH 7.5)/5 mM DTT/10 mM Mg Cl2 , followed by desalting and concentration using Zip-TipC18 (Millipore), and analysis by MALDI-MS.

    Techniques: High Performance Liquid Chromatography, Mass Spectrometry, Incubation, Produced

    Metabolite patterns detected in biocrust soil water. Metabolite dynamics (85 metabolites displayed as the average peak area, normalized across each row) were observed in biocrust soil water across wetting and successional stages. Unique patterns are indicated by cluster 1 (early metabolites including fatty acids), cluster 2 (early-to-mid time point metabolites) and cluster 3 (late metabolites). Putative metabolites are indicated by parentheses. n = 2–5 for each group

    Journal: Nature Communications

    Article Title: Linking soil biology and chemistry in biological soil crust using isolate exometabolomics

    doi: 10.1038/s41467-017-02356-9

    Figure Lengend Snippet: Metabolite patterns detected in biocrust soil water. Metabolite dynamics (85 metabolites displayed as the average peak area, normalized across each row) were observed in biocrust soil water across wetting and successional stages. Unique patterns are indicated by cluster 1 (early metabolites including fatty acids), cluster 2 (early-to-mid time point metabolites) and cluster 3 (late metabolites). Putative metabolites are indicated by parentheses. n = 2–5 for each group

    Article Snippet: Metabolite extraction and LC/MS analysis Biocrust soil water samples (1.5 mL) were lyophilized and resuspended in methanol (200 μL) containing internal standards (2–10 μg/mL) and filtered through 96-well Millipore filter plates (0.2 μm PVDF) by centrifuging at 1500 × g for 2 min.

    Techniques:

    Simplified biocrust foodweb for three dominant biocrust bacteria based on combining isolate exometabolomics data with in situ microbe–metabolite relationships. This network displays the relationships between metabolites and three dominant bacteria (or metabolically similar organisms) as they increase and decrease in relative abundance across wetting and successional stages in biocrust. The lower line plot corresponds to real relative abundance measurements for the three bacteria in level C successional stage biocrust. As Microcoleus sp. increases in relative abundance immediately after wetting, many metabolites released by the closest-related isolate are positively correlated with Microcoleus sp. in biocrust (solid red arrows) and as the two Bacilli increase in relative abundance (first Bacillus sp. 1 then Bacillus sp. 2), most metabolites consumed by the closest-related isolate decrease and are negatively correlated with these bacteria in biocrust (solid blue arrows) and most released metabolites are positively correlated (solid red arrows). Dotted arrows indicate metabolites that are released (red) or consumed (blue) by isolates, but did not display the expected relationship with that microorganism in situ. The thickness of the line corresponds to the absolute value of the Spearman’s rho correlation coefficient. The overall expected directionality (solid lines vs. dotted lines) was significant as determined by the exact binomial test (two-tailed p -value = 0.01). *FDR-adjusted p

    Journal: Nature Communications

    Article Title: Linking soil biology and chemistry in biological soil crust using isolate exometabolomics

    doi: 10.1038/s41467-017-02356-9

    Figure Lengend Snippet: Simplified biocrust foodweb for three dominant biocrust bacteria based on combining isolate exometabolomics data with in situ microbe–metabolite relationships. This network displays the relationships between metabolites and three dominant bacteria (or metabolically similar organisms) as they increase and decrease in relative abundance across wetting and successional stages in biocrust. The lower line plot corresponds to real relative abundance measurements for the three bacteria in level C successional stage biocrust. As Microcoleus sp. increases in relative abundance immediately after wetting, many metabolites released by the closest-related isolate are positively correlated with Microcoleus sp. in biocrust (solid red arrows) and as the two Bacilli increase in relative abundance (first Bacillus sp. 1 then Bacillus sp. 2), most metabolites consumed by the closest-related isolate decrease and are negatively correlated with these bacteria in biocrust (solid blue arrows) and most released metabolites are positively correlated (solid red arrows). Dotted arrows indicate metabolites that are released (red) or consumed (blue) by isolates, but did not display the expected relationship with that microorganism in situ. The thickness of the line corresponds to the absolute value of the Spearman’s rho correlation coefficient. The overall expected directionality (solid lines vs. dotted lines) was significant as determined by the exact binomial test (two-tailed p -value = 0.01). *FDR-adjusted p

    Article Snippet: Metabolite extraction and LC/MS analysis Biocrust soil water samples (1.5 mL) were lyophilized and resuspended in methanol (200 μL) containing internal standards (2–10 μg/mL) and filtered through 96-well Millipore filter plates (0.2 μm PVDF) by centrifuging at 1500 × g for 2 min.

    Techniques: In Situ, Metabolic Labelling, Two Tailed Test

    Experimental workflow and biocrust microbe–metabolite relationship predictions. a Biocrust wetup metabolomics and metagenomics experimental setup and analysis. To study microbe–metabolite relationships in situ, biocrusts from four successional stages were wetup and sampled at five time points (total n = 100). Biocrust soil water was removed and analyzed by liquid chromatography/ mass spectrometry ( n = 5 for each group) and biocrust DNA was extracted for shotgun sequencing ( n = 1 for each group). Metagenome-estimated genome and metabolite abundances were analyzed through Spearman rank correlations to determine microbe–metabolite relationships and compared to the expected relationships based on isolate exometabolomic studies. b Exometabolomics-based in situ microbe–metabolite relationship prediction. The hypothesis is that isolate exometabolomics can be used to predict microbe–metabolite patterns in situ based on microbial abundance: Across wetting and successional stages, microbes change in abundance and negatively correlate with metabolites that they consume and positively correlate with metabolites that they release (metabolites are indicated by dotted lines)

    Journal: Nature Communications

    Article Title: Linking soil biology and chemistry in biological soil crust using isolate exometabolomics

    doi: 10.1038/s41467-017-02356-9

    Figure Lengend Snippet: Experimental workflow and biocrust microbe–metabolite relationship predictions. a Biocrust wetup metabolomics and metagenomics experimental setup and analysis. To study microbe–metabolite relationships in situ, biocrusts from four successional stages were wetup and sampled at five time points (total n = 100). Biocrust soil water was removed and analyzed by liquid chromatography/ mass spectrometry ( n = 5 for each group) and biocrust DNA was extracted for shotgun sequencing ( n = 1 for each group). Metagenome-estimated genome and metabolite abundances were analyzed through Spearman rank correlations to determine microbe–metabolite relationships and compared to the expected relationships based on isolate exometabolomic studies. b Exometabolomics-based in situ microbe–metabolite relationship prediction. The hypothesis is that isolate exometabolomics can be used to predict microbe–metabolite patterns in situ based on microbial abundance: Across wetting and successional stages, microbes change in abundance and negatively correlate with metabolites that they consume and positively correlate with metabolites that they release (metabolites are indicated by dotted lines)

    Article Snippet: Metabolite extraction and LC/MS analysis Biocrust soil water samples (1.5 mL) were lyophilized and resuspended in methanol (200 μL) containing internal standards (2–10 μg/mL) and filtered through 96-well Millipore filter plates (0.2 μm PVDF) by centrifuging at 1500 × g for 2 min.

    Techniques: In Situ, Liquid Chromatography, Mass Spectrometry, Shotgun Sequencing

    Csx/Nkx2.5 phosphorylation increases DNA binding affinity. Csx/Nkx2.5 protein expressed in COS cells was dephosphorylated with CIAP, and DNA binding activity was compared to that of phosphorylated Csx/Nkx2.5. (A) Transfected-cell lysates were separated by SDS-PAGE and blotted with Csx/Nkx2.5 MAb. The majority of Csx/Nkx2.5 protein was dephosphorylated by CIAP treatment (lane 2). A 5-μl aliquot of each cell lysate (lanes 1 and 2) and 5 ng of MBP-Csx/Nkx2.5 fusion protein contained equivalent amounts of Csx/Nkx2.5 protein. (B) EMSAs with ANF Csx/Nkx2.5 binding site (TGAAGTG). The DNA-Csx/Nkx2.5 protein complex (arrowhead) was supershifted by anti-Csx/Nkx2.5 MAb (arrow) in a dose-dependent manner. ANF probe was end-labeled in this experiment. (C) EMSA with ANF Csx/Nkx2.5 binding site. Lanes CIAP+ contain twofold serially diluted CIAP-treated Csx/Nkx2.5 protein; lanes CIAP− contain untreated Csx/Nkx2.5 protein. The shifted bands were scanned and plotted against protein concentration (lower panel). The plotted line was shifted downward by CIAP treatment, indicating phosphorylated Csx/Nkx2.5 has higher DNA binding affinity than dephosphorylated protein. (D) DNA mobility shift assay (similar to that shown in panel C) using A20 Csx/Nkx2.5 binding site (AGTTAATTG). Phosphorylated protein (lanes CIAP−) showed higher DNA binding affinity than dephosphorylated protein.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    doi:

    Figure Lengend Snippet: Csx/Nkx2.5 phosphorylation increases DNA binding affinity. Csx/Nkx2.5 protein expressed in COS cells was dephosphorylated with CIAP, and DNA binding activity was compared to that of phosphorylated Csx/Nkx2.5. (A) Transfected-cell lysates were separated by SDS-PAGE and blotted with Csx/Nkx2.5 MAb. The majority of Csx/Nkx2.5 protein was dephosphorylated by CIAP treatment (lane 2). A 5-μl aliquot of each cell lysate (lanes 1 and 2) and 5 ng of MBP-Csx/Nkx2.5 fusion protein contained equivalent amounts of Csx/Nkx2.5 protein. (B) EMSAs with ANF Csx/Nkx2.5 binding site (TGAAGTG). The DNA-Csx/Nkx2.5 protein complex (arrowhead) was supershifted by anti-Csx/Nkx2.5 MAb (arrow) in a dose-dependent manner. ANF probe was end-labeled in this experiment. (C) EMSA with ANF Csx/Nkx2.5 binding site. Lanes CIAP+ contain twofold serially diluted CIAP-treated Csx/Nkx2.5 protein; lanes CIAP− contain untreated Csx/Nkx2.5 protein. The shifted bands were scanned and plotted against protein concentration (lower panel). The plotted line was shifted downward by CIAP treatment, indicating phosphorylated Csx/Nkx2.5 has higher DNA binding affinity than dephosphorylated protein. (D) DNA mobility shift assay (similar to that shown in panel C) using A20 Csx/Nkx2.5 binding site (AGTTAATTG). Phosphorylated protein (lanes CIAP−) showed higher DNA binding affinity than dephosphorylated protein.

    Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

    Techniques: Binding Assay, Activity Assay, Transfection, SDS Page, Labeling, Protein Concentration, Mobility Shift

    Csx/Nkx2.5 is phosphorylated in vivo and in vitro. (A) COS cells transfected with Csx/Nkx2.5 expression vector (lanes 1 and 3) or pcDNA3 vector alone (lanes 2 and 4) were 32 P labeled, lysed, and immunoprecipitated (IP) with Csx/Nkx2.5 MAb (lanes 1 and 2) or control IgG1 (lanes 3 and 4). The 37-kDa Csx/Nkx2.5 band was specifically immunoprecipitated with Csx/Nkx2.5 MAb (lane 1) but not with control IgG1 (lane 3). (B) MBP alone (MBP), MBP-homeodomain Csx/Nkx2.5 (HD), and MBP-full length Csx/Nkx2.5 (Csx) were phosphorylated with [γ- 32 P]ATP and 2 μg of cytoplasmic (Cyt) or nuclear (Nuc) extract of NIH 3T3 cells. Samples were subjected to SDS-PAGE, and phosphorylated proteins were visualized by autoradiography. Both cytoplasmic (lanes 1 to 3) and nuclear lysates (lanes 4 to 6) approximately equally phosphorylated Csx/Nkx2.5 proteins (lane 2 versus lane 5; lane 3 versus lane 6). MBP was not phosphorylated by either cytoplasmic (lane 1) or nuclear (lane 4) extracts.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    doi:

    Figure Lengend Snippet: Csx/Nkx2.5 is phosphorylated in vivo and in vitro. (A) COS cells transfected with Csx/Nkx2.5 expression vector (lanes 1 and 3) or pcDNA3 vector alone (lanes 2 and 4) were 32 P labeled, lysed, and immunoprecipitated (IP) with Csx/Nkx2.5 MAb (lanes 1 and 2) or control IgG1 (lanes 3 and 4). The 37-kDa Csx/Nkx2.5 band was specifically immunoprecipitated with Csx/Nkx2.5 MAb (lane 1) but not with control IgG1 (lane 3). (B) MBP alone (MBP), MBP-homeodomain Csx/Nkx2.5 (HD), and MBP-full length Csx/Nkx2.5 (Csx) were phosphorylated with [γ- 32 P]ATP and 2 μg of cytoplasmic (Cyt) or nuclear (Nuc) extract of NIH 3T3 cells. Samples were subjected to SDS-PAGE, and phosphorylated proteins were visualized by autoradiography. Both cytoplasmic (lanes 1 to 3) and nuclear lysates (lanes 4 to 6) approximately equally phosphorylated Csx/Nkx2.5 proteins (lane 2 versus lane 5; lane 3 versus lane 6). MBP was not phosphorylated by either cytoplasmic (lane 1) or nuclear (lane 4) extracts.

    Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

    Techniques: In Vivo, In Vitro, Transfection, Expressing, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Autoradiography

    Phosphorylation of Csx/Nkx2.5 by CKII within the homeodomain. (A) In-gel kinase assays. NIH 3T3 cell nuclear extracts (25 μg) were separated in a gel containing either the homeodomain-MBP fusion protein (HD), the full-length Csx/Nkx2.5-MBP fusion protein (Csx), or MBP. After denaturation and renaturation, gels were incubated in kinase buffer containing [γ- 32 P]ATP. One kinase with a molecular mass of ∼40 kDa (arrowheads) phosphorylated the Csx/Nkx2.5 homeodomain, Csx/Nkx2.5 (lane 1), and the full-length Csx/Nkx2.5 (lane 2), but not MBP (lane 3). (B) Schematic representation of Csx/Nkx2.5 mutants carrying the consensus CKII sites and serine-to-alanine substitutions. (C) In vitro CKII phosphorylation of Csx/Nkx2.5. Three different Csx/Nkx2.5 fusion proteins described in panel B were incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of purified CKII. Each sample was subjected to SDS-PAGE and transferred to a PVDF membrane. Autoradiography (upper panel) revealed that CKII phosphorylated the full-length Csx/Nkx2.5 (lane 1) and the homeodomain (lane 3) but neither the C-terminally deleted mutant (lane 5) nor MBP (lane 7). Loaded fusion proteins are shown by Western blotting of the same PVDF membrane using anti-MBP antibody (lower panel). (D) Serine 163 was mutated into alanine (Csx/Nkx2.5 163S-A ) and the kinase assay was performed. Lane 1 contains the wild-type Csx/Nkx2.5 and lane 2 contains the mutant Csx/Nkx2.5 163S-A . Equal amounts of proteins were subjected to the kinase reaction as shown in the MBP Western blot (lower panel).

    Journal: Molecular and Cellular Biology

    Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    doi:

    Figure Lengend Snippet: Phosphorylation of Csx/Nkx2.5 by CKII within the homeodomain. (A) In-gel kinase assays. NIH 3T3 cell nuclear extracts (25 μg) were separated in a gel containing either the homeodomain-MBP fusion protein (HD), the full-length Csx/Nkx2.5-MBP fusion protein (Csx), or MBP. After denaturation and renaturation, gels were incubated in kinase buffer containing [γ- 32 P]ATP. One kinase with a molecular mass of ∼40 kDa (arrowheads) phosphorylated the Csx/Nkx2.5 homeodomain, Csx/Nkx2.5 (lane 1), and the full-length Csx/Nkx2.5 (lane 2), but not MBP (lane 3). (B) Schematic representation of Csx/Nkx2.5 mutants carrying the consensus CKII sites and serine-to-alanine substitutions. (C) In vitro CKII phosphorylation of Csx/Nkx2.5. Three different Csx/Nkx2.5 fusion proteins described in panel B were incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of purified CKII. Each sample was subjected to SDS-PAGE and transferred to a PVDF membrane. Autoradiography (upper panel) revealed that CKII phosphorylated the full-length Csx/Nkx2.5 (lane 1) and the homeodomain (lane 3) but neither the C-terminally deleted mutant (lane 5) nor MBP (lane 7). Loaded fusion proteins are shown by Western blotting of the same PVDF membrane using anti-MBP antibody (lower panel). (D) Serine 163 was mutated into alanine (Csx/Nkx2.5 163S-A ) and the kinase assay was performed. Lane 1 contains the wild-type Csx/Nkx2.5 and lane 2 contains the mutant Csx/Nkx2.5 163S-A . Equal amounts of proteins were subjected to the kinase reaction as shown in the MBP Western blot (lower panel).

    Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

    Techniques: Incubation, In Vitro, Purification, SDS Page, Autoradiography, Mutagenesis, Western Blot, Kinase Assay

    CKII phosphorylation increases DNA binding affinity. (A) EMSA with ANF Csx/Nkx2.5 binding site. Csx/Nkx2.5 homeodomain fusion protein (MBP-HD) (1 ng) incubated with either CKII (lanes CKII+) or heat-inactivated CKII (lanes CKII−) and their twofold serially diluted proteins were assayed for DNA binding. The shifted bands were scanned and plotted against the protein concentration (lower panel). CKII treatment shifted the plotted line upward, indicating that CKII-phosphorylated protein has higher DNA binding affinity. Slower-migrating bands might represent the dimerized protein. The first lane contains the gel shift with non-fused MBP protein; the last lane contains the free probe without added fusion protein. (B) A result similar to that shown in panel A is observed with A20 Csx/Nkx2.5 binding site.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    doi:

    Figure Lengend Snippet: CKII phosphorylation increases DNA binding affinity. (A) EMSA with ANF Csx/Nkx2.5 binding site. Csx/Nkx2.5 homeodomain fusion protein (MBP-HD) (1 ng) incubated with either CKII (lanes CKII+) or heat-inactivated CKII (lanes CKII−) and their twofold serially diluted proteins were assayed for DNA binding. The shifted bands were scanned and plotted against the protein concentration (lower panel). CKII treatment shifted the plotted line upward, indicating that CKII-phosphorylated protein has higher DNA binding affinity. Slower-migrating bands might represent the dimerized protein. The first lane contains the gel shift with non-fused MBP protein; the last lane contains the free probe without added fusion protein. (B) A result similar to that shown in panel A is observed with A20 Csx/Nkx2.5 binding site.

    Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

    Techniques: Binding Assay, Incubation, Protein Concentration, Electrophoretic Mobility Shift Assay

    Phosphopeptide and phosphoamino acid analysis of Csx/Nkx2.5. (A) Tryptic phosphopeptide mapping of wild-type Csx/Nkx2.5 (a), CKII mutant (b), and in vitro CKII-phosphorylated MBP-homeodomain protein (c). Metabolically 32 P-labeled Csx/Nkx2.5 and the CKII mutant (Csx/Nkx2.5 163S-A ) were immunoprecipitated with anti-Csx/Nkx2.5 MAb (2D10), resolved by SDS-PAGE, transferred to a PVDF membrane, and autoradiographed. In vitro CKII-phosphorylated MBP-homeodomain protein was also electrophoresed and autoradiographed. These protein bands were cut out, digested with trypsin, and resolved on TLC plates by electrophoresis in the first dimension and chromatography in the second dimension. In vitro CKII-phosphorylated peptide (c [arrow]) which corresponded to peptide 1 in the wild type (a [arrow]), was markedly reduced in the CKII mutant (b). Arrowheads, sample loaded points; X, lysine marker. (B) Phosphoamino acid analysis of wild-type Csx/Nkx2.5 showed Csx/Nkx2.5 is phosphorylated predominantly in S and weakly in T.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    doi:

    Figure Lengend Snippet: Phosphopeptide and phosphoamino acid analysis of Csx/Nkx2.5. (A) Tryptic phosphopeptide mapping of wild-type Csx/Nkx2.5 (a), CKII mutant (b), and in vitro CKII-phosphorylated MBP-homeodomain protein (c). Metabolically 32 P-labeled Csx/Nkx2.5 and the CKII mutant (Csx/Nkx2.5 163S-A ) were immunoprecipitated with anti-Csx/Nkx2.5 MAb (2D10), resolved by SDS-PAGE, transferred to a PVDF membrane, and autoradiographed. In vitro CKII-phosphorylated MBP-homeodomain protein was also electrophoresed and autoradiographed. These protein bands were cut out, digested with trypsin, and resolved on TLC plates by electrophoresis in the first dimension and chromatography in the second dimension. In vitro CKII-phosphorylated peptide (c [arrow]) which corresponded to peptide 1 in the wild type (a [arrow]), was markedly reduced in the CKII mutant (b). Arrowheads, sample loaded points; X, lysine marker. (B) Phosphoamino acid analysis of wild-type Csx/Nkx2.5 showed Csx/Nkx2.5 is phosphorylated predominantly in S and weakly in T.

    Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

    Techniques: Phosphoamino Acid Analysis, Mutagenesis, In Vitro, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Thin Layer Chromatography, Electrophoresis, Chromatography, Marker

    Fig. 6. Co-localization of Δγ1 with paxillin. ( A ) Co-immunoprecipitation of paxillin and PP2A B56γ isoforms by PP2A C subunit Ab. Cells were lysed 30 min after non-adherent cell cultivation and immunoprecipitated with PP2A C subunit Ab. The blot was probed with a mixture of paxillin (mAb) and PP2A C antibodies (upper panel), or B56γ Ab alone (lower panel). ( B ) Cells were lysed 30 min after being plated on FN, and immunoprecipitated with paxillin mAb. The immunoprecipitates and whole-cell lysate (50 μg) of 3T3 Δγ1 -1 cells were blotted and probed with anti-B56γ Ab. The positions of the B56γ isoforms, γ3, γ2, γ1 and Δγ1, are indicated on the left of the blots in (A) and (B). NC indicates a representative result of negative controls in (A) and (B). Arrows in (A) and (B) indicate the position of the IgG heavy chain. ( C ) Subcellular localization of HA-tagged B56γ3, γ2, γ1 and Δγ1 proteins. COS-7 cells were transiently transfected with HA-tagged full length B56γ3 (COS-7 HA-γ3 ), γ2 (COS-7 HA-γ2 ), γ1 (COS-7 HA-γ1 ) or Δγ1 (COS-7 HA-Δγ1 ) vector. After transfection, cells were replaced on chamber slides, reacted with anti-HA and paxillin polyclonal Ab, and stained with Cy2 (green; upper row) and Cy3 (red; center row), respectively. Cy2 and Cy3 images were merged into one (lower row).

    Journal: The EMBO Journal

    Article Title: A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation

    doi: 10.1093/emboj/19.4.562

    Figure Lengend Snippet: Fig. 6. Co-localization of Δγ1 with paxillin. ( A ) Co-immunoprecipitation of paxillin and PP2A B56γ isoforms by PP2A C subunit Ab. Cells were lysed 30 min after non-adherent cell cultivation and immunoprecipitated with PP2A C subunit Ab. The blot was probed with a mixture of paxillin (mAb) and PP2A C antibodies (upper panel), or B56γ Ab alone (lower panel). ( B ) Cells were lysed 30 min after being plated on FN, and immunoprecipitated with paxillin mAb. The immunoprecipitates and whole-cell lysate (50 μg) of 3T3 Δγ1 -1 cells were blotted and probed with anti-B56γ Ab. The positions of the B56γ isoforms, γ3, γ2, γ1 and Δγ1, are indicated on the left of the blots in (A) and (B). NC indicates a representative result of negative controls in (A) and (B). Arrows in (A) and (B) indicate the position of the IgG heavy chain. ( C ) Subcellular localization of HA-tagged B56γ3, γ2, γ1 and Δγ1 proteins. COS-7 cells were transiently transfected with HA-tagged full length B56γ3 (COS-7 HA-γ3 ), γ2 (COS-7 HA-γ2 ), γ1 (COS-7 HA-γ1 ) or Δγ1 (COS-7 HA-Δγ1 ) vector. After transfection, cells were replaced on chamber slides, reacted with anti-HA and paxillin polyclonal Ab, and stained with Cy2 (green; upper row) and Cy3 (red; center row), respectively. Cy2 and Cy3 images were merged into one (lower row).

    Article Snippet: The immunoprecipitates, total cell or tissue lysates (50 μg per lane), were separated on 10% SDS–polyacrylamide gels, transferred to Immobilon (Millipore) and reacted with primary and POD-labeled secondary antibodies.

    Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Staining

    Fig. 5. Enhanced paxillin phosphorylation on serine residues in 3T3 Δγ1 cells. ( A ) Cells were lysed at 30 min after non-adherent culture (suspension), and 30 (FN 30min) and 90 (FN 90min) min after plating on FN. Immunoprecipitates with paxillin mAb (upper and center panels) and whole-cell lysates (lower panels) were separated by SDS–PAGE. The blots of immunoprecipitates were probed with a mixture of antibodies against FAK, paxillin (mAb) and PP2A C subunit (upper panel), or anti-phosphotyrosine Ab alone (4G10, center panel). Protein expression in the whole-cell lysates was also examined with antibodies against PP2A C subunit, FAK and vinculin (lower panels). NC indicates a representative result of negative controls. White and black arrowheads beside paxillin indicate phosphorylated and unphosphorylated paxillin, respectively. The arrow on the right indicates the position of the IgG heavy chain. ( B ) Phosphoamino acid analysis of 3T3 γ1 and 3T3 Δγ1 -1 cells. Immunoprecipitated paxillin with a radioactivity of 500 c.p.m. was hydrolyzed and loaded onto a thin-layer chromatography plate. Co-migration of ninhydrin-stained phosphoamino acid standards is indicated on the right. P i indicates the position of free 32 P-labeled inorganic phosphate. Lower spots are partial hydrolysis products (part.). ( C ) Quantification of phosphorylation on serine, threonine and tyrosine residues. Signal intensity for each residue, P i or partial hydrolysis products in (B) was quantified and expressed as a percentage of the total intensity.

    Journal: The EMBO Journal

    Article Title: A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation

    doi: 10.1093/emboj/19.4.562

    Figure Lengend Snippet: Fig. 5. Enhanced paxillin phosphorylation on serine residues in 3T3 Δγ1 cells. ( A ) Cells were lysed at 30 min after non-adherent culture (suspension), and 30 (FN 30min) and 90 (FN 90min) min after plating on FN. Immunoprecipitates with paxillin mAb (upper and center panels) and whole-cell lysates (lower panels) were separated by SDS–PAGE. The blots of immunoprecipitates were probed with a mixture of antibodies against FAK, paxillin (mAb) and PP2A C subunit (upper panel), or anti-phosphotyrosine Ab alone (4G10, center panel). Protein expression in the whole-cell lysates was also examined with antibodies against PP2A C subunit, FAK and vinculin (lower panels). NC indicates a representative result of negative controls. White and black arrowheads beside paxillin indicate phosphorylated and unphosphorylated paxillin, respectively. The arrow on the right indicates the position of the IgG heavy chain. ( B ) Phosphoamino acid analysis of 3T3 γ1 and 3T3 Δγ1 -1 cells. Immunoprecipitated paxillin with a radioactivity of 500 c.p.m. was hydrolyzed and loaded onto a thin-layer chromatography plate. Co-migration of ninhydrin-stained phosphoamino acid standards is indicated on the right. P i indicates the position of free 32 P-labeled inorganic phosphate. Lower spots are partial hydrolysis products (part.). ( C ) Quantification of phosphorylation on serine, threonine and tyrosine residues. Signal intensity for each residue, P i or partial hydrolysis products in (B) was quantified and expressed as a percentage of the total intensity.

    Article Snippet: The immunoprecipitates, total cell or tissue lysates (50 μg per lane), were separated on 10% SDS–polyacrylamide gels, transferred to Immobilon (Millipore) and reacted with primary and POD-labeled secondary antibodies.

    Techniques: SDS Page, Expressing, Phosphoamino Acid Analysis, Immunoprecipitation, Radioactivity, Thin Layer Chromatography, Migration, Staining, Labeling