in vitro phosphorylation assays in vitro radioactive assays  (PerkinElmer)

 
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    Structured Review

    PerkinElmer in vitro phosphorylation assays in vitro radioactive assays
    <t>In</t> <t>vitro</t> kinase assay. (A) Coomassie gel showing the purified proteins used in the in vitro kinase activity <t>assays.</t> (B) Recombinant ATG4B or ATG4B S34A was incubated with recombinant AKT2 and ATP γ- 32 P and incorporation of labeled γ- 32 P was measured by auto-radiography. The upper band corresponds to AKT2 <t>auto-phosphorylation</t> and the lower band corresponds to ATG4B. (C) Recombinant ATG4B wild-type (WT), S121A or S262A were incubated with (+) or without (–) recombinant AKT2 and incorporation of labeled γ- 32 P was measured by auto-radiography. On the left side, ULK1 mediated phosphorylation of ATG4B was included as control. On the right side, CLK2 (CLK2 catalytic domain with GST-tagged ( Prak et al., 2016 )) was included as another protein control to show that in the absence of ATG4B, AKT2 resulted in auto-phosphorylation.
    In Vitro Phosphorylation Assays In Vitro Radioactive Assays, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells"

    Article Title: Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2018.00148

    In vitro kinase assay. (A) Coomassie gel showing the purified proteins used in the in vitro kinase activity assays. (B) Recombinant ATG4B or ATG4B S34A was incubated with recombinant AKT2 and ATP γ- 32 P and incorporation of labeled γ- 32 P was measured by auto-radiography. The upper band corresponds to AKT2 auto-phosphorylation and the lower band corresponds to ATG4B. (C) Recombinant ATG4B wild-type (WT), S121A or S262A were incubated with (+) or without (–) recombinant AKT2 and incorporation of labeled γ- 32 P was measured by auto-radiography. On the left side, ULK1 mediated phosphorylation of ATG4B was included as control. On the right side, CLK2 (CLK2 catalytic domain with GST-tagged ( Prak et al., 2016 )) was included as another protein control to show that in the absence of ATG4B, AKT2 resulted in auto-phosphorylation.
    Figure Legend Snippet: In vitro kinase assay. (A) Coomassie gel showing the purified proteins used in the in vitro kinase activity assays. (B) Recombinant ATG4B or ATG4B S34A was incubated with recombinant AKT2 and ATP γ- 32 P and incorporation of labeled γ- 32 P was measured by auto-radiography. The upper band corresponds to AKT2 auto-phosphorylation and the lower band corresponds to ATG4B. (C) Recombinant ATG4B wild-type (WT), S121A or S262A were incubated with (+) or without (–) recombinant AKT2 and incorporation of labeled γ- 32 P was measured by auto-radiography. On the left side, ULK1 mediated phosphorylation of ATG4B was included as control. On the right side, CLK2 (CLK2 catalytic domain with GST-tagged ( Prak et al., 2016 )) was included as another protein control to show that in the absence of ATG4B, AKT2 resulted in auto-phosphorylation.

    Techniques Used: In Vitro, Kinase Assay, Purification, Activity Assay, Recombinant, Incubation, Labeling

    2) Product Images from "Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease"

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

    Journal: Molecules

    doi: 10.3390/molecules21060766

    Time course of ERCC1-XPF cleavage. The 32 P-labeled substrates (400 fmol) containing ( A ) 3′-OH; ( B ) ddC; ( C ) ACV; ( D ) ABC; ( E ) CBV; and ( F ) (−)3TC, were incubated at 30 °C for 0 (lanes 1), 30 (lanes 2), 60 (lanes 3) and 90 min (lanes 4), in the presence of ERCC1-XPF (92 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM MnCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. The experiments were performed in triplicate, and the increases in the amounts of the cleaved products shorter than the intact substrates were plotted. The data points are shown as mean ± SD.
    Figure Legend Snippet: Time course of ERCC1-XPF cleavage. The 32 P-labeled substrates (400 fmol) containing ( A ) 3′-OH; ( B ) ddC; ( C ) ACV; ( D ) ABC; ( E ) CBV; and ( F ) (−)3TC, were incubated at 30 °C for 0 (lanes 1), 30 (lanes 2), 60 (lanes 3) and 90 min (lanes 4), in the presence of ERCC1-XPF (92 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM MnCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. The experiments were performed in triplicate, and the increases in the amounts of the cleaved products shorter than the intact substrates were plotted. The data points are shown as mean ± SD.

    Techniques Used: Labeling, Incubation

    Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of dNTPs. ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM NaCl, 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.
    Figure Legend Snippet: Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of dNTPs. ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM NaCl, 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.

    Techniques Used: Activity Assay, Labeling, Incubation

    Removal of the CTNAs attached to the 3′-termini of oligonucleotides by human ERCC1-XPF endonuclease. The 32 P-labeled substrates (400 fmol) containing ( A ) 3′-OH; ( B ) ddC; ( C ) ACV; ( D ) ABC; ( E ) CBV; and ( F ) (−)3TC were treated with increasing amounts of ERCC1-XPF (0, 230 and 920 fmol for lanes 1, 2 and 3, respectively) at 30 °C for 90 min, in 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM MnCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 bovine serum albumin (BSA). The cleaved products in lanes 3 were quantified, and the remarkable cleavage sites are indicated by black, gray and open triangles, depending on the yield of each product ( > 20%, 10%–20%, 5%–10%, respectively). The quantified values of the products are summarized in Table S2 .
    Figure Legend Snippet: Removal of the CTNAs attached to the 3′-termini of oligonucleotides by human ERCC1-XPF endonuclease. The 32 P-labeled substrates (400 fmol) containing ( A ) 3′-OH; ( B ) ddC; ( C ) ACV; ( D ) ABC; ( E ) CBV; and ( F ) (−)3TC were treated with increasing amounts of ERCC1-XPF (0, 230 and 920 fmol for lanes 1, 2 and 3, respectively) at 30 °C for 90 min, in 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM MnCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 bovine serum albumin (BSA). The cleaved products in lanes 3 were quantified, and the remarkable cleavage sites are indicated by black, gray and open triangles, depending on the yield of each product ( > 20%, 10%–20%, 5%–10%, respectively). The quantified values of the products are summarized in Table S2 .

    Techniques Used: Labeling

    Repair of the CTNA-containing oligonucleotides by human ERCC1-XPF endonuclease and DNA polymerase. Panels A and B represent the reaction scheme and the results, respectively. First, the 32 P-labeled substrates (400 fmol) were incubated at 25 °C for 16 h, in the absence (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21 and 22) or presence (the other lanes) of ERCC1-XPF (230 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. Then, a polymerase reaction mixture (5 µL, containing 30 mM Tris-HCl, (pH 7.9), 150 mM NaCl, 30 mM MgCl 2 , 30 mM DTT, 300 µM dNTPs and 0.1 unit of KF − for even lanes) or 5 mM EDTA (5 µL for odd lanes) was added to the reaction mixture, and the total reaction mixtures (15 µL) were incubated at 37 °C for 10 min. The cleavage sites observed with ERCC1-XPF are indicated by black triangles. The fully extended products were quantified, and the values are shown.
    Figure Legend Snippet: Repair of the CTNA-containing oligonucleotides by human ERCC1-XPF endonuclease and DNA polymerase. Panels A and B represent the reaction scheme and the results, respectively. First, the 32 P-labeled substrates (400 fmol) were incubated at 25 °C for 16 h, in the absence (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21 and 22) or presence (the other lanes) of ERCC1-XPF (230 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. Then, a polymerase reaction mixture (5 µL, containing 30 mM Tris-HCl, (pH 7.9), 150 mM NaCl, 30 mM MgCl 2 , 30 mM DTT, 300 µM dNTPs and 0.1 unit of KF − for even lanes) or 5 mM EDTA (5 µL for odd lanes) was added to the reaction mixture, and the total reaction mixtures (15 µL) were incubated at 37 °C for 10 min. The cleavage sites observed with ERCC1-XPF are indicated by black triangles. The fully extended products were quantified, and the values are shown.

    Techniques Used: Labeling, Incubation

    3) Product Images from "Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein"

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2013.10.015

    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.
    Figure Legend Snippet: PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Techniques Used: In Vitro, Incubation, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, In Vivo, Modification, Sequencing, Ion Chromatography, Mutagenesis, Derivative Assay

    4) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    Journal: RNA

    doi: 10.1261/rna.5247704

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).
    Figure Legend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Techniques Used: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    5) Product Images from "Kinetic and structural analyses reveal residues in phosphoinositide 3-kinase α that are critical for catalysis and substrate recognition"

    Article Title: Kinetic and structural analyses reveal residues in phosphoinositide 3-kinase α that are critical for catalysis and substrate recognition

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.772426

    Lys-776 and His-917 are required for autophosphorylation on p85α . A , SDS-PAGE analysis and autoradiography for WT and mutant PI3Kα proteins. Autophosphorylation of p85α using radiolabeled ATP as substrate in the absence of PIP 2 . The negative control is unphosphorylated p85α in the absence of p110α. The positive control is PTEN-phosphorylated by CK2 kinase. B , autophosphorylation of PI3Kα with and without PIP 2 . C , relative quantification of Ser-608 phosphorylation in p85α for WT PI3Kα and mutants by tandem mass spectrometry (MS/MS) of TMT-labeled peptides. D , tandem mass spectrum of TMT labeled peptides showing phosphorylation of Ser-608 (marked by arrows on y15) and reporter ions region (boxed in blue ).
    Figure Legend Snippet: Lys-776 and His-917 are required for autophosphorylation on p85α . A , SDS-PAGE analysis and autoradiography for WT and mutant PI3Kα proteins. Autophosphorylation of p85α using radiolabeled ATP as substrate in the absence of PIP 2 . The negative control is unphosphorylated p85α in the absence of p110α. The positive control is PTEN-phosphorylated by CK2 kinase. B , autophosphorylation of PI3Kα with and without PIP 2 . C , relative quantification of Ser-608 phosphorylation in p85α for WT PI3Kα and mutants by tandem mass spectrometry (MS/MS) of TMT-labeled peptides. D , tandem mass spectrum of TMT labeled peptides showing phosphorylation of Ser-608 (marked by arrows on y15) and reporter ions region (boxed in blue ).

    Techniques Used: SDS Page, Autoradiography, Mutagenesis, Negative Control, Positive Control, Mass Spectrometry, Labeling

    6) Product Images from "Transforming Growth Factor-?1 (TGF-?1) Regulates Cell Junction Restructuring via Smad-Mediated Repression and Clathrin-Mediated Endocytosis of Nectin-like Molecule 2 (Necl-2)"

    Article Title: Transforming Growth Factor-?1 (TGF-?1) Regulates Cell Junction Restructuring via Smad-Mediated Repression and Clathrin-Mediated Endocytosis of Nectin-like Molecule 2 (Necl-2)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064316

    EMSA of MyoD and CCAATa motifs and ChIP assay. (A) and (D) Dose-dependent and competition assay for EMSA of MyoD motif (A) and CCAATa motif (D). Double stranded oligonucleotides containing the respective motif were radiolabeled with [γ- 32 P]ATP and incubated with nuclear extract (1–15 µg) alone or in the presence of cold competitors (100- to 500-fold excess). (B–C) and (E–F) Labeled probes were incubated with vehicle or TGF-β1 treated nuclear extract (15 µg for MyoD motif and 10 µg for CCAATa motif) in the presence of specified antibodies or rabbit serum (Rb serum). (G) A schematic drawing illustrating the relative location of MyoD and CCAAT cis -acting motifs in the Necl-2 promoter and chromatin immunoprecipitation assay. TGF-β1-treated genomic DNA-protein samples were immunopreciptated with antibodies against Smad3 and Smad4 (2 µg) or rabbit serum. Precipitated DNA-protein complexes were subjected to DNA purification. The promoter region and the open reading frame of Necl-2 gene were amplified using specific primer pairs no. A287/A288 (for promoter region) and no. A107/A108 (for open reading frame), respectively, followed by agarose gel electrophoresis. Rb, Rabbit. Cpx, complex.
    Figure Legend Snippet: EMSA of MyoD and CCAATa motifs and ChIP assay. (A) and (D) Dose-dependent and competition assay for EMSA of MyoD motif (A) and CCAATa motif (D). Double stranded oligonucleotides containing the respective motif were radiolabeled with [γ- 32 P]ATP and incubated with nuclear extract (1–15 µg) alone or in the presence of cold competitors (100- to 500-fold excess). (B–C) and (E–F) Labeled probes were incubated with vehicle or TGF-β1 treated nuclear extract (15 µg for MyoD motif and 10 µg for CCAATa motif) in the presence of specified antibodies or rabbit serum (Rb serum). (G) A schematic drawing illustrating the relative location of MyoD and CCAAT cis -acting motifs in the Necl-2 promoter and chromatin immunoprecipitation assay. TGF-β1-treated genomic DNA-protein samples were immunopreciptated with antibodies against Smad3 and Smad4 (2 µg) or rabbit serum. Precipitated DNA-protein complexes were subjected to DNA purification. The promoter region and the open reading frame of Necl-2 gene were amplified using specific primer pairs no. A287/A288 (for promoter region) and no. A107/A108 (for open reading frame), respectively, followed by agarose gel electrophoresis. Rb, Rabbit. Cpx, complex.

    Techniques Used: Chromatin Immunoprecipitation, Competitive Binding Assay, Incubation, Labeling, DNA Purification, Amplification, Agarose Gel Electrophoresis

    Effect of TGF-β1 on Necl-2 mRNA stability and promoter activity. (A) and (B) Analysis of Necl-2 mRNA stability was performed by actinomycin D (ActD) assay. Cells were pre-treated with ActD (5 µg/ml) for 2 h before vehicle or TGF-β1 treatment. RT-PCR (A) and real-time PCR (B) were performed to determine Necl-2 mRNA level. (C) Progressive 5′-deletion analysis of mouse Necl-2 promoter was performed between nt -502 and -1. A series 5′-deletion constructs and pEGFP vector were co-transfected into GC-1 spg cells followed by TGF-β1 treatment (5 ng/ml, 18 h). (D) Three putative cis -acting elements including MyoD, CCAATa and CCAATb motifs are located within the region between nt -159 and -1 (upper panel). Site-directed mutagenic constructs containing single, double or triple mutations and pEGFP vector were co-transfected into GC-1 spg cells followed by TGF-β1 treatment. pEGFP activity was used to normalize transfection efficiency. Promoter activity was represented as the fold change when compared with pGL-3 vector. (E) pGL-3 vector, p(-159/−1)Luc and pEGFP vector were co-transfected with si-Smad3 (#1/#2, 20 nM) or/and si-Smad4 (#1/#2, 20 nM) for 48 h followed by TGF-β1 treatment. pEGFP activity was used to normalize transfection efficiency. Smad3 and Smad4 protein levels were examined by Western blotting. (F) Wild-type and single mutated constructs of p(-159/−1)Luc were co-transfected with pcDNA3.1 vector, Smad3 or/and Smad4 expression constructs into GC-1 spg cells. The promoter activity was presented as a percentage of that of pcDNA3.1-transfected cells. (A–F), Results are expressed as the mean±S.D. of three independent experiments. ns, not significant vs vehicle control (A–E) or p(-159/−1)Luc (F); *, p
    Figure Legend Snippet: Effect of TGF-β1 on Necl-2 mRNA stability and promoter activity. (A) and (B) Analysis of Necl-2 mRNA stability was performed by actinomycin D (ActD) assay. Cells were pre-treated with ActD (5 µg/ml) for 2 h before vehicle or TGF-β1 treatment. RT-PCR (A) and real-time PCR (B) were performed to determine Necl-2 mRNA level. (C) Progressive 5′-deletion analysis of mouse Necl-2 promoter was performed between nt -502 and -1. A series 5′-deletion constructs and pEGFP vector were co-transfected into GC-1 spg cells followed by TGF-β1 treatment (5 ng/ml, 18 h). (D) Three putative cis -acting elements including MyoD, CCAATa and CCAATb motifs are located within the region between nt -159 and -1 (upper panel). Site-directed mutagenic constructs containing single, double or triple mutations and pEGFP vector were co-transfected into GC-1 spg cells followed by TGF-β1 treatment. pEGFP activity was used to normalize transfection efficiency. Promoter activity was represented as the fold change when compared with pGL-3 vector. (E) pGL-3 vector, p(-159/−1)Luc and pEGFP vector were co-transfected with si-Smad3 (#1/#2, 20 nM) or/and si-Smad4 (#1/#2, 20 nM) for 48 h followed by TGF-β1 treatment. pEGFP activity was used to normalize transfection efficiency. Smad3 and Smad4 protein levels were examined by Western blotting. (F) Wild-type and single mutated constructs of p(-159/−1)Luc were co-transfected with pcDNA3.1 vector, Smad3 or/and Smad4 expression constructs into GC-1 spg cells. The promoter activity was presented as a percentage of that of pcDNA3.1-transfected cells. (A–F), Results are expressed as the mean±S.D. of three independent experiments. ns, not significant vs vehicle control (A–E) or p(-159/−1)Luc (F); *, p

    Techniques Used: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Construct, Plasmid Preparation, Transfection, Western Blot, Expressing

    7) Product Images from "The Uve1 Endonuclease Is Regulated by the White Collar Complex to Protect Cryptococcus neoformans from UV Damage"

    Article Title: The Uve1 Endonuclease Is Regulated by the White Collar Complex to Protect Cryptococcus neoformans from UV Damage

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003769

    Uve1 is required for efficient repair of mitochondrial DNA damage post UV stress. Two independent experiments were carried out on different days. (A, C) Agarose gels for the PCR amplification of mitochondrial and nuclear DNA on template DNA from undamaged control cells and template DNA from cells exposed to UV stress 50 J/m 2 . C. neoformans var. grubii strains KN99α (WT) and AI191 ( uve1 Δ) PCR amplification pattern for no stress, 0 hour, 1 hour, 4 hour and 6 hour post UV stress recovery. DNA amplification of long mitochondrial (LM) DNA, short mitochondrial (SM) DNA, long nuclear (LN) DNA and short nuclear (SN) DNA. (B, D) Graphical representations of DNA damage experiments in panels A and C, respectively. X-axis represents number of lesions/10 kb and Y-axis represents time in hours.
    Figure Legend Snippet: Uve1 is required for efficient repair of mitochondrial DNA damage post UV stress. Two independent experiments were carried out on different days. (A, C) Agarose gels for the PCR amplification of mitochondrial and nuclear DNA on template DNA from undamaged control cells and template DNA from cells exposed to UV stress 50 J/m 2 . C. neoformans var. grubii strains KN99α (WT) and AI191 ( uve1 Δ) PCR amplification pattern for no stress, 0 hour, 1 hour, 4 hour and 6 hour post UV stress recovery. DNA amplification of long mitochondrial (LM) DNA, short mitochondrial (SM) DNA, long nuclear (LN) DNA and short nuclear (SN) DNA. (B, D) Graphical representations of DNA damage experiments in panels A and C, respectively. X-axis represents number of lesions/10 kb and Y-axis represents time in hours.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Bwc2 binds to the UVE1 promoter. (A) SDS-PAGE gel for C. neoformans recombinant (His) 6 -Bwc2. Left to right, lane 1 is the low molecular weight Bio-Rad protein ladder, lane 2, 3, 4 are purified recombinant Bwc2 eluted fractions. Lane 5 is crude unpurified protein extract. (B) Gel mobility shift assay for a fragment of the JEC21 UVE1 promoter with purified Bwc2. Left to right, lane 1 contains just the UVE1 promoter P 1 (P UVE1 ), lane 2, 3 and 4 contains P 1 +Bwc2 20 µg, 50 µg and 20 µg+Zn 2+ respectively. Lane 5 is loaded with the non-specific radiolabeled probe P 2 . Lane 6, 7 are P 1 +Bwc2 20 µg and 50 µg. Lane 8, 9 are competition with the increasing amount of specific probe cold P 1 to negate any binding observed in lane 7. Lane 10 is Bwc2 and P 2 with Zn 2+ .
    Figure Legend Snippet: Bwc2 binds to the UVE1 promoter. (A) SDS-PAGE gel for C. neoformans recombinant (His) 6 -Bwc2. Left to right, lane 1 is the low molecular weight Bio-Rad protein ladder, lane 2, 3, 4 are purified recombinant Bwc2 eluted fractions. Lane 5 is crude unpurified protein extract. (B) Gel mobility shift assay for a fragment of the JEC21 UVE1 promoter with purified Bwc2. Left to right, lane 1 contains just the UVE1 promoter P 1 (P UVE1 ), lane 2, 3 and 4 contains P 1 +Bwc2 20 µg, 50 µg and 20 µg+Zn 2+ respectively. Lane 5 is loaded with the non-specific radiolabeled probe P 2 . Lane 6, 7 are P 1 +Bwc2 20 µg and 50 µg. Lane 8, 9 are competition with the increasing amount of specific probe cold P 1 to negate any binding observed in lane 7. Lane 10 is Bwc2 and P 2 with Zn 2+ .

    Techniques Used: SDS Page, Recombinant, Molecular Weight, Purification, Mobility Shift, Binding Assay

    Light regulation of UVE1 homologs through the White Collar complex is conserved in fungi. Northern blots for UVE1 homologs from N. crassa [FGSC 4200 (WT), FGSC 4398 ( wc-1 )], P. blakesleeanus [NRRL1555 (WT), L51 ( madA madB )], and S. pombe [L972 (WT)]. All experiments were 1 h light exposure (Light) or constant darkness (Dark). Blots were stripped and reprobed with actin as a loading control. For clarity, the gene name UVE1 is used to refer to all homologs ( mus-18 N. crassa ; uvdE P. blakesleeanus ; uve1 S. pombe ).
    Figure Legend Snippet: Light regulation of UVE1 homologs through the White Collar complex is conserved in fungi. Northern blots for UVE1 homologs from N. crassa [FGSC 4200 (WT), FGSC 4398 ( wc-1 )], P. blakesleeanus [NRRL1555 (WT), L51 ( madA madB )], and S. pombe [L972 (WT)]. All experiments were 1 h light exposure (Light) or constant darkness (Dark). Blots were stripped and reprobed with actin as a loading control. For clarity, the gene name UVE1 is used to refer to all homologs ( mus-18 N. crassa ; uvdE P. blakesleeanus ; uve1 S. pombe ).

    Techniques Used: Northern Blot

    Subcellular localization of Uve1 (L)-GFP and Uve1 (D)-GFP from C. neoformans var. neoformans in the vegetative yeast cells of C. neoformans var. grubii (A) Uve1 (L)-GFP localization and (B) Uve1 (D)-GFP localization.
    Figure Legend Snippet: Subcellular localization of Uve1 (L)-GFP and Uve1 (D)-GFP from C. neoformans var. neoformans in the vegetative yeast cells of C. neoformans var. grubii (A) Uve1 (L)-GFP localization and (B) Uve1 (D)-GFP localization.

    Techniques Used:

    UVE1 overexpression rescues the UV sensitive phenotype of bwc1 Δ mutants in C. neoformans . Ten-fold serial dilutions for different strains of C. neoformans var. neoformans grown at 30°C for 2 days. Left panel untreated control and right panel treated with UV dose of 120 J/m 2 . Top three strains (JEC21, AI5 and AISVCN53) grown overnight in 2% glucose and bottom three strains (JEC21, AI5 and AISVCN53) grown overnight in 2% galactose before inoculating onto YPD plates.
    Figure Legend Snippet: UVE1 overexpression rescues the UV sensitive phenotype of bwc1 Δ mutants in C. neoformans . Ten-fold serial dilutions for different strains of C. neoformans var. neoformans grown at 30°C for 2 days. Left panel untreated control and right panel treated with UV dose of 120 J/m 2 . Top three strains (JEC21, AI5 and AISVCN53) grown overnight in 2% glucose and bottom three strains (JEC21, AI5 and AISVCN53) grown overnight in 2% galactose before inoculating onto YPD plates.

    Techniques Used: Over Expression

    UVE1 is a Bwc1-regulated gene in Cryptococcus . Northern blots of C. n. var. neoformans , C. n. var. grubii and C. gattii . From left to right, panel 1 is for JEC21 (WT) and AI5 ( bwc1 ); the upper band is for UVE1 light (L) isoform and the lower band is for UVE1 dark (D) isoform. Panel 2 is for KN99α (WT) and AI81 ( bwc1 ), panel 3 is for R265 (WT). All experiments were either 23 h dark+1 h light, (Light) or 24 h constant darkness (Dark). Blots were stripped and reprobed with actin as a loading control.
    Figure Legend Snippet: UVE1 is a Bwc1-regulated gene in Cryptococcus . Northern blots of C. n. var. neoformans , C. n. var. grubii and C. gattii . From left to right, panel 1 is for JEC21 (WT) and AI5 ( bwc1 ); the upper band is for UVE1 light (L) isoform and the lower band is for UVE1 dark (D) isoform. Panel 2 is for KN99α (WT) and AI81 ( bwc1 ), panel 3 is for R265 (WT). All experiments were either 23 h dark+1 h light, (Light) or 24 h constant darkness (Dark). Blots were stripped and reprobed with actin as a loading control.

    Techniques Used: Northern Blot

    Two mRNA isoforms of UVE1 are produced in C. neoformans var. neoformans . Boxes indicate coding regions, with the long light-induced isoform encoding a 660 amino acid residue protein and the shorter dark-expressed isoform encoding a 291 amino acid residue protein. Dark blue encompasses the pfam03851 domain that represents the conserved and active site of the endonuclease. The position of the 70 mer probe used in the C. neoformans microarray, which spans two exons common to both isoforms, is indicated above this region. The green region encodes the predicted mitochondrial localization signal. The orange arrows indicate the start of the UVE1 light and dark transcripts.
    Figure Legend Snippet: Two mRNA isoforms of UVE1 are produced in C. neoformans var. neoformans . Boxes indicate coding regions, with the long light-induced isoform encoding a 660 amino acid residue protein and the shorter dark-expressed isoform encoding a 291 amino acid residue protein. Dark blue encompasses the pfam03851 domain that represents the conserved and active site of the endonuclease. The position of the 70 mer probe used in the C. neoformans microarray, which spans two exons common to both isoforms, is indicated above this region. The green region encodes the predicted mitochondrial localization signal. The orange arrows indicate the start of the UVE1 light and dark transcripts.

    Techniques Used: Produced, Microarray

    UVE1 of C. neoformans confers resistance to UV stress. Ten-fold serial dilutions for different Cryptococcus strains grown at 30°C for 2 days. (A) Left panel untreated control and right panel treated with UV dose of 120 J/m 2 . Order of C. neoformans var. grubii strains from top to bottom KN99α, ST239E6, AI191, AI198. (B) Left panel untreated control and right panel treated with UV dose of 120 J/m 2 . Order of C. neoformans var. neoformans strains from top to bottom JEC21, AISVCN101.
    Figure Legend Snippet: UVE1 of C. neoformans confers resistance to UV stress. Ten-fold serial dilutions for different Cryptococcus strains grown at 30°C for 2 days. (A) Left panel untreated control and right panel treated with UV dose of 120 J/m 2 . Order of C. neoformans var. grubii strains from top to bottom KN99α, ST239E6, AI191, AI198. (B) Left panel untreated control and right panel treated with UV dose of 120 J/m 2 . Order of C. neoformans var. neoformans strains from top to bottom JEC21, AISVCN101.

    Techniques Used:

    C. neoformans Uve1 is functionally similar to S. pombe UVDE. (A) The C. neoformans light (L) and dark (D) isoforms of UVE1 were expressed in an uve1 deletion strain of S. pombe . (A) Ten-fold serial dilutions for different strains of S. pombe grown at 30°C for 2 days. Left panel untreated control and right panel treated with UV dose of 120 J/m 2 . The strains used, from top to bottom are L972, AISVSP2, AISVSP4, AISVSP3 and AISVSP1. For the lower portion of the figure strains used are L972, AISVSP2, AISVSP15. (B) Graph of survival of strains L972, AISVSP4, AISVSP15, AISVSP2, AISVSP1 and AISVSP3 in response to UV stress of 0, 60, 120 and 180 J/m 2 . (C) Subcellular localization of C. neoformans Uve1 (L)-GFP compared to nuclei (Hoechst) in strain AISVSP1. (D) Localization of Uve1 (L)-GFP compared to mitochondria (MitoTracker). Scale bar = 10 µm.
    Figure Legend Snippet: C. neoformans Uve1 is functionally similar to S. pombe UVDE. (A) The C. neoformans light (L) and dark (D) isoforms of UVE1 were expressed in an uve1 deletion strain of S. pombe . (A) Ten-fold serial dilutions for different strains of S. pombe grown at 30°C for 2 days. Left panel untreated control and right panel treated with UV dose of 120 J/m 2 . The strains used, from top to bottom are L972, AISVSP2, AISVSP4, AISVSP3 and AISVSP1. For the lower portion of the figure strains used are L972, AISVSP2, AISVSP15. (B) Graph of survival of strains L972, AISVSP4, AISVSP15, AISVSP2, AISVSP1 and AISVSP3 in response to UV stress of 0, 60, 120 and 180 J/m 2 . (C) Subcellular localization of C. neoformans Uve1 (L)-GFP compared to nuclei (Hoechst) in strain AISVSP1. (D) Localization of Uve1 (L)-GFP compared to mitochondria (MitoTracker). Scale bar = 10 µm.

    Techniques Used:

    The involvement of Uve1 in the photosensory response of C. neoformans . (A) Bwc1-Bwc2 influences three aspects of C. neoformans biology: mating, UV tolerance, and virulence. Uve1 impacts one of these three traits. (B) Uve1 endonuclease protects C. neoformans under UV radiation stress by protecting its mitochondrial genome. Photoreceptor Bwc1 senses blue/UV light, undergoes change in its flavin-binding domain, is activated, and forms a complex with Bwc2, a zinc finger transcription factor. Bwc2 with Bwc1 binds the UVE1 promoter to activate its transcription. Uve1 protein has a mitochondrial localization signal (green), and is transported to mitochondria. In mitochondria, on sensing the UV-induced DNA damage (star) Uve1 binds to damaged DNA and initiates repair by the UVDE DNA damage repair pathway. N nucleus; C cytoplasm; M mitochondria.
    Figure Legend Snippet: The involvement of Uve1 in the photosensory response of C. neoformans . (A) Bwc1-Bwc2 influences three aspects of C. neoformans biology: mating, UV tolerance, and virulence. Uve1 impacts one of these three traits. (B) Uve1 endonuclease protects C. neoformans under UV radiation stress by protecting its mitochondrial genome. Photoreceptor Bwc1 senses blue/UV light, undergoes change in its flavin-binding domain, is activated, and forms a complex with Bwc2, a zinc finger transcription factor. Bwc2 with Bwc1 binds the UVE1 promoter to activate its transcription. Uve1 protein has a mitochondrial localization signal (green), and is transported to mitochondria. In mitochondria, on sensing the UV-induced DNA damage (star) Uve1 binds to damaged DNA and initiates repair by the UVDE DNA damage repair pathway. N nucleus; C cytoplasm; M mitochondria.

    Techniques Used: Binding Assay

    8) Product Images from "Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase"

    Article Title: Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

    Journal: Nature

    doi: 10.1038/nature10560

    The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.
    Figure Legend Snippet: The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.

    Techniques Used: Incubation, Binding Assay, SDS Page, Sequencing, Labeling

    9) Product Images from "Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress"

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038246

    p65 interaction with ATM and p65 Ser 547 phosphorylation by ATM. ( A ) Confirmation of a direct interaction between ATM and p65 . Bacterially expressed GST or GST-ATM (1–247) fusion protein were purified on glutathione agarose beads and used to pull-down p65 from HEK-293 over-expressing HA-p65 cell lysate. Pulled down p65 was detected by immunoblotting with a p65 antibody (upper panel) and GST proteins were stained with coomassie bleu on the PVDF membrane. ( B ) Identification of the ATM target residue . Schematic representation of the different GST-p65 substrates used in the kinase assay. ( C ) In vitro kinase assay . Immunoprecipitated ATM from HEK-293 cells was incubated with GST-p53 and different GST-p65 proteins in presence of [γ− 32 P]ATP. The radiolabelled bands (upper panels) represent auto-phosphorylated ATM, phosphorylated GST-p53 or GST-p65. Levels of ATM and of substrate present in each reaction were determined by western blotting and by coomassie blue staining respectively (lower panels). ( D ) As in (C) In vitro kinase assay but with wt or mutated GST-p53 and GST-p65 proteins as substrates. ATM inhibitor KU55933 was added in some reaction samples as indicated. The same detection methodology than in ( C ) was used. ( E ) As in (D) in vitro kinase assay, but with purified recombinant ATM wt or kd instead of immunoprecipitated ATM and KU55933 utilization. ( F ) Conservation of Ser 547 among different species. Alignment of p65 C-terminal sequence from different mammalian and bird species.
    Figure Legend Snippet: p65 interaction with ATM and p65 Ser 547 phosphorylation by ATM. ( A ) Confirmation of a direct interaction between ATM and p65 . Bacterially expressed GST or GST-ATM (1–247) fusion protein were purified on glutathione agarose beads and used to pull-down p65 from HEK-293 over-expressing HA-p65 cell lysate. Pulled down p65 was detected by immunoblotting with a p65 antibody (upper panel) and GST proteins were stained with coomassie bleu on the PVDF membrane. ( B ) Identification of the ATM target residue . Schematic representation of the different GST-p65 substrates used in the kinase assay. ( C ) In vitro kinase assay . Immunoprecipitated ATM from HEK-293 cells was incubated with GST-p53 and different GST-p65 proteins in presence of [γ− 32 P]ATP. The radiolabelled bands (upper panels) represent auto-phosphorylated ATM, phosphorylated GST-p53 or GST-p65. Levels of ATM and of substrate present in each reaction were determined by western blotting and by coomassie blue staining respectively (lower panels). ( D ) As in (C) In vitro kinase assay but with wt or mutated GST-p53 and GST-p65 proteins as substrates. ATM inhibitor KU55933 was added in some reaction samples as indicated. The same detection methodology than in ( C ) was used. ( E ) As in (D) in vitro kinase assay, but with purified recombinant ATM wt or kd instead of immunoprecipitated ATM and KU55933 utilization. ( F ) Conservation of Ser 547 among different species. Alignment of p65 C-terminal sequence from different mammalian and bird species.

    Techniques Used: Purification, Expressing, Staining, Kinase Assay, In Vitro, Immunoprecipitation, Incubation, Western Blot, Recombinant, Sequencing

    10) Product Images from "Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA"

    Article Title: Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp525

    IFN-β, IDO, and IP-10 gene expression and activation of IRF3 by misplaced U1-snRNA. ( A ) A549 cells were either kept as mock-transfected control or were stimulated with the indicated concentrations of U1-snRNA for 8 h. Alternatively, cells were stimulated with U1-snRNA at 0.1 µg/ml or U1 ctr (0.003 µg/ml) for the indicated time periods ( B ). Thereafter, cells were harvested and IP-10, IDO and IFN-β mRNA was assessed by RT-PCR analysis. (A and B) One representative of three independently performed experiments for each experimental setup is shown. ( C and D ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 µg/ml) or U1ctr (0.003 µg/ml) for 12 h. Thereafter, IFN-β (C) and IP-10 (D) secretion was detected by ELISA analysis. IFN-β ( n = 3) and IP-10 ( n = 4) levels are expressed as means ± SD; ** P
    Figure Legend Snippet: IFN-β, IDO, and IP-10 gene expression and activation of IRF3 by misplaced U1-snRNA. ( A ) A549 cells were either kept as mock-transfected control or were stimulated with the indicated concentrations of U1-snRNA for 8 h. Alternatively, cells were stimulated with U1-snRNA at 0.1 µg/ml or U1 ctr (0.003 µg/ml) for the indicated time periods ( B ). Thereafter, cells were harvested and IP-10, IDO and IFN-β mRNA was assessed by RT-PCR analysis. (A and B) One representative of three independently performed experiments for each experimental setup is shown. ( C and D ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 µg/ml) or U1ctr (0.003 µg/ml) for 12 h. Thereafter, IFN-β (C) and IP-10 (D) secretion was detected by ELISA analysis. IFN-β ( n = 3) and IP-10 ( n = 4) levels are expressed as means ± SD; ** P

    Techniques Used: Expressing, Activation Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Induction of IFN-β by U1-snRNA is dependent on the ISRE-like PRDIII IFN-β promoter element. ( A ) For all experimental conditions, A549 cells were transfected with 0.25 µg of pGL3-IFNβ together with 0.1 µg of pRL-TK (Promega) as described in the ‘Materials and Methods’ section, followed by a 20 h period of rest. Thereafter, cells were either kept as mock-transfected control or were stimulated by additional transfection with U1-snRNA (0.1 µg/ml) or U1 ctr (0.003 µg/ml), respectively. Where indicated, cells were pre-incubated (0.5 h) with bafilomycin A1 at 1 µM. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for bafilomycin A1) throughout the experiment. After removal of liposomes (at 4 h, see ‘Materials and Methods’ section) fresh bafilomycin A1 was added where indicated. After an incubation period of 24 h (total experiment: 24.5 h), cells were harvested and luciferase reporter assays were performed. Data are shown as means ± SD of fold-induction of IFNβ promoter activity compared to cells transfected with pGL3-IFNβ but without subsequent transfection with U1-snRNA for stimulation (mock-transfection) ( n = 3); ** P
    Figure Legend Snippet: Induction of IFN-β by U1-snRNA is dependent on the ISRE-like PRDIII IFN-β promoter element. ( A ) For all experimental conditions, A549 cells were transfected with 0.25 µg of pGL3-IFNβ together with 0.1 µg of pRL-TK (Promega) as described in the ‘Materials and Methods’ section, followed by a 20 h period of rest. Thereafter, cells were either kept as mock-transfected control or were stimulated by additional transfection with U1-snRNA (0.1 µg/ml) or U1 ctr (0.003 µg/ml), respectively. Where indicated, cells were pre-incubated (0.5 h) with bafilomycin A1 at 1 µM. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for bafilomycin A1) throughout the experiment. After removal of liposomes (at 4 h, see ‘Materials and Methods’ section) fresh bafilomycin A1 was added where indicated. After an incubation period of 24 h (total experiment: 24.5 h), cells were harvested and luciferase reporter assays were performed. Data are shown as means ± SD of fold-induction of IFNβ promoter activity compared to cells transfected with pGL3-IFNβ but without subsequent transfection with U1-snRNA for stimulation (mock-transfection) ( n = 3); ** P

    Techniques Used: Transfection, Incubation, Concentration Assay, Luciferase, Activity Assay

    Activation by U1-snRNA is unlikely mediated by TLR4, TLR5, TLR7/8 and PKR. (A and B) A549 cells were either kept as mock-transfected control or were stimulated with U1-snRNA (0.1 µg/ml). Where indicated, cells were stimulated with LPS (10 µg/ml), flagellin (Flg) (100 ng/ml), resiquimod (Rq) (10 µg/ml), or total eukaryotic RNA (0.1 µg/ml) by using the U1-snRNA transfection protocol. After 24 h ( A ) and 4 h ( B ), expression of IDO and IFN-β mRNA was analyzed by RT-PCR (A) and cellular pIRF3 content was determined by immunoblot analysis (B), respectively. For each experimental setup, one representative of three independently performed experiments is shown. ( C ) TLR3 expression by mock-transfected A549 cells was analyzed immunohistochemically using confocal microscopy (TLR3/Cy3; nuclei/DAPI). Insets, negative controls where immunohistochemistry was performed in the absence of the primary antibody. (DE) A549 cells were either kept as mock-transfected control or stimulated by transfection with U1-snRNA (0.1 µg/ml), U1 ctr (0.003 µg/ml), or poly(I:C) (0.1 µg/ml). ( D ) After 4 h, cellular content of pIRF3 and p-eIF2α was determined by immunoblot analysis. For that purpose the blot was cut in half. ( E ) After 24 h, expression of IDO and IFN-β mRNA was analyzed by RT-PCR. (D and E) One representative of three independently performed experiments is shown for each experimental setup.
    Figure Legend Snippet: Activation by U1-snRNA is unlikely mediated by TLR4, TLR5, TLR7/8 and PKR. (A and B) A549 cells were either kept as mock-transfected control or were stimulated with U1-snRNA (0.1 µg/ml). Where indicated, cells were stimulated with LPS (10 µg/ml), flagellin (Flg) (100 ng/ml), resiquimod (Rq) (10 µg/ml), or total eukaryotic RNA (0.1 µg/ml) by using the U1-snRNA transfection protocol. After 24 h ( A ) and 4 h ( B ), expression of IDO and IFN-β mRNA was analyzed by RT-PCR (A) and cellular pIRF3 content was determined by immunoblot analysis (B), respectively. For each experimental setup, one representative of three independently performed experiments is shown. ( C ) TLR3 expression by mock-transfected A549 cells was analyzed immunohistochemically using confocal microscopy (TLR3/Cy3; nuclei/DAPI). Insets, negative controls where immunohistochemistry was performed in the absence of the primary antibody. (DE) A549 cells were either kept as mock-transfected control or stimulated by transfection with U1-snRNA (0.1 µg/ml), U1 ctr (0.003 µg/ml), or poly(I:C) (0.1 µg/ml). ( D ) After 4 h, cellular content of pIRF3 and p-eIF2α was determined by immunoblot analysis. For that purpose the blot was cut in half. ( E ) After 24 h, expression of IDO and IFN-β mRNA was analyzed by RT-PCR. (D and E) One representative of three independently performed experiments is shown for each experimental setup.

    Techniques Used: Activation Assay, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Confocal Microscopy, Immunohistochemistry

    Activation of DLD-1 cells by misplaced U1-snRNA. ( A ) DLD-1 cells were either kept as mock-transfected control or stimulated with U1-snRNA (2.5 μg/ml). Where indicated, cells were pre-incubated (0.5 h) either with bafilomycin A1 (Baf, 1 μM) or CHX (10 μg/ml). All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for Baf and CHX) throughout the experiment. After an incubation period of 4 h (total experiment: 4.5 h) cellular expression of pIRF3 was analyzed by immunoblot analysis. One representative of three independently performed experiments is shown. ( B ) DLD-1 cells were either kept as mock-transfected control or were stimulated with U1-snRNA at 2.5 µg/ml or U1 ctr (0.075 µg/ml) for 6 h or 12 h. Thereafter, cells were harvested and IDO and IFN-β mRNA was assessed by RT-PCR analysis. One representative of three independently performed experiments is shown. (C) DLD-1 cells were either kept as mock-transfected control or stimulated with U1-snRNA (2.5 µg/ml) for 6 h or 12 h. Thereafter, IDO protein expression was determined by immunoblot analysis. One representative of three independently performed experiments is shown. ( D ) U1-snRNA was digested by treatment with benzonase as outlined in the ‘Materials and Methods’ section. DLD-1 cells were either kept as mock-transfected control or stimulated with intact or digested U1-snRNA (each at 2.5 μg/ml). After 20 h, secretion of IP-10 was determined by ELISA analysis. IP-10 levels ( n = 4) are expressed as means ± SD; ** P
    Figure Legend Snippet: Activation of DLD-1 cells by misplaced U1-snRNA. ( A ) DLD-1 cells were either kept as mock-transfected control or stimulated with U1-snRNA (2.5 μg/ml). Where indicated, cells were pre-incubated (0.5 h) either with bafilomycin A1 (Baf, 1 μM) or CHX (10 μg/ml). All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for Baf and CHX) throughout the experiment. After an incubation period of 4 h (total experiment: 4.5 h) cellular expression of pIRF3 was analyzed by immunoblot analysis. One representative of three independently performed experiments is shown. ( B ) DLD-1 cells were either kept as mock-transfected control or were stimulated with U1-snRNA at 2.5 µg/ml or U1 ctr (0.075 µg/ml) for 6 h or 12 h. Thereafter, cells were harvested and IDO and IFN-β mRNA was assessed by RT-PCR analysis. One representative of three independently performed experiments is shown. (C) DLD-1 cells were either kept as mock-transfected control or stimulated with U1-snRNA (2.5 µg/ml) for 6 h or 12 h. Thereafter, IDO protein expression was determined by immunoblot analysis. One representative of three independently performed experiments is shown. ( D ) U1-snRNA was digested by treatment with benzonase as outlined in the ‘Materials and Methods’ section. DLD-1 cells were either kept as mock-transfected control or stimulated with intact or digested U1-snRNA (each at 2.5 μg/ml). After 20 h, secretion of IP-10 was determined by ELISA analysis. IP-10 levels ( n = 4) are expressed as means ± SD; ** P

    Techniques Used: Activation Assay, Transfection, Incubation, Concentration Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Activation of A549 cells under the influence of U1-snRNA is inhibited by bafilomycin A1. ( A ) Mock-transfected A549 cells were analyzed for expression of TLR3, TLR4, TLR5, TLR7, TLR8, RIG-I, MDA5 and PKR by RT–PCR. ( B ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 μg/ml). Where indicated, cells were pre-incubated (0.5 h) with either bafilomycin A1 (Baf, 1 μM) or CHX (10 μg/ml). All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for Baf and CHX) throughout the experiment. After an incubation period of 4 h (total experiment: 4.5 h) cellular expression of pIRF3 was analyzed by immunoblot analysis. One representative of three independently performed experiments is shown. ( C–E ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 μg/ml). In Figure 3 C, cells were also exposed to U1 ctr (0.003 µg/ml). Where indicated, cells were pre-incubated (0.5 h) with bafilomycin A1 at 1 μM. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for Baf) throughout the experiment. After removal of liposomes (at 4 h, see ‘Materials and Methods’ section) fresh bafilomycin A1 was added where indicated. (C) After an incubation period of 12 h (total experiment: 12.5 h), cellular mRNA expression of IDO, IFN-β, and IP-10 was determined by RT-PCR. One representative of five independently performed experiments is shown. (DE) After 12 h, also secretion of IFN-β and IP-10 was determined by ELISA analysis. IFN-β ( n = 3) and IP-10 (n = 4) levels are expressed as means ± SD; ** P
    Figure Legend Snippet: Activation of A549 cells under the influence of U1-snRNA is inhibited by bafilomycin A1. ( A ) Mock-transfected A549 cells were analyzed for expression of TLR3, TLR4, TLR5, TLR7, TLR8, RIG-I, MDA5 and PKR by RT–PCR. ( B ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 μg/ml). Where indicated, cells were pre-incubated (0.5 h) with either bafilomycin A1 (Baf, 1 μM) or CHX (10 μg/ml). All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for Baf and CHX) throughout the experiment. After an incubation period of 4 h (total experiment: 4.5 h) cellular expression of pIRF3 was analyzed by immunoblot analysis. One representative of three independently performed experiments is shown. ( C–E ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 μg/ml). In Figure 3 C, cells were also exposed to U1 ctr (0.003 µg/ml). Where indicated, cells were pre-incubated (0.5 h) with bafilomycin A1 at 1 μM. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for Baf) throughout the experiment. After removal of liposomes (at 4 h, see ‘Materials and Methods’ section) fresh bafilomycin A1 was added where indicated. (C) After an incubation period of 12 h (total experiment: 12.5 h), cellular mRNA expression of IDO, IFN-β, and IP-10 was determined by RT-PCR. One representative of five independently performed experiments is shown. (DE) After 12 h, also secretion of IFN-β and IP-10 was determined by ELISA analysis. IFN-β ( n = 3) and IP-10 (n = 4) levels are expressed as means ± SD; ** P

    Techniques Used: Activation Assay, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Biological activity of U1-snRNA is blunted by digestion using RNases A/T1 or benzonase and is dependent on U1-snRNA transfection. U1-snRNA was digested by RNases A/T1 ( A and C ) or benzonase ( B and D ) as outlined in the ‘Materials and Methods’ section. A549 cells were either kept as mock-transfected control or stimulated with intact or digested U1-snRNA (each at 0.1 μg/ml). (A and B) After 24 h, cells were harvested and mRNA coding for IDO and IFN-β was determined by RT-PCR. For each experimental setup, one representative of five independently performed experiments is shown. (C and D) After 6 h, cells were harvested and cellular pIRF3 content was determined by immunoblot analysis. For each experimental setup, one representative of three independently performed experiments is shown. ( E ) A549 cells were kept as unstimulated control or exposed to U1-snRNA (0.1 μg/ml) without transfection. In addition, cells were either kept as mock-transfected control or stimulated by standard transfection with U1-snRNA (0.1 μg/ml). After 2 h, cells were harvested and cellular pIRF3 content was determined by immunoblot analysis. For each experimental setup, one representative of three independently performed experiments is shown. ( F ) 32 P-labeled U1-snRNA (0.1 μg/ml) was transfected by standard protocol into A549 cells. After 4 h, total cellular RNA was isolated. Thereafter, integrity of transfected U1-snRNA was assessed by gel electrophoresis and subsequent analysis using a PhosphoImager. Left lane (ivt), 1000 cpm of in vitro transcribed 32 P-labeled U1-snRNA (not transfected into cells); middle lane, 1000 cpm of total RNA isolated 4 h after transfection of 32 P-labeled U1-snRNA into A549 cells; right lane 32 P-labeled GAPDH probe (184 nt) serving as size-control (U1-snRNA: 164 nt).
    Figure Legend Snippet: Biological activity of U1-snRNA is blunted by digestion using RNases A/T1 or benzonase and is dependent on U1-snRNA transfection. U1-snRNA was digested by RNases A/T1 ( A and C ) or benzonase ( B and D ) as outlined in the ‘Materials and Methods’ section. A549 cells were either kept as mock-transfected control or stimulated with intact or digested U1-snRNA (each at 0.1 μg/ml). (A and B) After 24 h, cells were harvested and mRNA coding for IDO and IFN-β was determined by RT-PCR. For each experimental setup, one representative of five independently performed experiments is shown. (C and D) After 6 h, cells were harvested and cellular pIRF3 content was determined by immunoblot analysis. For each experimental setup, one representative of three independently performed experiments is shown. ( E ) A549 cells were kept as unstimulated control or exposed to U1-snRNA (0.1 μg/ml) without transfection. In addition, cells were either kept as mock-transfected control or stimulated by standard transfection with U1-snRNA (0.1 μg/ml). After 2 h, cells were harvested and cellular pIRF3 content was determined by immunoblot analysis. For each experimental setup, one representative of three independently performed experiments is shown. ( F ) 32 P-labeled U1-snRNA (0.1 μg/ml) was transfected by standard protocol into A549 cells. After 4 h, total cellular RNA was isolated. Thereafter, integrity of transfected U1-snRNA was assessed by gel electrophoresis and subsequent analysis using a PhosphoImager. Left lane (ivt), 1000 cpm of in vitro transcribed 32 P-labeled U1-snRNA (not transfected into cells); middle lane, 1000 cpm of total RNA isolated 4 h after transfection of 32 P-labeled U1-snRNA into A549 cells; right lane 32 P-labeled GAPDH probe (184 nt) serving as size-control (U1-snRNA: 164 nt).

    Techniques Used: Activity Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Labeling, Isolation, Nucleic Acid Electrophoresis, In Vitro

    Activation of A549 cells by misplaced U2-snRNA. A549 cells were either kept as mock-transfected control or were stimulated with either U1-snRNA or U2-snRNA (both at 0.1 µg/ml) or with U1 ctr or U2 ctr (both at 0.003 µg/ml). After 6 h, cellular pIRF3 content was determined by immunoblot analysis. One representative of three independently performed experiments is shown.
    Figure Legend Snippet: Activation of A549 cells by misplaced U2-snRNA. A549 cells were either kept as mock-transfected control or were stimulated with either U1-snRNA or U2-snRNA (both at 0.1 µg/ml) or with U1 ctr or U2 ctr (both at 0.003 µg/ml). After 6 h, cellular pIRF3 content was determined by immunoblot analysis. One representative of three independently performed experiments is shown.

    Techniques Used: Activation Assay, Transfection

    Misplaced U1-snRNA mediates anti-inflammatory effects as detected in A549 cell/PBMC co-culture experiments. A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 µg/ml) or U1 ctr (0.003 µg/ml), respectively. After removal of liposomes at 4 h, PBMC were seeded into transwell inserts (9 × 10 6 cells per insert) and placed on top A549 cells for co-culturing as described in the ‘Materials and Methods’ section. Where indicated, PBMC were stimulated with PHA (1 µg/ml). After 72 h, co-culture supernatants were harvested and production of IL-10 ( A ) and TNF-α ( B ) was assessed by ELISA analysis. Data are expressed as means ± SEM with n = 4 (A) and 6 (B), respectively; ** P
    Figure Legend Snippet: Misplaced U1-snRNA mediates anti-inflammatory effects as detected in A549 cell/PBMC co-culture experiments. A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 µg/ml) or U1 ctr (0.003 µg/ml), respectively. After removal of liposomes at 4 h, PBMC were seeded into transwell inserts (9 × 10 6 cells per insert) and placed on top A549 cells for co-culturing as described in the ‘Materials and Methods’ section. Where indicated, PBMC were stimulated with PHA (1 µg/ml). After 72 h, co-culture supernatants were harvested and production of IL-10 ( A ) and TNF-α ( B ) was assessed by ELISA analysis. Data are expressed as means ± SEM with n = 4 (A) and 6 (B), respectively; ** P

    Techniques Used: Co-Culture Assay, Transfection, Enzyme-linked Immunosorbent Assay

    Activation of STAT1 and STAT3 by U1-snRNA. ( A and B ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 µg/ml) or U1 ctr (0.003 µg/ml). After the indicated time periods, cellular content of pIRF3 (A and B), pSTAT1 (A) and pSTAT3 (B) was determined by immunoblot analysis. For detection of pIRF3 and pSTAT1/3 on the same blot, blots were cut in half. For each experimental setup, one representative of three independently performed experiments is shown. ( C ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 µg/ml) or U1 ctr (0.003 µg/ml). After 8 h, cellular IL-18BP mRNA expression was assessed by quantitative realtime PCR analysis. IL-18BP mRNA was normalized to that of GAPDH and is shown as fold induction compared with mock-stimulated control ± SD ( n = 3); ** P
    Figure Legend Snippet: Activation of STAT1 and STAT3 by U1-snRNA. ( A and B ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 µg/ml) or U1 ctr (0.003 µg/ml). After the indicated time periods, cellular content of pIRF3 (A and B), pSTAT1 (A) and pSTAT3 (B) was determined by immunoblot analysis. For detection of pIRF3 and pSTAT1/3 on the same blot, blots were cut in half. For each experimental setup, one representative of three independently performed experiments is shown. ( C ) A549 cells were either kept as mock-transfected control or stimulated with U1-snRNA (0.1 µg/ml) or U1 ctr (0.003 µg/ml). After 8 h, cellular IL-18BP mRNA expression was assessed by quantitative realtime PCR analysis. IL-18BP mRNA was normalized to that of GAPDH and is shown as fold induction compared with mock-stimulated control ± SD ( n = 3); ** P

    Techniques Used: Activation Assay, Transfection, Expressing, Polymerase Chain Reaction

    11) Product Images from "?-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling"

    Article Title: ?-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E10-07-0636

    Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.
    Figure Legend Snippet: Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Techniques Used: Expressing, Copurification, Western Blot, Fluorescence, Microscopy, Purification, Positron Emission Tomography, Incubation, SDS Page, Staining

    12) Product Images from "LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus"

    Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124058

    Phosphotransfer reaction between Brucella LOVHK and RRs. Purified LOVHK protein was illuminated in phosphorylation buffer containing [γ- 32 P] ATP. After 15 min at 37°C, purified response regulators were added to the mixture to a final concentration of 2.5 μM each for the three proteins. The final concentration of LOVHK was also 2.5 μM. At the indicated times after addition of the corresponding response regulators, aliquots were drawn and separated by 15% SDS-PAGE. Autoradiograms are shown on the left, and the graphs on the right side indicate the relative intensity of each band to the total intensity at time 20 seconds. The experiment was repeated three times, and a representative experiment is shown. Numbers above the autoradiograms indicate the time in seconds (columns 1 and 2) or in minutes (columns from 3 to 9) respectively. A. Phosphotransfer between LOVHK and PhyR. B. Phosphotransfer between LOVHK and LovR. C. Phosphotransfer between LOVHK, PhyR and LovR simultaneously. LOVHK: blue circles, PhyR: green triangles, LovR: red rhomboids, total intensity: black squares. Molecular weights of protein constructions are indicated in Fig 1A .
    Figure Legend Snippet: Phosphotransfer reaction between Brucella LOVHK and RRs. Purified LOVHK protein was illuminated in phosphorylation buffer containing [γ- 32 P] ATP. After 15 min at 37°C, purified response regulators were added to the mixture to a final concentration of 2.5 μM each for the three proteins. The final concentration of LOVHK was also 2.5 μM. At the indicated times after addition of the corresponding response regulators, aliquots were drawn and separated by 15% SDS-PAGE. Autoradiograms are shown on the left, and the graphs on the right side indicate the relative intensity of each band to the total intensity at time 20 seconds. The experiment was repeated three times, and a representative experiment is shown. Numbers above the autoradiograms indicate the time in seconds (columns 1 and 2) or in minutes (columns from 3 to 9) respectively. A. Phosphotransfer between LOVHK and PhyR. B. Phosphotransfer between LOVHK and LovR. C. Phosphotransfer between LOVHK, PhyR and LovR simultaneously. LOVHK: blue circles, PhyR: green triangles, LovR: red rhomboids, total intensity: black squares. Molecular weights of protein constructions are indicated in Fig 1A .

    Techniques Used: Purification, Concentration Assay, SDS Page

    13) Product Images from "Plk4-dependent phosphorylation of STIL is required for centriole duplication"

    Article Title: Plk4-dependent phosphorylation of STIL is required for centriole duplication

    Journal: Biology Open

    doi: 10.1242/bio.201411023

    Phosphorylation of STIL by Plk4 triggers centriole duplication. (A) Flag-STIL full-length or 5A mutant (S871A/S873A/S874A/S1116A/T1250A) expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Co immunoprecipitation of Flag-STIL wt/5A and Myc-Plk4. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation using anti-Flag antibodies. Input and IP samples were analyzed by western blotting with antibodies against Flag- and Myc-tag. The asterisk marks an unspecific band recognized by the anti-Myc antibody. The dividing lane indicates grouping of images from different parts of the same gel, as an intervening lane was removed for presentation purposes. (C) U2OS cells transiently expressing Flag EV, Flag-STIL wt or Flag-STIL 5A were analyzed by indirect immunofluorescence using staining with anti-CP110 and mouse anti-Flag antibodies 72 h after transfection. The number of transfected cells with more than four centrioles was determined based on CP110 staining. Values in the graph are mean percentages±s.d. from three independent experiments, 50 transfected cells were analyzed in each experiment (***P
    Figure Legend Snippet: Phosphorylation of STIL by Plk4 triggers centriole duplication. (A) Flag-STIL full-length or 5A mutant (S871A/S873A/S874A/S1116A/T1250A) expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Co immunoprecipitation of Flag-STIL wt/5A and Myc-Plk4. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation using anti-Flag antibodies. Input and IP samples were analyzed by western blotting with antibodies against Flag- and Myc-tag. The asterisk marks an unspecific band recognized by the anti-Myc antibody. The dividing lane indicates grouping of images from different parts of the same gel, as an intervening lane was removed for presentation purposes. (C) U2OS cells transiently expressing Flag EV, Flag-STIL wt or Flag-STIL 5A were analyzed by indirect immunofluorescence using staining with anti-CP110 and mouse anti-Flag antibodies 72 h after transfection. The number of transfected cells with more than four centrioles was determined based on CP110 staining. Values in the graph are mean percentages±s.d. from three independent experiments, 50 transfected cells were analyzed in each experiment (***P

    Techniques Used: Mutagenesis, Immunoprecipitation, Incubation, In Vitro, Kinase Assay, SDS Page, Staining, Autoradiography, Transfection, Western Blot, Expressing, Immunofluorescence

    Phosphorylation of STIL by Plk4. (A) Full-length Flag-STIL expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Indicated Flag-STIL fragments were expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies. Immunoprecipitation fractions were incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP, followed by SDS-PAGE and autoradiography. In vitro kinase assay with Flag-STIL fragments or Plk4 alone is shown as control. The asterisk indicates phosphorylated Flag-STIL 781-1287. 10% of each precipitation fraction was analyzed by western blotting using anti-Plk4 and anti-Flag antibodies. (C) Plk4 phosphorylation sites in the STIL protein identified by mass spectrometry analysis of bacterially purified GST-STIL 1-619 and 619-1287 phosphorylated in vitro by Zz-Plk4. Alignment of the identified sites in human, mouse, Xenopus and zebrafish STIL and Drosophila Ana2 is shown.
    Figure Legend Snippet: Phosphorylation of STIL by Plk4. (A) Full-length Flag-STIL expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Indicated Flag-STIL fragments were expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies. Immunoprecipitation fractions were incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP, followed by SDS-PAGE and autoradiography. In vitro kinase assay with Flag-STIL fragments or Plk4 alone is shown as control. The asterisk indicates phosphorylated Flag-STIL 781-1287. 10% of each precipitation fraction was analyzed by western blotting using anti-Plk4 and anti-Flag antibodies. (C) Plk4 phosphorylation sites in the STIL protein identified by mass spectrometry analysis of bacterially purified GST-STIL 1-619 and 619-1287 phosphorylated in vitro by Zz-Plk4. Alignment of the identified sites in human, mouse, Xenopus and zebrafish STIL and Drosophila Ana2 is shown.

    Techniques Used: Immunoprecipitation, Incubation, In Vitro, Kinase Assay, SDS Page, Staining, Autoradiography, Western Blot, Mass Spectrometry, Purification

    14) Product Images from "The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals"

    Article Title: The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004799

    Membrane vesicles harboring the linker peptide mutant SaeS exhibit altered phosphotransferase activity. (A) Levels of SaeR-P following incubation of SaeR, [γ- 32 P] ATP, and membrane vesicles (300 μg) harboring the wild-type or the various linker mutant SaeS proteins. (B) Quantitation of the phosphotransfer assay shown in (A). Each datum of the plot depicts the level of SaeR-P relative to that by the wild-type SaeS at the initial time (1 min). Data correspond to the mean values of three independent experiments, and error bars show standard deviation.
    Figure Legend Snippet: Membrane vesicles harboring the linker peptide mutant SaeS exhibit altered phosphotransferase activity. (A) Levels of SaeR-P following incubation of SaeR, [γ- 32 P] ATP, and membrane vesicles (300 μg) harboring the wild-type or the various linker mutant SaeS proteins. (B) Quantitation of the phosphotransfer assay shown in (A). Each datum of the plot depicts the level of SaeR-P relative to that by the wild-type SaeS at the initial time (1 min). Data correspond to the mean values of three independent experiments, and error bars show standard deviation.

    Techniques Used: Mutagenesis, Activity Assay, Incubation, Quantitation Assay, Standard Deviation

    Alanine substitutions in the linker peptide alter the kinase and phosphotransferase activities of SaeS. (A) The autokinase activity of wild type (WT) and select linker peptide mutants of SaeS. The purified MBP-SaeS proteins (5 μM) were incubated with [γ- 32 P] ATP at RT for 20 min. The autoradiograph of the phosphorylated MBP-SaeS (upper panel) is shown with its quantification results in a bar graph (lower panel). (B) Assessment of the autokinase activity of wild type (WT) and select linker peptide mutants for 30 min. The wild-type or the linker peptide mutant MBP-SaeS proteins (5 μM) were mixed with [γ- 32 P] ATP and, at the indicated times, the level of phosphorylated MBP-SaeS was analyzed by phosphor imager analysis. (C) Quantitation of the autophosphorylation assays shown in (B). The plot depicts the levels of MBP-SaeS-P relative to the wild-type MBP-SaeS-P at time 1 min as a function of time. (D) Phosphotransferase activity of the wild type (WT) and select linker peptide mutants of SaeS. Phosphorylated MBP-SaeS (5 μM) was mixed with SaeR (10 μM). At the times indicated, the reaction was stopped and the phosphorylated proteins were analyzed by SDS-PAGE and phosphor imager analysis. (E) Quantification of the phosphotransfer assays shown in (D). Each datum on the plot depicts the level of SaeR-P relative to that of the wild-type SaeS at the initial time (1 min). All data correspond to the mean values of three independent experiments, and error bars show standard deviation. For statistical analyses, unpaired two-tailed student’s t-test was used. ***, p
    Figure Legend Snippet: Alanine substitutions in the linker peptide alter the kinase and phosphotransferase activities of SaeS. (A) The autokinase activity of wild type (WT) and select linker peptide mutants of SaeS. The purified MBP-SaeS proteins (5 μM) were incubated with [γ- 32 P] ATP at RT for 20 min. The autoradiograph of the phosphorylated MBP-SaeS (upper panel) is shown with its quantification results in a bar graph (lower panel). (B) Assessment of the autokinase activity of wild type (WT) and select linker peptide mutants for 30 min. The wild-type or the linker peptide mutant MBP-SaeS proteins (5 μM) were mixed with [γ- 32 P] ATP and, at the indicated times, the level of phosphorylated MBP-SaeS was analyzed by phosphor imager analysis. (C) Quantitation of the autophosphorylation assays shown in (B). The plot depicts the levels of MBP-SaeS-P relative to the wild-type MBP-SaeS-P at time 1 min as a function of time. (D) Phosphotransferase activity of the wild type (WT) and select linker peptide mutants of SaeS. Phosphorylated MBP-SaeS (5 μM) was mixed with SaeR (10 μM). At the times indicated, the reaction was stopped and the phosphorylated proteins were analyzed by SDS-PAGE and phosphor imager analysis. (E) Quantification of the phosphotransfer assays shown in (D). Each datum on the plot depicts the level of SaeR-P relative to that of the wild-type SaeS at the initial time (1 min). All data correspond to the mean values of three independent experiments, and error bars show standard deviation. For statistical analyses, unpaired two-tailed student’s t-test was used. ***, p

    Techniques Used: Activity Assay, Purification, Incubation, Autoradiography, Mutagenesis, Quantitation Assay, SDS Page, Standard Deviation, Two Tailed Test

    15) Product Images from "Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress"

    Article Title: Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000275

    In vitro phosphorylation of lamin A by GST-UL97. (A) Recombinant His-tagged lamin A was incubated in kinase reaction buffer in the presence of γ- 32 P-ATP either alone (no kinase), with catalytically deficient GST-UL97 K355Q (K355Q), or with wild-type GST-UL97 (GST97 WT). GST-UL97 K335Q or wild-type GST-UL97 were also incubated in kinase buffer without lamin A. Following termination of kinase reactions, proteins were resolved by SDS-PAGE. Signal from incorporation of 32 P was detected by exposure to a phosphorscreen (top panel), and total protein was detected by Coomassie brilliant blue staining (bottom panel). The positions of radiolabeled GST-UL97 (GST97) and lamin A, and Coomassie stained lamin A are indicated. (The amounts of GST-UL97 were too small to see on the stained gel.) (B) UL97 autophosphorylation and labeling of lamin A were quantified following in in vitro kinase reactions in the presence of varying concentrations of maribavir (MBV). Signal detected from 32 P incorporation for autophosphorylation of GST-UL97 and phosphorylation of His-tagged lamin A are plotted as a percent of the signal detected in the absence of drug. The results taken together show that UL97 phosphorylates lamin A in vitro.
    Figure Legend Snippet: In vitro phosphorylation of lamin A by GST-UL97. (A) Recombinant His-tagged lamin A was incubated in kinase reaction buffer in the presence of γ- 32 P-ATP either alone (no kinase), with catalytically deficient GST-UL97 K355Q (K355Q), or with wild-type GST-UL97 (GST97 WT). GST-UL97 K335Q or wild-type GST-UL97 were also incubated in kinase buffer without lamin A. Following termination of kinase reactions, proteins were resolved by SDS-PAGE. Signal from incorporation of 32 P was detected by exposure to a phosphorscreen (top panel), and total protein was detected by Coomassie brilliant blue staining (bottom panel). The positions of radiolabeled GST-UL97 (GST97) and lamin A, and Coomassie stained lamin A are indicated. (The amounts of GST-UL97 were too small to see on the stained gel.) (B) UL97 autophosphorylation and labeling of lamin A were quantified following in in vitro kinase reactions in the presence of varying concentrations of maribavir (MBV). Signal detected from 32 P incorporation for autophosphorylation of GST-UL97 and phosphorylation of His-tagged lamin A are plotted as a percent of the signal detected in the absence of drug. The results taken together show that UL97 phosphorylates lamin A in vitro.

    Techniques Used: In Vitro, Recombinant, Incubation, SDS Page, Staining, Labeling

    16) Product Images from "Diverse cell stresses induce unique patterns of tRNA up- and down-regulation: tRNA-seq for quantifying changes in tRNA copy number"

    Article Title: Diverse cell stresses induce unique patterns of tRNA up- and down-regulation: tRNA-seq for quantifying changes in tRNA copy number

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku945

    Two-step ligation method for the conversion of tRNA to cDNA. Bulk tRNAs extracted from S. cerevisiae are first 3′-end ligated with a DNA adaptor. After reverse transcription, a second DNA adaptor is ligated to the 3′-end of the resulting cDNA, and the resulting species are PCR amplified followed by standard Illumina sample preparation and analysis.
    Figure Legend Snippet: Two-step ligation method for the conversion of tRNA to cDNA. Bulk tRNAs extracted from S. cerevisiae are first 3′-end ligated with a DNA adaptor. After reverse transcription, a second DNA adaptor is ligated to the 3′-end of the resulting cDNA, and the resulting species are PCR amplified followed by standard Illumina sample preparation and analysis.

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification, Sample Prep

    17) Product Images from "Physicochemical analysis of rotavirus segment 11 supports a 'modified panhandle' structure and not the predicted alternative tRNA-like structure (TRLS)"

    Article Title: Physicochemical analysis of rotavirus segment 11 supports a 'modified panhandle' structure and not the predicted alternative tRNA-like structure (TRLS)

    Journal: Archives of Virology

    doi: 10.1007/s00705-013-1802-8

    RNase T1 cleavage results of single-stranded guanines in the 5’-terminal sequence of rotavirus RNA11. Single-stranded rotavirus RNA11 was labelled at the 5’ terminus using [γ- 32 P]ATP, subjected to partial digestion by RNase T1, and resolved on a 12 % polyacrylamide 7 M urea gel. The dark bands in the last column on the right show the positions of the single-stranded guanines cleaved by RNase T1, compared to the third column from the left (no-enzyme control). The first and second columns from the left represent an RNA11 sequence ladder generated from the same RNA by alkaline hydrolysis for 5 and 10 min, respectively
    Figure Legend Snippet: RNase T1 cleavage results of single-stranded guanines in the 5’-terminal sequence of rotavirus RNA11. Single-stranded rotavirus RNA11 was labelled at the 5’ terminus using [γ- 32 P]ATP, subjected to partial digestion by RNase T1, and resolved on a 12 % polyacrylamide 7 M urea gel. The dark bands in the last column on the right show the positions of the single-stranded guanines cleaved by RNase T1, compared to the third column from the left (no-enzyme control). The first and second columns from the left represent an RNA11 sequence ladder generated from the same RNA by alkaline hydrolysis for 5 and 10 min, respectively

    Techniques Used: Sequencing, Generated

    18) Product Images from "Novel VCP modulators mitigate major pathologies of rd10, a mouse model of retinitis pigmentosa"

    Article Title: Novel VCP modulators mitigate major pathologies of rd10, a mouse model of retinitis pigmentosa

    Journal: Scientific Reports

    doi: 10.1038/srep05970

    Structures and characterization of KUSs, novel VCP modulators. (a) Structures and IC 50 values of KUS11, KUS31, KUS69, KUS94, KUS121, and KUS187. Note that KUS11 did not inhibit the ATPase activity of recombinant VCP, and it did not share a common structure with the other KUSs. (b) ATPase activity assays of recombinant human NSF, comparing KUSs and DBeQ. (c) Immunoblot analysis of ubiquitinated proteins, an ER stress marker (CHOP), and an autophagy indicator (LC3), comparing KUSs and DBeQ. As a control, MG132, a proteasome inhibitor, was used for the analysis. Actin served as a loading control. Complete scans of the different blots are presented in Supplementary Fig. 7 . (d) Comparison of KUSs and DBeQ for cell death-inducing activities. HeLa cells were treated with 50 μM DBeQ or KUSs for 24 hours.
    Figure Legend Snippet: Structures and characterization of KUSs, novel VCP modulators. (a) Structures and IC 50 values of KUS11, KUS31, KUS69, KUS94, KUS121, and KUS187. Note that KUS11 did not inhibit the ATPase activity of recombinant VCP, and it did not share a common structure with the other KUSs. (b) ATPase activity assays of recombinant human NSF, comparing KUSs and DBeQ. (c) Immunoblot analysis of ubiquitinated proteins, an ER stress marker (CHOP), and an autophagy indicator (LC3), comparing KUSs and DBeQ. As a control, MG132, a proteasome inhibitor, was used for the analysis. Actin served as a loading control. Complete scans of the different blots are presented in Supplementary Fig. 7 . (d) Comparison of KUSs and DBeQ for cell death-inducing activities. HeLa cells were treated with 50 μM DBeQ or KUSs for 24 hours.

    Techniques Used: Activity Assay, Recombinant, Marker

    19) Product Images from "Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii"

    Article Title: Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii

    Journal: Scientific Reports

    doi: 10.1038/srep24278

    Effects of mutations on the phosphorylation activities of LtrK and LtrR. To assess the phosphorylation state of proteins, samples were electrophoresed on a SDS-polyacrylamide gel, and autoradiography performed by phosphorimaging (panels a–d ). ( a ) Autophosphorylation of LtrK mutant proteins. Proteins (7 μg) were incubated with [γ- 32 P]-ATP at room temperature for 30 min. Histidine residues were replaced with arginine, including the double mutant (DM) H443R/H448R. ( b ) Autophosphorylation of LtrK mutant proteins. Histidine residues were replaced with alanine, including the double mutant (DM) H443A/H448A. ( c ) Phosphatase activity of LtrK mutant proteins. LtrR-P was incubated with wild-type (lane 1) and mutant proteins H367R (lane 2), H443R (lane 3), H443R/H448R (lane 4), H502R (lane 5) for 30 min at room temperature. ( d ) Phosphotransfer from LtrK-P to LtrR mutant proteins. LtrR (80 μg) wild-type and mutant proteins were phosphorylated by GST-LtrK-P (20 μg) immobilized on a gravity flow column containing glutathione agarose beads as described for Fig. 2c .
    Figure Legend Snippet: Effects of mutations on the phosphorylation activities of LtrK and LtrR. To assess the phosphorylation state of proteins, samples were electrophoresed on a SDS-polyacrylamide gel, and autoradiography performed by phosphorimaging (panels a–d ). ( a ) Autophosphorylation of LtrK mutant proteins. Proteins (7 μg) were incubated with [γ- 32 P]-ATP at room temperature for 30 min. Histidine residues were replaced with arginine, including the double mutant (DM) H443R/H448R. ( b ) Autophosphorylation of LtrK mutant proteins. Histidine residues were replaced with alanine, including the double mutant (DM) H443A/H448A. ( c ) Phosphatase activity of LtrK mutant proteins. LtrR-P was incubated with wild-type (lane 1) and mutant proteins H367R (lane 2), H443R (lane 3), H443R/H448R (lane 4), H502R (lane 5) for 30 min at room temperature. ( d ) Phosphotransfer from LtrK-P to LtrR mutant proteins. LtrR (80 μg) wild-type and mutant proteins were phosphorylated by GST-LtrK-P (20 μg) immobilized on a gravity flow column containing glutathione agarose beads as described for Fig. 2c .

    Techniques Used: Autoradiography, Mutagenesis, Incubation, Activity Assay, Flow Cytometry

    Autophosphorylation and phosphotransfer (kinase and phosphatase) activities of LtrK with LtrR. To assess the phosphorylation state of proteins, samples were electrophoresed on a SDS-polyacrylamide gel, and autoradiography performed by phosphorimaging (panels a–e ). Incorporation for LtrK and/or LtrR shown as a percentage of the total radioactivity on the respective autoradiograms (panels b,d,e ). ( a ) Autophosphorylation of LtrK. LtrK fused to GST (GST-LtrK) and LtrK (1 μg) were incubated with [γ- 32 P]-ATP at room temperature. At indicated times, 10 μl samples were added to 5 μl of sample buffer, heated at 95 °C for 3 min and 3 μl of each mixture analysed by gel-phosphorimaging. ( b ) Time course of autophosphorylation. Plot showing autophosphorylation incorporation of GST-LtrK with [γ- 32 P]-ATP over a 60 min incubation at room temperature. The exponential fit curve (solid line) gave a calculated rate constant of 0.08. ( c ) Phosphotransfer from LtrK-P to LtrR. GST-LtrK (15 μg) bound to glutathione agarose beads in a gravity flow column was phosphorylated with [γ- 32 P]-ATP for 30 min at room temperature, free [γ- 32 P]-ATP washed off, LtrR (60 μg) passed through the column and LtrR-P collected in the flowthrough. The LtrR-P sample (10 μl) was added to 3 μl of sample buffer containing 100 mM EDTA, and 3 μl analysed by gel-phosphorimaging. ( d ) Stability of LtrR-P. LtrR-P generated from phosphotransfer from LtrK-P (panel c ) was incubated at room temperature and retention of γ- 32 P assessed over time (as for panel b ). The exponential fit curve (solid line) gave a calculated rate constant of 0.29, from which t 1/2 was calculated as ln2/k. ( e ) LtrK phosphatase activity. As for panel ( d ) except LtrR-P incubated with LtrK. The fit curve (solid line) represents two exponentials with calculated rate constants of 3.45 for the first phase and 0.3 for the second phase 2. Values of t 1/2 were calculated as ln2/k.
    Figure Legend Snippet: Autophosphorylation and phosphotransfer (kinase and phosphatase) activities of LtrK with LtrR. To assess the phosphorylation state of proteins, samples were electrophoresed on a SDS-polyacrylamide gel, and autoradiography performed by phosphorimaging (panels a–e ). Incorporation for LtrK and/or LtrR shown as a percentage of the total radioactivity on the respective autoradiograms (panels b,d,e ). ( a ) Autophosphorylation of LtrK. LtrK fused to GST (GST-LtrK) and LtrK (1 μg) were incubated with [γ- 32 P]-ATP at room temperature. At indicated times, 10 μl samples were added to 5 μl of sample buffer, heated at 95 °C for 3 min and 3 μl of each mixture analysed by gel-phosphorimaging. ( b ) Time course of autophosphorylation. Plot showing autophosphorylation incorporation of GST-LtrK with [γ- 32 P]-ATP over a 60 min incubation at room temperature. The exponential fit curve (solid line) gave a calculated rate constant of 0.08. ( c ) Phosphotransfer from LtrK-P to LtrR. GST-LtrK (15 μg) bound to glutathione agarose beads in a gravity flow column was phosphorylated with [γ- 32 P]-ATP for 30 min at room temperature, free [γ- 32 P]-ATP washed off, LtrR (60 μg) passed through the column and LtrR-P collected in the flowthrough. The LtrR-P sample (10 μl) was added to 3 μl of sample buffer containing 100 mM EDTA, and 3 μl analysed by gel-phosphorimaging. ( d ) Stability of LtrR-P. LtrR-P generated from phosphotransfer from LtrK-P (panel c ) was incubated at room temperature and retention of γ- 32 P assessed over time (as for panel b ). The exponential fit curve (solid line) gave a calculated rate constant of 0.29, from which t 1/2 was calculated as ln2/k. ( e ) LtrK phosphatase activity. As for panel ( d ) except LtrR-P incubated with LtrK. The fit curve (solid line) represents two exponentials with calculated rate constants of 3.45 for the first phase and 0.3 for the second phase 2. Values of t 1/2 were calculated as ln2/k.

    Techniques Used: Autoradiography, Radioactivity, Incubation, Flow Cytometry, Generated, Activity Assay

    Effect of temperature on kinase and phosphatase activities of LtrK. To assess the phosphorylation state of proteins, samples were electrophoresed on a SDS-polyacrylamide gel, and autoradiography performed by phosphorimaging (panels a,b ). ( a ) Effect of temperature on autophosphorylation. Autophosphorylation was performed (see Fig. 2b ) at different temperatures (0, 5, 10, 15, 20, 25, 30 °C) with aliquots withdrawn for analysis at different times of incubation (10 min, 30 min, 1 h, 2 h). Incorporation is shown as a percentage of the highest band intensity on autoradiograms across all samples (2 h at 10 °C). The mean values for two replicates are plotted for 30 min, 1 h and 2 h, and values for a single time course for 10 min. Error bars represent the standard error of the mean. ( b ) Effect of temperature on phosphatase activity. LtrR-P was incubated with LtrK in a 2 to 1 ratio for 10 min at different temperatures (0, 5, 10, 15, 20, 25, 30 °C) and the band intensity of LtrK-P plotted as a percentage of the highest band intensity on the autoradiograms (LtrK-P at 10 °C). The mean values for two replicates are plotted. Error bars represent the standard error of the mean. ( c ) Half-life of inactivation at 10 °C. LtrK was incubated at 10 °C for up to 4 d and residual autophosphorylation activity determined by incubating aliquots of the enzyme with [γ- 32 P]-ATP for 10 min at 10 °C (T opt ). The natural log (ln) of activity (band intensity) was plotted against incubation time. The straight line represents the linear fit to the data and the slope of the line was used to calculate t 1/2 (see Methods ). ( d ) Half-life of inactivation at 30 °C. As for panel ( c ) except LtrK was incubated at 30 °C.
    Figure Legend Snippet: Effect of temperature on kinase and phosphatase activities of LtrK. To assess the phosphorylation state of proteins, samples were electrophoresed on a SDS-polyacrylamide gel, and autoradiography performed by phosphorimaging (panels a,b ). ( a ) Effect of temperature on autophosphorylation. Autophosphorylation was performed (see Fig. 2b ) at different temperatures (0, 5, 10, 15, 20, 25, 30 °C) with aliquots withdrawn for analysis at different times of incubation (10 min, 30 min, 1 h, 2 h). Incorporation is shown as a percentage of the highest band intensity on autoradiograms across all samples (2 h at 10 °C). The mean values for two replicates are plotted for 30 min, 1 h and 2 h, and values for a single time course for 10 min. Error bars represent the standard error of the mean. ( b ) Effect of temperature on phosphatase activity. LtrR-P was incubated with LtrK in a 2 to 1 ratio for 10 min at different temperatures (0, 5, 10, 15, 20, 25, 30 °C) and the band intensity of LtrK-P plotted as a percentage of the highest band intensity on the autoradiograms (LtrK-P at 10 °C). The mean values for two replicates are plotted. Error bars represent the standard error of the mean. ( c ) Half-life of inactivation at 10 °C. LtrK was incubated at 10 °C for up to 4 d and residual autophosphorylation activity determined by incubating aliquots of the enzyme with [γ- 32 P]-ATP for 10 min at 10 °C (T opt ). The natural log (ln) of activity (band intensity) was plotted against incubation time. The straight line represents the linear fit to the data and the slope of the line was used to calculate t 1/2 (see Methods ). ( d ) Half-life of inactivation at 30 °C. As for panel ( c ) except LtrK was incubated at 30 °C.

    Techniques Used: Autoradiography, Incubation, Activity Assay

    Protein domains and structures predicted for LtrK and LtrR. ( a ) Schematic of LtrK and LtrR protein domains and sequence motifs drawn to scale. Protein domains identified using Pfam and NCBI BLAST (blue arrow boxes); predicted TMDs (hatched regions); H, N, G1, F, G2 and G3 blocks (white boxes) diagnostic of TCS histidine kinases 83 84 ; specific histidine residues H367 (H1), H443 (H2), H448 (H3), H502 (H4) of LtrK; specific aspartate residues D54 (D1), D55 (D2) and D98 (D3) of LtrR. ( b ) Homology model of the cytoplasmic domain of LtrK constructed using I-TASSER 78 . Only one subunit of the LtrK dimer is shown. The model with the highest confidence score best aligned with the structure of VicK (PDB 4I5S), a TCS SK from Streptococcus mutans , which has 37% sequence identity to the cytoplasmic domain of LtrK. The HisKA domain includes the α1 and α2 helices. The α1 helix contains the conserved H367 (red) and E368 (green) residues of the H block. The α4 helix (HATPase domain) contains the conserved N480 (orange) and R476 (blue) residues of the N block. A catalytic triad involved in autophosphorylation 45 46 is formed by R476 (blue), E368 (green) and N480 (orange). The α3 helix (between the HisKA and HATPase domains) contains the additional histidine residues H443 and H448 (magenta).
    Figure Legend Snippet: Protein domains and structures predicted for LtrK and LtrR. ( a ) Schematic of LtrK and LtrR protein domains and sequence motifs drawn to scale. Protein domains identified using Pfam and NCBI BLAST (blue arrow boxes); predicted TMDs (hatched regions); H, N, G1, F, G2 and G3 blocks (white boxes) diagnostic of TCS histidine kinases 83 84 ; specific histidine residues H367 (H1), H443 (H2), H448 (H3), H502 (H4) of LtrK; specific aspartate residues D54 (D1), D55 (D2) and D98 (D3) of LtrR. ( b ) Homology model of the cytoplasmic domain of LtrK constructed using I-TASSER 78 . Only one subunit of the LtrK dimer is shown. The model with the highest confidence score best aligned with the structure of VicK (PDB 4I5S), a TCS SK from Streptococcus mutans , which has 37% sequence identity to the cytoplasmic domain of LtrK. The HisKA domain includes the α1 and α2 helices. The α1 helix contains the conserved H367 (red) and E368 (green) residues of the H block. The α4 helix (HATPase domain) contains the conserved N480 (orange) and R476 (blue) residues of the N block. A catalytic triad involved in autophosphorylation 45 46 is formed by R476 (blue), E368 (green) and N480 (orange). The α3 helix (between the HisKA and HATPase domains) contains the additional histidine residues H443 and H448 (magenta).

    Techniques Used: Sequencing, Diagnostic Assay, Construct, Blocking Assay

    20) Product Images from "Abacavir, an anti–HIV-1 drug, targets TDP1-deficient adult T cell leukemia"

    Article Title: Abacavir, an anti–HIV-1 drug, targets TDP1-deficient adult T cell leukemia

    Journal: Science Advances

    doi: 10.1126/sciadv.1400203

    Specific lethality of ABC on ATL cells due to a defect in TDP1. ABC is phosphorylated in a unique stepwise anabolism and is converted to the triphosphate of CBV. During DNA synthesis, triphosphorylated ABC was incorporated into host chromosomal DNA by replicative DNA polymerases, leading to premature termination of DNA replication. In normal cells, TDP1 removes ABC quickly and DNA synthesis continues. However, in HTLV-1(+) cells, the collapse of the replication fork is induced because of the deficiency of TDP1, leading to DSB formation and apoptosis.
    Figure Legend Snippet: Specific lethality of ABC on ATL cells due to a defect in TDP1. ABC is phosphorylated in a unique stepwise anabolism and is converted to the triphosphate of CBV. During DNA synthesis, triphosphorylated ABC was incorporated into host chromosomal DNA by replicative DNA polymerases, leading to premature termination of DNA replication. In normal cells, TDP1 removes ABC quickly and DNA synthesis continues. However, in HTLV-1(+) cells, the collapse of the replication fork is induced because of the deficiency of TDP1, leading to DSB formation and apoptosis.

    Techniques Used: DNA Synthesis

    TDP1 removes ABC from DNA ends in vitro. ( A ) Schematic diagram of in vitro biochemical assays for TDP1 activity. Both substrates contained the same sequence and conjugated CBV or tyrosine as a 3′-blocking lesion via a phosphodiester linkage. The substrates (S) were radiolabeled at the 5′ end with 32 P. The products that are removed from the 3′-blocking lesion from the substrates by TDP1 are labeled “P.” Y: Tyr. ( B ) A representative gel demonstrating the processing of the indicated substrates by increasing amount of total cell lysates from Tdp1 −/− DT40 cells or Tdp1 −/− DT40 cells stably transfected with hTDP1 transgene. The substrates were incubated with serially diluted total cell lysates ranging from 0.03 to 7.5 μg. Reaction proceeded for 15 min at 25°C before being quenched and analyzed on 16% denaturing gels. ( C ) The percentage of product yield is plotted against increasing lysate concentration. Results are expressed as means ± SD of three independent experiments.
    Figure Legend Snippet: TDP1 removes ABC from DNA ends in vitro. ( A ) Schematic diagram of in vitro biochemical assays for TDP1 activity. Both substrates contained the same sequence and conjugated CBV or tyrosine as a 3′-blocking lesion via a phosphodiester linkage. The substrates (S) were radiolabeled at the 5′ end with 32 P. The products that are removed from the 3′-blocking lesion from the substrates by TDP1 are labeled “P.” Y: Tyr. ( B ) A representative gel demonstrating the processing of the indicated substrates by increasing amount of total cell lysates from Tdp1 −/− DT40 cells or Tdp1 −/− DT40 cells stably transfected with hTDP1 transgene. The substrates were incubated with serially diluted total cell lysates ranging from 0.03 to 7.5 μg. Reaction proceeded for 15 min at 25°C before being quenched and analyzed on 16% denaturing gels. ( C ) The percentage of product yield is plotted against increasing lysate concentration. Results are expressed as means ± SD of three independent experiments.

    Techniques Used: In Vitro, Activity Assay, Sequencing, Blocking Assay, Labeling, Stable Transfection, Transfection, Incubation, Concentration Assay

    21) Product Images from "NME7 is a functional component of the γ-tubulin ring complex"

    Article Title: NME7 is a functional component of the γ-tubulin ring complex

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E13-06-0339

    NME7 undergoes autophosphorylation. (A) Sequence alignment of the putative kinase domains (NME7A and NME7B) of NME7 with those of other members of the NME family. Asterisks indicate the residues targeted using site-directed mutagenesis. (B, C) Recombinant NME7 (WT) and its mutants were subjected to an autophosphorylation reaction in which either [γ- 32 P]ATP (B) or [γ- 32 P]GTP (C) was used as the phosphate donor. After the reaction, proteins were resolved using SDS–PAGE and examined by means of Coomassie blue staining and autoradiography. H206F, H355F, K173A, and R322A are the mutants.
    Figure Legend Snippet: NME7 undergoes autophosphorylation. (A) Sequence alignment of the putative kinase domains (NME7A and NME7B) of NME7 with those of other members of the NME family. Asterisks indicate the residues targeted using site-directed mutagenesis. (B, C) Recombinant NME7 (WT) and its mutants were subjected to an autophosphorylation reaction in which either [γ- 32 P]ATP (B) or [γ- 32 P]GTP (C) was used as the phosphate donor. After the reaction, proteins were resolved using SDS–PAGE and examined by means of Coomassie blue staining and autoradiography. H206F, H355F, K173A, and R322A are the mutants.

    Techniques Used: Sequencing, Mutagenesis, Recombinant, SDS Page, Staining, Autoradiography

    22) Product Images from "MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression"

    Article Title: MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002204

    Mapping the start site of transcription of the mrkA promoter by primer extension. Total cellular RNA was purified from E. coli MC4100 strains containing pMrkH with either pMU2385 (control) or mrkA-lacZ -2. The RNA samples were then hybridized with 32 P-labelled primer Px1mrkARev. Primer extension was performed using AMV reverse transcriptase in the presence of dNTPs. GA Ladder: GA sequence ladder prepared using the mrkA PCR fragment generated using primer pairs 32 P-Px1mrkARev and mrk295F. Lane 1: control experiment using RNA from E. coli MC4100 strain containing pMrkH and pMU2385. Lane 2: experiment using RNA from E. coli MC4100 strain containing pMrkH and mrkA-lacZ -2. The positions corresponding to 32 P-Px1mrkARev primer and the extension product are marked.
    Figure Legend Snippet: Mapping the start site of transcription of the mrkA promoter by primer extension. Total cellular RNA was purified from E. coli MC4100 strains containing pMrkH with either pMU2385 (control) or mrkA-lacZ -2. The RNA samples were then hybridized with 32 P-labelled primer Px1mrkARev. Primer extension was performed using AMV reverse transcriptase in the presence of dNTPs. GA Ladder: GA sequence ladder prepared using the mrkA PCR fragment generated using primer pairs 32 P-Px1mrkARev and mrk295F. Lane 1: control experiment using RNA from E. coli MC4100 strain containing pMrkH and pMU2385. Lane 2: experiment using RNA from E. coli MC4100 strain containing pMrkH and mrkA-lacZ -2. The positions corresponding to 32 P-Px1mrkARev primer and the extension product are marked.

    Techniques Used: Purification, Sequencing, Polymerase Chain Reaction, Generated

    Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA. The 32 P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32 P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.
    Figure Legend Snippet: Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA. The 32 P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32 P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Generated, Purification, Incubation

    23) Product Images from "Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity *"

    Article Title: Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.408450

    Membrane-inserted CFTR catalyzes phosphotransfer from [γ- 32 P]GTP to N 3 -AMP. A , Western blot ( WB ) probed with antibody 13-1. Letters label highly ( C ) and core glycosylated ( B ) CFTR. Each lane represents 30 μg of membrane protein. B , autoradiograph and Western blot (probed with antibody M3A7) of the same gel. Experiments were performed as illustrated in Fig. 1 . Experimental conditions are indicated below the lanes. N 3 -AMP concentration was 65 μ m . Comparing the autoradiograph and Western blot corroborated that the labeled band was CFTR. C , CFTR photolabeling with 8-N 3 -AMP and 2-N 3 -AMP. N 3 -AMP concentration was 65 μ m . To compare the results from different autoradiographs, data were normalized to CFTR radioactivity under conditions indicated below bar 4. Asterisks indicate p ≤ 0.001 when compared with bar 4, and double daggers indicate p ≤ 0.001 when compared with bar 3 (one-way analysis of variance followed by the Holm-Sidak method for multiple comparisons, n = 3).
    Figure Legend Snippet: Membrane-inserted CFTR catalyzes phosphotransfer from [γ- 32 P]GTP to N 3 -AMP. A , Western blot ( WB ) probed with antibody 13-1. Letters label highly ( C ) and core glycosylated ( B ) CFTR. Each lane represents 30 μg of membrane protein. B , autoradiograph and Western blot (probed with antibody M3A7) of the same gel. Experiments were performed as illustrated in Fig. 1 . Experimental conditions are indicated below the lanes. N 3 -AMP concentration was 65 μ m . Comparing the autoradiograph and Western blot corroborated that the labeled band was CFTR. C , CFTR photolabeling with 8-N 3 -AMP and 2-N 3 -AMP. N 3 -AMP concentration was 65 μ m . To compare the results from different autoradiographs, data were normalized to CFTR radioactivity under conditions indicated below bar 4. Asterisks indicate p ≤ 0.001 when compared with bar 4, and double daggers indicate p ≤ 0.001 when compared with bar 3 (one-way analysis of variance followed by the Holm-Sidak method for multiple comparisons, n = 3).

    Techniques Used: Western Blot, Autoradiography, Concentration Assay, Labeling, Radioactivity

    Model of CFTR labeling through phosphoryl group transfer between [γ- 32 P]GTP and N 3 -AMP followed by UV-mediated cross-linking of the resulting N 3 -[β- 32 P]ADP and solubilization and immunoprecipitation ( IP ) of CFTR. P * indicates a radioactive phosphoryl group containing 32 P. In each NBD, the open rectangle represents the Walker A motif, and the open triangle represents the signature motif. The binding site for AMP is not known.
    Figure Legend Snippet: Model of CFTR labeling through phosphoryl group transfer between [γ- 32 P]GTP and N 3 -AMP followed by UV-mediated cross-linking of the resulting N 3 -[β- 32 P]ADP and solubilization and immunoprecipitation ( IP ) of CFTR. P * indicates a radioactive phosphoryl group containing 32 P. In each NBD, the open rectangle represents the Walker A motif, and the open triangle represents the signature motif. The binding site for AMP is not known.

    Techniques Used: Labeling, Immunoprecipitation, Binding Assay

    CFTR has intrinsic adenylate kinase activity. A , autoradiograph of immunoprecipitated CFTR fractionated on a 6% SDS-polyacrylamide gel. Experiments were performed as illustrated in Fig. 1 . Membranes containing 30 μg of protein from CFTR-expressing HeLa cells ( lanes 3–5 ) or control membranes ( contr. membr. ) containing 30 μg of protein from HeLa cells not expressing recombinant CFTR ( lane 1 ) were used. In lane 6 , membranes containing 90 μg of protein from S1248F CFTR-expressing HeLa cells were employed. Membranes were incubated together with 50 μ m 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C followed by UV irradiation for 30 s (302 nm, 8-watt lamp) at a distance of 5 cm as described under “Experimental Procedures.” The sample of lane 4 was not UV-irradiated. In lane 2 , 30 μg of membranes from HeLa cells not expressing recombinant CFTR (control membranes) were incubated with 50 μ m 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C. Then 30 μg of membranes containing CFTR were added on ice before UV irradiation (30 s, 302 nm, 8-watt lamp). In all cases, CFTR was then solubilized and immunoprecipitated as described under “Experimental Procedures.” B , summary data. Radioactivity incorporated into CFTR was normalized to radioactivity for conditions indicated below bar 5. Asterisks indicate p = 0.029 when compared with bar 5 (Mann-Whitney rank sum test, n = 4). No significant differences were detected between bars 1–4 and 6 (Kruskal-Wallis one-way analysis of variance on ranks, n = 4). C , Western blot probed with CFTR antibody 13-1. 30 μg (control membranes and membranes with wild-type CFTR, lanes 1–3 ) and 90 μg (membranes with S1248F CFTR, lane 4 ) of protein were used.
    Figure Legend Snippet: CFTR has intrinsic adenylate kinase activity. A , autoradiograph of immunoprecipitated CFTR fractionated on a 6% SDS-polyacrylamide gel. Experiments were performed as illustrated in Fig. 1 . Membranes containing 30 μg of protein from CFTR-expressing HeLa cells ( lanes 3–5 ) or control membranes ( contr. membr. ) containing 30 μg of protein from HeLa cells not expressing recombinant CFTR ( lane 1 ) were used. In lane 6 , membranes containing 90 μg of protein from S1248F CFTR-expressing HeLa cells were employed. Membranes were incubated together with 50 μ m 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C followed by UV irradiation for 30 s (302 nm, 8-watt lamp) at a distance of 5 cm as described under “Experimental Procedures.” The sample of lane 4 was not UV-irradiated. In lane 2 , 30 μg of membranes from HeLa cells not expressing recombinant CFTR (control membranes) were incubated with 50 μ m 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C. Then 30 μg of membranes containing CFTR were added on ice before UV irradiation (30 s, 302 nm, 8-watt lamp). In all cases, CFTR was then solubilized and immunoprecipitated as described under “Experimental Procedures.” B , summary data. Radioactivity incorporated into CFTR was normalized to radioactivity for conditions indicated below bar 5. Asterisks indicate p = 0.029 when compared with bar 5 (Mann-Whitney rank sum test, n = 4). No significant differences were detected between bars 1–4 and 6 (Kruskal-Wallis one-way analysis of variance on ranks, n = 4). C , Western blot probed with CFTR antibody 13-1. 30 μg (control membranes and membranes with wild-type CFTR, lanes 1–3 ) and 90 μg (membranes with S1248F CFTR, lane 4 ) of protein were used.

    Techniques Used: Activity Assay, Autoradiography, Immunoprecipitation, Expressing, Recombinant, Incubation, Irradiation, Radioactivity, MANN-WHITNEY, Western Blot

    24) Product Images from "Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining"

    Article Title: Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining

    Journal: eLife

    doi: 10.7554/eLife.13740

    Polθ acts processively during alt-EJ in vitro. ( A ) Scheme for reconstitution of Polθ mediated alt-EJ in vitro with ssDNA trap (top). Sequences of alt-EJ products generated by Polθ in vitro using 10 mM Mg 2+ and 1 mM Mn 2+ (bottom). Red text, insertions; black text, original DNA sequence; grey underlines, sequences copied from original template; red underlines, complementary sequences due to snap-back replication; red sequence without underlines, random insertions; superscript 1, suggests sequences were copied from a template portion that was subsequently deleted during alt-EJ. Original DNA sequences indicated at top. Blue type, mutations. ( B ) Plot of insertion tract lengths generated in panel A. DOI: http://dx.doi.org/10.7554/eLife.13740.016
    Figure Legend Snippet: Polθ acts processively during alt-EJ in vitro. ( A ) Scheme for reconstitution of Polθ mediated alt-EJ in vitro with ssDNA trap (top). Sequences of alt-EJ products generated by Polθ in vitro using 10 mM Mg 2+ and 1 mM Mn 2+ (bottom). Red text, insertions; black text, original DNA sequence; grey underlines, sequences copied from original template; red underlines, complementary sequences due to snap-back replication; red sequence without underlines, random insertions; superscript 1, suggests sequences were copied from a template portion that was subsequently deleted during alt-EJ. Original DNA sequences indicated at top. Blue type, mutations. ( B ) Plot of insertion tract lengths generated in panel A. DOI: http://dx.doi.org/10.7554/eLife.13740.016

    Techniques Used: In Vitro, Generated, Sequencing

    Conserved residues contribute to Polθ processivity and template-independent terminal transferase activity. ( A ) Sequence alignment of Polθ and related A-family Pols. Conserved positively charged residues (2202, 2254) and loop 2 in Polθ are highlighted in yellow and grey, respectively. Black boxes indicate conserved motifs. * = identical residues,: = residues sharing very similar properties,. = residues sharing some properties. Red, small and hydrophobic; Blue, acidic; Magenta, basic; Green, hydroxyl, sulfhydryl, amine. ( B ) Structure of Polθ with ssDNA primer (PDB code 4X0P) ( Zahn et al., 2015 ). Residues R2202 and R2254 are indicated in blue. Dotted blue lines indicate ionic interactions. Loop 2 is indicated in dark red. Thumb and palm subdomains are indicated. ( C ) Denaturing gel showing PolθWT and PolθL2 extension of ssDNA with 5 mM Mn 2+ and all four dNTPs. ( D ) Denaturing gel showing PolθWT and PolθL2 extension of a primer-template with 5 mM Mn 2+ and all four dNTPs. Model of PolθWT-Mn 2+ and PolθL2-Mn 2+ activities on a primer-template (right). ( E ) Denaturing gel showing a time course of PolθWT and PolθRR extension of a primer-template in the presence of 10 mM Mg 2+ and all four dNTPs. ( F ) Denaturing gel showing PolθWT (left) and PolθRR (right) extension of poly-dC ssDNA with 5 mM Mn 2+ and the indicated dNTPs. ( G ) Schematic of assay (left). Denaturing gel showing PolθWT and PolθRR extension of an excess of radiolabeled primer-template with all four dNTPs and 10 mM Mg 2+ either in the presence or absence of 150-fold excess unlabeled DNA trap. DOI: http://dx.doi.org/10.7554/eLife.13740.023
    Figure Legend Snippet: Conserved residues contribute to Polθ processivity and template-independent terminal transferase activity. ( A ) Sequence alignment of Polθ and related A-family Pols. Conserved positively charged residues (2202, 2254) and loop 2 in Polθ are highlighted in yellow and grey, respectively. Black boxes indicate conserved motifs. * = identical residues,: = residues sharing very similar properties,. = residues sharing some properties. Red, small and hydrophobic; Blue, acidic; Magenta, basic; Green, hydroxyl, sulfhydryl, amine. ( B ) Structure of Polθ with ssDNA primer (PDB code 4X0P) ( Zahn et al., 2015 ). Residues R2202 and R2254 are indicated in blue. Dotted blue lines indicate ionic interactions. Loop 2 is indicated in dark red. Thumb and palm subdomains are indicated. ( C ) Denaturing gel showing PolθWT and PolθL2 extension of ssDNA with 5 mM Mn 2+ and all four dNTPs. ( D ) Denaturing gel showing PolθWT and PolθL2 extension of a primer-template with 5 mM Mn 2+ and all four dNTPs. Model of PolθWT-Mn 2+ and PolθL2-Mn 2+ activities on a primer-template (right). ( E ) Denaturing gel showing a time course of PolθWT and PolθRR extension of a primer-template in the presence of 10 mM Mg 2+ and all four dNTPs. ( F ) Denaturing gel showing PolθWT (left) and PolθRR (right) extension of poly-dC ssDNA with 5 mM Mn 2+ and the indicated dNTPs. ( G ) Schematic of assay (left). Denaturing gel showing PolθWT and PolθRR extension of an excess of radiolabeled primer-template with all four dNTPs and 10 mM Mg 2+ either in the presence or absence of 150-fold excess unlabeled DNA trap. DOI: http://dx.doi.org/10.7554/eLife.13740.023

    Techniques Used: Activity Assay, Sequencing

    Polθ template-independent activity is stimulated by physiological concentrations of Mn 2+ and Mg 2+ . ( A ) Denaturing gels showing Polθ extension of poly-dT in the presence of dCTP with indicated concentrations of Mn 2+ and Mg 2+ . ( B ) Plots of percent ssDNA extension observed in panel A. Percent extension was calculated by dividing the intensity of the sum of the extended products by the sum of the intensity of all DNA in each lane. DOI: http://dx.doi.org/10.7554/eLife.13740.004
    Figure Legend Snippet: Polθ template-independent activity is stimulated by physiological concentrations of Mn 2+ and Mg 2+ . ( A ) Denaturing gels showing Polθ extension of poly-dT in the presence of dCTP with indicated concentrations of Mn 2+ and Mg 2+ . ( B ) Plots of percent ssDNA extension observed in panel A. Percent extension was calculated by dividing the intensity of the sum of the extended products by the sum of the intensity of all DNA in each lane. DOI: http://dx.doi.org/10.7554/eLife.13740.004

    Techniques Used: Activity Assay

    Polθ-Mn 2+ oscillates between different terminal transferase activites in the presence of a DNA trap. ( A ) Scheme of experiment performed in solid-phase. ( B ) Bar graph depicting ssDNA product lengths generated by Polθ in the presence (orange) and absence (grey) of excess ssDNA with 10 mM Mg 2+ and 1 mM Mn 2+ . ( C,D ) Sequences generated by Polθ incubated with the indicated ssDNA substrate in the presence ( D ) and absence ( C ) of excess ssDNA trap with 10 mM Mg 2+ , 1 mM Mn 2+ , and all four dNTPs. Black underlines, sequences identical or complementary to initial ssDNA substrate; red underlines, sequences complementary to ssDNA trap; colored lines above text, complementary sequences within individual ssDNA products. DOI: http://dx.doi.org/10.7554/eLife.13740.012
    Figure Legend Snippet: Polθ-Mn 2+ oscillates between different terminal transferase activites in the presence of a DNA trap. ( A ) Scheme of experiment performed in solid-phase. ( B ) Bar graph depicting ssDNA product lengths generated by Polθ in the presence (orange) and absence (grey) of excess ssDNA with 10 mM Mg 2+ and 1 mM Mn 2+ . ( C,D ) Sequences generated by Polθ incubated with the indicated ssDNA substrate in the presence ( D ) and absence ( C ) of excess ssDNA trap with 10 mM Mg 2+ , 1 mM Mn 2+ , and all four dNTPs. Black underlines, sequences identical or complementary to initial ssDNA substrate; red underlines, sequences complementary to ssDNA trap; colored lines above text, complementary sequences within individual ssDNA products. DOI: http://dx.doi.org/10.7554/eLife.13740.012

    Techniques Used: Generated, Incubation

    25) Product Images from "Novel high-throughput electrochemiluminescent assay for identification of human tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors and characterization of furamidine (NSC 305831) as an inhibitor of Tdp1"

    Article Title: Novel high-throughput electrochemiluminescent assay for identification of human tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors and characterization of furamidine (NSC 305831) as an inhibitor of Tdp1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm463

    Inhibition of Tdp1 by furamidine is independent of the presence of thymidines at the 3′-end of the substrate. ( A ) Sequences of the oligonucletotide substrates 14Y and 14Y-CC, which differ in their 3′-terminal bases (–TT or –CC) linked to the phosphotyrosine. ( B ) Reactions (100 µl) containing either 25 nM 14Y or 14Y-CC and 5 ng of Tdp1 were incubated at 25°C. Aliquots were taken at the indicated times (min). Reaction products were analyzed by denaturing PAGE. ( C ) Densitometry analysis of the gel shown in B. Tdp1 activity measured as the percentage of DNA substrates 14Y-CC (left panel) or 14Y (right panel) converted to their corresponding products as a function of reaction time. ( D ) Reactions (20 µl) containing 25 nM 14Y or 14Y-CC and 1 ng Tdp1 were carried out in the presence of indicated concentrations (µM) of furamidine at 25°C, pH 8, for 20 min. A representative gel is shown. ( E ) Densitometry analysis of the gel shown in D. Tdp1 activity was calculated as the percentage of DNA substrates 14Y or 14Y-CC converted to their product. The horizontal line corresponds to 50% inhibition of Tdp1 activity.
    Figure Legend Snippet: Inhibition of Tdp1 by furamidine is independent of the presence of thymidines at the 3′-end of the substrate. ( A ) Sequences of the oligonucletotide substrates 14Y and 14Y-CC, which differ in their 3′-terminal bases (–TT or –CC) linked to the phosphotyrosine. ( B ) Reactions (100 µl) containing either 25 nM 14Y or 14Y-CC and 5 ng of Tdp1 were incubated at 25°C. Aliquots were taken at the indicated times (min). Reaction products were analyzed by denaturing PAGE. ( C ) Densitometry analysis of the gel shown in B. Tdp1 activity measured as the percentage of DNA substrates 14Y-CC (left panel) or 14Y (right panel) converted to their corresponding products as a function of reaction time. ( D ) Reactions (20 µl) containing 25 nM 14Y or 14Y-CC and 1 ng Tdp1 were carried out in the presence of indicated concentrations (µM) of furamidine at 25°C, pH 8, for 20 min. A representative gel is shown. ( E ) Densitometry analysis of the gel shown in D. Tdp1 activity was calculated as the percentage of DNA substrates 14Y or 14Y-CC converted to their product. The horizontal line corresponds to 50% inhibition of Tdp1 activity.

    Techniques Used: Inhibition, Incubation, Polyacrylamide Gel Electrophoresis, Activity Assay

    Competitive inhibition of Tdp1 by furamidine. ( A ) A 100-µl reaction mixture containing 25 nM 14Y and 5 ng of Tdp1 was incubated at pH 8.0 at 25°C in the absence of drug, or in the presence of the indicated concentrations of furamidine. Aliquots were taken at the indicated times (min). Reaction products were analyzed by denaturing PAGE. ( B ) Densitometry analysis of the gel shown in A. Tdp1 activity measured as the percentage of DNA substrate 14Y converted to 14P (left panel) or substrate 14Y remaining (right panel) as a function of reaction time. ( C ) Reactions (20 µl) containing 25 nM 14Y and the indicated amounts (ng) of Tdp1 were carried out in the absence or presence of increasing concentrations of furamidine for 20 min. A representative gel is shown. ( D ) Densitometry analysis of the gel shown in C. Tdp1 activity was calculated as the percentage of DNA substrate 14Y converted to 14P. The horizontal line corresponds to 50% inhibition of Tdp1 activity.
    Figure Legend Snippet: Competitive inhibition of Tdp1 by furamidine. ( A ) A 100-µl reaction mixture containing 25 nM 14Y and 5 ng of Tdp1 was incubated at pH 8.0 at 25°C in the absence of drug, or in the presence of the indicated concentrations of furamidine. Aliquots were taken at the indicated times (min). Reaction products were analyzed by denaturing PAGE. ( B ) Densitometry analysis of the gel shown in A. Tdp1 activity measured as the percentage of DNA substrate 14Y converted to 14P (left panel) or substrate 14Y remaining (right panel) as a function of reaction time. ( C ) Reactions (20 µl) containing 25 nM 14Y and the indicated amounts (ng) of Tdp1 were carried out in the absence or presence of increasing concentrations of furamidine for 20 min. A representative gel is shown. ( D ) Densitometry analysis of the gel shown in C. Tdp1 activity was calculated as the percentage of DNA substrate 14Y converted to 14P. The horizontal line corresponds to 50% inhibition of Tdp1 activity.

    Techniques Used: Inhibition, Incubation, Polyacrylamide Gel Electrophoresis, Activity Assay

    Inhibition of Tdp1 activity by furamidine. ( A ) Schematic representation of the Tdp1 biochemical assays. The partially duplex oligopeptide D14Y or single-stranded 14Y were used as substrates. 32 P-Radiolabeling (*) was at the 5′ terminus of the 14-mer strand. Tdp1 catalyzes the hydrolysis of the 3′-phosphotyrosine bond and converts 14Y and D14Y to an oligonucleotide with 3′-phosphate, 14P or D14P, respectively. ( B ) Representative gel showing Tdp1 inhibition by furamidine with single strand (14Y) and partially duplex (D14Y) substrates. Reactions were performed at 25°C for 20 min. Arrows indicate the 3′-phosphate oligonucleotide product (14P) that runs quicker than the corresponding tyrosyl oligonucleotide substrate (14Y) in a denaturing PAGE ( 18 ). The duplex D14Y substrate and D14P product are detected on the gel by their corresponding labeled single strands (14Y and 14P), as they are no longer annealed under the denatured conditions. ( C ) Densitometry analysis of the gel shown in panel B. Tdp1 activity was calculated as the percentage of 14Y converted to 14P as a function of the concentration of furamidine. The horizontal line corresponds to 50% inhibition of Tdp1 activity.
    Figure Legend Snippet: Inhibition of Tdp1 activity by furamidine. ( A ) Schematic representation of the Tdp1 biochemical assays. The partially duplex oligopeptide D14Y or single-stranded 14Y were used as substrates. 32 P-Radiolabeling (*) was at the 5′ terminus of the 14-mer strand. Tdp1 catalyzes the hydrolysis of the 3′-phosphotyrosine bond and converts 14Y and D14Y to an oligonucleotide with 3′-phosphate, 14P or D14P, respectively. ( B ) Representative gel showing Tdp1 inhibition by furamidine with single strand (14Y) and partially duplex (D14Y) substrates. Reactions were performed at 25°C for 20 min. Arrows indicate the 3′-phosphate oligonucleotide product (14P) that runs quicker than the corresponding tyrosyl oligonucleotide substrate (14Y) in a denaturing PAGE ( 18 ). The duplex D14Y substrate and D14P product are detected on the gel by their corresponding labeled single strands (14Y and 14P), as they are no longer annealed under the denatured conditions. ( C ) Densitometry analysis of the gel shown in panel B. Tdp1 activity was calculated as the percentage of 14Y converted to 14P as a function of the concentration of furamidine. The horizontal line corresponds to 50% inhibition of Tdp1 activity.

    Techniques Used: Inhibition, Activity Assay, Radioactivity, Polyacrylamide Gel Electrophoresis, Labeling, Concentration Assay

    High-throughput electrochemiluminescene assay developed to identify novel Tdp1 inhibitors. ( A ) Generation of the electrochemiluminescent (ECL) substrate (BV-14Y). The ruthenium-containing tag (NHS ester BV-Tag; from BioVeris Corp.) is coupled to the 3′-end of the tyrosyl-containing DNA substrate [14Y (sequence as in 3A) linked to a biotin at its 5′ end]. After coupling, the BV Tag is attached to the phosphotyrosine of the 14Y DNA forming the BV-14Y DNA after the release of a succinimide group. ( B ) Processing of the ECL substrate by Tdp1. The ECL substrate BV-14Y bound to magnetic beads was generated as described in the Materials and Methods section. Upon addition of Tdp1, the tyrosine-BV-Tag group is hydrolyzed away leaving behind a 3′ phosphate on the DNA bound to the beads. After stopping the reactions, the samples are analyzed by the ECL analyzer. During the analysis, only the magnetic beads are retained in the analysis chamber by magnetic field while the rest of the sample is washed away. In the absence of Tdp1, the ECL signal is maximum as the BV-14Y DNA is retained on the magnetic beads. Upon addition of Tdp1, the tyrosine-BV-Tag group is cleaved away resulting in the loss of ECL signal (arrow). Potential Tdp1 inhibitors prevent this loss of signal. ( C ) Signal response curve in the presence of increasing concentrations of Tdp1. The ECL signal is lost when the Tdp1 concentration is increased.
    Figure Legend Snippet: High-throughput electrochemiluminescene assay developed to identify novel Tdp1 inhibitors. ( A ) Generation of the electrochemiluminescent (ECL) substrate (BV-14Y). The ruthenium-containing tag (NHS ester BV-Tag; from BioVeris Corp.) is coupled to the 3′-end of the tyrosyl-containing DNA substrate [14Y (sequence as in 3A) linked to a biotin at its 5′ end]. After coupling, the BV Tag is attached to the phosphotyrosine of the 14Y DNA forming the BV-14Y DNA after the release of a succinimide group. ( B ) Processing of the ECL substrate by Tdp1. The ECL substrate BV-14Y bound to magnetic beads was generated as described in the Materials and Methods section. Upon addition of Tdp1, the tyrosine-BV-Tag group is hydrolyzed away leaving behind a 3′ phosphate on the DNA bound to the beads. After stopping the reactions, the samples are analyzed by the ECL analyzer. During the analysis, only the magnetic beads are retained in the analysis chamber by magnetic field while the rest of the sample is washed away. In the absence of Tdp1, the ECL signal is maximum as the BV-14Y DNA is retained on the magnetic beads. Upon addition of Tdp1, the tyrosine-BV-Tag group is cleaved away resulting in the loss of ECL signal (arrow). Potential Tdp1 inhibitors prevent this loss of signal. ( C ) Signal response curve in the presence of increasing concentrations of Tdp1. The ECL signal is lost when the Tdp1 concentration is increased.

    Techniques Used: High Throughput Screening Assay, Sequencing, Magnetic Beads, Generated, Concentration Assay

    Differential activities of furamidine, berenil and pentamidine against Tdp1. ( A ) Chemical structures of furamidine, berenil and pentamidine. Dashed lines indicate the variable chemical moiety. ( B ) Reactions were performed with the indicated concentrations (µM) of furamidine, berenil and pentamidine. Reactions were for 20 min at pH 8.0 and 25°C in the presence of 25 nM 14Y substrate and 1 ng of Tdp1. Samples were separated on a 20% urea–PAGE gel and visualized.
    Figure Legend Snippet: Differential activities of furamidine, berenil and pentamidine against Tdp1. ( A ) Chemical structures of furamidine, berenil and pentamidine. Dashed lines indicate the variable chemical moiety. ( B ) Reactions were performed with the indicated concentrations (µM) of furamidine, berenil and pentamidine. Reactions were for 20 min at pH 8.0 and 25°C in the presence of 25 nM 14Y substrate and 1 ng of Tdp1. Samples were separated on a 20% urea–PAGE gel and visualized.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    26) Product Images from "Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach"

    Article Title: Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach

    Journal: Scientific Reports

    doi: 10.1038/srep42741

    Oligonucleotides identified by ADAPT reveal aptamer-like characteristics. ( a ) Filter retention analysis of C1Q-binding by the ssODNs H1 (dark grey columns) and H11 (pale grey columns) at indicated C1Q concentrations. As a control, the reverse complement of H1, H1RC, was used (black columns). ( b ) ELONA analysis of C1Q-binding by the ssODNs H11 (circles) at indicated concentrations and a fixed C1Q concentration (0.625 nM). As a control, the reverse complement of H11, H11RC, was used (squares). “No aptamer” control (triangles) shows low background binding of detector Streptavidin-HRP. H11 specifically binds C1Q (estimated K D around 40 nM). ( c ) PAGE analysis of PEG-precipitated and ssODN-associated proteins pulled-down with L2. Lane 1: Molecular weight marker; lane 2: Input library L2; lane 3: Fraction pulled down by biotinylated L2; lane 4: Fraction pulled down by non-biotinylated L2; lane 5: Fraction found in the absence of DNA library; red arrows indicate the ssODN library; yellow arrows indicate protein bands cut out and analysed by LC-MS/MS; black arrows indicate streptavidin monomers leaking from beads. ( d ) Four-way Venn diagram of proteins detected by LC-MS/MS from Unfractionated plasma, PEG-precipitated plasma, PEG-precipitated plasma in presence of L0 or L2, respectively, purified by streptavidin magnetic beads (background-subtracted; i.e. biotinylated minus non-biotinylated libraries). ( e ) Gene ontology (GO) cellular component enrichment analysis of the subset of proteins associated with L0 (red bars) and L2 (grey bars) that show a p value of at least 6 × 10 −12 . Proteins listed are: 1 extracellular region part, 2 extracellular region, 3 extracellular exosome, 4 extracellular membrane-bounded organelle, 5 extracellular organelle, 6 extracellular vesicle, 7 membrane-bounded vesicle, 8 vesicle, 9 organelle, 10 membrane-bounded organelle, 11 extracellular space, 12 focal adhesion, 13 cell-substrate adherence junction, 14 cell-substrate junction, 15 adherence junction, 16 anchoring junction, 17 cellular component, 18 cell junction, 19 blood micro particle. Inset: 108 proteins pulled down by L2 (inset, 96 + 12), cut from the gel shown in ( c ), and analysed by LC-MS/MS, 13 proteins unique to background-subtracted L0 (inset, 13), and 12 overlapping proteins (inset, 12).
    Figure Legend Snippet: Oligonucleotides identified by ADAPT reveal aptamer-like characteristics. ( a ) Filter retention analysis of C1Q-binding by the ssODNs H1 (dark grey columns) and H11 (pale grey columns) at indicated C1Q concentrations. As a control, the reverse complement of H1, H1RC, was used (black columns). ( b ) ELONA analysis of C1Q-binding by the ssODNs H11 (circles) at indicated concentrations and a fixed C1Q concentration (0.625 nM). As a control, the reverse complement of H11, H11RC, was used (squares). “No aptamer” control (triangles) shows low background binding of detector Streptavidin-HRP. H11 specifically binds C1Q (estimated K D around 40 nM). ( c ) PAGE analysis of PEG-precipitated and ssODN-associated proteins pulled-down with L2. Lane 1: Molecular weight marker; lane 2: Input library L2; lane 3: Fraction pulled down by biotinylated L2; lane 4: Fraction pulled down by non-biotinylated L2; lane 5: Fraction found in the absence of DNA library; red arrows indicate the ssODN library; yellow arrows indicate protein bands cut out and analysed by LC-MS/MS; black arrows indicate streptavidin monomers leaking from beads. ( d ) Four-way Venn diagram of proteins detected by LC-MS/MS from Unfractionated plasma, PEG-precipitated plasma, PEG-precipitated plasma in presence of L0 or L2, respectively, purified by streptavidin magnetic beads (background-subtracted; i.e. biotinylated minus non-biotinylated libraries). ( e ) Gene ontology (GO) cellular component enrichment analysis of the subset of proteins associated with L0 (red bars) and L2 (grey bars) that show a p value of at least 6 × 10 −12 . Proteins listed are: 1 extracellular region part, 2 extracellular region, 3 extracellular exosome, 4 extracellular membrane-bounded organelle, 5 extracellular organelle, 6 extracellular vesicle, 7 membrane-bounded vesicle, 8 vesicle, 9 organelle, 10 membrane-bounded organelle, 11 extracellular space, 12 focal adhesion, 13 cell-substrate adherence junction, 14 cell-substrate junction, 15 adherence junction, 16 anchoring junction, 17 cellular component, 18 cell junction, 19 blood micro particle. Inset: 108 proteins pulled down by L2 (inset, 96 + 12), cut from the gel shown in ( c ), and analysed by LC-MS/MS, 13 proteins unique to background-subtracted L0 (inset, 13), and 12 overlapping proteins (inset, 12).

    Techniques Used: Binding Assay, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight, Marker, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Purification, Magnetic Beads

    Enriched ssODNs interact with the surface of exosomes. ( a ) The enriched 5′-biotinylated ssODN library L2 (blue curves) reveals enhanced binding, measured by flow-cytometry, to UC isolated exosomes, compared to the 5′-biotinylated starting library L0 (cyan curves), or absence of ssODNs (w/o; black curves) using SA-PE as a staining agent, and gated only on double-positive CFSE+DiD+ events ( Supplementary Fig. 2 ). The curves show the distribution of relative fluorescence intensities for observed events. Each curve represents an independent binding experiment (n = 3). ( b ) Total number of positive SA-PE staining events from ( a ) (number of events > RFU 400) of biotinylated L0 (cyan), and biotinylated L2 (blue), compared to absence of ssODNs (w/o; black), in exosome binding. In each binding reaction 12 nM of each library was used. ( c ) Post-ADAPT quantification of binding of individual aptamers as part of the library and individually as illustrated in Supplementary Fig. S3 . The top panel shows 23 representative individual aptamers, selected either with high “H” or low “L” normalized counts from L3 NGS data after binding pooled plasma from breast biopsy negative donors. There is at least a 5-fold difference between counts of H and L ssODNs. These 23 sequences were re-synthesized and tested individually in the same binding assay, but in equal concentrations, unlike their original representation in L3. PEG-precipitated aptamer/plasma complexes were directly subjected to qPCR (bottom panel). Inlay: Magnification of qPCR results of ssODNs incubated with PBS instead of plasma (red). ( d ) Silver-stained reducing SDS-PA gel of pulled down proteins from PPT plasma with the indicated ssODNs (H1, H11, L4, L15), immobilized on streptavidin magnetic beads, and the control without ssODN (NO). Dashed arrows indicate pulled down proteins C1QA, C1QB, and C1QC. The dotted arrow indicates the heavy chain of IgM (IgM HC ). SA: streptavidin. RC: reverse complement sequence.
    Figure Legend Snippet: Enriched ssODNs interact with the surface of exosomes. ( a ) The enriched 5′-biotinylated ssODN library L2 (blue curves) reveals enhanced binding, measured by flow-cytometry, to UC isolated exosomes, compared to the 5′-biotinylated starting library L0 (cyan curves), or absence of ssODNs (w/o; black curves) using SA-PE as a staining agent, and gated only on double-positive CFSE+DiD+ events ( Supplementary Fig. 2 ). The curves show the distribution of relative fluorescence intensities for observed events. Each curve represents an independent binding experiment (n = 3). ( b ) Total number of positive SA-PE staining events from ( a ) (number of events > RFU 400) of biotinylated L0 (cyan), and biotinylated L2 (blue), compared to absence of ssODNs (w/o; black), in exosome binding. In each binding reaction 12 nM of each library was used. ( c ) Post-ADAPT quantification of binding of individual aptamers as part of the library and individually as illustrated in Supplementary Fig. S3 . The top panel shows 23 representative individual aptamers, selected either with high “H” or low “L” normalized counts from L3 NGS data after binding pooled plasma from breast biopsy negative donors. There is at least a 5-fold difference between counts of H and L ssODNs. These 23 sequences were re-synthesized and tested individually in the same binding assay, but in equal concentrations, unlike their original representation in L3. PEG-precipitated aptamer/plasma complexes were directly subjected to qPCR (bottom panel). Inlay: Magnification of qPCR results of ssODNs incubated with PBS instead of plasma (red). ( d ) Silver-stained reducing SDS-PA gel of pulled down proteins from PPT plasma with the indicated ssODNs (H1, H11, L4, L15), immobilized on streptavidin magnetic beads, and the control without ssODN (NO). Dashed arrows indicate pulled down proteins C1QA, C1QB, and C1QC. The dotted arrow indicates the heavy chain of IgM (IgM HC ). SA: streptavidin. RC: reverse complement sequence.

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Isolation, Staining, Fluorescence, Next-Generation Sequencing, Synthesized, Real-time Polymerase Chain Reaction, Incubation, Magnetic Beads, Sequencing

    Design of the ssODN library and forward strand patterns of the index PCR product. ( a ) Design of the ssODN library. Reverse was used in the enrichment and is shown here. The primer highlighted in blue contains a sequence, complementary to the Illumina sequencing primer, which allows skipping one PCR in the NGS preparation (see details in NGS part below). The ssODN library has a staggered design to ensure the diversity while sequencing the constant region. The resulting library consists of 4 ssODNs, which differ from each other in length by+1, +2, and +3 nucleotides (shown in green; see details in NGS part below). There is no substantial homo-dimerization possible between constant regions, which ensures that primarily the variable region will drive conformational changes in the complete aptamer sequences. Additional balancing was introduced to the design of the 5′ constant region to ensure the diversity during NGS (shown in red). ( b ) Patterns of the forward strands of the index PCR product. The 3′-end was attached to the surface of the flow cell. Arrow: beginning of the sequencing.
    Figure Legend Snippet: Design of the ssODN library and forward strand patterns of the index PCR product. ( a ) Design of the ssODN library. Reverse was used in the enrichment and is shown here. The primer highlighted in blue contains a sequence, complementary to the Illumina sequencing primer, which allows skipping one PCR in the NGS preparation (see details in NGS part below). The ssODN library has a staggered design to ensure the diversity while sequencing the constant region. The resulting library consists of 4 ssODNs, which differ from each other in length by+1, +2, and +3 nucleotides (shown in green; see details in NGS part below). There is no substantial homo-dimerization possible between constant regions, which ensures that primarily the variable region will drive conformational changes in the complete aptamer sequences. Additional balancing was introduced to the design of the 5′ constant region to ensure the diversity during NGS (shown in red). ( b ) Patterns of the forward strands of the index PCR product. The 3′-end was attached to the surface of the flow cell. Arrow: beginning of the sequencing.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Next-Generation Sequencing, Flow Cytometry

    27) Product Images from "The senescence-associated secretory phenotype is potentiated by feedforward regulatory mechanisms involving Zscan4 and TAK1"

    Article Title: The senescence-associated secretory phenotype is potentiated by feedforward regulatory mechanisms involving Zscan4 and TAK1

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04010-4

    TAK1 activation engages p38 but its inhibition does not influence DDR signaling. a PSC27 cells were treated with bleomycin with or without increasing concentrations of the TAK1 inhibitor 5Z-7. Cell lysates were IPed with anti-p-TAK1, and subject to in vitro kinase assay with MKK6 as a substrate. Activation of kinase p38 was analyzed, with GAPDH as a loading control. Alternatively, IL-1α (20 ng/ml) was used to stimulate cells with or without 5Z-7, with the lysates subsequently analyzed. b IL-1α was eliminated from PSC27 cells by shRNAs. TAK1/MKK6 interaction and p38 activation in the cytosol were assessed by IP in the same conditions of a . c PSC27 cells were treated by bleomycin, or 5Z-7, or both, and subject to immunofluorescence (IF) staining of γ-H2AX. DNA damage extent in stromal cells was depicted as DDR statistics by counting the number of DDR foci per cell. Right, representative images of IF staining. Green, γ-H2AX; Blue, DAPI. Scale bars, 10 μm. Data analyzed by two-way ANOVA. d Top, representative images for clonogenic assay. PSC27 cells were treated by bleomycin alone for 6 h then released until 7d later, or treated by 5Z-7 alone continuously for 7d, or treated by both agents for individual time length in culture, after which clonogenic staining was performed. Bottom, statistics of stromal cell clonogenic growth. Data analyzed by Student’s t -test. e PSC27 cells were treated with bleomycin and/or 5Z-7, and cell lysates were collected 7d after drug treatment for immunoblot analysis. JNK1 phosphorylation and IL-8 expression were used to probe TAK1 activation and SASP development, respectively. Activation of mTOR and Akt were assayed with the same set of lysates, as well. Data in all bar plots are shown as mean ± SD. All data are representative of 3 biological replicates. * P
    Figure Legend Snippet: TAK1 activation engages p38 but its inhibition does not influence DDR signaling. a PSC27 cells were treated with bleomycin with or without increasing concentrations of the TAK1 inhibitor 5Z-7. Cell lysates were IPed with anti-p-TAK1, and subject to in vitro kinase assay with MKK6 as a substrate. Activation of kinase p38 was analyzed, with GAPDH as a loading control. Alternatively, IL-1α (20 ng/ml) was used to stimulate cells with or without 5Z-7, with the lysates subsequently analyzed. b IL-1α was eliminated from PSC27 cells by shRNAs. TAK1/MKK6 interaction and p38 activation in the cytosol were assessed by IP in the same conditions of a . c PSC27 cells were treated by bleomycin, or 5Z-7, or both, and subject to immunofluorescence (IF) staining of γ-H2AX. DNA damage extent in stromal cells was depicted as DDR statistics by counting the number of DDR foci per cell. Right, representative images of IF staining. Green, γ-H2AX; Blue, DAPI. Scale bars, 10 μm. Data analyzed by two-way ANOVA. d Top, representative images for clonogenic assay. PSC27 cells were treated by bleomycin alone for 6 h then released until 7d later, or treated by 5Z-7 alone continuously for 7d, or treated by both agents for individual time length in culture, after which clonogenic staining was performed. Bottom, statistics of stromal cell clonogenic growth. Data analyzed by Student’s t -test. e PSC27 cells were treated with bleomycin and/or 5Z-7, and cell lysates were collected 7d after drug treatment for immunoblot analysis. JNK1 phosphorylation and IL-8 expression were used to probe TAK1 activation and SASP development, respectively. Activation of mTOR and Akt were assayed with the same set of lysates, as well. Data in all bar plots are shown as mean ± SD. All data are representative of 3 biological replicates. * P

    Techniques Used: Activation Assay, Inhibition, In Vitro, Kinase Assay, Immunofluorescence, Staining, Clonogenic Assay, Expressing

    Targeting TAK1 restricts in vivo SASP and minimizes drug resistance acquired from the treatment-damaged TME. a Experimental design of severe combined immunodeficient (SCID) mouse-based in vivo studies. Two weeks after tumour implantation and stable uptake, mice received either single or combinational agents administered as metronomic treatments composed of several cycles. b Comparative statistics of tumour volumes. PC3 cells were implanted alone or with stromal cells subcutaneously to animals, which were then subject to cyclic treatments. Tumour volumes were measured at the end of an 8-week preclinical regimen. c Representative images of tumour-bearing animals in the preclinical trial. Digital signals were proportional to in vivo luciferase activities measured by an IVIS device. d Mice were sacrificed upon presence of advanced bulky diseases. Survival duration was calculated from the time of tissue recombinant injection until the day of death. Data compared with log-rank (Mantel–Cox) test. e Transcript assessment of several canonical SASP factors expressed in stromal cells isolated from PC3 tumours. Animals that had both stromal and cancer cells in the tumour foci were selected for analysis, with stromal cells acquired by LCM. f Graphical summary. DNA damage triggers an acute response of stromal cells, during which Zscan4 is expressed via the ATM-TRAF6-TAK1 axis and translocates to the nucleus. Zscan4 promotes the expression of a subset of ASAP-associated factors through NF-κB signaling, forming the first positive feedback loop. Further, TAK1 collaterally activates p38, a kinase that subsequently engages the PI3K/Akt/mTOR pathway. mTOR subsequently activates the NF-κB machinery both directly via interaction with IKKα and indirectly by promoting the translation of IL-1α, a cytokine that strengthens TAK1 phosphorylation in the chronic SASP. Together, several feedforward mechanisms favor the SASP development until its culmination in stromal cells, which confers substantial resistance on surviving cancer cells in the damaged TME and enables disease recurrence post-therapy. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. For animal treatments, n = 10/group. Data in b , e were analyzed by Student’s t -test. * P
    Figure Legend Snippet: Targeting TAK1 restricts in vivo SASP and minimizes drug resistance acquired from the treatment-damaged TME. a Experimental design of severe combined immunodeficient (SCID) mouse-based in vivo studies. Two weeks after tumour implantation and stable uptake, mice received either single or combinational agents administered as metronomic treatments composed of several cycles. b Comparative statistics of tumour volumes. PC3 cells were implanted alone or with stromal cells subcutaneously to animals, which were then subject to cyclic treatments. Tumour volumes were measured at the end of an 8-week preclinical regimen. c Representative images of tumour-bearing animals in the preclinical trial. Digital signals were proportional to in vivo luciferase activities measured by an IVIS device. d Mice were sacrificed upon presence of advanced bulky diseases. Survival duration was calculated from the time of tissue recombinant injection until the day of death. Data compared with log-rank (Mantel–Cox) test. e Transcript assessment of several canonical SASP factors expressed in stromal cells isolated from PC3 tumours. Animals that had both stromal and cancer cells in the tumour foci were selected for analysis, with stromal cells acquired by LCM. f Graphical summary. DNA damage triggers an acute response of stromal cells, during which Zscan4 is expressed via the ATM-TRAF6-TAK1 axis and translocates to the nucleus. Zscan4 promotes the expression of a subset of ASAP-associated factors through NF-κB signaling, forming the first positive feedback loop. Further, TAK1 collaterally activates p38, a kinase that subsequently engages the PI3K/Akt/mTOR pathway. mTOR subsequently activates the NF-κB machinery both directly via interaction with IKKα and indirectly by promoting the translation of IL-1α, a cytokine that strengthens TAK1 phosphorylation in the chronic SASP. Together, several feedforward mechanisms favor the SASP development until its culmination in stromal cells, which confers substantial resistance on surviving cancer cells in the damaged TME and enables disease recurrence post-therapy. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. For animal treatments, n = 10/group. Data in b , e were analyzed by Student’s t -test. * P

    Techniques Used: In Vivo, Mouse Assay, Luciferase, Recombinant, Injection, Isolation, Laser Capture Microdissection, Expressing

    mTOR is activated and interacts with the IKK complex upon genotoxic treatment. a Immunoblot analysis of ATM, TAK1, Akt and mTOR activation at different time points after bleomycin treatment of PSC27 cells. Total proteins of these factors and GAPDH were loading controls. b Representative IF images for mTOR phosphorylation (S2448) in PSC27 cells treated by bleomycin with or without RAD001 (50 nM) (blue, DAPI; red, γH2AX; green, p-mTOR). Cells were stained 7d after bleomycin treatment. Scale bars, 15 μm. c Immunoblot analysis of mTOR activation and downstream substrate (S6K1/4E-BP1) phosphorylation in cells treated by bleomycin with or without RAD001. Lysates were collected 7d after initiation of treatment, the same time point for sample acquirement of following assays. d DNA synthesis assay by BrdU incorporation for cells treated in different conditions, with positive ratios displayed in percentage. Right, representative images (green, BrdU; blue, DAPI). Scale bars, 15 μm. Data analyzed by Student’s t -test. e Stromal cells were stained in culture for SA-β-Gal, with results presented as positivity ratio. Right, representative images. Scale bars, 20 μm. Data analyzed by Student’s t -test. f Immunoblot examination of NF-κB pathway in stromal cells treated by bleomycin, with or without RAD001. Cell lysates were fractionated into cytoplasmic and nuclear extracts for comparative analysis. g Cells were transfected with a reporter construct that encodes NF-κB binding sites in the promoter region of a luciferase transgene. Signals were normalized as readings of firefly/renilla ratio. Data analyzed by Student’s t -test. h Co-IP assay to determine the interaction between mTOR and IKK complex. PSC27 cells were treated with bleomycin, and cell lysates were collected 7d later for IP analysis with anti-mTOR or anti-IKKα. IgG, control. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. * P
    Figure Legend Snippet: mTOR is activated and interacts with the IKK complex upon genotoxic treatment. a Immunoblot analysis of ATM, TAK1, Akt and mTOR activation at different time points after bleomycin treatment of PSC27 cells. Total proteins of these factors and GAPDH were loading controls. b Representative IF images for mTOR phosphorylation (S2448) in PSC27 cells treated by bleomycin with or without RAD001 (50 nM) (blue, DAPI; red, γH2AX; green, p-mTOR). Cells were stained 7d after bleomycin treatment. Scale bars, 15 μm. c Immunoblot analysis of mTOR activation and downstream substrate (S6K1/4E-BP1) phosphorylation in cells treated by bleomycin with or without RAD001. Lysates were collected 7d after initiation of treatment, the same time point for sample acquirement of following assays. d DNA synthesis assay by BrdU incorporation for cells treated in different conditions, with positive ratios displayed in percentage. Right, representative images (green, BrdU; blue, DAPI). Scale bars, 15 μm. Data analyzed by Student’s t -test. e Stromal cells were stained in culture for SA-β-Gal, with results presented as positivity ratio. Right, representative images. Scale bars, 20 μm. Data analyzed by Student’s t -test. f Immunoblot examination of NF-κB pathway in stromal cells treated by bleomycin, with or without RAD001. Cell lysates were fractionated into cytoplasmic and nuclear extracts for comparative analysis. g Cells were transfected with a reporter construct that encodes NF-κB binding sites in the promoter region of a luciferase transgene. Signals were normalized as readings of firefly/renilla ratio. Data analyzed by Student’s t -test. h Co-IP assay to determine the interaction between mTOR and IKK complex. PSC27 cells were treated with bleomycin, and cell lysates were collected 7d later for IP analysis with anti-mTOR or anti-IKKα. IgG, control. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. * P

    Techniques Used: Activation Assay, Staining, DNA Synthesis, BrdU Incorporation Assay, Transfection, Construct, Binding Assay, Luciferase, Co-Immunoprecipitation Assay

    TAK1 inhibition abrogates the SASP development and deprives cancer cells of stroma-conferred malignancy. a Heatmap profiling of stromal cell transcriptomics after treatment by bleomycin alone, or together with each of 5Z-7, SB203580 (SB), and RAD001. Cells were collected 7d after drug treatment. The SASP-associated soluble factors are selectively displayed on the top list per expression fold change. b Transcript expression assay of several SASP canonical factors. Cells were treated by bleomycin alone or co-treated by bleomycin/SASP inhibitors. Signals were normalized to untreated samples per factor. Data analyzed by Student’s t -test. c GSEA profiling with significant enrichment scores showing a SASP-specific expression signature composed of canonical soluble factors. Head-to-head comparison of differentially expressed SASP factors was performed between cells treated by bleomycin only and cells co-treated by bleomycin/SASP inhibitors. P values determined by GSEA software. d The prostate cancer (PCa) cell line PC3 was subject to treatment by mitoxantrone (MIT) while being incubated with several types of conditioned media (CM). Upper, representative images of PC3 cells upon different treatments. Arrows, apoptotic cells; arrowheads, damaged cells. Scale bars, 50 μm. Lower, viability assessment of several cell lines incubated with various types of CM and exposed to MIT at the IC50 concentration pre-determined per line. Data analyzed by Student’s t -test. e Measurement of apoptosis in culture conditions. Results were depicted in relative luminescence units (RLUs) representing signal readings proportional to caspase-3/7 activity of PC3 cells. Data analyzed by Student’s t -test. f Dose response curves (non-linear regression fit) of PC3 cells treated by PSC27-derived CM. The concentration of MIT was plotted in an exponential scale, with cell viability assessed in relative to the untreated group and calculated in percentage. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates.* P
    Figure Legend Snippet: TAK1 inhibition abrogates the SASP development and deprives cancer cells of stroma-conferred malignancy. a Heatmap profiling of stromal cell transcriptomics after treatment by bleomycin alone, or together with each of 5Z-7, SB203580 (SB), and RAD001. Cells were collected 7d after drug treatment. The SASP-associated soluble factors are selectively displayed on the top list per expression fold change. b Transcript expression assay of several SASP canonical factors. Cells were treated by bleomycin alone or co-treated by bleomycin/SASP inhibitors. Signals were normalized to untreated samples per factor. Data analyzed by Student’s t -test. c GSEA profiling with significant enrichment scores showing a SASP-specific expression signature composed of canonical soluble factors. Head-to-head comparison of differentially expressed SASP factors was performed between cells treated by bleomycin only and cells co-treated by bleomycin/SASP inhibitors. P values determined by GSEA software. d The prostate cancer (PCa) cell line PC3 was subject to treatment by mitoxantrone (MIT) while being incubated with several types of conditioned media (CM). Upper, representative images of PC3 cells upon different treatments. Arrows, apoptotic cells; arrowheads, damaged cells. Scale bars, 50 μm. Lower, viability assessment of several cell lines incubated with various types of CM and exposed to MIT at the IC50 concentration pre-determined per line. Data analyzed by Student’s t -test. e Measurement of apoptosis in culture conditions. Results were depicted in relative luminescence units (RLUs) representing signal readings proportional to caspase-3/7 activity of PC3 cells. Data analyzed by Student’s t -test. f Dose response curves (non-linear regression fit) of PC3 cells treated by PSC27-derived CM. The concentration of MIT was plotted in an exponential scale, with cell viability assessed in relative to the untreated group and calculated in percentage. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates.* P

    Techniques Used: Inhibition, Expressing, Software, Incubation, Concentration Assay, Activity Assay, Derivative Assay

    Zscan4 expression is induced by DNA damage and regulated by the ATM/TRAF6/TAK1/p65 signaling axis. a PSC27 cells were treated by bleomycin (50 μg/ml) with or without KU55933 (KU, 10 μM) in culture. Cell lysates were immunoprecipitated with anti-p-ATM, with the immunoprecipitates (IPs) analyzed with anti-p-ATM and anti-TRAF6, respectively. b PSC27 cells lentivirally infected with scramble or TRAF6-specific shRNAs were treated with bleomycin, with lysates collected at indicated time points and subject to IP and immunoblot assays. c Anti-TAK1-based IP of PSC27 cells treated with bleomycin in the presence or absence of 5Z-7 (500 nM), followed by immunoblot analysis. d Primary PSC27 cells or those stably expressing shRNA to TRAF6 were treated with bleomycin, with IPs pulled down by anti-TRAF6 and examined by immunoblots. e Cytoplasmic and nuclear protein samples from control and bleomycin-treated PSC27 cells were analyzed for TAK1 activation and NF-κB nuclear translocation. GAPDH and Histone H1, cytoplasmic and nuclear loading controls, respectively. f Chromatin immunoprecipitation (ChIP) was performed to identify potential NF-κB binding sites in Zscan4 proximal promoter. Zscan4-p1/p2/p3 denotes 3 representative genomic sites in promoter region, with known NF-κB binding sites from WNT16B, SFRP2, IL-6, and IL-8 selected as positive controls. g Zscan4 transcript expression in PSC27 cells stably expressing an NF-κB-null mutant and treated by bleomycin, mitoxantrone or radiation. Signals normalized to untreated cells. h Expression profiling of typical ASAP factors (IL-6/Timp-1) and Zscan4 in stromal cells exposed to bleomycin. Left (histograms), cells were pretreated with inhibitors of NF-κB, ATM, or TAK1 (Bay, KU or 5Z-7, respectively) before addition of bleomycin, with transcripts collected for analysis 24 h after genotoxic treatment (data normalized to untreated sample per factor set). Right (immunoblots), protein level assessment of IL-6, Timp-1, and Zscan4 24 h after cell exposure to bleomycin. GAPDH, loading control. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. BLEO bleomycin, KU KU55933, 5Z-7 5 Z -7-oxozeaenol, Bay Bay 11–7082. Data in g , h were analyzed by Student’s t -test. * P
    Figure Legend Snippet: Zscan4 expression is induced by DNA damage and regulated by the ATM/TRAF6/TAK1/p65 signaling axis. a PSC27 cells were treated by bleomycin (50 μg/ml) with or without KU55933 (KU, 10 μM) in culture. Cell lysates were immunoprecipitated with anti-p-ATM, with the immunoprecipitates (IPs) analyzed with anti-p-ATM and anti-TRAF6, respectively. b PSC27 cells lentivirally infected with scramble or TRAF6-specific shRNAs were treated with bleomycin, with lysates collected at indicated time points and subject to IP and immunoblot assays. c Anti-TAK1-based IP of PSC27 cells treated with bleomycin in the presence or absence of 5Z-7 (500 nM), followed by immunoblot analysis. d Primary PSC27 cells or those stably expressing shRNA to TRAF6 were treated with bleomycin, with IPs pulled down by anti-TRAF6 and examined by immunoblots. e Cytoplasmic and nuclear protein samples from control and bleomycin-treated PSC27 cells were analyzed for TAK1 activation and NF-κB nuclear translocation. GAPDH and Histone H1, cytoplasmic and nuclear loading controls, respectively. f Chromatin immunoprecipitation (ChIP) was performed to identify potential NF-κB binding sites in Zscan4 proximal promoter. Zscan4-p1/p2/p3 denotes 3 representative genomic sites in promoter region, with known NF-κB binding sites from WNT16B, SFRP2, IL-6, and IL-8 selected as positive controls. g Zscan4 transcript expression in PSC27 cells stably expressing an NF-κB-null mutant and treated by bleomycin, mitoxantrone or radiation. Signals normalized to untreated cells. h Expression profiling of typical ASAP factors (IL-6/Timp-1) and Zscan4 in stromal cells exposed to bleomycin. Left (histograms), cells were pretreated with inhibitors of NF-κB, ATM, or TAK1 (Bay, KU or 5Z-7, respectively) before addition of bleomycin, with transcripts collected for analysis 24 h after genotoxic treatment (data normalized to untreated sample per factor set). Right (immunoblots), protein level assessment of IL-6, Timp-1, and Zscan4 24 h after cell exposure to bleomycin. GAPDH, loading control. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. BLEO bleomycin, KU KU55933, 5Z-7 5 Z -7-oxozeaenol, Bay Bay 11–7082. Data in g , h were analyzed by Student’s t -test. * P

    Techniques Used: Expressing, Immunoprecipitation, Infection, Stable Transfection, shRNA, Western Blot, Activation Assay, Translocation Assay, Chromatin Immunoprecipitation, Binding Assay, Mutagenesis

    28) Product Images from "The Src family kinase Hck couples BCR/ABL to STAT5 activation in myeloid leukemia cells"

    Article Title: The Src family kinase Hck couples BCR/ABL to STAT5 activation in myeloid leukemia cells

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdf562

    Fig. 2. Pattern of phosphotyrosine proteins co-immunoprecipitating with STAT5. STAT5 was immunoprecipitated from the lysates of 32Dcl3 cells (Parental) and cells expressing BCR/ABL or the BCR/ABLΔΔ mutant, after being starved of IL-3 for 12 h. The immunoprecipitates were analyzed by western analysis with anti-P.Tyr antibodies (upper panel). Subsequent blotting with anti-STAT5, anti-ABL and anti-Hck confirmed the localization of the indicated proteins (lower panels). An equal amount of STAT5 was immunoprecipitated in each sample. Results represent two independent experiments.
    Figure Legend Snippet: Fig. 2. Pattern of phosphotyrosine proteins co-immunoprecipitating with STAT5. STAT5 was immunoprecipitated from the lysates of 32Dcl3 cells (Parental) and cells expressing BCR/ABL or the BCR/ABLΔΔ mutant, after being starved of IL-3 for 12 h. The immunoprecipitates were analyzed by western analysis with anti-P.Tyr antibodies (upper panel). Subsequent blotting with anti-STAT5, anti-ABL and anti-Hck confirmed the localization of the indicated proteins (lower panels). An equal amount of STAT5 was immunoprecipitated in each sample. Results represent two independent experiments.

    Techniques Used: Immunoprecipitation, Expressing, Mutagenesis, Western Blot

    Fig. 4. The BCR/ABL SH3 + SH2 region is required for activation of Hck and phosphorylation of STAT5B on Y699. ( A ) Hck was immunoprecipitated from cell lysates obtained from 32Dcl3 parental cells (Parental) and from cells expressing BCR/ABL or the BCR/ABLΔΔ mutant after being starved of IL-3 for 5 h. An in vitro kinase reaction was performed using [γ- 32 P]ATP and 5 µg of the substrates: Sam68 (lower panel) or GST–C5 (upper panel). ( B ) Hck was immunoprecipitated from BCR/ABL cells. GST–C5 (C5) or GST–C5[Y699F] mutant (C5-YF) proteins were added as substrates along with [γ- 32 P]ATP. ( C ) Wild-type Hck and Hck-KE were immunopurified from infected Sf9 cell lysates and incubated in vitro with [γ- 32 P]ATP alone (Control) or together with the C5 or C5-YF proteins. The kinase reactions were resolved by SDS–PAGE. Phosphorylation of GST fusion proteins was assessed by autoradiography ( 32 P.C5 and 32 P.Sam68 boxes). The presence of Hck in each reaction was verified by immunoblotting (Hck panel). Results represent three independent experiments.
    Figure Legend Snippet: Fig. 4. The BCR/ABL SH3 + SH2 region is required for activation of Hck and phosphorylation of STAT5B on Y699. ( A ) Hck was immunoprecipitated from cell lysates obtained from 32Dcl3 parental cells (Parental) and from cells expressing BCR/ABL or the BCR/ABLΔΔ mutant after being starved of IL-3 for 5 h. An in vitro kinase reaction was performed using [γ- 32 P]ATP and 5 µg of the substrates: Sam68 (lower panel) or GST–C5 (upper panel). ( B ) Hck was immunoprecipitated from BCR/ABL cells. GST–C5 (C5) or GST–C5[Y699F] mutant (C5-YF) proteins were added as substrates along with [γ- 32 P]ATP. ( C ) Wild-type Hck and Hck-KE were immunopurified from infected Sf9 cell lysates and incubated in vitro with [γ- 32 P]ATP alone (Control) or together with the C5 or C5-YF proteins. The kinase reactions were resolved by SDS–PAGE. Phosphorylation of GST fusion proteins was assessed by autoradiography ( 32 P.C5 and 32 P.Sam68 boxes). The presence of Hck in each reaction was verified by immunoblotting (Hck panel). Results represent three independent experiments.

    Techniques Used: Activation Assay, Immunoprecipitation, Expressing, Mutagenesis, In Vitro, Infection, Incubation, SDS Page, Autoradiography

    Fig. 3. The BCR/ABL SH3 + SH2 region interacts with the Hck–STAT5 complex. 32Dcl3 cells (Parental) and cells expressing BCR/ABL, or the indicated BCR/ABL mutants, were starved of IL-3 for 5 h. STAT5 immunoprecipitates ( A ) and Hck immunoprecipitates ( B ) were examined by SDS–PAGE followed by western analysis with anti-STAT5, anti-Hck, anti-P.Tyr and anti-ABL antibodies. Hck immuno precipitation and kinase assay ( C ) were performed to detect its association with BCR/ABL and to measure its catalytic activity. Hck immunoprecipitates were examined by SDS–PAGE followed by western analysis with anti-ABL (upper panel) and anti-anti-Hck (bottom panel) antibody. An in vitro kinase reaction was performed in these immunoprecipitates using [γ- 32 P]ATP and 5 µg of the standard substrate for the Src-related family of tyrosine kinases, Sam68 (middle panel). Results represent two or three independent experiments.
    Figure Legend Snippet: Fig. 3. The BCR/ABL SH3 + SH2 region interacts with the Hck–STAT5 complex. 32Dcl3 cells (Parental) and cells expressing BCR/ABL, or the indicated BCR/ABL mutants, were starved of IL-3 for 5 h. STAT5 immunoprecipitates ( A ) and Hck immunoprecipitates ( B ) were examined by SDS–PAGE followed by western analysis with anti-STAT5, anti-Hck, anti-P.Tyr and anti-ABL antibodies. Hck immuno precipitation and kinase assay ( C ) were performed to detect its association with BCR/ABL and to measure its catalytic activity. Hck immunoprecipitates were examined by SDS–PAGE followed by western analysis with anti-ABL (upper panel) and anti-anti-Hck (bottom panel) antibody. An in vitro kinase reaction was performed in these immunoprecipitates using [γ- 32 P]ATP and 5 µg of the standard substrate for the Src-related family of tyrosine kinases, Sam68 (middle panel). Results represent two or three independent experiments.

    Techniques Used: Expressing, SDS Page, Western Blot, Immunoprecipitation, Kinase Assay, Activity Assay, In Vitro

    Fig. 1. STAT5 is phosphorylated by BCR/ABL immunoprecipitates. ( A ) STAT5 phosphorylation was examined in anti-ABL immunoprecipitates obtained from IL-3- and serum-starved 32Dcl3 cells expressing BCR/ABL wild-type (WT) or the kinase-defective mutant (K1172R), using full-length STAT5 or the C5 (amino acids 547–787) and N5 (amino acids 1–546) fragments conjugated to GST as substrates and [γ- 32 P]ATP (upper box). GST alone was not phosphorylated (data not shown). GST–STAT5 proteins loaded onto the gel were detected by western blot using anti-STAT5B antibodies (C-17 recognizes the C- terminus and N-20 recognizes the N-terminus) (middle box). Arrows 1, 2 and 3 indicate the position of full-length GST–STAT5, GST–N5 and GST–C5, respectively. GST was detected by Ponceau red staining (not shown). Immunoprecipitated BCR/ABL proteins (arrow 4) were visualized by western analysis using anti-ABL antibody (bottom box). ( B ) Phosphorylation of GST–C5 (C5) and enolase (Eno) was examined in anti-ABL immunoprecipitates obtained from IL-3- and serum-starved 32Dcl3 cells (Parental) and cells expressing BCR/ABL kinase in the absence (–) or presence (+) of 1 µM STI571. C5 and enolase substrates were detected by Coomassie Blue staining of the gel. Results represent three independent experiments.
    Figure Legend Snippet: Fig. 1. STAT5 is phosphorylated by BCR/ABL immunoprecipitates. ( A ) STAT5 phosphorylation was examined in anti-ABL immunoprecipitates obtained from IL-3- and serum-starved 32Dcl3 cells expressing BCR/ABL wild-type (WT) or the kinase-defective mutant (K1172R), using full-length STAT5 or the C5 (amino acids 547–787) and N5 (amino acids 1–546) fragments conjugated to GST as substrates and [γ- 32 P]ATP (upper box). GST alone was not phosphorylated (data not shown). GST–STAT5 proteins loaded onto the gel were detected by western blot using anti-STAT5B antibodies (C-17 recognizes the C- terminus and N-20 recognizes the N-terminus) (middle box). Arrows 1, 2 and 3 indicate the position of full-length GST–STAT5, GST–N5 and GST–C5, respectively. GST was detected by Ponceau red staining (not shown). Immunoprecipitated BCR/ABL proteins (arrow 4) were visualized by western analysis using anti-ABL antibody (bottom box). ( B ) Phosphorylation of GST–C5 (C5) and enolase (Eno) was examined in anti-ABL immunoprecipitates obtained from IL-3- and serum-starved 32Dcl3 cells (Parental) and cells expressing BCR/ABL kinase in the absence (–) or presence (+) of 1 µM STI571. C5 and enolase substrates were detected by Coomassie Blue staining of the gel. Results represent three independent experiments.

    Techniques Used: Expressing, Mutagenesis, Western Blot, Staining, Immunoprecipitation

    29) Product Images from "Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5"

    Article Title: Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

    Journal: Biochemical Journal

    doi: 10.1042/BJ20040324

    Distribution of the cdk5 phosphorylation sites in DCX ( a ) DCX was phosphorylated for 10 min in the presence of [γ- 32 P]ATP and for 90 min in the presence of excess non-radioactive ATP and the samples were mixed. After SDS/PAGE, DCX was digested with trypsin and the fragments were separated by HPLC. A plot of Cerenkov radiation versus fraction number for the reversed-phase HPLC separation is shown. The percentage concentration of the organic phase (phase B) is also shown on the right axis. The fractions that showed a specific increase in phosphorylation were pooled into seven (i–vii) samples. ( b – e ) Part of the MALDI-MS spectra of samples v and vi before (Control) and after their treatment with alkaline phosphatase to dephosphorylate the peptides. Sample v before ( b ) and after ( c ) dephosphorylation. An 80 Da mass shift from m / z 1507.70 to 1427.75 and m / z 1490.64 to 1410.64 indicates the loss of a phosphate group. The dephosphorylated peptides match the mass of DCX 331–344 and the N-terminal pyroglutamic acid modified DCX 331–344 respectively. A region of the MALDI-MS spectra from sample vi is shown before ( d ) and after ( e ) alkaline phosphatase treatment. The 80 Da mass shift from m / z 2072.82 to 1992.87 and m / z 2056.81 to 1976.86 indicates loss of a phosphate group. The dephosphorylated peptides match oxidized and non-oxidized DCX 23–39 respectively.
    Figure Legend Snippet: Distribution of the cdk5 phosphorylation sites in DCX ( a ) DCX was phosphorylated for 10 min in the presence of [γ- 32 P]ATP and for 90 min in the presence of excess non-radioactive ATP and the samples were mixed. After SDS/PAGE, DCX was digested with trypsin and the fragments were separated by HPLC. A plot of Cerenkov radiation versus fraction number for the reversed-phase HPLC separation is shown. The percentage concentration of the organic phase (phase B) is also shown on the right axis. The fractions that showed a specific increase in phosphorylation were pooled into seven (i–vii) samples. ( b – e ) Part of the MALDI-MS spectra of samples v and vi before (Control) and after their treatment with alkaline phosphatase to dephosphorylate the peptides. Sample v before ( b ) and after ( c ) dephosphorylation. An 80 Da mass shift from m / z 1507.70 to 1427.75 and m / z 1490.64 to 1410.64 indicates the loss of a phosphate group. The dephosphorylated peptides match the mass of DCX 331–344 and the N-terminal pyroglutamic acid modified DCX 331–344 respectively. A region of the MALDI-MS spectra from sample vi is shown before ( d ) and after ( e ) alkaline phosphatase treatment. The 80 Da mass shift from m / z 2072.82 to 1992.87 and m / z 2056.81 to 1976.86 indicates loss of a phosphate group. The dephosphorylated peptides match oxidized and non-oxidized DCX 23–39 respectively.

    Techniques Used: SDS Page, High Performance Liquid Chromatography, Concentration Assay, Mass Spectrometry, De-Phosphorylation Assay, Modification

    Comparative phosphorylation of wt and mutant DCX by cdk5 GST-fusion protein (approx. 1 μg) of wt DCX, serine to alanine mutants and DCX truncations was phosphorylated by recombinant GST-cdk5/p25 in the presence of [γ- 32 or 33 P]ATP for 5 min at 37 °C and then analysed by SDS/PAGE and autoradiography. Phosphorylation was quantified by a STORM PhosphorImager. Results are represented as a percentage of wt DCX for the corrected activity (see the Methods section). Means±S.E.M. for six or seven replicated reactions per protein are presented.
    Figure Legend Snippet: Comparative phosphorylation of wt and mutant DCX by cdk5 GST-fusion protein (approx. 1 μg) of wt DCX, serine to alanine mutants and DCX truncations was phosphorylated by recombinant GST-cdk5/p25 in the presence of [γ- 32 or 33 P]ATP for 5 min at 37 °C and then analysed by SDS/PAGE and autoradiography. Phosphorylation was quantified by a STORM PhosphorImager. Results are represented as a percentage of wt DCX for the corrected activity (see the Methods section). Means±S.E.M. for six or seven replicated reactions per protein are presented.

    Techniques Used: Mutagenesis, Recombinant, SDS Page, Autoradiography, Activity Assay

    30) Product Images from "Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p"

    Article Title: Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0184236

    ATPase activity of liposome-reconstituted Pdr11p and Aus1p. Purified Pdr11p and Aus1p were reconstituted into different liposomes (containing Rho-PE as fluorescent lipid marker) and assayed for ATPase activity using [ γ - 32 P] ATP. A: SDS PAGE analysis of a flotation assay of Pdr11p proteoliposomes in a sucrose gradient. Detection of lipids and protein in the same low density fraction validated successful reconstitution. Proteins are visualised by silver staining and lipids by fluorescence from Rho-PE. B: Relative ATPase activity of Pdr11p reconstituted in PS liposomes in presence of the indicated inhibitors: orthovanadate, 1 mM; BeSO 4 , 1 mM; NaF, 5 mM. Data is based on at least two reconstitutions from one purification batch. C: Lipid effect on ATPase activity of reconstituted Pdr11p and Aus1p. All activities are corrected for protein amount in the proteoliposomes. Data is based on two reconstitutions from one purification batch of each protein. PC, PC only; PS, PC/PS (1:1); PG, PC/PG (7:3).
    Figure Legend Snippet: ATPase activity of liposome-reconstituted Pdr11p and Aus1p. Purified Pdr11p and Aus1p were reconstituted into different liposomes (containing Rho-PE as fluorescent lipid marker) and assayed for ATPase activity using [ γ - 32 P] ATP. A: SDS PAGE analysis of a flotation assay of Pdr11p proteoliposomes in a sucrose gradient. Detection of lipids and protein in the same low density fraction validated successful reconstitution. Proteins are visualised by silver staining and lipids by fluorescence from Rho-PE. B: Relative ATPase activity of Pdr11p reconstituted in PS liposomes in presence of the indicated inhibitors: orthovanadate, 1 mM; BeSO 4 , 1 mM; NaF, 5 mM. Data is based on at least two reconstitutions from one purification batch. C: Lipid effect on ATPase activity of reconstituted Pdr11p and Aus1p. All activities are corrected for protein amount in the proteoliposomes. Data is based on two reconstitutions from one purification batch of each protein. PC, PC only; PS, PC/PS (1:1); PG, PC/PG (7:3).

    Techniques Used: Activity Assay, Purification, Marker, SDS Page, Silver Staining, Fluorescence

    ATPase activity of solubilised Pdr11p. ATPase activity of the purified detergent-solubilised transporter was assayed as described under “Materials and Methods” using [ γ - 32 P] ATP. A: ATPase activity as a function of pH. Open and filled circles are data from two independent experiments. Values are normalised with respect to the values at pH 7.2 (open circles) or pH 7.4 (closed circles). The dashed line is included to guide the eye. B: Effect of various inhibitors: NaN 3 , 5 mM; ouabain, 5 mM; BeSO 4 , 1 mM; NaF, 5 mM; AlF 3 , 1 mM; orthovanadate, 1 mM; EDTA, 1 mM. C: ATPase activity as a function of orthovanadate concentration. Fitting of data to a dose-response/activity curve (see Material and methods ) gives IC 50 = 4 ± 2 mM, and a Hill coefficient = 0.8 ± 0.2. Results in B and C are the mean ± S.D. from at least two independent experiments relative to the value obtained for the purified detergent-solubilised protein in the absence of inhibitors (control).
    Figure Legend Snippet: ATPase activity of solubilised Pdr11p. ATPase activity of the purified detergent-solubilised transporter was assayed as described under “Materials and Methods” using [ γ - 32 P] ATP. A: ATPase activity as a function of pH. Open and filled circles are data from two independent experiments. Values are normalised with respect to the values at pH 7.2 (open circles) or pH 7.4 (closed circles). The dashed line is included to guide the eye. B: Effect of various inhibitors: NaN 3 , 5 mM; ouabain, 5 mM; BeSO 4 , 1 mM; NaF, 5 mM; AlF 3 , 1 mM; orthovanadate, 1 mM; EDTA, 1 mM. C: ATPase activity as a function of orthovanadate concentration. Fitting of data to a dose-response/activity curve (see Material and methods ) gives IC 50 = 4 ± 2 mM, and a Hill coefficient = 0.8 ± 0.2. Results in B and C are the mean ± S.D. from at least two independent experiments relative to the value obtained for the purified detergent-solubilised protein in the absence of inhibitors (control).

    Techniques Used: Activity Assay, Purification, Concentration Assay

    31) Product Images from "L290P/V mutations increase ERK3’s cytoplasmic localization and migration/invasion-promoting capability in cancer cells"

    Article Title: L290P/V mutations increase ERK3’s cytoplasmic localization and migration/invasion-promoting capability in cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15135-9

    L290P mutation leads to a decrease in S189 phosphorylation of ERK3 protein, whereas L290V mutation has no clear effect. ( a ) Characterization of a phospho-S189 specific ERK3 antibody. 293 T cells were transfected with wild type ERK3 (ERK3 WT) or ERK3 S189A. Total cell lysates were treated with or without λ phosphatase (PPase). Phosphorylation of ERK3 at S189 [p-ERK3 (S189)] and expression level of ERK3 were determined using a phospho-S189 specific ERK3 antibody and an ERK3 antibody, respectively. ( b and c ) Western blot analysis of ERK3 phosphorylation at S189 in HeLa cells ( b ) and in A549 cells ( c ). Cells were transected with a pSG5 empty vector, HA-tagged wild-type ERK3, or each of the ERK3 mutants as indicated. Two days post-transfection, cells were lysed and levels of total ERK3 and ERK3 phosphorylated at S189 were analyzed by Western blotting. β-actin was used as a loading control.
    Figure Legend Snippet: L290P mutation leads to a decrease in S189 phosphorylation of ERK3 protein, whereas L290V mutation has no clear effect. ( a ) Characterization of a phospho-S189 specific ERK3 antibody. 293 T cells were transfected with wild type ERK3 (ERK3 WT) or ERK3 S189A. Total cell lysates were treated with or without λ phosphatase (PPase). Phosphorylation of ERK3 at S189 [p-ERK3 (S189)] and expression level of ERK3 were determined using a phospho-S189 specific ERK3 antibody and an ERK3 antibody, respectively. ( b and c ) Western blot analysis of ERK3 phosphorylation at S189 in HeLa cells ( b ) and in A549 cells ( c ). Cells were transected with a pSG5 empty vector, HA-tagged wild-type ERK3, or each of the ERK3 mutants as indicated. Two days post-transfection, cells were lysed and levels of total ERK3 and ERK3 phosphorylated at S189 were analyzed by Western blotting. β-actin was used as a loading control.

    Techniques Used: Mutagenesis, Transfection, Expressing, Western Blot, Plasmid Preparation

    Both ERK3 L290P and L290V mutants have increased ability to promote lung cancer cell migration and invasion. ( a ) Western blot analysis of ERK3 and ERK3 mutants’ expression in H1299 cells transfected with an empty vector (EV), ERK3 wild type (ERK3), ERK3 L290P or ERK3 L290V plasmids as indicated. β-actin was used as a loading control. ( b ) The effect of L290P/V mutations on ERK3’s role in H1299 cell migration was analyzed by two-chamber transwell migration assay of H1299 cells transfected with an EV, wild type ERK3 or each of the ERK3 L290 mutants. Representative images are shown at the bottom. Quantitative results are presented as number of migrated cells per field. Values in bar graph represent mean ± S.D. of 3 separate experiments. *P
    Figure Legend Snippet: Both ERK3 L290P and L290V mutants have increased ability to promote lung cancer cell migration and invasion. ( a ) Western blot analysis of ERK3 and ERK3 mutants’ expression in H1299 cells transfected with an empty vector (EV), ERK3 wild type (ERK3), ERK3 L290P or ERK3 L290V plasmids as indicated. β-actin was used as a loading control. ( b ) The effect of L290P/V mutations on ERK3’s role in H1299 cell migration was analyzed by two-chamber transwell migration assay of H1299 cells transfected with an EV, wild type ERK3 or each of the ERK3 L290 mutants. Representative images are shown at the bottom. Quantitative results are presented as number of migrated cells per field. Values in bar graph represent mean ± S.D. of 3 separate experiments. *P

    Techniques Used: Migration, Western Blot, Expressing, Transfection, Plasmid Preparation, Transwell Migration Assay

    L290P/V mutations do not alter ERK3 kinase activity. ( a ) Coomassie staining of purified wild type or mutant ERK3 proteins. 293 T cells were transfected with HA-tagged wild type ERK3, ERK3 L290P, L290V or kinase dead (KD) plasmids. ERK3 proteins were immunoprecipitated using HA-antibody-conjugated agarose beads, followed by elution with HA peptide. The purified proteins (300 ng) were analyzed by SDS-PAGE gel followed by Coomassie staining. The molecular size of each protein marker is indicated on the right side. ( b ) In vitro ERK3 kinase assay was performed by incubating 100 ng of purified ERK3 or each of ERK3 mutants as indicated, together with 1 μg of recombinant GST-SRC3-CID (substrate) in the presence of γ- 32 P-ATP. Phosphorylation of GST-SRC3-CID by ERK3 proteins was detected by autoradiograph (the right panel). Total protein level of GST-SRC3-CID in the reactions is shown by Coomassie staining (the left panel). Please note that ERK3 proteins are hardly seen in the Coomassie-stained gel due to their small amount (100 ng). ( c ) Quantification of GST-SRC3-CID phosphorylation by wild type or mutant ERK3 proteins. The relative phosphorylation level of GST-SRC3-CID is represented by the ratio of the band intensity of phosphorylated GST-SRC3-CID (shown in the autoradiograph) over that of the corresponding total GST-SRC3-CID (shown in the commassie-stained gel). For the purpose of comparison, the nomalized phosphorylation level of GST-SRC3-CID by wild type ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of 3 independent experiments. *P
    Figure Legend Snippet: L290P/V mutations do not alter ERK3 kinase activity. ( a ) Coomassie staining of purified wild type or mutant ERK3 proteins. 293 T cells were transfected with HA-tagged wild type ERK3, ERK3 L290P, L290V or kinase dead (KD) plasmids. ERK3 proteins were immunoprecipitated using HA-antibody-conjugated agarose beads, followed by elution with HA peptide. The purified proteins (300 ng) were analyzed by SDS-PAGE gel followed by Coomassie staining. The molecular size of each protein marker is indicated on the right side. ( b ) In vitro ERK3 kinase assay was performed by incubating 100 ng of purified ERK3 or each of ERK3 mutants as indicated, together with 1 μg of recombinant GST-SRC3-CID (substrate) in the presence of γ- 32 P-ATP. Phosphorylation of GST-SRC3-CID by ERK3 proteins was detected by autoradiograph (the right panel). Total protein level of GST-SRC3-CID in the reactions is shown by Coomassie staining (the left panel). Please note that ERK3 proteins are hardly seen in the Coomassie-stained gel due to their small amount (100 ng). ( c ) Quantification of GST-SRC3-CID phosphorylation by wild type or mutant ERK3 proteins. The relative phosphorylation level of GST-SRC3-CID is represented by the ratio of the band intensity of phosphorylated GST-SRC3-CID (shown in the autoradiograph) over that of the corresponding total GST-SRC3-CID (shown in the commassie-stained gel). For the purpose of comparison, the nomalized phosphorylation level of GST-SRC3-CID by wild type ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of 3 independent experiments. *P

    Techniques Used: Activity Assay, Staining, Purification, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Marker, In Vitro, Kinase Assay, Recombinant, Autoradiography

    L290P/V mutations increase ERK3 protein’s cytoplasmic localization. ( a ) Subcellular distribution of wild type ERK3 and the L290P/V mutants in HeLa cells. HA-tagged wild type ERK3 or each of the L290P/V mutants was exogenously expressed in HeLa cells. Subcellular localization of ERK3 proteins was determined by immunofluorescent staining using an anti-HA antibody (red), and DNA was stained with DAPI (blue) to show the nucleus. Pictures were taken under 63X magnification and representative images are shown (left). For each transfection, at least 50 cells expressing ERK3 or each ERK3 mutant were analyzed and classified into three groups as follow: cells showing predominant cytoplasmic ERK3 localization (C > N), cells showing relatively equal distribution of ERK3 in the nucleus and cytoplasm (N = C) and cells showing predominant nuclear localization of ERK3 (N > C). The bar graph (right) represents the percentage of total transfected cells for each different group. ( b ) Subcellular distribution of wild type ERK3 and the ERK3 L290P/V mutants in A549 lung cancer cells. Experiments were done with the same procedures as described in ( a ), except that myc-tagged ERK3 was transduced into A549 cells by lentivirus and an anti-myc antibody was used for detecting the exogenously expressed ERK3 and ERK3 mutants.
    Figure Legend Snippet: L290P/V mutations increase ERK3 protein’s cytoplasmic localization. ( a ) Subcellular distribution of wild type ERK3 and the L290P/V mutants in HeLa cells. HA-tagged wild type ERK3 or each of the L290P/V mutants was exogenously expressed in HeLa cells. Subcellular localization of ERK3 proteins was determined by immunofluorescent staining using an anti-HA antibody (red), and DNA was stained with DAPI (blue) to show the nucleus. Pictures were taken under 63X magnification and representative images are shown (left). For each transfection, at least 50 cells expressing ERK3 or each ERK3 mutant were analyzed and classified into three groups as follow: cells showing predominant cytoplasmic ERK3 localization (C > N), cells showing relatively equal distribution of ERK3 in the nucleus and cytoplasm (N = C) and cells showing predominant nuclear localization of ERK3 (N > C). The bar graph (right) represents the percentage of total transfected cells for each different group. ( b ) Subcellular distribution of wild type ERK3 and the ERK3 L290P/V mutants in A549 lung cancer cells. Experiments were done with the same procedures as described in ( a ), except that myc-tagged ERK3 was transduced into A549 cells by lentivirus and an anti-myc antibody was used for detecting the exogenously expressed ERK3 and ERK3 mutants.

    Techniques Used: Staining, Transfection, Expressing, Mutagenesis

    Both ERK3 L290P and ERK3 L290V mutants have increased ability to promote HeLa cell migration as compared to wild type ERK3. ( a ) Western blot analysis of ERK3 and ERK3 mutants’ expression in HeLa cells transfected with either a pSG5 empty vector, wild type ERK3 (ERK3 WT), or each ERK3 L290 mutant as indicated. β-actin was used as a loading control. ( b ) The effect of L290P/V mutations on ERK3’s role in HeLa cell migration. HeLa cells were transfected with each different pSG5 plasmid as indicated. Two days post-transfection, cell migration was analyzed using a two-chamber transwell system. Migrated cells were stained with crystal violet, photographed and counted under a microscope at 50X magnifications. Quantitative results are presented as the number of migrated cells per field. Values in the bar graph represent mean ± S.D. of 3 separate experiments. *P
    Figure Legend Snippet: Both ERK3 L290P and ERK3 L290V mutants have increased ability to promote HeLa cell migration as compared to wild type ERK3. ( a ) Western blot analysis of ERK3 and ERK3 mutants’ expression in HeLa cells transfected with either a pSG5 empty vector, wild type ERK3 (ERK3 WT), or each ERK3 L290 mutant as indicated. β-actin was used as a loading control. ( b ) The effect of L290P/V mutations on ERK3’s role in HeLa cell migration. HeLa cells were transfected with each different pSG5 plasmid as indicated. Two days post-transfection, cell migration was analyzed using a two-chamber transwell system. Migrated cells were stained with crystal violet, photographed and counted under a microscope at 50X magnifications. Quantitative results are presented as the number of migrated cells per field. Values in the bar graph represent mean ± S.D. of 3 separate experiments. *P

    Techniques Used: Migration, Western Blot, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Staining, Microscopy

    As compared to wild type ERK3, both L290P and L290V mutants have increased interactions with CRM1. HA-tagged wild type ERK3, ERK3 L290P or ERK3 L290V was exogenously expressed in HeLa cells. ERK3 protein complexes were immunoprecipitated using agarose beads conjugated with anti-HA antibodies, followed by Western blotting of the proteins as indicated in the figure. Input: 2% of the amount for immunoprecipitation (IP). Numbers below the immunoblots of CRM1 and MK5 in HA-IP samples represent the relative binding capacity of ERK3 (or L290P or V mutants) with these proteins, which is determined by the ratio of the band intensity in HA-IP over that in the corresponding input. For the purpose of comparison, the relative binding capacity of wild type ERK3 with either CRM1 or MK5 was arbitrarily set as 1.0.
    Figure Legend Snippet: As compared to wild type ERK3, both L290P and L290V mutants have increased interactions with CRM1. HA-tagged wild type ERK3, ERK3 L290P or ERK3 L290V was exogenously expressed in HeLa cells. ERK3 protein complexes were immunoprecipitated using agarose beads conjugated with anti-HA antibodies, followed by Western blotting of the proteins as indicated in the figure. Input: 2% of the amount for immunoprecipitation (IP). Numbers below the immunoblots of CRM1 and MK5 in HA-IP samples represent the relative binding capacity of ERK3 (or L290P or V mutants) with these proteins, which is determined by the ratio of the band intensity in HA-IP over that in the corresponding input. For the purpose of comparison, the relative binding capacity of wild type ERK3 with either CRM1 or MK5 was arbitrarily set as 1.0.

    Techniques Used: Immunoprecipitation, Western Blot, Binding Assay

    32) Product Images from "L290P/V mutations increase ERK3’s cytoplasmic localization and migration/invasion-promoting capability in cancer cells"

    Article Title: L290P/V mutations increase ERK3’s cytoplasmic localization and migration/invasion-promoting capability in cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15135-9

    L290P/V mutations do not alter ERK3 kinase activity. ( a ) Coomassie staining of purified wild type or mutant ERK3 proteins. 293 T cells were transfected with HA-tagged wild type ERK3, ERK3 L290P, L290V or kinase dead (KD) plasmids. ERK3 proteins were immunoprecipitated using HA-antibody-conjugated agarose beads, followed by elution with HA peptide. The purified proteins (300 ng) were analyzed by SDS-PAGE gel followed by Coomassie staining. The molecular size of each protein marker is indicated on the right side. ( b ) In vitro ERK3 kinase assay was performed by incubating 100 ng of purified ERK3 or each of ERK3 mutants as indicated, together with 1 μg of recombinant GST-SRC3-CID (substrate) in the presence of γ- 32 P-ATP. Phosphorylation of GST-SRC3-CID by ERK3 proteins was detected by autoradiograph (the right panel). Total protein level of GST-SRC3-CID in the reactions is shown by Coomassie staining (the left panel). Please note that ERK3 proteins are hardly seen in the Coomassie-stained gel due to their small amount (100 ng). ( c ) Quantification of GST-SRC3-CID phosphorylation by wild type or mutant ERK3 proteins. The relative phosphorylation level of GST-SRC3-CID is represented by the ratio of the band intensity of phosphorylated GST-SRC3-CID (shown in the autoradiograph) over that of the corresponding total GST-SRC3-CID (shown in the commassie-stained gel). For the purpose of comparison, the nomalized phosphorylation level of GST-SRC3-CID by wild type ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of 3 independent experiments. *P
    Figure Legend Snippet: L290P/V mutations do not alter ERK3 kinase activity. ( a ) Coomassie staining of purified wild type or mutant ERK3 proteins. 293 T cells were transfected with HA-tagged wild type ERK3, ERK3 L290P, L290V or kinase dead (KD) plasmids. ERK3 proteins were immunoprecipitated using HA-antibody-conjugated agarose beads, followed by elution with HA peptide. The purified proteins (300 ng) were analyzed by SDS-PAGE gel followed by Coomassie staining. The molecular size of each protein marker is indicated on the right side. ( b ) In vitro ERK3 kinase assay was performed by incubating 100 ng of purified ERK3 or each of ERK3 mutants as indicated, together with 1 μg of recombinant GST-SRC3-CID (substrate) in the presence of γ- 32 P-ATP. Phosphorylation of GST-SRC3-CID by ERK3 proteins was detected by autoradiograph (the right panel). Total protein level of GST-SRC3-CID in the reactions is shown by Coomassie staining (the left panel). Please note that ERK3 proteins are hardly seen in the Coomassie-stained gel due to their small amount (100 ng). ( c ) Quantification of GST-SRC3-CID phosphorylation by wild type or mutant ERK3 proteins. The relative phosphorylation level of GST-SRC3-CID is represented by the ratio of the band intensity of phosphorylated GST-SRC3-CID (shown in the autoradiograph) over that of the corresponding total GST-SRC3-CID (shown in the commassie-stained gel). For the purpose of comparison, the nomalized phosphorylation level of GST-SRC3-CID by wild type ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of 3 independent experiments. *P

    Techniques Used: Activity Assay, Staining, Purification, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Marker, In Vitro, Kinase Assay, Recombinant, Autoradiography

    33) Product Images from "Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions"

    Article Title: Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.802587

    CK1δ induces the phosphorylation of Ser-780 and Thr-804 residues on HEF1, leading to the formation of the HEF1–Plk1 complex. A , expression of CK1δ induces the phosphorylation of Ser-780 and Thr-804 residues on HEF1. HEK293T cells co-transfected with FLAG-HEF1 WT and a Myc-empty vector (+ Vector ), Myc-tagged CK1δ (+ Myc-CK1 δ) vector, or CK1ϵ (+ Myc-CK1 ϵ) vector were subjected to an immunoprecipitation assay. Cell lysates were immunoprecipitated with either anti-phospho-Ser-780 or -Thr-804 antiserum, with 10 μg/ml non-phospho-Ser-780 (+ S780 pep ) or non-phospho-Thr-804 (+ T804 pep ) peptide, respectively, and then immunoblotted with an anti-FLAG antibody. Cell lysates were probed with either anti-FLAG or -Myc antibody.  B , expression of CK1δ induces HEF1–Plk1 complex formation. HEK293T cells co-transfected with FLAG-HEF1 T6 and a Myc-empty vector (+ Vector ), Myc-tagged CK1δ (+ Myc-CK1 δ) vector, or CK1ϵ (+ Myc-CK1 ϵ) vector were subjected to a PBD pulldown assay using GST-Plk1 PBD WT. The resulting precipitates were immunoblotted with the indicated antibodies. Note that the expression of CK1δ greatly enhances FLAG-HEF1 T6 binding to Plk1 PBD.  C , CK1δ directly phosphorylates HEF1. The bacterially purified GST-HEF1 WT proteins were reacted with either a bacterially purified GST-CK1δ WT or K38M (kinase-dead mutant) in the presence of [γ- 32 p]ATP, and the resulting samples were then separated by 10% SDS-PAGE and exposed on an X-ray film (Autorad). CBB represents the amount of loaded protein.  Asterisks  indicate degradation products of GST-HEF1 protein.  D , HEF1 S780A/T804A double mutant reduces HEF1 phosphorylation by CK1δ. The bacterially purified GST-CK1δ WT proteins were reacted with either a bacterially purified GST-HEF1 WT or S780A/T804A mutant in the presence of [γ- 32 p]ATP, and the resulting samples were then separated by 10% SDS-PAGE and exposed on an X-ray film (Autorad). CBB represents the amount of loaded protein.  Asterisks  indicate degradation products of GST-HEF1 protein.  E , time course of HEF1 WT and HEF1 S780A/T804A mutant phosphorylation by CK1δ. The kinase reaction was carried out as indicated in  D . A dried SDS-polyacrylamide gel band was excised and dissolved in 30% H 2 O 2 . Phosphorylation (cpm) was measured by liquid scintillation counting. **,  p
    Figure Legend Snippet: CK1δ induces the phosphorylation of Ser-780 and Thr-804 residues on HEF1, leading to the formation of the HEF1–Plk1 complex. A , expression of CK1δ induces the phosphorylation of Ser-780 and Thr-804 residues on HEF1. HEK293T cells co-transfected with FLAG-HEF1 WT and a Myc-empty vector (+ Vector ), Myc-tagged CK1δ (+ Myc-CK1 δ) vector, or CK1ϵ (+ Myc-CK1 ϵ) vector were subjected to an immunoprecipitation assay. Cell lysates were immunoprecipitated with either anti-phospho-Ser-780 or -Thr-804 antiserum, with 10 μg/ml non-phospho-Ser-780 (+ S780 pep ) or non-phospho-Thr-804 (+ T804 pep ) peptide, respectively, and then immunoblotted with an anti-FLAG antibody. Cell lysates were probed with either anti-FLAG or -Myc antibody. B , expression of CK1δ induces HEF1–Plk1 complex formation. HEK293T cells co-transfected with FLAG-HEF1 T6 and a Myc-empty vector (+ Vector ), Myc-tagged CK1δ (+ Myc-CK1 δ) vector, or CK1ϵ (+ Myc-CK1 ϵ) vector were subjected to a PBD pulldown assay using GST-Plk1 PBD WT. The resulting precipitates were immunoblotted with the indicated antibodies. Note that the expression of CK1δ greatly enhances FLAG-HEF1 T6 binding to Plk1 PBD. C , CK1δ directly phosphorylates HEF1. The bacterially purified GST-HEF1 WT proteins were reacted with either a bacterially purified GST-CK1δ WT or K38M (kinase-dead mutant) in the presence of [γ- 32 p]ATP, and the resulting samples were then separated by 10% SDS-PAGE and exposed on an X-ray film (Autorad). CBB represents the amount of loaded protein. Asterisks indicate degradation products of GST-HEF1 protein. D , HEF1 S780A/T804A double mutant reduces HEF1 phosphorylation by CK1δ. The bacterially purified GST-CK1δ WT proteins were reacted with either a bacterially purified GST-HEF1 WT or S780A/T804A mutant in the presence of [γ- 32 p]ATP, and the resulting samples were then separated by 10% SDS-PAGE and exposed on an X-ray film (Autorad). CBB represents the amount of loaded protein. Asterisks indicate degradation products of GST-HEF1 protein. E , time course of HEF1 WT and HEF1 S780A/T804A mutant phosphorylation by CK1δ. The kinase reaction was carried out as indicated in D . A dried SDS-polyacrylamide gel band was excised and dissolved in 30% H 2 O 2 . Phosphorylation (cpm) was measured by liquid scintillation counting. **, p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Purification, Mutagenesis, SDS Page

    34) Product Images from "Phosphorylation of β-arrestin2 at Thr383 by MEK underlies β-arrestin-dependent activation of Erk1/2 by GPCRs"

    Article Title: Phosphorylation of β-arrestin2 at Thr383 by MEK underlies β-arrestin-dependent activation of Erk1/2 by GPCRs

    Journal: eLife

    doi: 10.7554/eLife.23777

    In vitro phosphorylation of β-arrestin2 versus Erk2 by MEK1. YFP-tagged β-arrestin2 (wild-type or Thr 383 Ala mutant) purified from transfected HEK-293 cells or purified non-activated Erk2 (~1 µg each) were incubated with active MEK1 in presence of [γ- 32 P]-ATP (2 µCi/nmol) for 10 min at 37°C. Proteins were separated by SDS-PAGE and stained with Coomassie colloidal blue (top image) and 32 P incorporation into the different substrates was monitored by autoradiography (bottom image). The data in the histogram, expressed in nmol/min/mg enzyme, represent the means ± SD of 32 P incorporation into β-arrestin2 and Erk2 protein bands in the corresponding experiment after radioactive background subtraction for each lane. DOI: http://dx.doi.org/10.7554/eLife.23777.015 10.7554/eLife.23777.016 This file contains raw values used to build Figure 2—figure supplement 3 . DOI: http://dx.doi.org/10.7554/eLife.23777.016
    Figure Legend Snippet: In vitro phosphorylation of β-arrestin2 versus Erk2 by MEK1. YFP-tagged β-arrestin2 (wild-type or Thr 383 Ala mutant) purified from transfected HEK-293 cells or purified non-activated Erk2 (~1 µg each) were incubated with active MEK1 in presence of [γ- 32 P]-ATP (2 µCi/nmol) for 10 min at 37°C. Proteins were separated by SDS-PAGE and stained with Coomassie colloidal blue (top image) and 32 P incorporation into the different substrates was monitored by autoradiography (bottom image). The data in the histogram, expressed in nmol/min/mg enzyme, represent the means ± SD of 32 P incorporation into β-arrestin2 and Erk2 protein bands in the corresponding experiment after radioactive background subtraction for each lane. DOI: http://dx.doi.org/10.7554/eLife.23777.015 10.7554/eLife.23777.016 This file contains raw values used to build Figure 2—figure supplement 3 . DOI: http://dx.doi.org/10.7554/eLife.23777.016

    Techniques Used: In Vitro, Mutagenesis, Purification, Transfection, Incubation, SDS Page, Staining, Autoradiography

    35) Product Images from "Extended N-terminal region of the essential phosphorelay signaling protein Ypd1 from Cryptococcus neoformans contributes to structural stability, phosphostability and binding of calcium ions"

    Article Title: Extended N-terminal region of the essential phosphorelay signaling protein Ypd1 from Cryptococcus neoformans contributes to structural stability, phosphostability and binding of calcium ions

    Journal: FEMS Yeast Research

    doi: 10.1093/femsyr/fow068

    Phosphorylation of CnYpd1 from a heterologous phosphodonor. The HK and RR domains from a heterologous donor, Sln1 from S. cerevisiae (Sln1-HKR1), were used to phosphorylate CnYpd1. ScSln1-HKR1 was autophosphorylated using 0.1 μM γ- 32 P-labeled ATP (lane 1). ScSln1-HKR1 was incubated with CnYpd1 alone (lane 2) or with ScSsk1-R2 (lane 3), CnYpd1-H138Q alone (lane 4) or with ScSsk1-R2 (lane 5).
    Figure Legend Snippet: Phosphorylation of CnYpd1 from a heterologous phosphodonor. The HK and RR domains from a heterologous donor, Sln1 from S. cerevisiae (Sln1-HKR1), were used to phosphorylate CnYpd1. ScSln1-HKR1 was autophosphorylated using 0.1 μM γ- 32 P-labeled ATP (lane 1). ScSln1-HKR1 was incubated with CnYpd1 alone (lane 2) or with ScSsk1-R2 (lane 3), CnYpd1-H138Q alone (lane 4) or with ScSsk1-R2 (lane 5).

    Techniques Used: Labeling, Incubation

    36) Product Images from "CTD-dependent and -independent mechanisms govern co-transcriptional capping of Pol II transcripts"

    Article Title: CTD-dependent and -independent mechanisms govern co-transcriptional capping of Pol II transcripts

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05923-w

    Co-transcriptional capping activation in a defined enzyme system. a Biotinylated DNA templates used for promoter-dependent transcription. Both contain the Adenovirus 2 Major Late core promoter (AdML) followed by one (G21) or two (G23) G-less cassettes. b 23mer transcripts in washed ternary complexes were prepared according to the diagram and incubated with GTP (lane 1), GTP and 5 ng of capping enzyme (CE) (lane 2), or ATP, CTP, and UTP (lane 3). In this and subsequent figures, radiolabeled transcripts were resolved by denaturing gel electrophoresis and detected using a phosphorimager. c Kinetics of co-transcriptional capping and capping of free RNA. Free RNA or washed ternary complexes containing 21mers were incubated for varying lengths of time with 50 µM GTP and the indicated amounts of capping enzyme (CE). % Capped RNA is the quantification of a single representative experiment
    Figure Legend Snippet: Co-transcriptional capping activation in a defined enzyme system. a Biotinylated DNA templates used for promoter-dependent transcription. Both contain the Adenovirus 2 Major Late core promoter (AdML) followed by one (G21) or two (G23) G-less cassettes. b 23mer transcripts in washed ternary complexes were prepared according to the diagram and incubated with GTP (lane 1), GTP and 5 ng of capping enzyme (CE) (lane 2), or ATP, CTP, and UTP (lane 3). In this and subsequent figures, radiolabeled transcripts were resolved by denaturing gel electrophoresis and detected using a phosphorimager. c Kinetics of co-transcriptional capping and capping of free RNA. Free RNA or washed ternary complexes containing 21mers were incubated for varying lengths of time with 50 µM GTP and the indicated amounts of capping enzyme (CE). % Capped RNA is the quantification of a single representative experiment

    Techniques Used: Activation Assay, Incubation, Nucleic Acid Electrophoresis

    TFIIH-dependent activation of co-transcriptional capping in artificial ternary complexes assembled on DNA:RNA scaffolds. a Scheme for assembly of artificial ternary elongation complexes on DNA:RNA scaffolds. See text and “Methods” for details. b Representative gel image showing step-wise increase of RNA length following addition of appropriate combinations of rNTPs. 20-nt synthetic RNA in artificial ternary complexes was extended to position +23 with 5 µM ATP and 10 µCi α- 32 P-UTP (first lane), washed and walked to position +25 with 20 µM CTP and ATP (second lane), and finally washed and walked to position +29 with 20 µM ATP and GTP (last lane). Sequence on left denotes RNA sequence from +21 (bottom) to +29 (top). c 23mers in artificial ternary complexes were incubated with buffer or 300 ng of purified TFIIH for 10 min, washed, and incubated for 1, 2, or 4 min with 50 µM GTP and the indicated amounts of capping enzyme. Graph shows mean and range of data from two independent reactions
    Figure Legend Snippet: TFIIH-dependent activation of co-transcriptional capping in artificial ternary complexes assembled on DNA:RNA scaffolds. a Scheme for assembly of artificial ternary elongation complexes on DNA:RNA scaffolds. See text and “Methods” for details. b Representative gel image showing step-wise increase of RNA length following addition of appropriate combinations of rNTPs. 20-nt synthetic RNA in artificial ternary complexes was extended to position +23 with 5 µM ATP and 10 µCi α- 32 P-UTP (first lane), washed and walked to position +25 with 20 µM CTP and ATP (second lane), and finally washed and walked to position +29 with 20 µM ATP and GTP (last lane). Sequence on left denotes RNA sequence from +21 (bottom) to +29 (top). c 23mers in artificial ternary complexes were incubated with buffer or 300 ng of purified TFIIH for 10 min, washed, and incubated for 1, 2, or 4 min with 50 µM GTP and the indicated amounts of capping enzyme. Graph shows mean and range of data from two independent reactions

    Techniques Used: Activation Assay, Sequencing, Incubation, Purification

    37) Product Images from "Pre-assembled tau filaments phosphorylated by GSK-3? form large tangle-like structures"

    Article Title: Pre-assembled tau filaments phosphorylated by GSK-3? form large tangle-like structures

    Journal:

    doi: 10.1016/j.nbd.2008.05.011

    Inhibition of GSK-3β activity by arachidonic acid
    Figure Legend Snippet: Inhibition of GSK-3β activity by arachidonic acid

    Techniques Used: Inhibition, Activity Assay

    Electron micrographs of GSK-3β phosphorylated tau filaments
    Figure Legend Snippet: Electron micrographs of GSK-3β phosphorylated tau filaments

    Techniques Used:

    Tau monomer and polymer are phosphorylated by GSK-3β
    Figure Legend Snippet: Tau monomer and polymer are phosphorylated by GSK-3β

    Techniques Used:

    38) Product Images from "Analysis of a Growth-Phase-Regulated Two-Component Regulatory System in the Periodontal Pathogen Treponema denticola ▿"

    Article Title: Analysis of a Growth-Phase-Regulated Two-Component Regulatory System in the Periodontal Pathogen Treponema denticola ▿

    Journal:

    doi: 10.1128/JB.00046-08

    Autophosphorylation and phosphotransfer activities of AtcS. The ability of AtcS to autophosphorylate (A) was assessed as described in the text. AtcS (lanes 2, 4, and 6) and the negative control factor H binding protein FhbB (lanes 1, 3, and 5) were incubated
    Figure Legend Snippet: Autophosphorylation and phosphotransfer activities of AtcS. The ability of AtcS to autophosphorylate (A) was assessed as described in the text. AtcS (lanes 2, 4, and 6) and the negative control factor H binding protein FhbB (lanes 1, 3, and 5) were incubated

    Techniques Used: Negative Control, Binding Assay, Incubation

    39) Product Images from "Inositol polyphosphate multikinase is a nuclear PI3-kinase with transcriptional regulatory activity"

    Article Title: Inositol polyphosphate multikinase is a nuclear PI3-kinase with transcriptional regulatory activity

    Journal:

    doi: 10.1073/pnas.0506184102

    Yeast and mammalian IPMKs are wortmannin-insensitive PI3Ks. Human PI3K p110γ ( A ), rat IPMK ( B ), and yeast IPMK ( C ) were incubated with PI(4,5)P 2 and [γ- 32 P]ATP in the presence of increasing concentrations of wortmannin. Reaction products
    Figure Legend Snippet: Yeast and mammalian IPMKs are wortmannin-insensitive PI3Ks. Human PI3K p110γ ( A ), rat IPMK ( B ), and yeast IPMK ( C ) were incubated with PI(4,5)P 2 and [γ- 32 P]ATP in the presence of increasing concentrations of wortmannin. Reaction products

    Techniques Used: Incubation

    40) Product Images from "Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA"

    Article Title: Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA

    Journal:

    doi: 10.1261/rna.67906

    Ribosomes associate with the 5′-end to reach the two AUGs. ( A ) Diagram showing the sequence of the 13-bp stem–loop (−26.5 kcal) located adjacent to the 5′-cap and used to suppress the association of ribosomes with the 5′-end
    Figure Legend Snippet: Ribosomes associate with the 5′-end to reach the two AUGs. ( A ) Diagram showing the sequence of the 13-bp stem–loop (−26.5 kcal) located adjacent to the 5′-cap and used to suppress the association of ribosomes with the 5′-end

    Techniques Used: Sequencing

    41) Product Images from "The Translation Inhibitor Rocaglamide Targets a Biomolecular Cavity between eIF4A and Polypurine RNA"

    Article Title: The Translation Inhibitor Rocaglamide Targets a Biomolecular Cavity between eIF4A and Polypurine RNA

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.11.026

    Phe163Leu-Ile199Met mutations in eIF4A1 confer RocA-resistance on HEK293 cells. A. Western blot analyses of eIF4A1 from SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1 SINI ). B. Cell viability of SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1 SINI HEK293 cells upon RocA treatment. C. Histogram representing the distribution of mRNAs along translation change by RocA. Naïve HEK293 cells and SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1 SINI ). Bin width is 0.1. D. Scatter plot representing mRNA translation change by RocA treatment. Dots indicate translation changes of the mRNAs in naïve and SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1 SINI HEK293 cells. RocA-high sensitivity mRNAs defined in naïve HEK cells are highlighted. E. Reporter assays with mRNAs possessing CAA-repeat or polypurine motifs in 5′ UTR. In B and E, data represent mean and S.D. (n = 3). .
    Figure Legend Snippet: Phe163Leu-Ile199Met mutations in eIF4A1 confer RocA-resistance on HEK293 cells. A. Western blot analyses of eIF4A1 from SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1 SINI ). B. Cell viability of SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1 SINI HEK293 cells upon RocA treatment. C. Histogram representing the distribution of mRNAs along translation change by RocA. Naïve HEK293 cells and SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1 SINI ). Bin width is 0.1. D. Scatter plot representing mRNA translation change by RocA treatment. Dots indicate translation changes of the mRNAs in naïve and SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1 SINI HEK293 cells. RocA-high sensitivity mRNAs defined in naïve HEK cells are highlighted. E. Reporter assays with mRNAs possessing CAA-repeat or polypurine motifs in 5′ UTR. In B and E, data represent mean and S.D. (n = 3). .

    Techniques Used: Western Blot

    Double mutant eIF4A1 is defective in ATP-independent polypurine RNA binding upon RocA treatment. A. Conventional RNA binding assays between recombinant wild-type (WT) and mutated eIF4A1 proteins and polypurine RNA, in the presence of ADP, Pi, and RocA. Affinities were measured by fluorescence polarization with 5′ FAM-labeled (AG) 10 RNAs. B and C. The formation of eIF4A1•RocA complex on polypurine motifs on reporter mRNAs monitored by toeprinting assays (B), and following in vitro translation assays in rabbit rabbit reticulocyte lysates (C). D. In vitro translation assays by the reconstituted pure system with human factors under a series of RocA concentrations. WT or double mutant eIF4A1 proteins were supplemented into the reactions. mRNA reporter with polypurine motifs in 5′ UTR was used. In A, C, and D, data represent mean and S.D. (n = 3).
    Figure Legend Snippet: Double mutant eIF4A1 is defective in ATP-independent polypurine RNA binding upon RocA treatment. A. Conventional RNA binding assays between recombinant wild-type (WT) and mutated eIF4A1 proteins and polypurine RNA, in the presence of ADP, Pi, and RocA. Affinities were measured by fluorescence polarization with 5′ FAM-labeled (AG) 10 RNAs. B and C. The formation of eIF4A1•RocA complex on polypurine motifs on reporter mRNAs monitored by toeprinting assays (B), and following in vitro translation assays in rabbit rabbit reticulocyte lysates (C). D. In vitro translation assays by the reconstituted pure system with human factors under a series of RocA concentrations. WT or double mutant eIF4A1 proteins were supplemented into the reactions. mRNA reporter with polypurine motifs in 5′ UTR was used. In A, C, and D, data represent mean and S.D. (n = 3).

    Techniques Used: Mutagenesis, RNA Binding Assay, Recombinant, Fluorescence, Labeling, In Vitro

    Distinctive amino acid substitutions in Aglaia eIF4A revealed by de novo transcriptome assembly. A. Normalized Kabat conservation matrix calculated from 94 eIF4A homologs registered in Uniprot and NCBI homologene. B. Alignment of eIF4A sequences among representative eukaryotes and Meliaceae family plants, including Aglaia odorata and Azadirachita indica . Amino acid position in human eIF4A1 is shown. C. Conventional RNA binding assays between recombinant Aglaia eIF4A protein and polypurine RNA. Affinities were measured by fluorescence polarization with 5′ FAM-labeled (AG) 10 RNAs. D. RocA and five possible rotamers of modeled Leu residues (blue colors) at Phe163 residues (wall-eyed stereo view). In C, data represent mean and S.D. (n = 3). .
    Figure Legend Snippet: Distinctive amino acid substitutions in Aglaia eIF4A revealed by de novo transcriptome assembly. A. Normalized Kabat conservation matrix calculated from 94 eIF4A homologs registered in Uniprot and NCBI homologene. B. Alignment of eIF4A sequences among representative eukaryotes and Meliaceae family plants, including Aglaia odorata and Azadirachita indica . Amino acid position in human eIF4A1 is shown. C. Conventional RNA binding assays between recombinant Aglaia eIF4A protein and polypurine RNA. Affinities were measured by fluorescence polarization with 5′ FAM-labeled (AG) 10 RNAs. D. RocA and five possible rotamers of modeled Leu residues (blue colors) at Phe163 residues (wall-eyed stereo view). In C, data represent mean and S.D. (n = 3). .

    Techniques Used: RNA Binding Assay, Recombinant, Fluorescence, Labeling

    RocA targets a cavity formed between eIF4A1 and purines. (A and B) NMR assays of 15 N-labeled eIF4A NTD of WT and double-mutant proteins in the absence or presence of RocA (A), and quantification of peaks (B). Arrows indicate the regions of chemical shifts observed upon RocA treatment. C. Pulldown assays via biotinylated RocA with recombinant eIF4A1 (WT or double mutant) and 5′ FAM-labeled RNA [(AG) 10 or (CAA) 6 CA]. D. Schematic representations of biomolecular-cavity targeting by RocA. Red and blue dotted circles represent steric hindrance and the weakened contact, respectively .
    Figure Legend Snippet: RocA targets a cavity formed between eIF4A1 and purines. (A and B) NMR assays of 15 N-labeled eIF4A NTD of WT and double-mutant proteins in the absence or presence of RocA (A), and quantification of peaks (B). Arrows indicate the regions of chemical shifts observed upon RocA treatment. C. Pulldown assays via biotinylated RocA with recombinant eIF4A1 (WT or double mutant) and 5′ FAM-labeled RNA [(AG) 10 or (CAA) 6 CA]. D. Schematic representations of biomolecular-cavity targeting by RocA. Red and blue dotted circles represent steric hindrance and the weakened contact, respectively .

    Techniques Used: Nuclear Magnetic Resonance, Labeling, Mutagenesis, Recombinant

    42) Product Images from "Prospective Estimation of Recombination Signal Efficiency and Identification of Functional Cryptic Signals in the Genome by Statistical Modeling"

    Article Title: Prospective Estimation of Recombination Signal Efficiency and Identification of Functional Cryptic Signals in the Genome by Statistical Modeling

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20020250

    (A) Table of γ-satellite cRS within cloned LM-PCR products. BW indicates the BW-linker sequence. * identifies groups of identical cRS. Sequence logos (reference 72 ) depict the information content for each position in an alignment of the Tet on and Tet off groups of cloned cRS. The sequence logos were generated at http://www.bio.cam.ac.uk/cgi-bin/seqlogo/logo.cgi . (B) cRS in γ-satellite DNA. The consensus γ-satellite sequence was generated in Vector NTI from the 31 published γ-satellite repeat elements (reference 34 ). Nucleotide position in the consensus is shown on the x axis. The squares indicate the location of CA dinucleotides in the consensus and the RIC score (y axis, right) for the 39-bp sequence beginning with that CA. The location of CAC trinucleotides is indicated by the horizontal black lines. The height of the bars (gray, Tet off ; black, Tet on ) indicates the number of LM-PCR products (y axis, left) that align to that location in the γ-satellite consensus.
    Figure Legend Snippet: (A) Table of γ-satellite cRS within cloned LM-PCR products. BW indicates the BW-linker sequence. * identifies groups of identical cRS. Sequence logos (reference 72 ) depict the information content for each position in an alignment of the Tet on and Tet off groups of cloned cRS. The sequence logos were generated at http://www.bio.cam.ac.uk/cgi-bin/seqlogo/logo.cgi . (B) cRS in γ-satellite DNA. The consensus γ-satellite sequence was generated in Vector NTI from the 31 published γ-satellite repeat elements (reference 34 ). Nucleotide position in the consensus is shown on the x axis. The squares indicate the location of CA dinucleotides in the consensus and the RIC score (y axis, right) for the 39-bp sequence beginning with that CA. The location of CAC trinucleotides is indicated by the horizontal black lines. The height of the bars (gray, Tet off ; black, Tet on ) indicates the number of LM-PCR products (y axis, left) that align to that location in the γ-satellite consensus.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Sequencing, Generated, Chick Chorioallantoic Membrane Assay, Plasmid Preparation

    LM-PCR detects in vivo RAG-mediated double strand breaks in the γ-satellite repeat. For detailed protocol, see Materials and Methods. 5B3 cells were incubated for 48 h with or without tetracycline and subsequently analyzed by flow cytometry (A) for expression of the RAG2-GFP fusion protein. (B) Genomic DNA was isolated, ligated (+ Ligase) to the BW-LC linker, and LM-PCR of genomic DNA was performed on serial 2-fold diluted samples. The initial amount of template used for the CD14 PCR and γ-satellite LM-PCR was ∼5 ng. The CD14 PCR is included to show normalization of template. The Dβ and Vλ LM-PCR use an initial template of 40 ng. No ligase controls are indicated as (−Ligase), while Tet on or Tet off refers to the presence or absence of tetracycline in the media, respectively. γ-sat, Dβ, and Vλ LM-PCR products are detected by radioactive-oligonucleotide hybridization.
    Figure Legend Snippet: LM-PCR detects in vivo RAG-mediated double strand breaks in the γ-satellite repeat. For detailed protocol, see Materials and Methods. 5B3 cells were incubated for 48 h with or without tetracycline and subsequently analyzed by flow cytometry (A) for expression of the RAG2-GFP fusion protein. (B) Genomic DNA was isolated, ligated (+ Ligase) to the BW-LC linker, and LM-PCR of genomic DNA was performed on serial 2-fold diluted samples. The initial amount of template used for the CD14 PCR and γ-satellite LM-PCR was ∼5 ng. The CD14 PCR is included to show normalization of template. The Dβ and Vλ LM-PCR use an initial template of 40 ng. No ligase controls are indicated as (−Ligase), while Tet on or Tet off refers to the presence or absence of tetracycline in the media, respectively. γ-sat, Dβ, and Vλ LM-PCR products are detected by radioactive-oligonucleotide hybridization.

    Techniques Used: Polymerase Chain Reaction, In Vivo, Incubation, Flow Cytometry, Cytometry, Expressing, Isolation, Hybridization

    43) Product Images from "Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan"

    Article Title: Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-10-7

    Promoter regions characterized in this work . (A) Fragment of the promoter region of csnN106 gene variants. Pr-WT: native promoter region, the putative -35 and -10 boxes are indicated in blue. Pr-Ph: a construct in which the native promoter has been replaced by a double promoter from Streptomyces ghanaensis phage I19, the respective -35 and -10 boxes are over and underlined. Low case letters indicate nucleotide changes between Pr-WT and Pr-PH. (*): start points of transcription. Arrows: inverted repeats of the palindromic box. (B) Alignment of palindromic sequences present in the promoter regions of chitosanase genes in actinomycetes. Nucleotides are numbered relative to the center of symmetry. In the consensus sequence, nucleotides are coloured according to their importance for DNA-protein interaction established by equilibrium competition experiments [ 39 ]: red: nucleotides critical for interaction; green: nucleotides moderately important for interaction; black: nucleotides without apparent effect on interaction. (↑): base pairs mutated in the Pr-Pa construct. GH: glycoside hydrolase family.
    Figure Legend Snippet: Promoter regions characterized in this work . (A) Fragment of the promoter region of csnN106 gene variants. Pr-WT: native promoter region, the putative -35 and -10 boxes are indicated in blue. Pr-Ph: a construct in which the native promoter has been replaced by a double promoter from Streptomyces ghanaensis phage I19, the respective -35 and -10 boxes are over and underlined. Low case letters indicate nucleotide changes between Pr-WT and Pr-PH. (*): start points of transcription. Arrows: inverted repeats of the palindromic box. (B) Alignment of palindromic sequences present in the promoter regions of chitosanase genes in actinomycetes. Nucleotides are numbered relative to the center of symmetry. In the consensus sequence, nucleotides are coloured according to their importance for DNA-protein interaction established by equilibrium competition experiments [ 39 ]: red: nucleotides critical for interaction; green: nucleotides moderately important for interaction; black: nucleotides without apparent effect on interaction. (↑): base pairs mutated in the Pr-Pa construct. GH: glycoside hydrolase family.

    Techniques Used: Construct, Sequencing

    Primer extension analysis of csnN106 transcripts . The apparent 5' terminus for the csnN106 transcript was identified by annealing a radiolabeled primer complementary to the mRNA of csnN106 and extension with reverse transcriptase. 40 μg of total RNA, from GlcN-chitosan oligomers induced S. lividans TK24(pHPr-WT), were used for extension reaction. The same primer was used for DNA sequencing reactions with the pHPr-WT plasmid. (→): primer extension product; (*): apparent transcription start site. Vertical arrows: palindromic sequence.
    Figure Legend Snippet: Primer extension analysis of csnN106 transcripts . The apparent 5' terminus for the csnN106 transcript was identified by annealing a radiolabeled primer complementary to the mRNA of csnN106 and extension with reverse transcriptase. 40 μg of total RNA, from GlcN-chitosan oligomers induced S. lividans TK24(pHPr-WT), were used for extension reaction. The same primer was used for DNA sequencing reactions with the pHPr-WT plasmid. (→): primer extension product; (*): apparent transcription start site. Vertical arrows: palindromic sequence.

    Techniques Used: DNA Sequencing, Plasmid Preparation, Sequencing

    Effect of csnR deletion on DNA-protein interaction at the csnN106 gene operator . Gel retardation experiment was set up combining 0.1 nM double strand oligonucleotide probe covering the palindromic box of csnN106 with 10 μg of crude protein extracts from S. lividans TK24 strain (WT) or the csnR deleted strain ( ΔcsnR ) cultivated in medium with 0.125% GlcN and 0.375% chitosan oligomers for the time (hours) indicated. P: probe only; T+: control reaction with 2 μg of partially purified protein from Kitasatospora sp. N106 [ 39 ].
    Figure Legend Snippet: Effect of csnR deletion on DNA-protein interaction at the csnN106 gene operator . Gel retardation experiment was set up combining 0.1 nM double strand oligonucleotide probe covering the palindromic box of csnN106 with 10 μg of crude protein extracts from S. lividans TK24 strain (WT) or the csnR deleted strain ( ΔcsnR ) cultivated in medium with 0.125% GlcN and 0.375% chitosan oligomers for the time (hours) indicated. P: probe only; T+: control reaction with 2 μg of partially purified protein from Kitasatospora sp. N106 [ 39 ].

    Techniques Used: Electrophoretic Mobility Shift Assay, Purification

    Effect of mutations in csnN106 gene and S. lividans host on chitosanase production . Chitosanase activity was assayed in supernatants sampled from 16 h cultures. Media: M14 M with 0.5% mannitol (empty columns) or M14 M with 0.125% GlcN and 0.375% chitosan oligomers (filled columns). Data and error bars are the mean of three experiments. ** P ≤ 0.01, * P ≤ 0.05 obtained with an unpaired t test (GraphPad Prism version 5.00 for Windows; GraphPad Software, San Diego, CA). The table lists the genotypes of strains for each pair of columns. Variants of csnN106 gene were introduced in one copy per genome via an integrative vector. Symbols: WT: wild type; Δ: ΔcsnR mutant host; M: mutated palindromic box; Ph: phage-type promoter. The induction ratio represents the chitosanase activity of culture induced with GlcN and chitosan oligomer divided by the activity of culture in mannitol medium.
    Figure Legend Snippet: Effect of mutations in csnN106 gene and S. lividans host on chitosanase production . Chitosanase activity was assayed in supernatants sampled from 16 h cultures. Media: M14 M with 0.5% mannitol (empty columns) or M14 M with 0.125% GlcN and 0.375% chitosan oligomers (filled columns). Data and error bars are the mean of three experiments. ** P ≤ 0.01, * P ≤ 0.05 obtained with an unpaired t test (GraphPad Prism version 5.00 for Windows; GraphPad Software, San Diego, CA). The table lists the genotypes of strains for each pair of columns. Variants of csnN106 gene were introduced in one copy per genome via an integrative vector. Symbols: WT: wild type; Δ: ΔcsnR mutant host; M: mutated palindromic box; Ph: phage-type promoter. The induction ratio represents the chitosanase activity of culture induced with GlcN and chitosan oligomer divided by the activity of culture in mannitol medium.

    Techniques Used: Activity Assay, Software, Plasmid Preparation, Mutagenesis

    44) Product Images from "A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression"

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.6.2423-2443.2004

    RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.
    Figure Legend Snippet: RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Techniques Used: Plasmid Preparation, Sequencing, Construct, Polymerase Chain Reaction, Amplification, Labeling, Footprinting, DNA Sequencing, Marker, Autoradiography

    45) Product Images from "Bacillus subtilis RarA modulates replication restart"

    Article Title: Bacillus subtilis RarA modulates replication restart

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky541

    RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 P]-dCTP) and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.
    Figure Legend Snippet: RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 P]-dCTP) and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.

    Techniques Used: Nucleic Acid Electrophoresis, Autoradiography

    RarA does not inhibit SPP1 DNA replication. Quantification of leading ( A ) and lagging ( B ) strand synthesis obtained in standard SPP1 rolling circle DNA replication assays in the absence or in the presence of 100 nM RarA. Reaction mixes contained the SPP1 replisome, which is composed by SPP1 preprimosomal proteins (G 38 P and G 39 P) and DNA helicase G 40 P, and host proteins (DnaG, τ-complex, β, PolC and DnaE). The SPP1 replisome works with both SSB proteins (SsbA or G 36 P) and the effect of RarA on reactions having either viral G 36 P or host SbsA was tested. An enzyme mix consisting of all proteins except the SSB was generated, and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and the indicated SSB (none, 30 nM G 36 P, or, 90 nM SsbA). Then reactions were placed at 37°C and incubated for 10 min. Leading strand synthesis was quantified by [α- 32 P]-dCTP incorporation and lagging strand synthesis by [α- 32 P]-dGTP incorporation. The results are expressed as the mean ± SEM of > 3 independent experiments.
    Figure Legend Snippet: RarA does not inhibit SPP1 DNA replication. Quantification of leading ( A ) and lagging ( B ) strand synthesis obtained in standard SPP1 rolling circle DNA replication assays in the absence or in the presence of 100 nM RarA. Reaction mixes contained the SPP1 replisome, which is composed by SPP1 preprimosomal proteins (G 38 P and G 39 P) and DNA helicase G 40 P, and host proteins (DnaG, τ-complex, β, PolC and DnaE). The SPP1 replisome works with both SSB proteins (SsbA or G 36 P) and the effect of RarA on reactions having either viral G 36 P or host SbsA was tested. An enzyme mix consisting of all proteins except the SSB was generated, and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and the indicated SSB (none, 30 nM G 36 P, or, 90 nM SsbA). Then reactions were placed at 37°C and incubated for 10 min. Leading strand synthesis was quantified by [α- 32 P]-dCTP incorporation and lagging strand synthesis by [α- 32 P]-dGTP incorporation. The results are expressed as the mean ± SEM of > 3 independent experiments.

    Techniques Used: Generated, Incubation

    SsbA-dependent RarA-mediated inhibition of B. subtilis PriA-dependent DNA replication. ( A ) Total DNA synthesis obtained in the presence of increasing RarA concentrations (15 min, 37°C). Reaction mixes contained all replisome components (preprimosomal proteins [PriA, DnaB, DnaD, DnaI), DnaC, DnaG, SsbA, τ-complex, β, PolC, DnaE), the indicated RarA concentration, template DNA, rNTPs, dNTPs and [α- 32 P]-dCTP and [α- 32 P]-dGTP. An enzyme mix consisting of all proteins except SsbA was generated and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and SsbA. Then, samples were placed at 37°C. ( B ) Visualization of products obtained in the presence of 100 nM RarA or RarAK51A (15 min, 37°C). In the presence of [α- 32 P]-dCTP very large DNA fragments derived from rolling circle leading strand DNA synthesis is observed. A parallel reaction in the presence of [α- 32 P]-dGTP renders visible the small Okazaki fragments due to lagging strand DNA synthesis. Quantification of leading ( C ) and lagging strand ( D ) synthesis in the absence/presence of 100nM RarA and the indicated SsbA concentrations (15 min, 37°C). The quantification of the results is expressed as the mean ± SEM of six independent experiments. On the right part, a representative alkaline gel visualized by autoradiography showing the products of the DNA synthesis obtained in the presence or absence of RarA and SsbA.
    Figure Legend Snippet: SsbA-dependent RarA-mediated inhibition of B. subtilis PriA-dependent DNA replication. ( A ) Total DNA synthesis obtained in the presence of increasing RarA concentrations (15 min, 37°C). Reaction mixes contained all replisome components (preprimosomal proteins [PriA, DnaB, DnaD, DnaI), DnaC, DnaG, SsbA, τ-complex, β, PolC, DnaE), the indicated RarA concentration, template DNA, rNTPs, dNTPs and [α- 32 P]-dCTP and [α- 32 P]-dGTP. An enzyme mix consisting of all proteins except SsbA was generated and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and SsbA. Then, samples were placed at 37°C. ( B ) Visualization of products obtained in the presence of 100 nM RarA or RarAK51A (15 min, 37°C). In the presence of [α- 32 P]-dCTP very large DNA fragments derived from rolling circle leading strand DNA synthesis is observed. A parallel reaction in the presence of [α- 32 P]-dGTP renders visible the small Okazaki fragments due to lagging strand DNA synthesis. Quantification of leading ( C ) and lagging strand ( D ) synthesis in the absence/presence of 100nM RarA and the indicated SsbA concentrations (15 min, 37°C). The quantification of the results is expressed as the mean ± SEM of six independent experiments. On the right part, a representative alkaline gel visualized by autoradiography showing the products of the DNA synthesis obtained in the presence or absence of RarA and SsbA.

    Techniques Used: Inhibition, DNA Synthesis, Concentration Assay, Generated, Derivative Assay, Autoradiography

    46) Product Images from "Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein ? Subunit *"

    Article Title: Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein ? Subunit *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.239343

    Elm1 is required for Gpa1 polyubiquitination. Temperature-sensitive proteasome-deficient ( cim3-1 or cim3-1/elm1 Δ) or isogenic wild-type cells overexpressing the indicated form of plasmid-borne GPA1 (pAD4M-GPA1, GPA1 ) grown at the restrictive temperature.
    Figure Legend Snippet: Elm1 is required for Gpa1 polyubiquitination. Temperature-sensitive proteasome-deficient ( cim3-1 or cim3-1/elm1 Δ) or isogenic wild-type cells overexpressing the indicated form of plasmid-borne GPA1 (pAD4M-GPA1, GPA1 ) grown at the restrictive temperature.

    Techniques Used: Plasmid Preparation

    Yeast kinome screen reveals Elm1 as a Gpa1 kinase. The abundance of phosphorylated Gpa1 was determined by immunoblotting 109 different kinase gene deletion strains. A single kinase, Elm1, is required for detection of phosphorylated Gpa1. A , validation
    Figure Legend Snippet: Yeast kinome screen reveals Elm1 as a Gpa1 kinase. The abundance of phosphorylated Gpa1 was determined by immunoblotting 109 different kinase gene deletion strains. A single kinase, Elm1, is required for detection of phosphorylated Gpa1. A , validation

    Techniques Used:

    Model of cell cycle G protein regulation. Elm1 phosphorylates a subpopulation of Gpa1 during S and G 2 /M phase. Phosphorylated Gpa1 is stable until entrance into the following G 1 phase, when it is targeted for polyubiquitination by the SCF Cdc4 ubiquitin
    Figure Legend Snippet: Model of cell cycle G protein regulation. Elm1 phosphorylates a subpopulation of Gpa1 during S and G 2 /M phase. Phosphorylated Gpa1 is stable until entrance into the following G 1 phase, when it is targeted for polyubiquitination by the SCF Cdc4 ubiquitin

    Techniques Used:

    Elm1 is necessary and sufficient for Gpa1 phosphorylation. The association between Elm1 and Gpa1 as determined by copurification and by in vitro kinase assay. A , in vitro kinase assay with purified GST-Elm1 or catalytically inactive GST-Elm1 K117R and
    Figure Legend Snippet: Elm1 is necessary and sufficient for Gpa1 phosphorylation. The association between Elm1 and Gpa1 as determined by copurification and by in vitro kinase assay. A , in vitro kinase assay with purified GST-Elm1 or catalytically inactive GST-Elm1 K117R and

    Techniques Used: Copurification, In Vitro, Kinase Assay, Purification

    47) Product Images from "Radiation-generated short DNA fragments may perturb non-homologous end-joining and induce genomic instability"

    Article Title: Radiation-generated short DNA fragments may perturb non-homologous end-joining and induce genomic instability

    Journal: Journal of radiation research

    doi:

    DNA-PK kinase activity inhibition by synthesized short DNA fragments. Recombinant p53 protein was incubated with DNA-PK in the presence of γ- 32 P-ATP and various lengths of DNA: synthesized double-stranded oligos (14 mer, 20 mer, 24 mer, 28 mer,
    Figure Legend Snippet: DNA-PK kinase activity inhibition by synthesized short DNA fragments. Recombinant p53 protein was incubated with DNA-PK in the presence of γ- 32 P-ATP and various lengths of DNA: synthesized double-stranded oligos (14 mer, 20 mer, 24 mer, 28 mer,

    Techniques Used: Activity Assay, Inhibition, Synthesized, Recombinant, Incubation

    Comparison of kinase activations stimulated by fragments generated by Co-60 γ-rays and 0.75 MeV fission-neutron irradiation generated short DNA fragments. Recombinant p53 protein was incubated with DNA-PK in the presence of γ- 32 P-ATP and
    Figure Legend Snippet: Comparison of kinase activations stimulated by fragments generated by Co-60 γ-rays and 0.75 MeV fission-neutron irradiation generated short DNA fragments. Recombinant p53 protein was incubated with DNA-PK in the presence of γ- 32 P-ATP and

    Techniques Used: Generated, Irradiation, Recombinant, Incubation

    48) Product Images from "The Two-Component Sensor KinB Acts as a Phosphatase To Regulate Pseudomonas aeruginosa Virulence"

    Article Title: The Two-Component Sensor KinB Acts as a Phosphatase To Regulate Pseudomonas aeruginosa Virulence

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01168-12

    KinB acts as a phosphatase to regulate AlgB activity. Autophosphorylation and phosphotransfer assays were performed in vitro with radiolabeled ATP and labeled proteins were separate by SDS-PAGE. (A) Autophosphorylation of HC-KinB and HC-KinBP390S. (B)
    Figure Legend Snippet: KinB acts as a phosphatase to regulate AlgB activity. Autophosphorylation and phosphotransfer assays were performed in vitro with radiolabeled ATP and labeled proteins were separate by SDS-PAGE. (A) Autophosphorylation of HC-KinB and HC-KinBP390S. (B)

    Techniques Used: Activity Assay, In Vitro, Labeling, SDS Page

    49) Product Images from "In Vivo Recognition of the fecA3 Target Promoter by Helicobacter pylori NikR ▿"

    Article Title: In Vivo Recognition of the fecA3 Target Promoter by Helicobacter pylori NikR ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01153-10

    Hydroxyl radical footprinting of Ni- Hp NikR on the P nikR-exbB divergent promoter locus. (A and B) Profile of base protection of the coding (A) and noncoding (B) strands of the nikR - exbB promoter region with increasing amounts of holo- Hp NikR. Lanes 1, G+A
    Figure Legend Snippet: Hydroxyl radical footprinting of Ni- Hp NikR on the P nikR-exbB divergent promoter locus. (A and B) Profile of base protection of the coding (A) and noncoding (B) strands of the nikR - exbB promoter region with increasing amounts of holo- Hp NikR. Lanes 1, G+A

    Techniques Used: Footprinting

    Bases contacted by Ni- Hp NikR within P fecA3 mutant promoters. (A to F) Pattern of protection from hydroxyl radicals for the probes P fecA3.1 (A), P fecA3.15 (B), P fecA3.16 (C), P fecA3.17 (D), P fecA3.19 (E), and P fecA3.21 (F) by increasing concentrations
    Figure Legend Snippet: Bases contacted by Ni- Hp NikR within P fecA3 mutant promoters. (A to F) Pattern of protection from hydroxyl radicals for the probes P fecA3.1 (A), P fecA3.15 (B), P fecA3.16 (C), P fecA3.17 (D), P fecA3.19 (E), and P fecA3.21 (F) by increasing concentrations

    Techniques Used: Mutagenesis

    Determination of Ni- Hp NikR K 50 on P fecA3 wild type. (A) Electrophoretic mobility shifts of the wild-type promoter in the presence of increasing amounts of holo- Hp NikR and 200 ng of nonspecific competitor DNA. Lane 1, no protein added; lane 2, 0.34 nM;
    Figure Legend Snippet: Determination of Ni- Hp NikR K 50 on P fecA3 wild type. (A) Electrophoretic mobility shifts of the wild-type promoter in the presence of increasing amounts of holo- Hp NikR and 200 ng of nonspecific competitor DNA. Lane 1, no protein added; lane 2, 0.34 nM;

    Techniques Used:

    Hydroxyl radical footprinting of Ni- Hp NikR on the P fecA3 region. (A) The fecA3 gene of H. pylori G27 (HPG27_1459, nucleotides 1586410 to 1583885, minus strand) is separated by 352 nucleotides from the upstream gene rocF (HPG27_1460, nucleotides 1587730
    Figure Legend Snippet: Hydroxyl radical footprinting of Ni- Hp NikR on the P fecA3 region. (A) The fecA3 gene of H. pylori G27 (HPG27_1459, nucleotides 1586410 to 1583885, minus strand) is separated by 352 nucleotides from the upstream gene rocF (HPG27_1460, nucleotides 1587730

    Techniques Used: Footprinting

    50) Product Images from "Glucose-Dependent Activation of Bacillus anthracis Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ †"

    Article Title: Glucose-Dependent Activation of Bacillus anthracis Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01656-09

    Electrophoretic mobility shift assay to determine conditions of CcpA binding to atxA , citZ , and BAS3893 promoter regions. Fragments were generated by PCR amplification and end labeled with [γ- 32 P]ATP via previous phosphorylation with PNK of one
    Figure Legend Snippet: Electrophoretic mobility shift assay to determine conditions of CcpA binding to atxA , citZ , and BAS3893 promoter regions. Fragments were generated by PCR amplification and end labeled with [γ- 32 P]ATP via previous phosphorylation with PNK of one

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Generated, Polymerase Chain Reaction, Amplification, Labeling

    51) Product Images from "Human Mitochondrial RNA Polymerase: Evaluation of the Single-Nucleotide-Addition Cycle on Synthetic RNA/DNA Scaffolds"

    Article Title: Human Mitochondrial RNA Polymerase: Evaluation of the Single-Nucleotide-Addition Cycle on Synthetic RNA/DNA Scaffolds

    Journal: Biochemistry

    doi: 10.1021/bi200350d

    Characterization of h-mtRNAP-catalyzed pyropho sphorolysis. (a) Experimental design. h-mtRNAP (1 μ ) (0.5 μ M) and [α- 32 P]ATP (0.45 μ M) for 5
    Figure Legend Snippet: Characterization of h-mtRNAP-catalyzed pyropho sphorolysis. (a) Experimental design. h-mtRNAP (1 μ ) (0.5 μ M) and [α- 32 P]ATP (0.45 μ M) for 5

    Techniques Used:

    52) Product Images from "Circadian Autodephosphorylation of Cyanobacterial Clock Protein KaiC Occurs via Formation of ATP as Intermediate *"

    Article Title: Circadian Autodephosphorylation of Cyanobacterial Clock Protein KaiC Occurs via Formation of ATP as Intermediate *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.350660

    Preparation of 32 P-labeled KaiC monomers for autodephosphorylation assays. A , KaiC hexamers were phosphorylated by incubating the samples on ice in the presence of 1 m m [γ- 32 P]ATP. At each time point, an aliquot of the reaction mixture was collected
    Figure Legend Snippet: Preparation of 32 P-labeled KaiC monomers for autodephosphorylation assays. A , KaiC hexamers were phosphorylated by incubating the samples on ice in the presence of 1 m m [γ- 32 P]ATP. At each time point, an aliquot of the reaction mixture was collected

    Techniques Used: Labeling

    53) Product Images from "The profile of snoRNA-derived microRNAs that regulate expression of variant surface proteins in Giardia lamblia"

    Article Title: The profile of snoRNA-derived microRNAs that regulate expression of variant surface proteins in Giardia lamblia

    Journal: Cellular microbiology

    doi: 10.1111/j.1462-5822.2012.01811.x

    miR6 and miR10 can repress the expression of myc-VSP1267 in Giardia
    Figure Legend Snippet: miR6 and miR10 can repress the expression of myc-VSP1267 in Giardia

    Techniques Used: Expressing

    miR6 and miR10 inhibit the expression of an RLuc mRNA carrying the dual target sites
    Figure Legend Snippet: miR6 and miR10 inhibit the expression of an RLuc mRNA carrying the dual target sites

    Techniques Used: Expressing

    Repression of myc-VSP-213 expression by miR2, miR4 and miR10
    Figure Legend Snippet: Repression of myc-VSP-213 expression by miR2, miR4 and miR10

    Techniques Used: Expressing

    The ASOs of miR6 and miR10 can suppress the endogenously repressed myc-VSP1267 expression
    Figure Legend Snippet: The ASOs of miR6 and miR10 can suppress the endogenously repressed myc-VSP1267 expression

    Techniques Used: Expressing

    The vsp genes carrying putative target sites for both miR6 and miR10
    Figure Legend Snippet: The vsp genes carrying putative target sites for both miR6 and miR10

    Techniques Used:

    54) Product Images from "A 5?-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli"

    Article Title: A 5?-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

    Journal: RNA

    doi: 10.1261/rna.027698.111

    Hammerhead ribozyme sequence, proposed structure, and site of cleavage (arrow). Shaded regions represent areas of base pairing. ( A ) Structure resulting in leaderless c I- lac Z mRNA with a 5′-OH after cleavage. ( B ) Structure resulting in lac -leadered
    Figure Legend Snippet: Hammerhead ribozyme sequence, proposed structure, and site of cleavage (arrow). Shaded regions represent areas of base pairing. ( A ) Structure resulting in leaderless c I- lac Z mRNA with a 5′-OH after cleavage. ( B ) Structure resulting in lac -leadered

    Techniques Used: Sequencing

    55) Product Images from "Differential expression of multidrug resistance protein 5 and phosphodiesterase 5 and regulation of cGMP levels in phasic and tonic smooth muscle"

    Article Title: Differential expression of multidrug resistance protein 5 and phosphodiesterase 5 and regulation of cGMP levels in phasic and tonic smooth muscle

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00457.2012

    cGMP transport in plasma membrane vesicle. A : membrane vesicle (100 μg protein) prepared from muscle cells isolated from fundus were incubated with [ 3 H]cGMP (5 μM) in the presence of ATP (5 mM) or 5′-AMP (5 mM) for different times.
    Figure Legend Snippet: cGMP transport in plasma membrane vesicle. A : membrane vesicle (100 μg protein) prepared from muscle cells isolated from fundus were incubated with [ 3 H]cGMP (5 μM) in the presence of ATP (5 mM) or 5′-AMP (5 mM) for different times.

    Techniques Used: Isolation, Incubation

    ATP-dependent cGMP efflux. A and B : 1 ml of cell suspension (2 × 10 6 cells/ml) of freshly dispersed muscle cells from antrum and fundus was treated with GSNO for different times in the presence of nonspecific PDE inhibitor, 100 μM IBMX
    Figure Legend Snippet: ATP-dependent cGMP efflux. A and B : 1 ml of cell suspension (2 × 10 6 cells/ml) of freshly dispersed muscle cells from antrum and fundus was treated with GSNO for different times in the presence of nonspecific PDE inhibitor, 100 μM IBMX

    Techniques Used:

    56) Product Images from "Fission Yeast Rad51 and Dmc1, Two Efficient DNA Recombinases Forming Helical Nucleoprotein Filaments"

    Article Title: Fission Yeast Rad51 and Dmc1, Two Efficient DNA Recombinases Forming Helical Nucleoprotein Filaments

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.11.4377-4387.2005

    Strand exchange catalyzed by S. pombe Rad51 (A) and Dmc1 (B). (A) DNA strand exchange as a function of Rad51 concentration (lanes a to h). (B) Cofactor dependence of Rad51-mediated strand exchange. Reactions were carried out in standard buffer with 6 μM Rad51 (lane b) or in buffer in which ATP was replaced with ATPγS (lane c) or AMP-PNP (lane d). Lane e, standard buffer lacking Mg 2+ ; lane f, pPB4.3 ssDNA was replaced with heterologous φX174 ssDNA; lane a, without protein. (C) Strand exchange promoted by Dmc1 (lanes a to i) and effect of SSB on Dmc1 strand exchange (lanes j to r). (D) Effect of cofactors on Dmc1 strand exchange. Reactions were carried out in buffer containing an ATP regeneration system with 12.5 μM Dmc1 (lane b), in standard buffer (lane c), or in standard buffer in which ATP was replaced with ATPγS (lane d) or AMP-PNP (lane e). Lane f, standard buffer lacking Mg 2+ ; lane g, pPB4.3 ssDNA was replaced with heterologous X174 ssDNA; lane a, without protein.
    Figure Legend Snippet: Strand exchange catalyzed by S. pombe Rad51 (A) and Dmc1 (B). (A) DNA strand exchange as a function of Rad51 concentration (lanes a to h). (B) Cofactor dependence of Rad51-mediated strand exchange. Reactions were carried out in standard buffer with 6 μM Rad51 (lane b) or in buffer in which ATP was replaced with ATPγS (lane c) or AMP-PNP (lane d). Lane e, standard buffer lacking Mg 2+ ; lane f, pPB4.3 ssDNA was replaced with heterologous φX174 ssDNA; lane a, without protein. (C) Strand exchange promoted by Dmc1 (lanes a to i) and effect of SSB on Dmc1 strand exchange (lanes j to r). (D) Effect of cofactors on Dmc1 strand exchange. Reactions were carried out in buffer containing an ATP regeneration system with 12.5 μM Dmc1 (lane b), in standard buffer (lane c), or in standard buffer in which ATP was replaced with ATPγS (lane d) or AMP-PNP (lane e). Lane f, standard buffer lacking Mg 2+ ; lane g, pPB4.3 ssDNA was replaced with heterologous X174 ssDNA; lane a, without protein.

    Techniques Used: Concentration Assay

    Electron microscopic visualization of fission yeast Rad51 and Dmc1. (A) Electron micrograph indicating the binding of Rad51 (0.6 μM) to a linear duplex (5 μM) containing a single-stranded tail. The bound ssDNA tail is indicated by the white arrow, while the black arrow indicates unbound dsDNA. (B) Enlargement of a typical Rad51 ring. (C) Electron microscopic visualization of Rad51 (1.65 μM) bound to φX174 ssDNA (5 μM). (D) Electron micrograph of Dmc1 (2 μM) bound to a linear duplex with a single-stranded tail at both ends (5 μM). The white arrows indicate two short helical regions on ssDNA. (E) Magnification of a characteristic Dmc1 ring. (F) Longer helical filaments formed by Dmc1 (2 μM) on tailed DNA (5 μM) and single-strand DNA (insert). (G) Electron microscopic visualization of Dmc1 (10 μM) bound to pPB4.3 ssDNA (5 μM). A region of stacked rings is indicated by the black arrow. (H) Close-up view of a human Dmc1-dsDNA complex consisting of a series of stacked rings as a comparison. The magnification bars represent 50 nm.
    Figure Legend Snippet: Electron microscopic visualization of fission yeast Rad51 and Dmc1. (A) Electron micrograph indicating the binding of Rad51 (0.6 μM) to a linear duplex (5 μM) containing a single-stranded tail. The bound ssDNA tail is indicated by the white arrow, while the black arrow indicates unbound dsDNA. (B) Enlargement of a typical Rad51 ring. (C) Electron microscopic visualization of Rad51 (1.65 μM) bound to φX174 ssDNA (5 μM). (D) Electron micrograph of Dmc1 (2 μM) bound to a linear duplex with a single-stranded tail at both ends (5 μM). The white arrows indicate two short helical regions on ssDNA. (E) Magnification of a characteristic Dmc1 ring. (F) Longer helical filaments formed by Dmc1 (2 μM) on tailed DNA (5 μM) and single-strand DNA (insert). (G) Electron microscopic visualization of Dmc1 (10 μM) bound to pPB4.3 ssDNA (5 μM). A region of stacked rings is indicated by the black arrow. (H) Close-up view of a human Dmc1-dsDNA complex consisting of a series of stacked rings as a comparison. The magnification bars represent 50 nm.

    Techniques Used: Binding Assay

    57) Product Images from "Bacterial Degradation of Benzoate: CROSS-REGULATION BETWEEN AEROBIC AND ANAEROBIC PATHWAYS*"

    Article Title: Bacterial Degradation of Benzoate: CROSS-REGULATION BETWEEN AEROBIC AND ANAEROBIC PATHWAYS*

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.309005

    The boxR gene encodes a transcriptional repressor of the box genes. A , agarose gel electrophoresis of RT-PCR products is shown. Total RNA was isolated from Azoarcus sp. CIB ( CIBwt ) or Azoarcus sp. CIBd boxR ( CIBdboxR ) cells grown in alanine (0.4%)-containing
    Figure Legend Snippet: The boxR gene encodes a transcriptional repressor of the box genes. A , agarose gel electrophoresis of RT-PCR products is shown. Total RNA was isolated from Azoarcus sp. CIB ( CIBwt ) or Azoarcus sp. CIBd boxR ( CIBdboxR ) cells grown in alanine (0.4%)-containing

    Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Isolation

    58) Product Images from "Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination 1Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination 1 [OPEN]"

    Article Title: Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination 1Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.16.01219

    In vitro transphosphorylation activities between EMS1 and SERK1/2. A and B, In vitro kinase assays were performed using EMS1-CD, SERK1-CD, and SERK2-CD in the presence of [γ- 32 P]ATP. Top gels, Input proteins stained with Coomassie Brilliant Blue. Bottom gels, Phosphorylation changes analyzed by autoradiography. EMS1-CD T930A and SERK1-CD K330E are inactive forms of EMS1 and SERK1 kinases, respectively. Consistent results were obtained from three independent repeats. C, Identified in vitro autophosphorylation (in black) and transphosphorylation (in blue) sites in the EMS1-CD via mass spectrometry. S, Ser; T, Thr. D, Relative phosphorylation level changes of specific residues in autophosphorylated and transphosphorylated EMS1-CD. Relative phosphorylation was calculated based on the ratio of spectral counts for total versus phosphorylated peptides identified by mass spectrometry analysis.
    Figure Legend Snippet: In vitro transphosphorylation activities between EMS1 and SERK1/2. A and B, In vitro kinase assays were performed using EMS1-CD, SERK1-CD, and SERK2-CD in the presence of [γ- 32 P]ATP. Top gels, Input proteins stained with Coomassie Brilliant Blue. Bottom gels, Phosphorylation changes analyzed by autoradiography. EMS1-CD T930A and SERK1-CD K330E are inactive forms of EMS1 and SERK1 kinases, respectively. Consistent results were obtained from three independent repeats. C, Identified in vitro autophosphorylation (in black) and transphosphorylation (in blue) sites in the EMS1-CD via mass spectrometry. S, Ser; T, Thr. D, Relative phosphorylation level changes of specific residues in autophosphorylated and transphosphorylated EMS1-CD. Relative phosphorylation was calculated based on the ratio of spectral counts for total versus phosphorylated peptides identified by mass spectrometry analysis.

    Techniques Used: In Vitro, Staining, Autoradiography, Mass Spectrometry

    59) Product Images from "Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity"

    Article Title: Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni166

    ( A ) Denaturing gel electrophoresis pattern of the cleavage reaction products. The same double labelled duplexes used for FCCS experiments are run. (A) TR, single labelled Texas red-30mer; RG, single labelled Rhodamine green-30mer; lanes 0–7, double labelled DNA reacted with 0, 5, 11, 21, 32, 43, 100, 200 ng of HeLa cell extracts. The uncleaved fragments reveal the co-migration of the two colors (yellow band). Lane DL, A + G reaction on the double labelled strand. ( B ) A + G reaction on single labelled oligonucleotides (3′ end, Texas red; 5′ end, Rhodamine green). Bases are always numbered from the 5′ end.
    Figure Legend Snippet: ( A ) Denaturing gel electrophoresis pattern of the cleavage reaction products. The same double labelled duplexes used for FCCS experiments are run. (A) TR, single labelled Texas red-30mer; RG, single labelled Rhodamine green-30mer; lanes 0–7, double labelled DNA reacted with 0, 5, 11, 21, 32, 43, 100, 200 ng of HeLa cell extracts. The uncleaved fragments reveal the co-migration of the two colors (yellow band). Lane DL, A + G reaction on the double labelled strand. ( B ) A + G reaction on single labelled oligonucleotides (3′ end, Texas red; 5′ end, Rhodamine green). Bases are always numbered from the 5′ end.

    Techniques Used: Nucleic Acid Electrophoresis, Migration

    Comparison of the FCCS to the electrophoresis assay. Closed circles are the fraction of the uncleaved DNA fragments obtained from digestion with the HeLa cell extracts as measured by autoradiograph, open squares are from FCCS assay. Open squares are the estimates obtained from the cross-correlation amplitude extrapolated to zero lag time. The reference concentration for both sets is the one (150 nM) of the sample analysed by electrophoresis at 0 enzyme concentration. Bars indicate standard errors. Inset: percent standard error on the best fit cross-correlation data at zero delay time, G GR (0), versus the experiment duration, T D . The fitting of the cross-correlation functions has been performed by keeping the diffusion coefficient D constant to the value of the 30mer DNA. The solid line is a best fit with the function ≈ T D − 0.5 .
    Figure Legend Snippet: Comparison of the FCCS to the electrophoresis assay. Closed circles are the fraction of the uncleaved DNA fragments obtained from digestion with the HeLa cell extracts as measured by autoradiograph, open squares are from FCCS assay. Open squares are the estimates obtained from the cross-correlation amplitude extrapolated to zero lag time. The reference concentration for both sets is the one (150 nM) of the sample analysed by electrophoresis at 0 enzyme concentration. Bars indicate standard errors. Inset: percent standard error on the best fit cross-correlation data at zero delay time, G GR (0), versus the experiment duration, T D . The fitting of the cross-correlation functions has been performed by keeping the diffusion coefficient D constant to the value of the 30mer DNA. The solid line is a best fit with the function ≈ T D − 0.5 .

    Techniques Used: Electrophoresis, Autoradiography, Concentration Assay, Diffusion-based Assay

    60) Product Images from "Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress"

    Article Title: Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000275

    In vitro phosphorylation of lamin A by GST-UL97. (A) Recombinant His-tagged lamin A was incubated in kinase reaction buffer in the presence of γ- 32 P-ATP either alone (no kinase), with catalytically deficient GST-UL97 K355Q (K355Q), or with wild-type GST-UL97 (GST97 WT). GST-UL97 K335Q or wild-type GST-UL97 were also incubated in kinase buffer without lamin A. Following termination of kinase reactions, proteins were resolved by SDS-PAGE. Signal from incorporation of 32 P was detected by exposure to a phosphorscreen (top panel), and total protein was detected by Coomassie brilliant blue staining (bottom panel). The positions of radiolabeled GST-UL97 (GST97) and lamin A, and Coomassie stained lamin A are indicated. (The amounts of GST-UL97 were too small to see on the stained gel.) (B) UL97 autophosphorylation and labeling of lamin A were quantified following in in vitro kinase reactions in the presence of varying concentrations of maribavir (MBV). Signal detected from 32 P incorporation for autophosphorylation of GST-UL97 and phosphorylation of His-tagged lamin A are plotted as a percent of the signal detected in the absence of drug. The results taken together show that UL97 phosphorylates lamin A in vitro.
    Figure Legend Snippet: In vitro phosphorylation of lamin A by GST-UL97. (A) Recombinant His-tagged lamin A was incubated in kinase reaction buffer in the presence of γ- 32 P-ATP either alone (no kinase), with catalytically deficient GST-UL97 K355Q (K355Q), or with wild-type GST-UL97 (GST97 WT). GST-UL97 K335Q or wild-type GST-UL97 were also incubated in kinase buffer without lamin A. Following termination of kinase reactions, proteins were resolved by SDS-PAGE. Signal from incorporation of 32 P was detected by exposure to a phosphorscreen (top panel), and total protein was detected by Coomassie brilliant blue staining (bottom panel). The positions of radiolabeled GST-UL97 (GST97) and lamin A, and Coomassie stained lamin A are indicated. (The amounts of GST-UL97 were too small to see on the stained gel.) (B) UL97 autophosphorylation and labeling of lamin A were quantified following in in vitro kinase reactions in the presence of varying concentrations of maribavir (MBV). Signal detected from 32 P incorporation for autophosphorylation of GST-UL97 and phosphorylation of His-tagged lamin A are plotted as a percent of the signal detected in the absence of drug. The results taken together show that UL97 phosphorylates lamin A in vitro.

    Techniques Used: In Vitro, Recombinant, Incubation, SDS Page, Staining, Labeling

    Phosphorylation sites detected on lamin A/C from HCMV-infected cells and from UL97 treated lamin A in vitro. (A) Mass spectrum from electrospray ionization (ESI)-MS-MS of SGAQASSTPLpSPTR tryptic peptide (amino acids 12–25 of native lamin A) after phosphorylation of His-lamin A in vitro with GST-UL97 indicating phosphorylation at Ser 22 . Diagnostic fragment ions used to verify detection of the sequence and the phosphorylation site are indicated in shaded circles and asterisks. A diagram of predicted fragment ions is shown below the spectrum. (B) Mass spectrum from ESI-MS-MS of LRLpSPSPTSQR tryptic peptide (amino acids 386–397 of native lamin A) after phosphorylation of His-lamin A in vitro with GST-UL97, indicating phosphorylation at Ser 390 . Diagnostic fragment ions are indicated as above. (C) Mass spectrum from ESI-MS-MS of LRLpSPSPTSQR tryptic peptide (amino acids 386–397 of native lamin A) after phosphorylation of His-lamin A in vitro with recombinant human CDK1/cyclin B complex, indicating phosphorylation at Ser 390 . Diagnostic fragment ions are indicated as above. (D) Representative mass spectrum from ESI-MS-MS of SGAQASSTPLpSPTR tryptic peptide (amino acids 12–25 of native lamin A) after immunoprecipitation of lamin A/C from HCMV-infected cells and iTRAQ labeling. Diagnostic fragment ions are presented as above. Note that the iTRAQ label alters the mass of the b-ions. The masses of the ions from this spectrum are presented in Figure S3 . (E) Representative mass spectrum from ESI-MS-MS of LSPpSPTSQR tryptic peptide (amino acids 389–397 of native lamin A) after immunoprecipitation of lamin A/C from HCMV-infected cells. Diagnostic fragment ions are indicated as above. The results show that UL97, like CDK1 phosphorylates lamin A on Ser 22 and Ser 390 in vitro, but that phosphorylation occurs on Ser 392 (and Se r22 ) in infected cells.
    Figure Legend Snippet: Phosphorylation sites detected on lamin A/C from HCMV-infected cells and from UL97 treated lamin A in vitro. (A) Mass spectrum from electrospray ionization (ESI)-MS-MS of SGAQASSTPLpSPTR tryptic peptide (amino acids 12–25 of native lamin A) after phosphorylation of His-lamin A in vitro with GST-UL97 indicating phosphorylation at Ser 22 . Diagnostic fragment ions used to verify detection of the sequence and the phosphorylation site are indicated in shaded circles and asterisks. A diagram of predicted fragment ions is shown below the spectrum. (B) Mass spectrum from ESI-MS-MS of LRLpSPSPTSQR tryptic peptide (amino acids 386–397 of native lamin A) after phosphorylation of His-lamin A in vitro with GST-UL97, indicating phosphorylation at Ser 390 . Diagnostic fragment ions are indicated as above. (C) Mass spectrum from ESI-MS-MS of LRLpSPSPTSQR tryptic peptide (amino acids 386–397 of native lamin A) after phosphorylation of His-lamin A in vitro with recombinant human CDK1/cyclin B complex, indicating phosphorylation at Ser 390 . Diagnostic fragment ions are indicated as above. (D) Representative mass spectrum from ESI-MS-MS of SGAQASSTPLpSPTR tryptic peptide (amino acids 12–25 of native lamin A) after immunoprecipitation of lamin A/C from HCMV-infected cells and iTRAQ labeling. Diagnostic fragment ions are presented as above. Note that the iTRAQ label alters the mass of the b-ions. The masses of the ions from this spectrum are presented in Figure S3 . (E) Representative mass spectrum from ESI-MS-MS of LSPpSPTSQR tryptic peptide (amino acids 389–397 of native lamin A) after immunoprecipitation of lamin A/C from HCMV-infected cells. Diagnostic fragment ions are indicated as above. The results show that UL97, like CDK1 phosphorylates lamin A on Ser 22 and Ser 390 in vitro, but that phosphorylation occurs on Ser 392 (and Se r22 ) in infected cells.

    Techniques Used: Infection, In Vitro, Mass Spectrometry, Diagnostic Assay, Sequencing, Recombinant, Immunoprecipitation, Labeling

    61) Product Images from "A novel AMPK activator, WS070117, improves lipid metabolism discords in hamsters and HepG2 cells"

    Article Title: A novel AMPK activator, WS070117, improves lipid metabolism discords in hamsters and HepG2 cells

    Journal: Lipids in Health and Disease

    doi: 10.1186/1476-511X-10-67

    Effects of WS070117 on hepatic lipids accumulation in HFD fed hamsters . A : Representative frozen tissue sections of liver taken from hamsters fed chow diet, HDF diet, HFD+ simvastatin (2 mg/kg) and HFD+WS070117 (18 mg/kg) were stained with Oil Red-O to demonstrate the reduction in lipid droplets. B : Hepatic TC and TAG were measured in liver samples of control (n = 5), HFD, 8-weeks WS070117 (2, 6, 18 mg/kg) or simvastatin (positive control; 2 mg/kg) treated HFD fed hamsters (n = 8). ** P
    Figure Legend Snippet: Effects of WS070117 on hepatic lipids accumulation in HFD fed hamsters . A : Representative frozen tissue sections of liver taken from hamsters fed chow diet, HDF diet, HFD+ simvastatin (2 mg/kg) and HFD+WS070117 (18 mg/kg) were stained with Oil Red-O to demonstrate the reduction in lipid droplets. B : Hepatic TC and TAG were measured in liver samples of control (n = 5), HFD, 8-weeks WS070117 (2, 6, 18 mg/kg) or simvastatin (positive control; 2 mg/kg) treated HFD fed hamsters (n = 8). ** P

    Techniques Used: Staining, Positive Control

    A novel structure compound, WS070117, activated AMPK in HepG2 cells . A : Structure of WS070117; B : Effect of WS070117 on AMPK activity in HepG2 cells detected by AMPK activity assays with SAMS peptide and [γ-32P]ATP used as substrates. HepG2 cells were treated with WS070117 (10 μM) for 1h, 6h and 12h. C : Effect of 12h treatment of WS070117 (1,10,100 μM) or AICAR (1 mM) on AMPK activity of HepG2 cells. AMPK activities are expressed relative to activity detected in HepG2 cells lysate. Each point is the mean (± SEM) of 3 separate experiments. ** P
    Figure Legend Snippet: A novel structure compound, WS070117, activated AMPK in HepG2 cells . A : Structure of WS070117; B : Effect of WS070117 on AMPK activity in HepG2 cells detected by AMPK activity assays with SAMS peptide and [γ-32P]ATP used as substrates. HepG2 cells were treated with WS070117 (10 μM) for 1h, 6h and 12h. C : Effect of 12h treatment of WS070117 (1,10,100 μM) or AICAR (1 mM) on AMPK activity of HepG2 cells. AMPK activities are expressed relative to activity detected in HepG2 cells lysate. Each point is the mean (± SEM) of 3 separate experiments. ** P

    Techniques Used: Activity Assay

    Effects of WS070117 on steatosis and de novo lipids synthesis in OLA-HepG2 cells . A : Oil Red O stain of 0.25 mM OLA-induced cellular lipid accumulation in WS070117 treated HepG2 cells. 12h treatment of WS070117 (10 μM) reduced the lipid droplets amount in HepG2 cells. B : TAG and TC synthesis was measured by the lipid accumulated from [1- 14 C] acetic acid, and was measured in control and WS070117 (0.1, 1, 10 μM) 12-h-treated OLA-HepG2 cells. ** P
    Figure Legend Snippet: Effects of WS070117 on steatosis and de novo lipids synthesis in OLA-HepG2 cells . A : Oil Red O stain of 0.25 mM OLA-induced cellular lipid accumulation in WS070117 treated HepG2 cells. 12h treatment of WS070117 (10 μM) reduced the lipid droplets amount in HepG2 cells. B : TAG and TC synthesis was measured by the lipid accumulated from [1- 14 C] acetic acid, and was measured in control and WS070117 (0.1, 1, 10 μM) 12-h-treated OLA-HepG2 cells. ** P

    Techniques Used: Staining

    WS070117 treatment increases AMP-activated protein kinase (AMPK) phosphorylation in OLA-induced HepG2 cells and HFD fed hamster livers. Data depict at least 3 experiments . ** P
    Figure Legend Snippet: WS070117 treatment increases AMP-activated protein kinase (AMPK) phosphorylation in OLA-induced HepG2 cells and HFD fed hamster livers. Data depict at least 3 experiments . ** P

    Techniques Used:

    62) Product Images from "Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations"

    Article Title: Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

    Journal: Open biology

    doi: 10.1098/rsob.110012

    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Figure Legend Snippet: Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.

    Techniques Used: Mutagenesis, Activity Assay, Incubation, Staining

    Characterization of active insect orthologues of PINK1. ( a ) Assessment of activity of wild-type N-terminally truncated human PINK1 (125–581) expressed in E. coli and Sf9 cells, full-length D. melanogaster PINK1 (dPINK1, 1–721), T. castaneum PINK1 (TcPINK1, 1–570) and P. humanus corporis PINK1 (PhcPINK1, 1–575), and corresponding kinase-inactive mutants (HsPINK1-D384A, dPINK1-D501A, TcPINK1-D359A, PhcPINK1-D357A) against myelin basic protein (MBP). The indicated enzymes (1 µg) were incubated in the presence of 5 µg MBP and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting on P81 paper, washing in phosphoric acid and quantifying phosphorylation of myelin basic protein. The results are presented as ±s.d. for a representative experiment undertaken in duplicate (upper panel). In the lower panel, representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( b ) Assessment of kinase activity of wild-type or kinase inactive (D359A) full-length (1–570), N-terminal truncation (128–570 and 155–570) and N- and C-terminal truncation mutants (155–486) of TcPINK1. The indicated forms of TcPINK1 (1 µg) were incubated in the presence (+) or absence (−) of myelin basic protein (2 µM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by the addition of SDS sample buffer and separated by SDS-PAGE. Gels were analysed by Coomassie staining (upper panel) and incorporation of [γ- 32 P] ATP was detected by autoradiography (lower panel). Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( c ) Analysis of T. castaneum and P. humanus corporis PINK1 function in vivo . TcPINK1 or PhcPINK1 was ectopically expressed in Drosophila lacking endogenous PINK1. Flight ability, climbing ability and presence of thoracic indentations were quantified. Genotypes are as follows. Control: PINK1 B9 /+, mutant: PINK1 B9 /Y; da-GAL4 /+, mutant rescue: PINK1 B9 /Y; da-GAL4 /+, UAS-Tb.PINK1 2a /+ or PINK1 B9 /Y; da-GAL4 /+, UAS-Phc.PINK1 1 /+. Data are presented as mean ± s.e.m.
    Figure Legend Snippet: Characterization of active insect orthologues of PINK1. ( a ) Assessment of activity of wild-type N-terminally truncated human PINK1 (125–581) expressed in E. coli and Sf9 cells, full-length D. melanogaster PINK1 (dPINK1, 1–721), T. castaneum PINK1 (TcPINK1, 1–570) and P. humanus corporis PINK1 (PhcPINK1, 1–575), and corresponding kinase-inactive mutants (HsPINK1-D384A, dPINK1-D501A, TcPINK1-D359A, PhcPINK1-D357A) against myelin basic protein (MBP). The indicated enzymes (1 µg) were incubated in the presence of 5 µg MBP and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting on P81 paper, washing in phosphoric acid and quantifying phosphorylation of myelin basic protein. The results are presented as ±s.d. for a representative experiment undertaken in duplicate (upper panel). In the lower panel, representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( b ) Assessment of kinase activity of wild-type or kinase inactive (D359A) full-length (1–570), N-terminal truncation (128–570 and 155–570) and N- and C-terminal truncation mutants (155–486) of TcPINK1. The indicated forms of TcPINK1 (1 µg) were incubated in the presence (+) or absence (−) of myelin basic protein (2 µM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by the addition of SDS sample buffer and separated by SDS-PAGE. Gels were analysed by Coomassie staining (upper panel) and incorporation of [γ- 32 P] ATP was detected by autoradiography (lower panel). Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( c ) Analysis of T. castaneum and P. humanus corporis PINK1 function in vivo . TcPINK1 or PhcPINK1 was ectopically expressed in Drosophila lacking endogenous PINK1. Flight ability, climbing ability and presence of thoracic indentations were quantified. Genotypes are as follows. Control: PINK1 B9 /+, mutant: PINK1 B9 /Y; da-GAL4 /+, mutant rescue: PINK1 B9 /Y; da-GAL4 /+, UAS-Tb.PINK1 2a /+ or PINK1 B9 /Y; da-GAL4 /+, UAS-Phc.PINK1 1 /+. Data are presented as mean ± s.e.m.

    Techniques Used: Activity Assay, Incubation, Staining, SDS Page, Autoradiography, In Vivo, Mutagenesis

    63) Product Images from "Protein Kinase A-Dependent Phosphorylation of Serine 119 in the Proto-Oncogenic Serine/Arginine-Rich Splicing Factor 1 Modulates Its Activity as a Splicing Enhancer Protein"

    Article Title: Protein Kinase A-Dependent Phosphorylation of Serine 119 in the Proto-Oncogenic Serine/Arginine-Rich Splicing Factor 1 Modulates Its Activity as a Splicing Enhancer Protein

    Journal: Genes & Cancer

    doi: 10.1177/1947601911430226

    Mutation of serine 119 decreases the ability of SRSF1 to interact with RNA. ( A ) 293T cells were transfected with SRSF1 (lanes 1 and 2) or SRSF1 S119A (lanes 3 and 4). Twenty hours posttransfection, the cells were UV cross-linked and lysed. The cell lysates were adjusted to equal protein concentration and treated with T1 RNase before IPs with magnetic beads conjugated with either mouse IgG (lanes 1 and 3) or anti-SRSF1 (lanes 2 and 4). Immunoprecipitated samples were dephosphorylated, labeled with γ-[ 32 P]-ATP by PNK kinase, and run on a denaturating polyacrylamide gel before analysis by autoradiography. The immunoblot (lower panel) shows the amount of SRSF1 in the cell lysate. The arrows indicate accumulation of specific RNA species. ( B ) Lanes in unsaturated images were manually detected in Adobe Photoshop using identical frames. The obtained intensities were adjusted for background and analyzed in GraphPad Prism by a paired t test ( n = 3).
    Figure Legend Snippet: Mutation of serine 119 decreases the ability of SRSF1 to interact with RNA. ( A ) 293T cells were transfected with SRSF1 (lanes 1 and 2) or SRSF1 S119A (lanes 3 and 4). Twenty hours posttransfection, the cells were UV cross-linked and lysed. The cell lysates were adjusted to equal protein concentration and treated with T1 RNase before IPs with magnetic beads conjugated with either mouse IgG (lanes 1 and 3) or anti-SRSF1 (lanes 2 and 4). Immunoprecipitated samples were dephosphorylated, labeled with γ-[ 32 P]-ATP by PNK kinase, and run on a denaturating polyacrylamide gel before analysis by autoradiography. The immunoblot (lower panel) shows the amount of SRSF1 in the cell lysate. The arrows indicate accumulation of specific RNA species. ( B ) Lanes in unsaturated images were manually detected in Adobe Photoshop using identical frames. The obtained intensities were adjusted for background and analyzed in GraphPad Prism by a paired t test ( n = 3).

    Techniques Used: Mutagenesis, Transfection, Protein Concentration, Magnetic Beads, Immunoprecipitation, Labeling, Autoradiography

    PKA phosphorylates SRSF1 at serine 119 in vitro . Purified SRSF1 (lanes 1 and 2), SRSF1 S119A (lanes 3 and 4), SRSF1 ΔRS (lanes 5 and 6), and SRSF1 ΔRS S119A (lanes 7 and 8) were incubated with active or heat-inactivated PKA Cα1 and γ-[ 32 P]-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE followed by Coomassie staining (lower panel) and autoradiography (upper panel).
    Figure Legend Snippet: PKA phosphorylates SRSF1 at serine 119 in vitro . Purified SRSF1 (lanes 1 and 2), SRSF1 S119A (lanes 3 and 4), SRSF1 ΔRS (lanes 5 and 6), and SRSF1 ΔRS S119A (lanes 7 and 8) were incubated with active or heat-inactivated PKA Cα1 and γ-[ 32 P]-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE followed by Coomassie staining (lower panel) and autoradiography (upper panel).

    Techniques Used: In Vitro, Purification, Incubation, SDS Page, Staining, Autoradiography

    64) Product Images from "Requirement for protein kinase A in the phosphorylation of the TGFss receptor-interacting protein km23-1 as a component of TGFss downstream effects"

    Article Title: Requirement for protein kinase A in the phosphorylation of the TGFss receptor-interacting protein km23-1 as a component of TGFss downstream effects

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2012.12.029

    PKA directly phosphorylates km23-1 on S73 in vitro A: Purified His-km23-1 fusion proteins were used as substrates in an in vitro kinase reaction with the recombinant catalytic subunit of PKA in the presence of [γ- 32 P] ATP. B: Similar studies were performed as for A except with increasing amounts of the recombinant catalytic subunit of PKA used. Equal loading of His-km23-1 fusion proteins and of the catalytic subunit of PKA were revealed by Coomassie blue staining in the lower panels. C: After pull-down using Ni-NTA agarose followed by SDS-PAGE as for A , the His-fusion proteins were detected by blotting with the S73 phospho-specific km23-1 Ab. Lower panel, Western blot analysis showing equal loading of His-km23-1 using km23-1 (27-43)w Ab. The results are representative of triplicate experiments.
    Figure Legend Snippet: PKA directly phosphorylates km23-1 on S73 in vitro A: Purified His-km23-1 fusion proteins were used as substrates in an in vitro kinase reaction with the recombinant catalytic subunit of PKA in the presence of [γ- 32 P] ATP. B: Similar studies were performed as for A except with increasing amounts of the recombinant catalytic subunit of PKA used. Equal loading of His-km23-1 fusion proteins and of the catalytic subunit of PKA were revealed by Coomassie blue staining in the lower panels. C: After pull-down using Ni-NTA agarose followed by SDS-PAGE as for A , the His-fusion proteins were detected by blotting with the S73 phospho-specific km23-1 Ab. Lower panel, Western blot analysis showing equal loading of His-km23-1 using km23-1 (27-43)w Ab. The results are representative of triplicate experiments.

    Techniques Used: In Vitro, Purification, Recombinant, Staining, SDS Page, Western Blot

    TGFß induces PKA phosphorylation of km23-1 on S73 in vivo, which is inhibited by H89 A: Mv1Lu cells were transfected with km23-1-Flag. 24h later cells were changed to SF conditions for 1 h prior to TGFß1 (10 ng/ml) treatment for 30 min. Cell lysates were subjected to IP using a Flag Ab, and blotted using the S73 phospho-specific anti-km23-1 antibody (S73-P-km23-1). Lower panel, Western blot analysis showing equal expression of km23-1-Flag using anti-Flag. The results are representative of triplicate experiments. B: Endogenous km23-1 was IP'd using hkm23-1 (1-15) Ab, and blotted using S73-P-km23-1. Lower panel, Western blot analysis showing equal expression of endogenous km23-1 using km23-1(27-43)w Ab. C: Mv1Lu cells were transfected with km23-1-Flag, and treated with TGFß1 as for A. For lane 3, cells were treated with H89 for 30 min prior to TGFß1 treatment for 30 min. Cell lysates were treated and analyzed as for A. Lane 4 is the IgG control. The results are representative of triplicate experiments.
    Figure Legend Snippet: TGFß induces PKA phosphorylation of km23-1 on S73 in vivo, which is inhibited by H89 A: Mv1Lu cells were transfected with km23-1-Flag. 24h later cells were changed to SF conditions for 1 h prior to TGFß1 (10 ng/ml) treatment for 30 min. Cell lysates were subjected to IP using a Flag Ab, and blotted using the S73 phospho-specific anti-km23-1 antibody (S73-P-km23-1). Lower panel, Western blot analysis showing equal expression of km23-1-Flag using anti-Flag. The results are representative of triplicate experiments. B: Endogenous km23-1 was IP'd using hkm23-1 (1-15) Ab, and blotted using S73-P-km23-1. Lower panel, Western blot analysis showing equal expression of endogenous km23-1 using km23-1(27-43)w Ab. C: Mv1Lu cells were transfected with km23-1-Flag, and treated with TGFß1 as for A. For lane 3, cells were treated with H89 for 30 min prior to TGFß1 treatment for 30 min. Cell lysates were treated and analyzed as for A. Lane 4 is the IgG control. The results are representative of triplicate experiments.

    Techniques Used: In Vivo, Transfection, Western Blot, Expressing

    km23-1 interacts with the R1ß regulatory subunit of PKA in a TGFß-dependent manner Mv1Lu cells were washed once with SF medium and incubated in SF medium for 1 h prior to TGFß1 (10 ng/ml) treatment for the indicated times. Cell lysates (500 μg) were used for IP with 1 μg of anti-PKA RIß subunit Ab, and were then blotted with an anti-hkm23-1 (27-43)w Ab (top panel). The same membrane was then blotted with the PKA RIß subunit Ab to show equal IP'd proteins (1st middle panel, lane 1-4). Lane 6 is the negative control for the IP Ab in which an RIß blocking peptide (b.p.) was added to the cell lysates during the IP incubation. 2nd middle panel, equal expression of endogenous km23-1 protein was demonstrated by IP/blot analysis using the hkm23-1(1-15) as the IP Ab, and the hkm23-1(27-43)w as the blotting Ab. Bottom panel, the blots were scanned and quantified by using Image J software. The results are representative of triplicate experiments.
    Figure Legend Snippet: km23-1 interacts with the R1ß regulatory subunit of PKA in a TGFß-dependent manner Mv1Lu cells were washed once with SF medium and incubated in SF medium for 1 h prior to TGFß1 (10 ng/ml) treatment for the indicated times. Cell lysates (500 μg) were used for IP with 1 μg of anti-PKA RIß subunit Ab, and were then blotted with an anti-hkm23-1 (27-43)w Ab (top panel). The same membrane was then blotted with the PKA RIß subunit Ab to show equal IP'd proteins (1st middle panel, lane 1-4). Lane 6 is the negative control for the IP Ab in which an RIß blocking peptide (b.p.) was added to the cell lysates during the IP incubation. 2nd middle panel, equal expression of endogenous km23-1 protein was demonstrated by IP/blot analysis using the hkm23-1(1-15) as the IP Ab, and the hkm23-1(27-43)w as the blotting Ab. Bottom panel, the blots were scanned and quantified by using Image J software. The results are representative of triplicate experiments.

    Techniques Used: Incubation, Negative Control, Blocking Assay, Expressing, Software

    S73 is required for TGFß induction of the km23-1-DIC complex A: 293T cells were transiently co-transfected with the indicated plasmids. 24 h after transfection, cells were incubated in SF medium for 60 min before addition of TGFß1 (10 ng/ml) for 0, or 15 min. Cell lysates were then IP'd with an anti-DIC Ab and were then blotted with an anti-Flag Ab (top panel). The same membrane was then blotted with the DIC Ab to show equal IP'd proteins (1st middle panel). Equal inputs and expression of RII-HA, and Flag-tagged proteins were verified by Western blotting (bottom two panels). B: The IP/blot analysis using the samples from A was performed in the opposite direction with Flag as the IP Ab. Results are representative of two experiments.
    Figure Legend Snippet: S73 is required for TGFß induction of the km23-1-DIC complex A: 293T cells were transiently co-transfected with the indicated plasmids. 24 h after transfection, cells were incubated in SF medium for 60 min before addition of TGFß1 (10 ng/ml) for 0, or 15 min. Cell lysates were then IP'd with an anti-DIC Ab and were then blotted with an anti-Flag Ab (top panel). The same membrane was then blotted with the DIC Ab to show equal IP'd proteins (1st middle panel). Equal inputs and expression of RII-HA, and Flag-tagged proteins were verified by Western blotting (bottom two panels). B: The IP/blot analysis using the samples from A was performed in the opposite direction with Flag as the IP Ab. Results are representative of two experiments.

    Techniques Used: Transfection, Incubation, Expressing, Western Blot

    S73-km23-1 inhibits TGFß induction of TGFß/Smad2-dependent transcription in ARE reporter assays, but has no effect on TGFß/Smad3-dependent transcriptional activation A: Mv1Lu cells were transfected with EV, km23-1-Flag, or S73A km23-1-Flag, along with 0.2 μg of ARE-Lux reporter and 0.2 μg of FAST-1. To normalize transfection efficiencies, 0.2 μg of Renilla was co-transfected as an internal control. Luciferase reporter assays were performed as described in “Materials and Methods.” Error bars represent the SEM. The results are representative of at least two experiments, each performed in triplicate. Asterisks indicate a statistically significant difference (p
    Figure Legend Snippet: S73-km23-1 inhibits TGFß induction of TGFß/Smad2-dependent transcription in ARE reporter assays, but has no effect on TGFß/Smad3-dependent transcriptional activation A: Mv1Lu cells were transfected with EV, km23-1-Flag, or S73A km23-1-Flag, along with 0.2 μg of ARE-Lux reporter and 0.2 μg of FAST-1. To normalize transfection efficiencies, 0.2 μg of Renilla was co-transfected as an internal control. Luciferase reporter assays were performed as described in “Materials and Methods.” Error bars represent the SEM. The results are representative of at least two experiments, each performed in triplicate. Asterisks indicate a statistically significant difference (p

    Techniques Used: Activation Assay, Transfection, Luciferase

    65) Product Images from "The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair"

    Article Title: The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.806638

    The conserved XRCC1 phenylalanine motif is required for the phosphorylation-independent stimulation of PNKP activity. A , stimulation of PNKP DNA kinase activity. 0.5 μ m PNKP was incubated in the presence of [γ- 32 P]ATP with 10 μ m oligonucleotide substrate and 4 μ m XRCC1-His, XRCC1-His FFF , His-XRCC1 161–406 , or His-XRCC1 161–406-RK for 2 min at 37 °C. The amount of radiolabeled 5′-phosphorylated 24-mer oligonucleotide was then quantified by gel electrophoresis and autoradiography. Data are the mean ± S.D. of three independent experiments. B , stimulation of PNKP DNA kinase enzyme-product turnover. DNA kinase reactions (50 μl) containing 2 μ m 1-nt gapped oligonucleotide substrate and 0.2 μ m PNKP were conducted as above in the absence of XRCC1 for 20 min and XRCC1-His or XRCC1-His FFF then added to 0.8 μ m for a further 20 min. Phosphorylated oligonucleotide product was quantified at the indicated times, as above. Data are the mean ± S.D. of three independent experiments. C , stimulation of PNKP DNA phosphatase activity. DNA phosphatase reactions (30 μl) containing 0.33 μ m 1-nt gapped oligonucleotide substrate and 0.86 μ m PNKP were incubated in the absence of any XRCC1 protein for 20 min and then, where indicated, in the additional presence of 1.65 μ m XRCC1-His or XRCC1-His FFF for a further 20 min. 3′-Dephosphorylated oligonucleotide product was quantified at the indicated times, as above. Data are the mean ± S.D. of three independent experiments. D , Interaction of XRCC1-His (■) and XRCC1-His FFF (●) with 1-nt gapped DNA. Proteins (30 n m ) were excited at 295 nm and the fluorescence intensity at 340 nm was monitored as a function of added 1 nt-gapped DNA substrate (see inset for data with XRCC1-His). The fraction bound, i.e. relative fluorescence ( Rel. Fluor .), versus ligand concentration is plotted.
    Figure Legend Snippet: The conserved XRCC1 phenylalanine motif is required for the phosphorylation-independent stimulation of PNKP activity. A , stimulation of PNKP DNA kinase activity. 0.5 μ m PNKP was incubated in the presence of [γ- 32 P]ATP with 10 μ m oligonucleotide substrate and 4 μ m XRCC1-His, XRCC1-His FFF , His-XRCC1 161–406 , or His-XRCC1 161–406-RK for 2 min at 37 °C. The amount of radiolabeled 5′-phosphorylated 24-mer oligonucleotide was then quantified by gel electrophoresis and autoradiography. Data are the mean ± S.D. of three independent experiments. B , stimulation of PNKP DNA kinase enzyme-product turnover. DNA kinase reactions (50 μl) containing 2 μ m 1-nt gapped oligonucleotide substrate and 0.2 μ m PNKP were conducted as above in the absence of XRCC1 for 20 min and XRCC1-His or XRCC1-His FFF then added to 0.8 μ m for a further 20 min. Phosphorylated oligonucleotide product was quantified at the indicated times, as above. Data are the mean ± S.D. of three independent experiments. C , stimulation of PNKP DNA phosphatase activity. DNA phosphatase reactions (30 μl) containing 0.33 μ m 1-nt gapped oligonucleotide substrate and 0.86 μ m PNKP were incubated in the absence of any XRCC1 protein for 20 min and then, where indicated, in the additional presence of 1.65 μ m XRCC1-His or XRCC1-His FFF for a further 20 min. 3′-Dephosphorylated oligonucleotide product was quantified at the indicated times, as above. Data are the mean ± S.D. of three independent experiments. D , Interaction of XRCC1-His (■) and XRCC1-His FFF (●) with 1-nt gapped DNA. Proteins (30 n m ) were excited at 295 nm and the fluorescence intensity at 340 nm was monitored as a function of added 1 nt-gapped DNA substrate (see inset for data with XRCC1-His). The fraction bound, i.e. relative fluorescence ( Rel. Fluor .), versus ligand concentration is plotted.

    Techniques Used: Activity Assay, Incubation, Field Flow Fractionation, Nucleic Acid Electrophoresis, Autoradiography, Fluorescence, Concentration Assay

    66) Product Images from "Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism"

    Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001527

    TGF-β activates HIPK2 by phosphorylating a highly conserved tyrosine residue on position 361. (A) Amino acid sequence alignment of the HIPK protein family from human and mouse reveals a stretch of highly conserved residues from position 346 to 371 in the activation segment of the subdomain VII in HIPK2. (B) Alignment of the similar regions of HIPK2 (346 to 371) from different species confirms that these amino acid residues are highly conserved from nematodes to the vertebrates. Conserved amino acids that can potentially be phosphorylated in MAPK signaling pathway are shown in bold. (C) The combined immunoprecipitation and in vitro kinase (IP-IVK) assays show that TGF-β treatment promotes the ability of wild-type HIPK2 to incorporate γ- 32 P-ATP. In contrast, kinase inactive HIPK2-K221A fails to incorporate γ- 32 P-ATP. While HIPK2-S359A and HIPK2-T360A mutant proteins can still incorporate γ- 32 P-ATP in response to TGF-β treatment, the Y361F mutation in HIPK2 completely eliminates its ability to incorporate γ- 32 P-ATP. (D) TGF-β and TAK1-induced phosphorylation of HIPK2 occurs primarily on Y361 residue in HIPK2. HIPK2-Y361F mutant completely loses its ability to incorporate γ- 32 P-ATP upon activation by TGF-β or TAK1. Data are shown as mean + s.e.m., n = 3. Statistics in (C) and (D) use Student's t test. * p
    Figure Legend Snippet: TGF-β activates HIPK2 by phosphorylating a highly conserved tyrosine residue on position 361. (A) Amino acid sequence alignment of the HIPK protein family from human and mouse reveals a stretch of highly conserved residues from position 346 to 371 in the activation segment of the subdomain VII in HIPK2. (B) Alignment of the similar regions of HIPK2 (346 to 371) from different species confirms that these amino acid residues are highly conserved from nematodes to the vertebrates. Conserved amino acids that can potentially be phosphorylated in MAPK signaling pathway are shown in bold. (C) The combined immunoprecipitation and in vitro kinase (IP-IVK) assays show that TGF-β treatment promotes the ability of wild-type HIPK2 to incorporate γ- 32 P-ATP. In contrast, kinase inactive HIPK2-K221A fails to incorporate γ- 32 P-ATP. While HIPK2-S359A and HIPK2-T360A mutant proteins can still incorporate γ- 32 P-ATP in response to TGF-β treatment, the Y361F mutation in HIPK2 completely eliminates its ability to incorporate γ- 32 P-ATP. (D) TGF-β and TAK1-induced phosphorylation of HIPK2 occurs primarily on Y361 residue in HIPK2. HIPK2-Y361F mutant completely loses its ability to incorporate γ- 32 P-ATP upon activation by TGF-β or TAK1. Data are shown as mean + s.e.m., n = 3. Statistics in (C) and (D) use Student's t test. * p

    Techniques Used: Sequencing, Activation Assay, Immunoprecipitation, In Vitro, Mutagenesis

    TGF-β–TAK1 promotes HIPK2 activity through protein–protein interaction and protects HIPK2 from proteasome-mediated degradation. (A) TGF-β promotes HIPK2 kinase activity in HEK293T cells, whereas kinase inactive HIPK2-K221A shows no incorporation of γ- 32 P-ATP upon TGF-β treatment. (B) The ability of TGF-β to activate HIPK2 kinase activity can be blocked by TGF-β type I receptor inhibitor SB431542. (C and D) TGF-β and wild-type TAK1 activate HIPK2 kinase and maintain the stability of HIPK2 protein. In contrast, dominant negative TAK1 (DN-TAK1) promotes HIPK2 degradation via the proteasome pathway.
    Figure Legend Snippet: TGF-β–TAK1 promotes HIPK2 activity through protein–protein interaction and protects HIPK2 from proteasome-mediated degradation. (A) TGF-β promotes HIPK2 kinase activity in HEK293T cells, whereas kinase inactive HIPK2-K221A shows no incorporation of γ- 32 P-ATP upon TGF-β treatment. (B) The ability of TGF-β to activate HIPK2 kinase activity can be blocked by TGF-β type I receptor inhibitor SB431542. (C and D) TGF-β and wild-type TAK1 activate HIPK2 kinase and maintain the stability of HIPK2 protein. In contrast, dominant negative TAK1 (DN-TAK1) promotes HIPK2 degradation via the proteasome pathway.

    Techniques Used: Activity Assay, Dominant Negative Mutation

    67) Product Images from "DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation"

    Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080313

    Differential phosphorylation pattern and total protein status after Dbait32Hc treatment. (A) SDS-polyacrylamide gel electrophoresis after isoelectric focusing (pH range 4.5–5.5) of MRC-5 lysates treated with Dbait32Hc or 8H. Total protein was detected by Sypro Ruby (red, SR) staining and phosphorylation was monitored by Pro-Q Diamond (green, Pro-Q) staining of the same gel. Spots displaying a marked increase in phosphorylation after treatment are highlighted (white arrows). (B) Higher magnification of selected spots from (A) showing higher levels of phosphorylation (arrows) of the indicated proteins after Dbait32Hc treatment than after transfection with the control, 8H. No difference in total protein levels was founds. Proteins displaying at least a 10-fold increase in Pro-Q Diamond staining that could be unambiguously assigned to proteins stained by Sypro Ruby were excised and analyzed by LC-MS/MS. (C) In vitro phosphorylation of vimentin by DNA-PK. Purified DNA-PK (DNA-PKcs and Ku) was incubated with [γ- 32 P]ATP and the indicated amounts of purified vimentin protein. Dbait32Hc was added where indicated, to activate DNA-PK, and the proteins were then denatured, separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography. (D) Peptides and phosphosites of in vitro DNA-PK-phosphorylated vimentin, as identified by LC-MS/MS with the LTQ-Orbitrap after trypsin digestion. (pT) and (pS) correspond to phosphorylated threonine and serine, respectively.
    Figure Legend Snippet: Differential phosphorylation pattern and total protein status after Dbait32Hc treatment. (A) SDS-polyacrylamide gel electrophoresis after isoelectric focusing (pH range 4.5–5.5) of MRC-5 lysates treated with Dbait32Hc or 8H. Total protein was detected by Sypro Ruby (red, SR) staining and phosphorylation was monitored by Pro-Q Diamond (green, Pro-Q) staining of the same gel. Spots displaying a marked increase in phosphorylation after treatment are highlighted (white arrows). (B) Higher magnification of selected spots from (A) showing higher levels of phosphorylation (arrows) of the indicated proteins after Dbait32Hc treatment than after transfection with the control, 8H. No difference in total protein levels was founds. Proteins displaying at least a 10-fold increase in Pro-Q Diamond staining that could be unambiguously assigned to proteins stained by Sypro Ruby were excised and analyzed by LC-MS/MS. (C) In vitro phosphorylation of vimentin by DNA-PK. Purified DNA-PK (DNA-PKcs and Ku) was incubated with [γ- 32 P]ATP and the indicated amounts of purified vimentin protein. Dbait32Hc was added where indicated, to activate DNA-PK, and the proteins were then denatured, separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography. (D) Peptides and phosphosites of in vitro DNA-PK-phosphorylated vimentin, as identified by LC-MS/MS with the LTQ-Orbitrap after trypsin digestion. (pT) and (pS) correspond to phosphorylated threonine and serine, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Transfection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, In Vitro, Purification, Incubation, Autoradiography

    68) Product Images from "Discovery of new substrates of the elongation factor-2 kinase suggests a broader role in the cellular nutrient response"

    Article Title: Discovery of new substrates of the elongation factor-2 kinase suggests a broader role in the cellular nutrient response

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2016.10.006

    Discovery and validation of two new substrates of eEF2K: IGBP1 (alpha 4) and NDRG1 Wild type and mutant IGBP1 and NDRG1 proteins were purified from HEK293T cells and phosphorylated using 32 P-γ-ATP. The samples were analyzed by SDS-PAGE followed by autoradiography. Samples were also analyzed by western blot for loading prior to addition of the 32 P-γ-ATP.
    Figure Legend Snippet: Discovery and validation of two new substrates of eEF2K: IGBP1 (alpha 4) and NDRG1 Wild type and mutant IGBP1 and NDRG1 proteins were purified from HEK293T cells and phosphorylated using 32 P-γ-ATP. The samples were analyzed by SDS-PAGE followed by autoradiography. Samples were also analyzed by western blot for loading prior to addition of the 32 P-γ-ATP.

    Techniques Used: Mutagenesis, Purification, SDS Page, Autoradiography, Western Blot

    69) Product Images from "The CroRS Two-Component Regulatory System Is Required for Intrinsic ?-Lactam Resistance in Enterococcus faecalis"

    Article Title: The CroRS Two-Component Regulatory System Is Required for Intrinsic ?-Lactam Resistance in Enterococcus faecalis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.24.7184-7192.2003

    Phosphotransfer reactions catalyzed by CroS S and CroR H . (A) Kinetics of CroS S autophosphorylation. CroS S was incubated with [γ 32 -P]ATP for 0, 5, 10, 30, and 60 min (lanes 1 to 5, respectively) and applied to an SDS-13.5% polyacrylamide gel. (B) Transfer of the phosphate group from the phosphorylated form of CroS S (P-CroS) to CroR H . Phospho-CroS S was prepared (lane 1) and incubated with CroR H for 2, 5, and 20 min (lanes 2, 3, and 4, respectively).
    Figure Legend Snippet: Phosphotransfer reactions catalyzed by CroS S and CroR H . (A) Kinetics of CroS S autophosphorylation. CroS S was incubated with [γ 32 -P]ATP for 0, 5, 10, 30, and 60 min (lanes 1 to 5, respectively) and applied to an SDS-13.5% polyacrylamide gel. (B) Transfer of the phosphate group from the phosphorylated form of CroS S (P-CroS) to CroR H . Phospho-CroS S was prepared (lane 1) and incubated with CroR H for 2, 5, and 20 min (lanes 2, 3, and 4, respectively).

    Techniques Used: Incubation

    70) Product Images from "Functional Analysis of the Mycobacterium tuberculosis MprAB Two-Component Signal Transduction System "

    Article Title: Functional Analysis of the Mycobacterium tuberculosis MprAB Two-Component Signal Transduction System

    Journal: Infection and Immunity

    doi: 10.1128/IAI.71.12.6962-6970.2003

    Transphosphorylation between GST-cMprB and M. tuberculosis response regulators. Wild-type GST-cMprB was autophosporylated with [γ- 32 P]ATP and then incubated in the absence of other proteins (lanes 1 and 2) or in the presence of wild-type His-MprA (lanes 3 to 7), the His-MprA (Asp48-Ala) mutant (lanes 8 to 12), or wild-type His-MtrA (lanes 13 to 17). Transphosphorylation reactions were allowed to proceed for 0 min (lanes 1, 3, 8, and 13), 5 min (lanes 4, 9, and 14), 10 min (lanes 5, 10, and 15), 20 min (lanes 6, 11, and 16), or 30 min (lanes 2, 7, 12, and 17). Closed arrows indicate the locations of full-length GST-cMprB and response regulator proteins. Open arrows indicate the locations of truncated (trunc.) forms of GST-cMprB. The asterisk indicates the position of phosphorylated His-MprA species. Transfer of radiolabel from GST-cMprB to response regulator proteins was detected by autoradiography. WT, wild type.
    Figure Legend Snippet: Transphosphorylation between GST-cMprB and M. tuberculosis response regulators. Wild-type GST-cMprB was autophosporylated with [γ- 32 P]ATP and then incubated in the absence of other proteins (lanes 1 and 2) or in the presence of wild-type His-MprA (lanes 3 to 7), the His-MprA (Asp48-Ala) mutant (lanes 8 to 12), or wild-type His-MtrA (lanes 13 to 17). Transphosphorylation reactions were allowed to proceed for 0 min (lanes 1, 3, 8, and 13), 5 min (lanes 4, 9, and 14), 10 min (lanes 5, 10, and 15), 20 min (lanes 6, 11, and 16), or 30 min (lanes 2, 7, 12, and 17). Closed arrows indicate the locations of full-length GST-cMprB and response regulator proteins. Open arrows indicate the locations of truncated (trunc.) forms of GST-cMprB. The asterisk indicates the position of phosphorylated His-MprA species. Transfer of radiolabel from GST-cMprB to response regulator proteins was detected by autoradiography. WT, wild type.

    Techniques Used: Incubation, Mutagenesis, Autoradiography

    In vitro autophosphorylation of GST-cMprB derivatives. (A) Purified GST-cMprB was incubated in the presence of [γ- 32 P]ATP (lanes 1, 3, 5, and 7) or [α- 32 P]ATP (lanes 2, 4, 6, and 8) and divalent cations including Mg 2+ (lanes 1 and 2), Mn 2+ (lanes 3 and 4), and Ca 2+ (lanes 5 and 6) or in the absence of metal (lanes 7 and 8). (B) Wild-type GST-cMprB (lanes 1 to 4) or the GST-cMprB (His249-Gln) mutant (lanes 5 to 8) was incubated in the presence of [γ- 32 P]ATP and Mg 2+ (lanes 1 and 5), Mn 2+ (lanes 2 and 6), or Ca 2+ (lanes 3 and 7) or in the absence of divalent cations (lanes 4 and 8). Phosphorylation of wild-type or mutant GST-cMprB was detected by autoradiography, and polyclonal antibody directed against cMprB was used in Western blotting to confirm similar loading amounts between reactions.
    Figure Legend Snippet: In vitro autophosphorylation of GST-cMprB derivatives. (A) Purified GST-cMprB was incubated in the presence of [γ- 32 P]ATP (lanes 1, 3, 5, and 7) or [α- 32 P]ATP (lanes 2, 4, 6, and 8) and divalent cations including Mg 2+ (lanes 1 and 2), Mn 2+ (lanes 3 and 4), and Ca 2+ (lanes 5 and 6) or in the absence of metal (lanes 7 and 8). (B) Wild-type GST-cMprB (lanes 1 to 4) or the GST-cMprB (His249-Gln) mutant (lanes 5 to 8) was incubated in the presence of [γ- 32 P]ATP and Mg 2+ (lanes 1 and 5), Mn 2+ (lanes 2 and 6), or Ca 2+ (lanes 3 and 7) or in the absence of divalent cations (lanes 4 and 8). Phosphorylation of wild-type or mutant GST-cMprB was detected by autoradiography, and polyclonal antibody directed against cMprB was used in Western blotting to confirm similar loading amounts between reactions.

    Techniques Used: In Vitro, Purification, Incubation, Mutagenesis, Autoradiography, Western Blot

    71) Product Images from "Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation *"

    Article Title: Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.727735

    In vitro phosphorylation of FACT and PAF-C by CKII. A , autoradiograph of a representative set of in vitro reactions performed with [γ 32 P]ATP in the presence or absence of Ctr9-FLAG, Spt16-TAP, or recombinant CKII, as indicated at the top . The
    Figure Legend Snippet: In vitro phosphorylation of FACT and PAF-C by CKII. A , autoradiograph of a representative set of in vitro reactions performed with [γ 32 P]ATP in the presence or absence of Ctr9-FLAG, Spt16-TAP, or recombinant CKII, as indicated at the top . The

    Techniques Used: In Vitro, Autoradiography, Recombinant

    72) Product Images from "Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1 *"

    Article Title: Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.666289

    ATP binding and hydrolysis by DMC1 and mutant proteins. A , schematic of the UV cross-linking analysis. DMC1 and mutant proteins were incubated with [γ- 32 P]ATP. Following UV cross-linking, radiolabeled proteins were run on a 13.5% denaturing polyacrylamide
    Figure Legend Snippet: ATP binding and hydrolysis by DMC1 and mutant proteins. A , schematic of the UV cross-linking analysis. DMC1 and mutant proteins were incubated with [γ- 32 P]ATP. Following UV cross-linking, radiolabeled proteins were run on a 13.5% denaturing polyacrylamide

    Techniques Used: Binding Assay, Mutagenesis, Incubation

    73) Product Images from "Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases *"

    Article Title: Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.614420

    GAP activity of sr-synGAP and r-synGAP. GTPase activities in the presence of sr-synGAP ( left panels ) and r-synGAP ( right panels ) were measured as a function of GTPase concentration in the absence and presence of purified sr- or r-synGAP (350 n m ) for 10
    Figure Legend Snippet: GAP activity of sr-synGAP and r-synGAP. GTPase activities in the presence of sr-synGAP ( left panels ) and r-synGAP ( right panels ) were measured as a function of GTPase concentration in the absence and presence of purified sr- or r-synGAP (350 n m ) for 10

    Techniques Used: Activity Assay, Concentration Assay, Purification

    Intrinsic GAP activities of alanine and aspartic acid mutants of r-synGAP. GAP activities of wild type and mutant r-synGAP (250 n m ) were measured by incubation with [γ- 32 P]GTP-loaded GTPases for 10 min at 25 °C as described under “Experimental
    Figure Legend Snippet: Intrinsic GAP activities of alanine and aspartic acid mutants of r-synGAP. GAP activities of wild type and mutant r-synGAP (250 n m ) were measured by incubation with [γ- 32 P]GTP-loaded GTPases for 10 min at 25 °C as described under “Experimental

    Techniques Used: Mutagenesis, Incubation

    74) Product Images from "The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control *"

    Article Title: The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.737619

    The final purities of WT Cdc34, Δ190 Cdc34 lacking the acidic tail, WT Ubc1, and full-length San1. Approximately 1 μg of protein was loaded onto a SDS-PAGE gel that, after electrophoresis, was stained with Coomassie Blue. The final purity for KR (No Lys) San1 is shown and is similar to the purities of all San1 constructs.
    Figure Legend Snippet: The final purities of WT Cdc34, Δ190 Cdc34 lacking the acidic tail, WT Ubc1, and full-length San1. Approximately 1 μg of protein was loaded onto a SDS-PAGE gel that, after electrophoresis, was stained with Coomassie Blue. The final purity for KR (No Lys) San1 is shown and is similar to the purities of all San1 constructs.

    Techniques Used: SDS Page, Electrophoresis, Staining, Construct

    Ubc1 activity is strongly stimulated in the presence of San1. A , diubiquitin synthesis assay for WT Ubc1 showing the time-dependent formation of diubiquitin product. B , same as in A except KR San1 was added to the reaction mixture before the addition of acceptor ubiquitin. C , same as in B except with N13K/R280A San1. D , same as in C except with Cul1-Rbx1. Notice that the presence of KR San1 resulted in the stimulation of Ubc1 activity; however, the addition of Cul1-Rbx1 had no effect on Ubc1 activity. E , quantification of diubiquitin formation for reactions in A–D. Error bars represent the S.E. of measurements from duplicate data points.
    Figure Legend Snippet: Ubc1 activity is strongly stimulated in the presence of San1. A , diubiquitin synthesis assay for WT Ubc1 showing the time-dependent formation of diubiquitin product. B , same as in A except KR San1 was added to the reaction mixture before the addition of acceptor ubiquitin. C , same as in B except with N13K/R280A San1. D , same as in C except with Cul1-Rbx1. Notice that the presence of KR San1 resulted in the stimulation of Ubc1 activity; however, the addition of Cul1-Rbx1 had no effect on Ubc1 activity. E , quantification of diubiquitin formation for reactions in A–D. Error bars represent the S.E. of measurements from duplicate data points.

    Techniques Used: Activity Assay

    Single lysine San1 is rapidly autoubiquitylated in the presence of WT Cdc34. A , time-course showing autoubiquitylation of radiolabeled N13K San1 in the presence of WT Cdc34, E1, and ubiquitin. Product is defined as any San1 protein that has been modified by one or more ubiquitins. B , quantitation of the fraction of N13K San1 that was converted to ubiquitylated product. Notice that the fraction of San1 product was linear with respect with time. Error bars represent the S.E. of measurements from duplicate data points. C , same as in A but with N444K San1. D , same as in B but with N444K San1. E , same as in A but with N13K/R280A San1.
    Figure Legend Snippet: Single lysine San1 is rapidly autoubiquitylated in the presence of WT Cdc34. A , time-course showing autoubiquitylation of radiolabeled N13K San1 in the presence of WT Cdc34, E1, and ubiquitin. Product is defined as any San1 protein that has been modified by one or more ubiquitins. B , quantitation of the fraction of N13K San1 that was converted to ubiquitylated product. Notice that the fraction of San1 product was linear with respect with time. Error bars represent the S.E. of measurements from duplicate data points. C , same as in A but with N444K San1. D , same as in B but with N444K San1. E , same as in A but with N13K/R280A San1.

    Techniques Used: Modification, Quantitation Assay

    Ubc1 has greater affinity for San1 than Cdc34. A , San1 peptide ubiquitylation reactions containing KR San1 and titrations of WT Cdc34 protein. Each lane represents a single ubiquitylation reaction that was quenched with 2× SDS-PAGE loading buffer after 10 min. Error bars represent the S.E. of measurements from duplicate data points. B , same as in A except with WT Ubc1. Reactions were quenched with SDS-PAGE loading buffer after 6 min. C , same as in A except with Δ190 Cdc34. Notice that deletion of the Cdc34 acidic tail does not affect the affinity of Cdc34 for San1.
    Figure Legend Snippet: Ubc1 has greater affinity for San1 than Cdc34. A , San1 peptide ubiquitylation reactions containing KR San1 and titrations of WT Cdc34 protein. Each lane represents a single ubiquitylation reaction that was quenched with 2× SDS-PAGE loading buffer after 10 min. Error bars represent the S.E. of measurements from duplicate data points. B , same as in A except with WT Ubc1. Reactions were quenched with SDS-PAGE loading buffer after 6 min. C , same as in A except with Δ190 Cdc34. Notice that deletion of the Cdc34 acidic tail does not affect the affinity of Cdc34 for San1.

    Techniques Used: SDS Page

    San1 functions preferentially with Ubc1 over WT Cdc34. A , San1 peptide multi-turnover ubiquitylation reactions were carried out in the presence of KR San1 and either WT Ubc1 alone, WT Cdc34 alone, or Ubc1 and WT Cdc34 in combination. Notice that reactions with Ubc1 show intense high molecular weight smears corresponding to long polyubiquitin chains on substrate in comparison with the reaction containing WT Cdc34 alone. B , quantification of the conversion of San1 peptide substrate into products containing one or more ubiquitins. Notice that substrate is converted to product more rapidly in the presence of Ubc1 than with WT Cdc34, and the reaction with both Ubc1 and WT Cdc34 does not further enhance product formation in comparison with Ubc1 alone. Error bars represent the S.E. of measurement derived from duplicate data points.
    Figure Legend Snippet: San1 functions preferentially with Ubc1 over WT Cdc34. A , San1 peptide multi-turnover ubiquitylation reactions were carried out in the presence of KR San1 and either WT Ubc1 alone, WT Cdc34 alone, or Ubc1 and WT Cdc34 in combination. Notice that reactions with Ubc1 show intense high molecular weight smears corresponding to long polyubiquitin chains on substrate in comparison with the reaction containing WT Cdc34 alone. B , quantification of the conversion of San1 peptide substrate into products containing one or more ubiquitins. Notice that substrate is converted to product more rapidly in the presence of Ubc1 than with WT Cdc34, and the reaction with both Ubc1 and WT Cdc34 does not further enhance product formation in comparison with Ubc1 alone. Error bars represent the S.E. of measurement derived from duplicate data points.

    Techniques Used: Molecular Weight, Derivative Assay

    San1 peptide is rapidly ubiquitylated in the presence of KR San1. A , multi-turnover ubiquitylation reactions were carried out in the presence of WT Cdc34 and either KR San1 or N13K/R280A San1. Notice that San1 peptide became ubiquitylated only in the presence of KR San1, indicating that the San1 RING domain and subsequent recruitment of Cdc34∼ubiquitin are required for substrate ubiquitylation. PO (peptide only) indicates a reaction containing San1 peptide where all additional reaction components were excluded. B , multi-turnover ubiquitylation reactions in either the presence of WT Cdc34 or WT Ubc1 and either WT ubiquitin or a Lys-48-only ubiquitin mutant ( K48O ). Notice that product formation is similar in reactions containing WT or K48O ubiquitin, indicating that both WT Cdc34 and Ubc1 likely generate polyubiquitin chains on San1 peptide that are Lys-48-specific.
    Figure Legend Snippet: San1 peptide is rapidly ubiquitylated in the presence of KR San1. A , multi-turnover ubiquitylation reactions were carried out in the presence of WT Cdc34 and either KR San1 or N13K/R280A San1. Notice that San1 peptide became ubiquitylated only in the presence of KR San1, indicating that the San1 RING domain and subsequent recruitment of Cdc34∼ubiquitin are required for substrate ubiquitylation. PO (peptide only) indicates a reaction containing San1 peptide where all additional reaction components were excluded. B , multi-turnover ubiquitylation reactions in either the presence of WT Cdc34 or WT Ubc1 and either WT ubiquitin or a Lys-48-only ubiquitin mutant ( K48O ). Notice that product formation is similar in reactions containing WT or K48O ubiquitin, indicating that both WT Cdc34 and Ubc1 likely generate polyubiquitin chains on San1 peptide that are Lys-48-specific.

    Techniques Used: Mutagenesis

    Single lysine San1 is autoubiquitylated more rapidly in the presence of WT Ubc1 than with Cdc34. A , time-course showing autoubiquitylation of radiolabeled N13K San1 in the presence of WT Ubc1, E1, and ubiquitin. B , quantitation of the fraction of N13K San1 that had been modified by one or more ubiquitins. Notice that product formation strays from linearity early in the time-course due to rapid conversion of San1 into product. Error bars represent the S.E. of measurements from duplicate data points. C , same as in A but with N13K/R280A San1. D , same as in A but with N444K San1. E , same as in B but with N444K San1.
    Figure Legend Snippet: Single lysine San1 is autoubiquitylated more rapidly in the presence of WT Ubc1 than with Cdc34. A , time-course showing autoubiquitylation of radiolabeled N13K San1 in the presence of WT Ubc1, E1, and ubiquitin. B , quantitation of the fraction of N13K San1 that had been modified by one or more ubiquitins. Notice that product formation strays from linearity early in the time-course due to rapid conversion of San1 into product. Error bars represent the S.E. of measurements from duplicate data points. C , same as in A but with N13K/R280A San1. D , same as in A but with N444K San1. E , same as in B but with N444K San1.

    Techniques Used: Quantitation Assay, Modification

    75) Product Images from "A Conserved Threonine Residue in the Juxtamembrane Domain of the XA21 Pattern Recognition Receptor Is Critical for Kinase Autophosphorylation and XA21-mediated Immunity *"

    Article Title: A Conserved Threonine Residue in the Juxtamembrane Domain of the XA21 Pattern Recognition Receptor Is Critical for Kinase Autophosphorylation and XA21-mediated Immunity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.093427

    Kinase autophosphorylation assay for the XA21 variants. A , the structures of XA21 variants. B , the autophosphorylation assay for the E. coli -expressed, GST-fused XA21 variant proteins. The upper panel shows the autoradiograph ( auto-rad ) and the lower panel is a photograph of the gel stained with Coomassie Brilliant Blue ( CBB ). K668 , K668 K736E , K690 , K705 , and JM denote GST-XA21K668, GST-XA21K668 K736E , GST-XA21K690, GST-XA21K705, and GST-XA21K668JM, respectively, and GST denotes GST alone.
    Figure Legend Snippet: Kinase autophosphorylation assay for the XA21 variants. A , the structures of XA21 variants. B , the autophosphorylation assay for the E. coli -expressed, GST-fused XA21 variant proteins. The upper panel shows the autoradiograph ( auto-rad ) and the lower panel is a photograph of the gel stained with Coomassie Brilliant Blue ( CBB ). K668 , K668 K736E , K690 , K705 , and JM denote GST-XA21K668, GST-XA21K668 K736E , GST-XA21K690, GST-XA21K705, and GST-XA21K668JM, respectively, and GST denotes GST alone.

    Techniques Used: Variant Assay, Autoradiography, Staining

    Kinase autophosphorylation assay for the GST-XA21K668 variants. The autophosphorylation assay was performed for the E. coli -expressed, GST-fused XA21K668 variant proteins. The upper panel shows the autoradiograph (autorad) and the lower panel is a photograph of the gel stained with Coomassie Brilliant Blue ( CBB ). K668, K668 K736E , K668 S697A , K668 S697D , K668 T705A , K668 T705E , K668 T680A , K668 S686A/T688A/S689A , and K668 S699A , denote their XA21 GST fusion proteins, respectively.
    Figure Legend Snippet: Kinase autophosphorylation assay for the GST-XA21K668 variants. The autophosphorylation assay was performed for the E. coli -expressed, GST-fused XA21K668 variant proteins. The upper panel shows the autoradiograph (autorad) and the lower panel is a photograph of the gel stained with Coomassie Brilliant Blue ( CBB ). K668, K668 K736E , K668 S697A , K668 S697D , K668 T705A , K668 T705E , K668 T680A , K668 S686A/T688A/S689A , and K668 S699A , denote their XA21 GST fusion proteins, respectively.

    Techniques Used: Variant Assay, Autoradiography, Staining

    Identification of Thr 705 as an autophosphorylation site by mass spectrometry. GST-XA21K668 protein was incubated with or without the ATPase His-tagged XB24 ( His-XB24 ). The purified GST-XA21K668 protein was subject to LC-MS/MS analysis. The top panel shows the spectrum of the peptide containing the Thr 705 residue from the GST-XA21K668 protein subjected to kinase autophosphorylation in the presence of His-XB24. The spectrum was generated using Scaffold Viewer 2.0. T + 80 , indicated by the arrows , designates phosphorylated Thr 705 . The bottom panel summarizes the highest probability of phosphorylation of the peptide containing Thr 705 . GST-K668 , GST-K668 + ATP , and GST-K668 + ATP + His-XB24 represent the different treatments of the GST-XA21K668 protein, namely GST-K668 only, GST-K668 subjected to kinase autophosphorylation, and GST-K668 subjected to kinase autophosphorylation in the presence of His-tagged XB24, respectively.
    Figure Legend Snippet: Identification of Thr 705 as an autophosphorylation site by mass spectrometry. GST-XA21K668 protein was incubated with or without the ATPase His-tagged XB24 ( His-XB24 ). The purified GST-XA21K668 protein was subject to LC-MS/MS analysis. The top panel shows the spectrum of the peptide containing the Thr 705 residue from the GST-XA21K668 protein subjected to kinase autophosphorylation in the presence of His-XB24. The spectrum was generated using Scaffold Viewer 2.0. T + 80 , indicated by the arrows , designates phosphorylated Thr 705 . The bottom panel summarizes the highest probability of phosphorylation of the peptide containing Thr 705 . GST-K668 , GST-K668 + ATP , and GST-K668 + ATP + His-XB24 represent the different treatments of the GST-XA21K668 protein, namely GST-K668 only, GST-K668 subjected to kinase autophosphorylation, and GST-K668 subjected to kinase autophosphorylation in the presence of His-tagged XB24, respectively.

    Techniques Used: Mass Spectrometry, Incubation, Purification, Liquid Chromatography with Mass Spectroscopy, Generated

    Kinase autophosphorylation analyses of XA21K668 and its single amino acid mutants T705A and T705E. A , kinase autophosphorylation was determined for E. coli -expressed wild-type GST-XA21K668 ( K668 ) and the GST-XA21K668 T705A ( T705A ), and GST-XA21K668 T705E ( T705E ) mutants. B , kinase autophosphorylation was determined for rice-expressed Myc-XA21 ( XA21 ) and mutants Myc-XA21 T705A ( T705A ) and Myc-XA21 T705E ( T705E ). The upper panel in A show a Coomassie Brilliant Blue-stained gel and the upper panel in B shows a Western blot ( WB ) probed with an anti-Myc antibody to normalize protein loading. The middle panels show typical autoradiographs of the indicated proteins, and the bottom panels show quantification of kinase autophosphorylation. Error bars indicate the standard deviations from three independent experiments.
    Figure Legend Snippet: Kinase autophosphorylation analyses of XA21K668 and its single amino acid mutants T705A and T705E. A , kinase autophosphorylation was determined for E. coli -expressed wild-type GST-XA21K668 ( K668 ) and the GST-XA21K668 T705A ( T705A ), and GST-XA21K668 T705E ( T705E ) mutants. B , kinase autophosphorylation was determined for rice-expressed Myc-XA21 ( XA21 ) and mutants Myc-XA21 T705A ( T705A ) and Myc-XA21 T705E ( T705E ). The upper panel in A show a Coomassie Brilliant Blue-stained gel and the upper panel in B shows a Western blot ( WB ) probed with an anti-Myc antibody to normalize protein loading. The middle panels show typical autoradiographs of the indicated proteins, and the bottom panels show quantification of kinase autophosphorylation. Error bars indicate the standard deviations from three independent experiments.

    Techniques Used: Staining, Western Blot

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    Activity Assay:

    Article Title: The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities
    Article Snippet: .. RT Assays RT activity was assayed for 45 min at 37°C in 14 µl of reaction medium containing 10 mM KCl, 10 mM MgCl2 , 50 mM Tris-HCl at pH 8.0, 5 mM DTT, 0.05% NP40, 1 µg of poly(rA)-oligo(dT)12–18 (Amersham), 10 µCi of [α-32 P]dTTP (3,000 Ci/mmole, Perkin-Elmer) and 1 µg of RNase A (Sigma Aldrich), as described . ..

    Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation
    Article Snippet: Paragraph title: In vitro BER-, DNA-glycosylase- and AP-site cleavage activity assays ... Excision activities by purified proteins were measured using recombinant human His-tagged UNG2, SMUG1, or TDG, 0.1 pmol oligonucleotide substrate in UDG buffer ( ) containing 50 mM NaCl and 0.1 pmol recombinant hAPE1 ( ) after incubation at 37°C for 30 min. BER incorporation assays were carried out in the same buffer as BER/MMR assays, supplemented with 3 µCi dCTP or dTTP (3000 Ci/mmol, Perkin-Elmer) essentially as described ( ).

    Expressing:

    Article Title: Identification of DNA primase inhibitors via a combined fragment-based and virtual screening
    Article Snippet: Paragraph title: Protein expression and purification ... [γ–32 P] dATP (800 Ci/mmol), [α–32 P] CTP, and dTTP (800 Ci/mmol) were from Perkin Elmer.

    Article Title: Impact of macromolecular crowding on DNA replication
    Article Snippet: Paragraph title: Protein expression and purification ... [γ–32 P] dATP (800 Ci/mmol), [α–32 P] CTP, and dTTP (800 Ci/mmol) were from Perkin Elmer.

    Modification:

    Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation
    Article Snippet: In vitro BER-, DNA-glycosylase- and AP-site cleavage activity assays U, FU- and HmU-DNA excision activities were measured using a 5′-end labelled FAM or 33 P-labelled 22-mer oligodeoxynucleotide containing a centrally positioned modified base (5′-GATCCTCTAGAGT-X-GACCTGCA-3′, where X = FU, HmU or U). .. Excision activities by purified proteins were measured using recombinant human His-tagged UNG2, SMUG1, or TDG, 0.1 pmol oligonucleotide substrate in UDG buffer ( ) containing 50 mM NaCl and 0.1 pmol recombinant hAPE1 ( ) after incubation at 37°C for 30 min. BER incorporation assays were carried out in the same buffer as BER/MMR assays, supplemented with 3 µCi dCTP or dTTP (3000 Ci/mmol, Perkin-Elmer) essentially as described ( ).

    Protease Inhibitor:

    Article Title: Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase ?
    Article Snippet: [α-32 P]dCTP (3000 Ci/mmol), [α-32 P] dTTP (3000 Ci/mmol), [α-32 P] Cordycepin (3000 Ci/mmol) from PerkinElmer Life Sciences (Waltham, MA, USA), dCTP, ddCTP and MicroSpin G-25 columns were from GE Healthcare (Piscataway, NJ, USA). .. Protease inhibitor cocktail was from Roche Diagnostic Corp. (Indianapolis, IN, USA).

    Footprinting:

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression
    Article Snippet: Plasmids pGL3-basic, pRL-TK, and pGEM-T Easy and a luciferase reporter assay system as well as a footprinting kit were purchased from Promega. .. Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences.

    Northern Blot:

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression
    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences. .. Probes for Northern analysis and oligonucleotides based on blr1 promoter sequences for electromobility shift assays (EMSAs) were ordered from Operon Qiagen.

    Oligonucleotide Labeling:

    Article Title: The Zn-finger domain of human PrimPol is required to stabilize the initiating nucleotide during DNA priming
    Article Snippet: Radiolabeled nucleotides [γ-32 P]ATP , [α-32 P]dGTP, [γ-32 P]GTP and [α-32 P]dTTP (250 μCi; 3000 Ci/mmol,) were purchased from Perkin Elmer (Waltham, MA, USA). .. T4 polynucleotide kinase, used for 5′ oligonucleotide labeling, was supplied by New England Biolabs (Ipswich, MA, USA).

    Article Title: The Zn-finger domain of human PrimPol is required to stabilize the initiating nucleotide during DNA priming
    Article Snippet: Radiolabeled nucleotides [γ-32 P] ATP , [α-32 P]dGTP, [γ-32 P] GTP and [α-32 P]dTTP (250 μCi; 3000 Ci/mmol,) were purchased from Perkin Elmer (Waltham, MA, USA). .. T4 polynucleotide kinase, used for 5′ oligonucleotide labeling, was supplied by New England Biolabs (Ipswich, MA, USA).

    other:

    Article Title: DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork *DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork * ♦
    Article Snippet: [α-32 P]dATP and [α-32 P]dTTP were purchased from PerkinElmer Life Sciences.

    Article Title: The Specificity and Flexibility of L1 Reverse Transcription Priming at Imperfect T-Tracts
    Article Snippet: Direct L1 extension assay (DLEA) Reverse transcriptase assays were carried out for 4 min at 37°C in 25 µL reactions containing 2 µg of RNPs, 400 nM of primer, 50 mM Tris-HCl [pH 7.5], 50 mM KCl, 5 mM MgCl2 , 10 mM DTT, 0.05% (v/v) Tween-20 and 10 µCi of α-32 P-dTTP (3000 Ci/mmol, PerkinElmer).

    Article Title: Kinetic Mechanism of DNA Polymerization Catalyzed by Human DNA Polymerase ε
    Article Snippet: These chemicals were purchased from the following companies: [α-32 P]dTTP and [γ-32 P]ATP from Perkin-Elmer Life Sciences (Boston, MA), dNTPs from Bioline (Taunton, MA), S p -dTTPαS and S p -dATPαS from Biolog-Life Science Institute (Bremen, Germany).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression
    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences. .. A TITANIUM One-Step reverse transcriptase PCR (RT-PCR) kit was purchased from BD Biosciences.

    Recombinant:

    Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation
    Article Snippet: .. Excision activities by purified proteins were measured using recombinant human His-tagged UNG2, SMUG1, or TDG, 0.1 pmol oligonucleotide substrate in UDG buffer ( ) containing 50 mM NaCl and 0.1 pmol recombinant hAPE1 ( ) after incubation at 37°C for 30 min. BER incorporation assays were carried out in the same buffer as BER/MMR assays, supplemented with 3 µCi dCTP or dTTP (3000 Ci/mmol, Perkin-Elmer) essentially as described ( ). ..

    Mutagenesis:

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression
    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences. .. A random primer labeling system and mutagenesis reagents were obtained from Stratagene.

    Labeling:

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression
    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences. .. A random primer labeling system and mutagenesis reagents were obtained from Stratagene.

    Purification:

    Article Title: Identification of DNA primase inhibitors via a combined fragment-based and virtual screening
    Article Snippet: Paragraph title: Protein expression and purification ... [γ–32 P] dATP (800 Ci/mmol), [α–32 P] CTP, and dTTP (800 Ci/mmol) were from Perkin Elmer.

    Article Title: Impact of macromolecular crowding on DNA replication
    Article Snippet: Paragraph title: Protein expression and purification ... [γ–32 P] dATP (800 Ci/mmol), [α–32 P] CTP, and dTTP (800 Ci/mmol) were from Perkin Elmer.

    Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation
    Article Snippet: .. Excision activities by purified proteins were measured using recombinant human His-tagged UNG2, SMUG1, or TDG, 0.1 pmol oligonucleotide substrate in UDG buffer ( ) containing 50 mM NaCl and 0.1 pmol recombinant hAPE1 ( ) after incubation at 37°C for 30 min. BER incorporation assays were carried out in the same buffer as BER/MMR assays, supplemented with 3 µCi dCTP or dTTP (3000 Ci/mmol, Perkin-Elmer) essentially as described ( ). ..

    Article Title: Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase ?
    Article Snippet: [α-32 P]dCTP (3000 Ci/mmol), [α-32 P] dTTP (3000 Ci/mmol), [α-32 P] Cordycepin (3000 Ci/mmol) from PerkinElmer Life Sciences (Waltham, MA, USA), dCTP, ddCTP and MicroSpin G-25 columns were from GE Healthcare (Piscataway, NJ, USA). .. Human pol β ( , ), UDG , APE , DNA ligase I ( and human pol θ domain ( were purified as described.

    Polymerase Chain Reaction:

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression
    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences. .. A TITANIUM One-Step reverse transcriptase PCR (RT-PCR) kit was purchased from BD Biosciences.

    SDS Page:

    Article Title: Identification of DNA primase inhibitors via a combined fragment-based and virtual screening
    Article Snippet: [γ–32 P] dATP (800 Ci/mmol), [α–32 P] CTP, and dTTP (800 Ci/mmol) were from Perkin Elmer. .. All chemical reagents were of molecular biology grade (Sigma); ATP and CTP (Roche Molecular Biochemicals). dNTPs and ddNTPs were purchased from USB Corp. Premade gels (10–20% linear gradients) used for SDS–PAGE and Precision Plus Protein prestained standards were purchased from BioRad (Hercules, CA).

    Software:

    Article Title: The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities
    Article Snippet: RT Assays RT activity was assayed for 45 min at 37°C in 14 µl of reaction medium containing 10 mM KCl, 10 mM MgCl2 , 50 mM Tris-HCl at pH 8.0, 5 mM DTT, 0.05% NP40, 1 µg of poly(rA)-oligo(dT)12–18 (Amersham), 10 µCi of [α-32 P]dTTP (3,000 Ci/mmole, Perkin-Elmer) and 1 µg of RNase A (Sigma Aldrich), as described . .. After an overnight exposure on a phosphor screen (Molecular Dynamics PhosphorImager System; GE Healthcare Bio-Sciences), radioactive spots were detected by the Storm system (GE-Healthcare Bio-Sciences) and data were analyzed with ImageQuant software (GE Healthcare Life Sciences).

    In Vitro:

    Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation
    Article Snippet: Paragraph title: In vitro BER-, DNA-glycosylase- and AP-site cleavage activity assays ... Excision activities by purified proteins were measured using recombinant human His-tagged UNG2, SMUG1, or TDG, 0.1 pmol oligonucleotide substrate in UDG buffer ( ) containing 50 mM NaCl and 0.1 pmol recombinant hAPE1 ( ) after incubation at 37°C for 30 min. BER incorporation assays were carried out in the same buffer as BER/MMR assays, supplemented with 3 µCi dCTP or dTTP (3000 Ci/mmol, Perkin-Elmer) essentially as described ( ).

    Article Title: Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *
    Article Snippet: .. For nucleotide specificity assays, in vitro reactions were conducted as above with α-32 P-labeled ATP, GTP, CTP, UTP, or dTTP (PerkinElmer Life Sciences) containing 1 mm of each respective cold dNTP. .. Adenylylation was visualized by autoradiography at various exposures.

    Staining:

    Article Title: Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *
    Article Snippet: Protein load was visualized by Ponceau S staining. .. For nucleotide specificity assays, in vitro reactions were conducted as above with α-32 P-labeled ATP, GTP, CTP, UTP, or dTTP (PerkinElmer Life Sciences) containing 1 mm of each respective cold dNTP.

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  • 99
    PerkinElmer γ 32 p atp
    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 <t>P]-ATP.</t> Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 10 article reviews
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    99
    PerkinElmer atp
    The phosphoadaptor subunit <t>Cks1</t> provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 <t>P-ATP.</t> The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.
    Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Journal: Biochemical pharmacology

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

    doi: 10.1016/j.bcp.2013.10.015

    Figure Lengend Snippet: PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Article Snippet: Different amount of His-PXR as indicated (1 µg or 2.5 µg) was incubated in kinase buffer with 20 ng Cdk2/cyclin E (EMD Millipore, Billerica, MA), 5 µCi [γ-32 P]ATP (Perkin-Elmer, Santa Clara, CA), and 5 µM cold ATP.

    Techniques: In Vitro, Incubation, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, In Vivo, Modification, Sequencing, Ion Chromatography, Mutagenesis, Derivative Assay

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Article Snippet: Radiolabeled RNAs were prepared with γ-32 P-ATP (PerkinElmer) and T4 PNK (New England Biolabs) and purified by 20% denaturing PAGE followed by ethanol precipitation.

    Techniques: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Journal: Molecular Biology of the Cell

    Article Title: ?-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

    doi: 10.1091/mbc.E10-07-0636

    Figure Lengend Snippet: Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Article Snippet: In Vitro Kinase Assays pET and pET-Aly2 were incubated for 30 min at 30°C in kinase buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2 , 1 mM DTT, 1 μM unlabeled ATP, aprotinin, and leupeptin) with 75 nM [γ-32 P]ATP (Perkin Elmer-Cetus) with or without Npr1 kinase (purified from Y258 yeast cells).

    Techniques: Expressing, Copurification, Western Blot, Fluorescence, Microscopy, Purification, Positron Emission Tomography, Incubation, SDS Page, Staining

    The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.

    Journal: Nature

    Article Title: Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

    doi: 10.1038/nature10560

    Figure Lengend Snippet: The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.

    Article Snippet: The general composition of the assay mixture was as follows: 50 mM Hepes pH 7.4, 100 mM NaCl, 0.1% NP-40, 20 mM imidazole, 2% glycerol, 2 mM EGTA, 0.2 mg/ml BSA, 500 nM Cks1 and 500 μM ATP (with added γ-32 P-ATP (Perkin Elmer)).

    Techniques: Incubation, Binding Assay, SDS Page, Sequencing, Labeling