atp biotin  (New England Biolabs)


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    Name:
    Adenosine 5 Triphosphate ATP
    Description:
    Adenosine 5 Triphosphate ATP 5 ml
    Catalog Number:
    p0756l
    Price:
    140
    Size:
    5 ml
    Category:
    Nucleotides
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    Structured Review

    New England Biolabs atp biotin
    Adenosine 5 Triphosphate ATP
    Adenosine 5 Triphosphate ATP 5 ml
    https://www.bioz.com/result/atp biotin/product/New England Biolabs
    Average 99 stars, based on 173 article reviews
    Price from $9.99 to $1999.99
    atp biotin - by Bioz Stars, 2021-02
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    Images

    1) Product Images from "Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: Implications for phosphoproteomics"

    Article Title: Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: Implications for phosphoproteomics

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201200626

    Gel analysis of phosphoproteins (ProQ, top) or biotinylated phosphoproteins (SA-Cy5, bottom) from HeLa cell lysates incubated with ATP or ATP-biotin in the absence or presence of kinase or phosphatase inhibitors.
    Figure Legend Snippet: Gel analysis of phosphoproteins (ProQ, top) or biotinylated phosphoproteins (SA-Cy5, bottom) from HeLa cell lysates incubated with ATP or ATP-biotin in the absence or presence of kinase or phosphatase inhibitors.

    Techniques Used: Incubation

    (A) Reactions of HeLa cell lysates and ATP without (untreated) or with phosphatase inhibitors (phosphatase inhibitor treated). Proteins were visualized with Pro-Q diamond phosphoprotein stain. (B) Reactions of HeLa cell lysates and ATP-biotin without
    Figure Legend Snippet: (A) Reactions of HeLa cell lysates and ATP without (untreated) or with phosphatase inhibitors (phosphatase inhibitor treated). Proteins were visualized with Pro-Q diamond phosphoprotein stain. (B) Reactions of HeLa cell lysates and ATP-biotin without

    Techniques Used: Staining

    2) Product Images from "RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling"

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    Journal: mBio

    doi: 10.1128/mBio.00833-16

    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
    Figure Legend Snippet: RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Techniques Used: Western Blot, Incubation, RNA Binding Assay, Protease Inhibitor

    RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.
    Figure Legend Snippet: RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Techniques Used: Western Blot

    3) Product Images from "Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication"

    Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Journal: Cancers

    doi: 10.3390/cancers6031464

    MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.
    Figure Legend Snippet: MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

    Techniques Used: Sequencing, Clone Assay, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Labeling, Western Blot, Purification, Mutagenesis, Affinity Purification, Negative Control

    4) Product Images from "Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain"

    Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

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

    doi: 10.1073/pnas.1516729112

    PELP1 is a novel substrate of GSK3β. Hippocampal lysates collected 24 h after GCI reperfusion were subjected to immunoprecipitation with PELP1 or IgG antibody. Immunoprecipitates were pulled down by adding dynabeads protein A. Immunoprecipitates were subjected to SDS/PAGE, followed by mass spectrometry analysis. ( A ) List of unique PELP1-interacting proteins. ( B ) Forebrain lysates were subjected to immunoprecipitation or GST pull-down assay, and PELP1 interaction with GSK3β was confirmed by Western blotting. ( C ) Forebrain lysates were used in pull-down assays using GST or various deletion fragments of PELP1, and interaction of PELP1 deletions with GSK3β was analyzed by Western blotting. ( D ) GST-tagged PELP1 deletions and full-length GST-PELP1 were incubated with purified GSK3β enzyme in kinase buffer with [ γ32 -P] ATP and 100 μM cold ATP for 30 min at 30 °C, and the phosphorylation of PELP1 fragments was analyzed by PhosphorImager.
    Figure Legend Snippet: PELP1 is a novel substrate of GSK3β. Hippocampal lysates collected 24 h after GCI reperfusion were subjected to immunoprecipitation with PELP1 or IgG antibody. Immunoprecipitates were pulled down by adding dynabeads protein A. Immunoprecipitates were subjected to SDS/PAGE, followed by mass spectrometry analysis. ( A ) List of unique PELP1-interacting proteins. ( B ) Forebrain lysates were subjected to immunoprecipitation or GST pull-down assay, and PELP1 interaction with GSK3β was confirmed by Western blotting. ( C ) Forebrain lysates were used in pull-down assays using GST or various deletion fragments of PELP1, and interaction of PELP1 deletions with GSK3β was analyzed by Western blotting. ( D ) GST-tagged PELP1 deletions and full-length GST-PELP1 were incubated with purified GSK3β enzyme in kinase buffer with [ γ32 -P] ATP and 100 μM cold ATP for 30 min at 30 °C, and the phosphorylation of PELP1 fragments was analyzed by PhosphorImager.

    Techniques Used: Immunoprecipitation, SDS Page, Mass Spectrometry, Pull Down Assay, Western Blot, Incubation, Purification

    5) Product Images from "Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP"

    Article Title: Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

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

    doi: 10.1073/pnas.1510132112

    Delayed maturation kinetics and suboptimal assembly of MHC class I molecules in cells expressing calreticulin mutants that disrupt ATP or high-affinity calcium binding: CRT −/− MEFs were infected with retroviruses encoding the indicated
    Figure Legend Snippet: Delayed maturation kinetics and suboptimal assembly of MHC class I molecules in cells expressing calreticulin mutants that disrupt ATP or high-affinity calcium binding: CRT −/− MEFs were infected with retroviruses encoding the indicated

    Techniques Used: Expressing, Binding Assay, Infection

    Proposed role for ATP in promoting the release of MHC class I from the PLC. Within the PLC, calreticulin functions as a chaperone to stabilize MHC class I interactions with the PLC. Interaction of calreticulin with ATP is predicted to destabilize the
    Figure Legend Snippet: Proposed role for ATP in promoting the release of MHC class I from the PLC. Within the PLC, calreticulin functions as a chaperone to stabilize MHC class I interactions with the PLC. Interaction of calreticulin with ATP is predicted to destabilize the

    Techniques Used: Planar Chromatography

    ATP induces dissociation of MHC class I from the PLC ( A ) and interacts with the globular domain of calreticulin ( B – E ). ( A ) Gel panel shows a representative anti-TAP IP of lysates from [ 35 S]-methionine–labeled CRT −/− MEFs
    Figure Legend Snippet: ATP induces dissociation of MHC class I from the PLC ( A ) and interacts with the globular domain of calreticulin ( B – E ). ( A ) Gel panel shows a representative anti-TAP IP of lysates from [ 35 S]-methionine–labeled CRT −/− MEFs

    Techniques Used: Planar Chromatography, Labeling

    6) Product Images from "Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis"

    Article Title: Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis

    Journal: Journal of proteome research

    doi: 10.1021/pr9004844

    Two-dimensional gel analysis of in vitro phosphorylated mitochondrial ribosomal proteins using [γ- 32 P] ATP. Bovine mitochondrial ribosomes (1.8 A 260 units) were incubated in the presence of 50 μCi [γ- 32 P] ATP for 1 hr at 30 °C
    Figure Legend Snippet: Two-dimensional gel analysis of in vitro phosphorylated mitochondrial ribosomal proteins using [γ- 32 P] ATP. Bovine mitochondrial ribosomes (1.8 A 260 units) were incubated in the presence of 50 μCi [γ- 32 P] ATP for 1 hr at 30 °C

    Techniques Used: Two-Dimensional Gel Electrophoresis, In Vitro, Incubation

    7) Product Images from "Tetrameric architecture of an active phenol-bound form of the AAA+ transcriptional regulator DmpR"

    Article Title: Tetrameric architecture of an active phenol-bound form of the AAA+ transcriptional regulator DmpR

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16562-5

    Oligomerization and ATPase activities of DmpR derivatives. a Schematic diagram of wild type DmpR (DmpR WT ) and truncated DmpR proteins (DmpR ΔD , residues 18–481; DmpR C , residues 232–481; DmpR BC , residues 205–481; and DmpR ΔS , residues 205–563). The molecular weights given include the N-terminal 6 × His tag. b BN-PAGE of truncated DmpR proteins after the addition of ATP or ATPγS. Bands corresponding to tetramers are marked by an arrow. Asterisks indicate an artefact band of DmpR ΔS . This data is representative of three replicates with similar results. c SEC combined with multi-angle light scattering (MALS) analysis to calculate the molecular weight (MW) of DmpR ΔS (dotted black line). d ATPase activity of DmpR WT and DmpR ΔD in the presence or absence of phenol. Hydrolysis of [γ-P 32 ] ATP to generate P 32 was visualised by thin-layer chromatography. ‘C’ represents the reaction with the reaction buffer as control. ‘P’ represents the reaction with the alkaline phosphatase as positive control. This data is representative of five replicates with similar results. e ATPase activities of truncated DmpR derivatives. This data is representative of five replicates with similar results.
    Figure Legend Snippet: Oligomerization and ATPase activities of DmpR derivatives. a Schematic diagram of wild type DmpR (DmpR WT ) and truncated DmpR proteins (DmpR ΔD , residues 18–481; DmpR C , residues 232–481; DmpR BC , residues 205–481; and DmpR ΔS , residues 205–563). The molecular weights given include the N-terminal 6 × His tag. b BN-PAGE of truncated DmpR proteins after the addition of ATP or ATPγS. Bands corresponding to tetramers are marked by an arrow. Asterisks indicate an artefact band of DmpR ΔS . This data is representative of three replicates with similar results. c SEC combined with multi-angle light scattering (MALS) analysis to calculate the molecular weight (MW) of DmpR ΔS (dotted black line). d ATPase activity of DmpR WT and DmpR ΔD in the presence or absence of phenol. Hydrolysis of [γ-P 32 ] ATP to generate P 32 was visualised by thin-layer chromatography. ‘C’ represents the reaction with the reaction buffer as control. ‘P’ represents the reaction with the alkaline phosphatase as positive control. This data is representative of five replicates with similar results. e ATPase activities of truncated DmpR derivatives. This data is representative of five replicates with similar results.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Molecular Weight, Activity Assay, Thin Layer Chromatography, Positive Control

    Phenol-binding pocket and the coiled-coil B-linkers. a Positions of phenol and zinc inside the core (β/α) 4 barrel scaffold of the sensory domain. b Key residues in the phenol-binding pocket and residues involved in zinc coordination are shown as sticks. c Depiction of the hydroxyl group of phenol interacting with His100 and Trp128 (left); sequence alignment of five aromatic compound-binding transcriptional activators (right). DmpR, P. putida KCTC 1452; PoxR, Ralstonia sp . E2; MopR, Acinetobacter guillouiae ; and XylR, Pseudomonas putida . d The interface of the coiled-coil B-linkers with the strips of leucine residues highlighted within the enlarged box. e B-factor putty tube representation of the B-linker in the ATPase domain connection loop. Orange and red colours and a wider tube indicate regions with high B-factors, whereas shades of green and narrower tubes indicate regions with low B-factors. The putative ATP-binding site is shown in dot representation.
    Figure Legend Snippet: Phenol-binding pocket and the coiled-coil B-linkers. a Positions of phenol and zinc inside the core (β/α) 4 barrel scaffold of the sensory domain. b Key residues in the phenol-binding pocket and residues involved in zinc coordination are shown as sticks. c Depiction of the hydroxyl group of phenol interacting with His100 and Trp128 (left); sequence alignment of five aromatic compound-binding transcriptional activators (right). DmpR, P. putida KCTC 1452; PoxR, Ralstonia sp . E2; MopR, Acinetobacter guillouiae ; and XylR, Pseudomonas putida . d The interface of the coiled-coil B-linkers with the strips of leucine residues highlighted within the enlarged box. e B-factor putty tube representation of the B-linker in the ATPase domain connection loop. Orange and red colours and a wider tube indicate regions with high B-factors, whereas shades of green and narrower tubes indicate regions with low B-factors. The putative ATP-binding site is shown in dot representation.

    Techniques Used: Binding Assay, Sequencing

    8) Product Images from "Intrinsic ubiquitin E3 ligase activity of histone acetyltransferase Hbo1 for estrogen receptor α"

    Article Title: Intrinsic ubiquitin E3 ligase activity of histone acetyltransferase Hbo1 for estrogen receptor α

    Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

    doi: 10.2183/pjab.93.030

    Self-ubiquitination of Hbo1. (A) E2 screen for Hbo1 E3 ligase assay. Ten different His-tagged E2s were incubated with (+) or without (−) commercial recombinant Hbo1 in the presence of His-tagged E1, ATP, and biotinylated ubiquitin. The reaction mixture was run on SDS-PAGE, transferred onto the membrane, and probed with Cy5-conjugated streptavidin to detect ubiquitin. Recombinant MurF1 protein (1.25 µg) served as a positive control for E3 ligase activity (lanes 1 and 2). (B) Recombinant Hbo1 protein from insect cells (lanes 1 and 4) and from bacteria (lanes 2 and 5), and recombinant ubiquitin (lanes 3 and 6), were blotted with anti-Hbo1 or anti-ubiquitin antibodies. Ub: ubiquitin. (C) Commercial recombinant Hbo1 protein was incubated with His-tagged E1, E2 (UbcH5c), ATP, and non-tagged ubiquitin. The reaction mixture was run on SDS-PAGE, transferred onto the membrane, and probed with anti-ubiquitin or anti-His antibodies. Recombinant MurF1 protein (1.25 µg) served as a positive control (lane 2). Ub: ubiquitin. (D) Ubiquitination reaction mixture performed in the listed combination using commercial Hbo1 protein and non-tagged ubiquitin, was blotted with anti-ubiquitin or anti-Hbo1 antibodies. (E) Hbo1 protein from Sf9 cells (1.2 µg) was incubated in the listed combinations for 3 hours. After removal of His-tagged E1 and E2 by mixing with Ni-NTA Agarose, the reaction mixture was blotted with Cy5-conjugated streptavidin to detect ubiquitin.
    Figure Legend Snippet: Self-ubiquitination of Hbo1. (A) E2 screen for Hbo1 E3 ligase assay. Ten different His-tagged E2s were incubated with (+) or without (−) commercial recombinant Hbo1 in the presence of His-tagged E1, ATP, and biotinylated ubiquitin. The reaction mixture was run on SDS-PAGE, transferred onto the membrane, and probed with Cy5-conjugated streptavidin to detect ubiquitin. Recombinant MurF1 protein (1.25 µg) served as a positive control for E3 ligase activity (lanes 1 and 2). (B) Recombinant Hbo1 protein from insect cells (lanes 1 and 4) and from bacteria (lanes 2 and 5), and recombinant ubiquitin (lanes 3 and 6), were blotted with anti-Hbo1 or anti-ubiquitin antibodies. Ub: ubiquitin. (C) Commercial recombinant Hbo1 protein was incubated with His-tagged E1, E2 (UbcH5c), ATP, and non-tagged ubiquitin. The reaction mixture was run on SDS-PAGE, transferred onto the membrane, and probed with anti-ubiquitin or anti-His antibodies. Recombinant MurF1 protein (1.25 µg) served as a positive control (lane 2). Ub: ubiquitin. (D) Ubiquitination reaction mixture performed in the listed combination using commercial Hbo1 protein and non-tagged ubiquitin, was blotted with anti-ubiquitin or anti-Hbo1 antibodies. (E) Hbo1 protein from Sf9 cells (1.2 µg) was incubated in the listed combinations for 3 hours. After removal of His-tagged E1 and E2 by mixing with Ni-NTA Agarose, the reaction mixture was blotted with Cy5-conjugated streptavidin to detect ubiquitin.

    Techniques Used: Incubation, Recombinant, SDS Page, Positive Control, Activity Assay

    Ubiquitination of ERα LBD by Hbo1. (A) GST-tagged p53 (2 µg) and GST-tagged Mdm2 (E3 ligase) were mixed in the indicated combination in the presence of E1, E2 (UbcH5c), biotinylated ubiquitin, and ATP for 2 hours, followed by affinity purification of GST-tagged p53 and Mdm2 proteins with Glutathione Sepharose under stringent condition and by immunoblotting with Cy5-strepavidion (left panel) or anti-GST antibody (right panel). Arrowhead: ubiquitinated E1. Bracket: ubiquitinated GST-p53. (B) Commercial Hbo1 protein (1.2 µg) was incubated in the listed combinations. GST-tagged ERα LBD protein was affinity-purified with Glutathione Sepharose under stringent condition, and assayed for ubiquitin (left panel) and GST (right panel). (C) No enzyme (lane 1), Recombinant His-Hbo1 (amino acids: 311–611) protein (lane 2: 1 µg; lane 3: 2.5 µg), and commercial Hbo1 (1.2 µg, lane 4) were assayed in the ubiquitination reaction, and GST-ERα LBD protein was stringently affinity-purified with Glutathione Sepharose. The purified proteins were separated, transferred onto membrane, and ubiquitin and GST were detected with Cy5-conjugated streptavidin (left panel) or with anti-GST antibody (right panel), respectively. The identity of the band between 50 and 75 kDa (*) is unknown.
    Figure Legend Snippet: Ubiquitination of ERα LBD by Hbo1. (A) GST-tagged p53 (2 µg) and GST-tagged Mdm2 (E3 ligase) were mixed in the indicated combination in the presence of E1, E2 (UbcH5c), biotinylated ubiquitin, and ATP for 2 hours, followed by affinity purification of GST-tagged p53 and Mdm2 proteins with Glutathione Sepharose under stringent condition and by immunoblotting with Cy5-strepavidion (left panel) or anti-GST antibody (right panel). Arrowhead: ubiquitinated E1. Bracket: ubiquitinated GST-p53. (B) Commercial Hbo1 protein (1.2 µg) was incubated in the listed combinations. GST-tagged ERα LBD protein was affinity-purified with Glutathione Sepharose under stringent condition, and assayed for ubiquitin (left panel) and GST (right panel). (C) No enzyme (lane 1), Recombinant His-Hbo1 (amino acids: 311–611) protein (lane 2: 1 µg; lane 3: 2.5 µg), and commercial Hbo1 (1.2 µg, lane 4) were assayed in the ubiquitination reaction, and GST-ERα LBD protein was stringently affinity-purified with Glutathione Sepharose. The purified proteins were separated, transferred onto membrane, and ubiquitin and GST were detected with Cy5-conjugated streptavidin (left panel) or with anti-GST antibody (right panel), respectively. The identity of the band between 50 and 75 kDa (*) is unknown.

    Techniques Used: Affinity Purification, Incubation, Recombinant, Purification

    9) Product Images from "An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme"

    Article Title: An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr480

    3 nM pBRsk1 plasmid DNA was digested with a substoichiometric amount of nuclease (1 nM) in the presence/absence of different amounts of R subunit. The leftmost lane shows DNA size markers as in Figure 1 . The next two lanes show uncut DNA and the partial cutting of the DNA when the ratio of nuclease to DNA was 1:3. The following lanes show the same ratio of nuclease to DNA of 1:3 but with additional R subunit being added. Ratio of nuclease to R subunit for Lanes 1–4 were 1:1, 1:3, 1:4 and 1:8, respectively. Lane 5, R subunit only. Lane 6, same as Lane 4 but no ATP. Lane 6, same as Lane 4 but no SAM.
    Figure Legend Snippet: 3 nM pBRsk1 plasmid DNA was digested with a substoichiometric amount of nuclease (1 nM) in the presence/absence of different amounts of R subunit. The leftmost lane shows DNA size markers as in Figure 1 . The next two lanes show uncut DNA and the partial cutting of the DNA when the ratio of nuclease to DNA was 1:3. The following lanes show the same ratio of nuclease to DNA of 1:3 but with additional R subunit being added. Ratio of nuclease to R subunit for Lanes 1–4 were 1:1, 1:3, 1:4 and 1:8, respectively. Lane 5, R subunit only. Lane 6, same as Lane 4 but no ATP. Lane 6, same as Lane 4 but no SAM.

    Techniques Used: Plasmid Preparation

    Gel assay to test reactivation of EcoKI by RecBCD. Lane 1, undigested pBRskI (3 nM); Lane 2, partial digest of plasmid using EcoKI (1.5 nM) showing some L DNA; Lane 3, partial digest of plasmid by 1.5 nM EcoKI as in Lane 2 but in the presence of RecBCD (10 nM) showing the removal of the L DNA; Lane 4, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest from Lane 2 and incubated for a further 5 min showing no further digestion but an increase in the amount of CCC DNA as expected; Lane 5, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing the increase in CCC DNA but no extra digestion; Lane 6, after 5 min an additional aliquot of ATP (2 nM), SAM (0.1 mM) and plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing that sufficient cofactors were present.
    Figure Legend Snippet: Gel assay to test reactivation of EcoKI by RecBCD. Lane 1, undigested pBRskI (3 nM); Lane 2, partial digest of plasmid using EcoKI (1.5 nM) showing some L DNA; Lane 3, partial digest of plasmid by 1.5 nM EcoKI as in Lane 2 but in the presence of RecBCD (10 nM) showing the removal of the L DNA; Lane 4, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest from Lane 2 and incubated for a further 5 min showing no further digestion but an increase in the amount of CCC DNA as expected; Lane 5, after 5 min digestion an additional aliquot of plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing the increase in CCC DNA but no extra digestion; Lane 6, after 5 min an additional aliquot of ATP (2 nM), SAM (0.1 mM) and plasmid DNA (3 nM) was added to the partial digest performed in the presence of RecBCD (Lane 3) and incubated for a further 5 min showing that sufficient cofactors were present.

    Techniques Used: Plasmid Preparation, Incubation, Countercurrent Chromatography

    ATP hydrolysis activity of EcoKI on various oligonucleotides duplexes as measured using the NADH-coupled enzyme assay. The assay contained 10 nM EcoKI, 5 nM of the appropriate DNA duplex given in Table 1 , 0.1 mM SAM and 2 mM ATP.
    Figure Legend Snippet: ATP hydrolysis activity of EcoKI on various oligonucleotides duplexes as measured using the NADH-coupled enzyme assay. The assay contained 10 nM EcoKI, 5 nM of the appropriate DNA duplex given in Table 1 , 0.1 mM SAM and 2 mM ATP.

    Techniques Used: Activity Assay, Nadh Coupled Enzyme Assay

    10) Product Images from "Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli"

    Article Title: Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli

    Journal: eLife

    doi: 10.7554/eLife.10807

    Recombination Intermediate Branch Migration. ( A ) Branch Migration of intermediates Mediated by RadA. Three-strand recombination reactions were stopped after 12 min and deproteinized and purified as described in the procedures. Branch migration assays contained DNA intermediates (100 ng), 1 µM RadA, 3 mM ATP, and 2.4 µM SSB when indicated. After incubation for the times incubated, reactions were stopped and products were resolved on an 0.8% TAE agarose gel. The No protein sample includes DNA intermediate fractions and ATP and was incubated for 30 min at 37 °C without RadA or SSB. ( B ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA. Amounts of each DNA species was determined from scanned digital photographs using ImageJ64 (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( C ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA and SSB.* Amounts of each DNA species was determined from scanned digital photographs using ImageJ64. (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( D ) Quantification of the Nicked Product (NP) and Duplex Linear Substrate (DLS) Formed in Branch Migration Assays. Graph shows the mean and standard deviation of the relative amounts of NP and DLS formed in three independent branch migration experiments. Two different RadA preparations and three different DNA Intermediate preparations were used in these experiments. ( E ) Nucleotide Dependence of the Branch Migration Assay. Reactions were performed as above except 1mM of the nucleotide indicated replaced 3mM ATP. Incubation was for 30 min at 37 °C. ( F ) Model Depicting RadA Directionality. In the absence of SSB, RadA (illustrated by wedge shape) preferentially migrates DNA, displacing a 5’ ssDNA flap. In the presence of SSB, the directional bias of RadA branch migration is largely eliminated. * No correction for the difference in binding affinity of ethidium bromide for single-strand and double-strand DNA was made. Thus, the absolute amount of the DNA species containing single-strand DNA may be underestimated. DOI: http://dx.doi.org/10.7554/eLife.10807.019
    Figure Legend Snippet: Recombination Intermediate Branch Migration. ( A ) Branch Migration of intermediates Mediated by RadA. Three-strand recombination reactions were stopped after 12 min and deproteinized and purified as described in the procedures. Branch migration assays contained DNA intermediates (100 ng), 1 µM RadA, 3 mM ATP, and 2.4 µM SSB when indicated. After incubation for the times incubated, reactions were stopped and products were resolved on an 0.8% TAE agarose gel. The No protein sample includes DNA intermediate fractions and ATP and was incubated for 30 min at 37 °C without RadA or SSB. ( B ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA. Amounts of each DNA species was determined from scanned digital photographs using ImageJ64 (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( C ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA and SSB.* Amounts of each DNA species was determined from scanned digital photographs using ImageJ64. (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( D ) Quantification of the Nicked Product (NP) and Duplex Linear Substrate (DLS) Formed in Branch Migration Assays. Graph shows the mean and standard deviation of the relative amounts of NP and DLS formed in three independent branch migration experiments. Two different RadA preparations and three different DNA Intermediate preparations were used in these experiments. ( E ) Nucleotide Dependence of the Branch Migration Assay. Reactions were performed as above except 1mM of the nucleotide indicated replaced 3mM ATP. Incubation was for 30 min at 37 °C. ( F ) Model Depicting RadA Directionality. In the absence of SSB, RadA (illustrated by wedge shape) preferentially migrates DNA, displacing a 5’ ssDNA flap. In the presence of SSB, the directional bias of RadA branch migration is largely eliminated. * No correction for the difference in binding affinity of ethidium bromide for single-strand and double-strand DNA was made. Thus, the absolute amount of the DNA species containing single-strand DNA may be underestimated. DOI: http://dx.doi.org/10.7554/eLife.10807.019

    Techniques Used: Migration, Purification, Incubation, Agarose Gel Electrophoresis, Standard Deviation, Binding Assay

    Four-strand Recombination Reactions in the Presence of RadA. ( A ) Diagram of the Four-strand Recombination Reaction. Gapped circular substrate (GS) prepared as described in the procedures was mixed with double-strand φX174 DNA linearized with Pst I (DLS) in the presence of RecA, SSB, RadA, ATP and an ATP regenerating system. Complex, largely duplex DNA intermediates are formed first. The final products are nicked circular double-DNA (NP) and Duplex Linear DNA with Single-strand Tails (DLP). Note: The tailed linear product species is not well-resolved from the duplex linear substrate (DLS). ( B ) Comparison of 3-strand and 4-strand Recombination Mediated by RecA in the Presence and Absence of RadA. Recombination reactions between either single-strand circular φX174 DNA (SCS) and double-strand φX174 DNA linearized with Pst I (DLS)-3-strand reactions or double-strand circular φX174 with a 1.3 kB single-strand gap (GS) and double-strand φX174 DNA linearized with Pst I (DLS)-4-strand reactions were performed as described. At the times indicated, reactions were stopped and de-proteinated. Products were resolved using an 1.0% agarose gel in TAE buffer. DOI: http://dx.doi.org/10.7554/eLife.10807.020
    Figure Legend Snippet: Four-strand Recombination Reactions in the Presence of RadA. ( A ) Diagram of the Four-strand Recombination Reaction. Gapped circular substrate (GS) prepared as described in the procedures was mixed with double-strand φX174 DNA linearized with Pst I (DLS) in the presence of RecA, SSB, RadA, ATP and an ATP regenerating system. Complex, largely duplex DNA intermediates are formed first. The final products are nicked circular double-DNA (NP) and Duplex Linear DNA with Single-strand Tails (DLP). Note: The tailed linear product species is not well-resolved from the duplex linear substrate (DLS). ( B ) Comparison of 3-strand and 4-strand Recombination Mediated by RecA in the Presence and Absence of RadA. Recombination reactions between either single-strand circular φX174 DNA (SCS) and double-strand φX174 DNA linearized with Pst I (DLS)-3-strand reactions or double-strand circular φX174 with a 1.3 kB single-strand gap (GS) and double-strand φX174 DNA linearized with Pst I (DLS)-4-strand reactions were performed as described. At the times indicated, reactions were stopped and de-proteinated. Products were resolved using an 1.0% agarose gel in TAE buffer. DOI: http://dx.doi.org/10.7554/eLife.10807.020

    Techniques Used: Agarose Gel Electrophoresis

    How branch migration assists homologous recombination. ( A ) Heteroduplex extension. In reactions between linear resected DNA and an intact chromosome, initial strand pairing and invasion may occur at a distance from the 3’ end. Branch migration of the D-loop (in direction of the arrow) allows the heteroduplex region to extend fully to the 3’ end, allowing it to be engaged by DNA polymerases. Branch migration also allows the D-loop to be extended, lengthening and stabilizing the region of heteroduplex and forming a 4-strand Holliday juntion. ( B ) Synthesis-dependent strand annealing (SDSA). After resection of a broken chromosome and strand invasion into a sister molecule, branch migration is required to dissolve the intermediate, allowing broken strands to anneal to one another and the break to be healed. Reactions contained 1 mM ATP and 20.3 µM (nucleotide) φX174 DNA. The values shown are the average of two experiments, except for the circular single-strand value. It is the average of five experiments. Standard deviations are reported. DOI: http://dx.doi.org/10.7554/eLife.10807.021
    Figure Legend Snippet: How branch migration assists homologous recombination. ( A ) Heteroduplex extension. In reactions between linear resected DNA and an intact chromosome, initial strand pairing and invasion may occur at a distance from the 3’ end. Branch migration of the D-loop (in direction of the arrow) allows the heteroduplex region to extend fully to the 3’ end, allowing it to be engaged by DNA polymerases. Branch migration also allows the D-loop to be extended, lengthening and stabilizing the region of heteroduplex and forming a 4-strand Holliday juntion. ( B ) Synthesis-dependent strand annealing (SDSA). After resection of a broken chromosome and strand invasion into a sister molecule, branch migration is required to dissolve the intermediate, allowing broken strands to anneal to one another and the break to be healed. Reactions contained 1 mM ATP and 20.3 µM (nucleotide) φX174 DNA. The values shown are the average of two experiments, except for the circular single-strand value. It is the average of five experiments. Standard deviations are reported. DOI: http://dx.doi.org/10.7554/eLife.10807.021

    Techniques Used: Migration, Homologous Recombination

    11) Product Images from "Characterization of Type II and III Restriction-Modification Systems from Bacillus cereus Strains ATCC 10987 and ATCC 14579"

    Article Title: Characterization of Type II and III Restriction-Modification Systems from Bacillus cereus Strains ATCC 10987 and ATCC 14579

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.06248-11

    Characterization of BceSIV endonuclease. (A) Schematic diagram of six ORFs of the BceSIV operon. The gene identifier (Bce_0364 to Bce_0370) is indicated below the ORFs. Bce_0364, a possible transcription regulator, similar to phage repressors; Bce_0365, M.BceSIV (GCWGC); Bce_0367, unknown function, no apparent endonuclease activity; Bce_0368, R1 subunit of BceSIV, NTPase/helicase; Bce_0369, BceSIV endonuclease catalytic subunit, R2 subunit; Bce_0370, putative transcription regular, similar to the controller (C) protein of other R-M systems. (B) Digestion of pBR322 DNA by cell extracts containing BceSIV R1, R2, and R1+R2 in the presence or absence of ATP (2 mM). Lanes 1 and 13, 1-kb DNA ladder; lanes 2 to 11 contain either R1, R2, or R1+R2 as indicated; lane 12, linear DNA; lane 14, nicked circular DNA; lane 15, uncut pBR322 DNA. (C) Purified BceSIV R1 subunit from a chitin column. The predicted molecular mass of R1 is 69.6 kDa. Lanes 1 to 9, eluted fractions containing R1; lane 10, total protein from the chitin column (R1-intein-CBD fusion protein, R1, and intein-CBD). (D) Purified BceSIV R1 and R2 subunits from a chitin column (lanes 1 to 9). The predicted molecular mass of R2 is 42.7 kDa. Lane 10, total protein from the chitin column. (E) BceSIV digestion of pBR322 in the presence of GTP, γ-S-GTP (nonhydrolyzable GTP analog), ATP, and UTP. (F) BceSIV digestion of pBR322 in the presence of ATP in the range of 0.4 mM to 20 mM. M, 1 kb DNA ladder. (G) BceSIV digestion of pBR322 in the presence of GTP in the range of 0.4 mM to 20 mM. Lanes M, 1-kb DNA ladder.
    Figure Legend Snippet: Characterization of BceSIV endonuclease. (A) Schematic diagram of six ORFs of the BceSIV operon. The gene identifier (Bce_0364 to Bce_0370) is indicated below the ORFs. Bce_0364, a possible transcription regulator, similar to phage repressors; Bce_0365, M.BceSIV (GCWGC); Bce_0367, unknown function, no apparent endonuclease activity; Bce_0368, R1 subunit of BceSIV, NTPase/helicase; Bce_0369, BceSIV endonuclease catalytic subunit, R2 subunit; Bce_0370, putative transcription regular, similar to the controller (C) protein of other R-M systems. (B) Digestion of pBR322 DNA by cell extracts containing BceSIV R1, R2, and R1+R2 in the presence or absence of ATP (2 mM). Lanes 1 and 13, 1-kb DNA ladder; lanes 2 to 11 contain either R1, R2, or R1+R2 as indicated; lane 12, linear DNA; lane 14, nicked circular DNA; lane 15, uncut pBR322 DNA. (C) Purified BceSIV R1 subunit from a chitin column. The predicted molecular mass of R1 is 69.6 kDa. Lanes 1 to 9, eluted fractions containing R1; lane 10, total protein from the chitin column (R1-intein-CBD fusion protein, R1, and intein-CBD). (D) Purified BceSIV R1 and R2 subunits from a chitin column (lanes 1 to 9). The predicted molecular mass of R2 is 42.7 kDa. Lane 10, total protein from the chitin column. (E) BceSIV digestion of pBR322 in the presence of GTP, γ-S-GTP (nonhydrolyzable GTP analog), ATP, and UTP. (F) BceSIV digestion of pBR322 in the presence of ATP in the range of 0.4 mM to 20 mM. M, 1 kb DNA ladder. (G) BceSIV digestion of pBR322 in the presence of GTP in the range of 0.4 mM to 20 mM. Lanes M, 1-kb DNA ladder.

    Techniques Used: Activity Assay, Purification

    12) Product Images from "T-LAK Cell-originated Protein Kinase (TOPK) Phosphorylation of MKP1 Protein Prevents Solar Ultraviolet Light-induced Inflammation through Inhibition of the p38 Protein Signaling Pathway *"

    Article Title: T-LAK Cell-originated Protein Kinase (TOPK) Phosphorylation of MKP1 Protein Prevents Solar Ultraviolet Light-induced Inflammation through Inhibition of the p38 Protein Signaling Pathway *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.225813

    TOPK phosphorylates MKP1 at Ser-359 in vitro and ex vivo . A , active TOPK phosphorylated MKP1 in vitro in the presence of [γ- 32 P]ATP and was visualized by autoradiography. ERK2 phosphorylation of MKP1 was used as a positive control. B , TOPK phosphorylated
    Figure Legend Snippet: TOPK phosphorylates MKP1 at Ser-359 in vitro and ex vivo . A , active TOPK phosphorylated MKP1 in vitro in the presence of [γ- 32 P]ATP and was visualized by autoradiography. ERK2 phosphorylation of MKP1 was used as a positive control. B , TOPK phosphorylated

    Techniques Used: In Vitro, Ex Vivo, Autoradiography, Positive Control

    13) Product Images from "K-BILDS: A kinase substrate discovery tool"

    Article Title: K-BILDS: A kinase substrate discovery tool

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201600511

    Kinase-catalyzed biotinylation and K-BILDS workflow. a) Kinase-catalyzed phosphorylation of substrates with ATP or the ATP-biotin analog. b) In the K-BILDS workflow, cell lysates were first treated with FSBA to inactivate kinases, followed by removal of excess FSBA by filtration. c) Kinase-inactivated lysates were treated with ATP-biotin and exogenous active kinase (top, Kinase reaction) to generate biotinylated kinase substrates. A negative control reaction containing ATP-biotin but no kinase (bottom) was also included. d) Biotinylated proteins were enriched using avidin resin, separated by SDS-PAGE, and subsequently analyzed by LC-MS/MS to identify biotinylated protein in the kinase but not control reaction. orange: endogenous kinase, red: exogenous active kinase, blue: substrates, grey: cellular proteins.
    Figure Legend Snippet: Kinase-catalyzed biotinylation and K-BILDS workflow. a) Kinase-catalyzed phosphorylation of substrates with ATP or the ATP-biotin analog. b) In the K-BILDS workflow, cell lysates were first treated with FSBA to inactivate kinases, followed by removal of excess FSBA by filtration. c) Kinase-inactivated lysates were treated with ATP-biotin and exogenous active kinase (top, Kinase reaction) to generate biotinylated kinase substrates. A negative control reaction containing ATP-biotin but no kinase (bottom) was also included. d) Biotinylated proteins were enriched using avidin resin, separated by SDS-PAGE, and subsequently analyzed by LC-MS/MS to identify biotinylated protein in the kinase but not control reaction. orange: endogenous kinase, red: exogenous active kinase, blue: substrates, grey: cellular proteins.

    Techniques Used: Filtration, Negative Control, Avidin-Biotin Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    14) Product Images from "Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)"

    Article Title: Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

    Journal: International Journal of Plant Genomics

    doi: 10.1155/2009/915061

    Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.
    Figure Legend Snippet: Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.

    Techniques Used: Ligation, Clone Assay, Blocking Assay

    15) Product Images from "Characterization of DNA Primase Complex Isolated from the Archaeon, Thermococcus kodakaraensis *"

    Article Title: Characterization of DNA Primase Complex Isolated from the Archaeon, Thermococcus kodakaraensis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.338145

    Analysis of DNA and RNA products formed by T. kodakaraensis primase complex. Reactions (100 μl), as described in the legend for , containing 100 μ m each of GTP, CTP, UTP, and [α- 32 P]ATP or 100 μ m each of dGTP, dCTP,
    Figure Legend Snippet: Analysis of DNA and RNA products formed by T. kodakaraensis primase complex. Reactions (100 μl), as described in the legend for , containing 100 μ m each of GTP, CTP, UTP, and [α- 32 P]ATP or 100 μ m each of dGTP, dCTP,

    Techniques Used:

    A , effects of dATP and ATP on RNA and DNA synthesis. Mixtures (20 μl), as indicated under “Experimental Procedures,” contained 100 μ m of each of the four dNTPs or rNTPs with [α- 32 P]dATP (1344 cpm/pmol) or [α-
    Figure Legend Snippet: A , effects of dATP and ATP on RNA and DNA synthesis. Mixtures (20 μl), as indicated under “Experimental Procedures,” contained 100 μ m of each of the four dNTPs or rNTPs with [α- 32 P]dATP (1344 cpm/pmol) or [α-

    Techniques Used: DNA Synthesis

    16) Product Images from "TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun"

    Article Title: TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6826

    TOPK phosphorylates c-Jun at serine 63 and serine 73 A. Active TOPK phosphorylates full-length c-Jun in vitro in the presence of [γ- 32 P] ATP. The incorporation of 32 P was visualized by autoradiography. Inactive p38 protein was used as a positive control. B. Active TOPK phosphorylation of wild-type c-Jun, c-Jun(S63A), c-Jun(S73A) and c-Jun (S63A S73A) was visualized by autoradiography. Equal protein loading was visualized by Coomassie Blue (CB) staining. C. Ectopic expression of TOPK increases c-Jun phosphorylation at the Ser63 and Ser73 sites. Cells were transfected with pcDNA3.1A-HA-TOPK and pcDNA4-His-c-Jun and cultured for 24 h. After starvation in DMEM supplemented with 0.1% FBS for 24 h, cells were stimulated with EGF (20 ng/mL) and harvested 15 min later. Whole cell lysates were then analyzed by Western blotting. A representative blot was presented. All protein levels were measured with densitometry and normalized to β-actin. Each bar represents the mean±SD from three experiments.* p
    Figure Legend Snippet: TOPK phosphorylates c-Jun at serine 63 and serine 73 A. Active TOPK phosphorylates full-length c-Jun in vitro in the presence of [γ- 32 P] ATP. The incorporation of 32 P was visualized by autoradiography. Inactive p38 protein was used as a positive control. B. Active TOPK phosphorylation of wild-type c-Jun, c-Jun(S63A), c-Jun(S73A) and c-Jun (S63A S73A) was visualized by autoradiography. Equal protein loading was visualized by Coomassie Blue (CB) staining. C. Ectopic expression of TOPK increases c-Jun phosphorylation at the Ser63 and Ser73 sites. Cells were transfected with pcDNA3.1A-HA-TOPK and pcDNA4-His-c-Jun and cultured for 24 h. After starvation in DMEM supplemented with 0.1% FBS for 24 h, cells were stimulated with EGF (20 ng/mL) and harvested 15 min later. Whole cell lysates were then analyzed by Western blotting. A representative blot was presented. All protein levels were measured with densitometry and normalized to β-actin. Each bar represents the mean±SD from three experiments.* p

    Techniques Used: In Vitro, Autoradiography, Positive Control, Staining, Expressing, Transfection, Cell Culture, Western Blot

    17) Product Images from "Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival"

    Article Title: Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival

    Journal: Nature cell biology

    doi: 10.1038/ncb3194

    Proposed working-model of PANX1-mediated ATP release as a regulator of intravascular metastatic cell survival
    Figure Legend Snippet: Proposed working-model of PANX1-mediated ATP release as a regulator of intravascular metastatic cell survival

    Techniques Used:

    Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch
    Figure Legend Snippet: Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch

    Techniques Used:

    PANX1 1–89 augments PANX1-mediated ATP release in metastatic breast cancer cells
    Figure Legend Snippet: PANX1 1–89 augments PANX1-mediated ATP release in metastatic breast cancer cells

    Techniques Used:

    18) Product Images from "Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured using the α-Hemolysin Nanopore"

    Article Title: Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured using the α-Hemolysin Nanopore

    Journal: ACS nano

    doi: 10.1021/acsnano.6b05995

    Detecting backbone damage in a DNA duplex. (a–b) Current histograms showing the measured blockade currents for 14 µM solutions of the (a) nicked and (b) repaired duplexes, respectively. (c) Current histogram of a mixture of the same amount (7 µM) of nicked and repaired duplexes. (d) Sample current-time traces generated during dsDNA residence events, showing the difference in blockade currents, I N and I R , for nicked DNA and repaired DNA, respectively. The trace was post-filtered at 1 kHz for presentation. An expanded view of the same I-t trace is also shown in (d) to present the difference in blockade current of the two types of events. The expanded I-t trace was post-filtered at 0.1 kHz for presentation. Experiments were carried out at 20.0 °C in a 100 mM KCl (7.5% PEG, 5 mM MgCl 2 , 1 mM ATP) solution buffered to pH 7.6 by 66 mM Tris-HCl. Counts indicate the number of individual dsDNA unzipping events.
    Figure Legend Snippet: Detecting backbone damage in a DNA duplex. (a–b) Current histograms showing the measured blockade currents for 14 µM solutions of the (a) nicked and (b) repaired duplexes, respectively. (c) Current histogram of a mixture of the same amount (7 µM) of nicked and repaired duplexes. (d) Sample current-time traces generated during dsDNA residence events, showing the difference in blockade currents, I N and I R , for nicked DNA and repaired DNA, respectively. The trace was post-filtered at 1 kHz for presentation. An expanded view of the same I-t trace is also shown in (d) to present the difference in blockade current of the two types of events. The expanded I-t trace was post-filtered at 0.1 kHz for presentation. Experiments were carried out at 20.0 °C in a 100 mM KCl (7.5% PEG, 5 mM MgCl 2 , 1 mM ATP) solution buffered to pH 7.6 by 66 mM Tris-HCl. Counts indicate the number of individual dsDNA unzipping events.

    Techniques Used: Generated

    19) Product Images from "Structural basis for transcript elongation control by NusG family universal regulators"

    Article Title: Structural basis for transcript elongation control by NusG family universal regulators

    Journal: Cell

    doi: 10.1016/j.cell.2018.05.017

    NusG and RfaH remodel and stabilize the upstream duplex DNA ). The RNAP is shown as a transparent molecular surface, revealing the nucleic acid scaffold inside (shown in cartoon format and colored according to the legend). The boxed region is magnified below. (bottom) Magnified view. Most of the β subunit (light cyan) has been removed to reveal the inside of the RNAP active site cleft. The β′ subunit is light pink but with the zipper, lid, and rudder highlighted in brown. The Bridge-Helix (BH) is also labeled. The nucleic acids are shown as sticks (with the first four base pairs of the upstream duplex, - 10 through −13, labeled). The RNAP active site Mg 2+ -ion is shown as a yellow sphere. The thin black arrows are drawn parallel to the downstream duplex DNA axis (nearly horizontal) and the upstream duplex, which subtends an angle of 124°. B. As in (A) but showing the NusG- ops EC structure. NusG is green. C. As in (A) but showing the RfaH- ops EC structure. RfaH is magenta. D. Probing the upstream fork junction by psoralen crosslinking. The ops ECs were assembled on the scaffold shown on top, with the TA intercalation motif (blue) positioned immediately upstream from the ops element; the t-DNA was labeled with [γ 32 P]-ATP. Following incubation with RfaH or NusG, the ECs were illuminated with 365 nm UV light. The crosslinked products were analyzed on 12 % gels and the fraction of t-DNA crosslinked to the nt-DNA was quantified (bottom). Error bars indicate the s.d. of triplicate measurements.
    Figure Legend Snippet: NusG and RfaH remodel and stabilize the upstream duplex DNA ). The RNAP is shown as a transparent molecular surface, revealing the nucleic acid scaffold inside (shown in cartoon format and colored according to the legend). The boxed region is magnified below. (bottom) Magnified view. Most of the β subunit (light cyan) has been removed to reveal the inside of the RNAP active site cleft. The β′ subunit is light pink but with the zipper, lid, and rudder highlighted in brown. The Bridge-Helix (BH) is also labeled. The nucleic acids are shown as sticks (with the first four base pairs of the upstream duplex, - 10 through −13, labeled). The RNAP active site Mg 2+ -ion is shown as a yellow sphere. The thin black arrows are drawn parallel to the downstream duplex DNA axis (nearly horizontal) and the upstream duplex, which subtends an angle of 124°. B. As in (A) but showing the NusG- ops EC structure. NusG is green. C. As in (A) but showing the RfaH- ops EC structure. RfaH is magenta. D. Probing the upstream fork junction by psoralen crosslinking. The ops ECs were assembled on the scaffold shown on top, with the TA intercalation motif (blue) positioned immediately upstream from the ops element; the t-DNA was labeled with [γ 32 P]-ATP. Following incubation with RfaH or NusG, the ECs were illuminated with 365 nm UV light. The crosslinked products were analyzed on 12 % gels and the fraction of t-DNA crosslinked to the nt-DNA was quantified (bottom). Error bars indicate the s.d. of triplicate measurements.

    Techniques Used: Labeling, Incubation

    20) Product Images from "The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma"

    Article Title: The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00578-5

    FAM46C interacts with RNA and is an active RNA poly(A) polymerase in vitro and in vivo. a Recombinant FAM46C WT (lanes 8–13), but not its catalytic mutant FAM46C mut (lanes 2–7), displays poly(A) polymerase activity in vitro. Reaction products (using 32 P-labeled (A) 15 as substrate) were separated in denaturing PAGE gels and visualized by autoradiography. b SDS-PAGE analysis of recombinant FAM46C WT and its catalytic mutant FAM46C mut . c FAM46D WT is an active poly(A) polymerase in vitro and requires Mn 2+ ions for its activity. Purified protein was incubated with 32 P-labeled (A) 15 primer in the presence of ATP and divalent cations as follows: Mg 2+ (lanes 4–6), both Mg 2+ /Mn 2+ (lanes 7–9), or Mn 2+ (lanes 10–12). Control reactions were carried out without the protein (lanes 1–3). d FAM46C interacts with RNA in human cells. Autoradiography of UV cross-linked 32 P-labeled RNAs co-purified with FAM46C WT GFP from HEK293 cells stably expressing the fusion protein (lanes 3–4) and from control empty cells (lanes 1–2). Immunoprecipitated RNA-protein complexes were separated by SDS-PAGE, transferred to nitrocellulose membrane, stained with Ponceau S, and subsequently autoradiographed. The right panel shows the Ponceau S stained blot merged with autoradiogram
    Figure Legend Snippet: FAM46C interacts with RNA and is an active RNA poly(A) polymerase in vitro and in vivo. a Recombinant FAM46C WT (lanes 8–13), but not its catalytic mutant FAM46C mut (lanes 2–7), displays poly(A) polymerase activity in vitro. Reaction products (using 32 P-labeled (A) 15 as substrate) were separated in denaturing PAGE gels and visualized by autoradiography. b SDS-PAGE analysis of recombinant FAM46C WT and its catalytic mutant FAM46C mut . c FAM46D WT is an active poly(A) polymerase in vitro and requires Mn 2+ ions for its activity. Purified protein was incubated with 32 P-labeled (A) 15 primer in the presence of ATP and divalent cations as follows: Mg 2+ (lanes 4–6), both Mg 2+ /Mn 2+ (lanes 7–9), or Mn 2+ (lanes 10–12). Control reactions were carried out without the protein (lanes 1–3). d FAM46C interacts with RNA in human cells. Autoradiography of UV cross-linked 32 P-labeled RNAs co-purified with FAM46C WT GFP from HEK293 cells stably expressing the fusion protein (lanes 3–4) and from control empty cells (lanes 1–2). Immunoprecipitated RNA-protein complexes were separated by SDS-PAGE, transferred to nitrocellulose membrane, stained with Ponceau S, and subsequently autoradiographed. The right panel shows the Ponceau S stained blot merged with autoradiogram

    Techniques Used: In Vitro, In Vivo, Recombinant, Mutagenesis, Activity Assay, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, SDS Page, Purification, Incubation, Stable Transfection, Expressing, Immunoprecipitation, Staining

    21) Product Images from "Phosphorylation of Ubc9 by Cdk1 Enhances SUMOylation Activity"

    Article Title: Phosphorylation of Ubc9 by Cdk1 Enhances SUMOylation Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034250

    CDK1/cyclin B phosphorylates Ubc9 in vitro . (A) In vitro phosphorylation of SAE1/SAE2 by CDK1/cyclin B. CDK1/cyclin B (3 nM, 6 nM and 12 nM) was incubated with (left) or without (right) SAE1/His 6 -SAE2 for 30 min at 30°C in the presence of [γ- 32 P] ATP. The reactions were analyzed by 10% SDS-PAGE followed by Coomassie blue staining and autoradiography. (B) In vitro phosphorylation of His 6 -SUMO1 by CDK1/cyclin B. Various concentrations of CDK1/cyclin B (as mentioned above) were incubated with (left) or without His 6 -SUMO1 (right) in the presence of [γ- 32 P] ATP for 30 min at 30°C. (C) In vitro phosphorylation of Ubc9 by CDK1/cyclin B. Various concentrations of CDK1/cyclin B (as mentioned above) were incubated with (left panel) or without His 6 -Ubc9 (right panel) in the presence of [γ- 32 P] ATP for 30 min at 30°C. (D) In vitro phosphorylation of GST-hTOP1 (110–125) AA. Various concentrations of CDK1/cyclin B (as mentioned above) were incubated with (left) or without GST-hTOP1 (110–125) AA (right) in the presence of [γ- 32 P] ATP. Reaction mixtures of B, C and D were analyzed by 15% SDS-PAGE followed by Coomassie blue staining and autoradiography.
    Figure Legend Snippet: CDK1/cyclin B phosphorylates Ubc9 in vitro . (A) In vitro phosphorylation of SAE1/SAE2 by CDK1/cyclin B. CDK1/cyclin B (3 nM, 6 nM and 12 nM) was incubated with (left) or without (right) SAE1/His 6 -SAE2 for 30 min at 30°C in the presence of [γ- 32 P] ATP. The reactions were analyzed by 10% SDS-PAGE followed by Coomassie blue staining and autoradiography. (B) In vitro phosphorylation of His 6 -SUMO1 by CDK1/cyclin B. Various concentrations of CDK1/cyclin B (as mentioned above) were incubated with (left) or without His 6 -SUMO1 (right) in the presence of [γ- 32 P] ATP for 30 min at 30°C. (C) In vitro phosphorylation of Ubc9 by CDK1/cyclin B. Various concentrations of CDK1/cyclin B (as mentioned above) were incubated with (left panel) or without His 6 -Ubc9 (right panel) in the presence of [γ- 32 P] ATP for 30 min at 30°C. (D) In vitro phosphorylation of GST-hTOP1 (110–125) AA. Various concentrations of CDK1/cyclin B (as mentioned above) were incubated with (left) or without GST-hTOP1 (110–125) AA (right) in the presence of [γ- 32 P] ATP. Reaction mixtures of B, C and D were analyzed by 15% SDS-PAGE followed by Coomassie blue staining and autoradiography.

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

    22) Product Images from "Endoplasmic Reticulum Ca2+ Release Modulates Endothelial Nitric-oxide Synthase via Extracellular Signal-regulated Kinase (ERK) 1/2-mediated Serine 635 Phosphorylation *"

    Article Title: Endoplasmic Reticulum Ca2+ Release Modulates Endothelial Nitric-oxide Synthase via Extracellular Signal-regulated Kinase (ERK) 1/2-mediated Serine 635 Phosphorylation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.220236

    ER Ca 2+ release mediated the effect of ATP on eNOS Ser-635 phosphorylation in BAECs. A , effects of BAPTA-AM (50 μ m ) on ATP-induced ERK1/2 activation and eNOS Ser-635 phosphorylation. BAECs were preloaded with BAPTA-AM for 30 min and then exposed
    Figure Legend Snippet: ER Ca 2+ release mediated the effect of ATP on eNOS Ser-635 phosphorylation in BAECs. A , effects of BAPTA-AM (50 μ m ) on ATP-induced ERK1/2 activation and eNOS Ser-635 phosphorylation. BAECs were preloaded with BAPTA-AM for 30 min and then exposed

    Techniques Used: Activation Assay

    Mutation of Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. A , effects of ATP (20 μ m ) on eNOS Ser-635 phosphorylation in WT eNOS and S635A eNOS-transfect cells. Representative blots are shown from three independent
    Figure Legend Snippet: Mutation of Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. A , effects of ATP (20 μ m ) on eNOS Ser-635 phosphorylation in WT eNOS and S635A eNOS-transfect cells. Representative blots are shown from three independent

    Techniques Used: Mutagenesis

    ATP triggered eNOS Ser-635 phosphorylation in BAECs. A , time course effects of ATP (20 μ m ) on eNOS phosphorylation. Representative blots are shown from five independent experiments. B , quantitative analyses of the effects of ATP on eNOS Ser-1179,
    Figure Legend Snippet: ATP triggered eNOS Ser-635 phosphorylation in BAECs. A , time course effects of ATP (20 μ m ) on eNOS phosphorylation. Representative blots are shown from five independent experiments. B , quantitative analyses of the effects of ATP on eNOS Ser-1179,

    Techniques Used:

    23) Product Images from "Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells"

    Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

    Journal: Oncology Reports

    doi: 10.3892/or.2014.3309

    UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P
    Figure Legend Snippet: UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P

    Techniques Used: Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

    24) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    25) Product Images from "Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: Implications for phosphoproteomics"

    Article Title: Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: Implications for phosphoproteomics

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201200626

    Gel analysis of phosphoproteins (ProQ, top) or biotinylated phosphoproteins (SA-Cy5, bottom) from HeLa cell lysates incubated with ATP or ATP-biotin in the absence or presence of kinase or phosphatase inhibitors.
    Figure Legend Snippet: Gel analysis of phosphoproteins (ProQ, top) or biotinylated phosphoproteins (SA-Cy5, bottom) from HeLa cell lysates incubated with ATP or ATP-biotin in the absence or presence of kinase or phosphatase inhibitors.

    Techniques Used: Incubation

    A) HPLC analysis of unmodified peptides (PKA peptide at ∼8.3 min; CK2 peptide at ∼7.5 min; Abl peptide at ∼13.8 min). B) HPLC analysis of ATP-phosphorylated peptides (top; PKA phosphopeptide at ∼7.5 min; CK2 phosphopeptide
    Figure Legend Snippet: A) HPLC analysis of unmodified peptides (PKA peptide at ∼8.3 min; CK2 peptide at ∼7.5 min; Abl peptide at ∼13.8 min). B) HPLC analysis of ATP-phosphorylated peptides (top; PKA phosphopeptide at ∼7.5 min; CK2 phosphopeptide

    Techniques Used: High Performance Liquid Chromatography

    26) Product Images from "Characterization of DNA Primase Complex Isolated from the Archaeon, Thermococcus kodakaraensis *"

    Article Title: Characterization of DNA Primase Complex Isolated from the Archaeon, Thermococcus kodakaraensis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.338145

    Analysis of DNA and RNA products formed by T. kodakaraensis primase complex. Reactions (100 μl), as described in the legend for , containing 100 μ m each of GTP, CTP, UTP, and [α- 32 P]ATP or 100 μ m each of dGTP, dCTP,
    Figure Legend Snippet: Analysis of DNA and RNA products formed by T. kodakaraensis primase complex. Reactions (100 μl), as described in the legend for , containing 100 μ m each of GTP, CTP, UTP, and [α- 32 P]ATP or 100 μ m each of dGTP, dCTP,

    Techniques Used:

    A , effects of dATP and ATP on RNA and DNA synthesis. Mixtures (20 μl), as indicated under “Experimental Procedures,” contained 100 μ m of each of the four dNTPs or rNTPs with [α- 32 P]dATP (1344 cpm/pmol) or [α-
    Figure Legend Snippet: A , effects of dATP and ATP on RNA and DNA synthesis. Mixtures (20 μl), as indicated under “Experimental Procedures,” contained 100 μ m of each of the four dNTPs or rNTPs with [α- 32 P]dATP (1344 cpm/pmol) or [α-

    Techniques Used: DNA Synthesis

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    Article Snippet: .. For in vitro ubiquitination assay, reactions were carried out using a ubiquitinylation kit purchased from Enzo Life Sciences (Farmingdale, NY, USA) containing 2.5 µM biotinylated or non-tagged ubiquitin, 100 nM His-tagged E1, approximately 1–2.5 µM His-tagged E2, and 5 mM Mg-ATP, and 20 U/mL yeast inorganic pyrophosphatase (New England Biolabs, Ipswich, Massachusetts, USA). .. Detection was made by immunoblotting with Cy5-conjugated streptavidin (Cell Signaling Technology) or anti-ubiquitin antibody.

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    Article Snippet: .. In vitro kinase assay was performed using the kinase buffer (60 mM Hepes⋅NaOH at pH 7.5, 3 mM MgCl2 , 3 mM MnCl2 , 3 μM Na-orthovanadate, 1.2 mM DTT), 10 μCi [γ32 -P] ATP, 100 μM cold ATP, and 100 ng GSK3 β enzyme (New England Biolabs). .. GST- or GST-tagged PELP1 deletions, as well as full-length PELP1, were used as substrates (∼2 μg) for the in vitro GSK3β kinase assays.

    Labeling:

    Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication
    Article Snippet: .. The purified probe (100 ng) was then 5' labeled with [32 P-γ] ATP with T4 Polynucleotide Kinase (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions. .. The labeled probe was then diluted 1:50 before being used in the EMSA.

    Purification:

    Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication
    Article Snippet: .. The purified probe (100 ng) was then 5' labeled with [32 P-γ] ATP with T4 Polynucleotide Kinase (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions. .. The labeled probe was then diluted 1:50 before being used in the EMSA.

    Ubiquitin Assay:

    Article Title: Intrinsic ubiquitin E3 ligase activity of histone acetyltransferase Hbo1 for estrogen receptor α
    Article Snippet: .. For in vitro ubiquitination assay, reactions were carried out using a ubiquitinylation kit purchased from Enzo Life Sciences (Farmingdale, NY, USA) containing 2.5 µM biotinylated or non-tagged ubiquitin, 100 nM His-tagged E1, approximately 1–2.5 µM His-tagged E2, and 5 mM Mg-ATP, and 20 U/mL yeast inorganic pyrophosphatase (New England Biolabs, Ipswich, Massachusetts, USA). .. Detection was made by immunoblotting with Cy5-conjugated streptavidin (Cell Signaling Technology) or anti-ubiquitin antibody.

    Incubation:

    Article Title: Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: Implications for phosphoproteomics
    Article Snippet: .. For the reactions consisting of lysates, HeLa cell lysates (100 μg) were incubated with either ATP or ATP-biotin (2 mM) in the presence kinase inhibitor (Staurosporine, 0.10 mM) or phosphatase inhibitors (sodium fluoride and sodium orthovanadate, 0.10 mM each) in the manufacturer-provided buffer for PKA (1×, NEB) for 2 hours at 30 °C. ..

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    Kinase Assay:

    Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain
    Article Snippet: .. In vitro kinase assay was performed using the kinase buffer (60 mM Hepes⋅NaOH at pH 7.5, 3 mM MgCl2 , 3 mM MnCl2 , 3 μM Na-orthovanadate, 1.2 mM DTT), 10 μCi [γ32 -P] ATP, 100 μM cold ATP, and 100 ng GSK3 β enzyme (New England Biolabs). .. GST- or GST-tagged PELP1 deletions, as well as full-length PELP1, were used as substrates (∼2 μg) for the in vitro GSK3β kinase assays.

    SDS Page:

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    New England Biolabs atp biotin
    Gel analysis of phosphoproteins (ProQ, top) or biotinylated phosphoproteins (SA-Cy5, bottom) from <t>HeLa</t> cell lysates incubated with <t>ATP</t> or ATP-biotin in the absence or presence of kinase or phosphatase inhibitors.
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    Gel analysis of phosphoproteins (ProQ, top) or biotinylated phosphoproteins (SA-Cy5, bottom) from HeLa cell lysates incubated with ATP or ATP-biotin in the absence or presence of kinase or phosphatase inhibitors.

    Journal: Chembiochem : a European journal of chemical biology

    Article Title: Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: Implications for phosphoproteomics

    doi: 10.1002/cbic.201200626

    Figure Lengend Snippet: Gel analysis of phosphoproteins (ProQ, top) or biotinylated phosphoproteins (SA-Cy5, bottom) from HeLa cell lysates incubated with ATP or ATP-biotin in the absence or presence of kinase or phosphatase inhibitors.

    Article Snippet: For the reactions consisting of lysates, HeLa cell lysates (100 μg) were incubated with either ATP or ATP-biotin (2 mM) in the presence kinase inhibitor (Staurosporine, 0.10 mM) or phosphatase inhibitors (sodium fluoride and sodium orthovanadate, 0.10 mM each) in the manufacturer-provided buffer for PKA (1×, NEB) for 2 hours at 30 °C.

    Techniques: Incubation

    (A) Reactions of HeLa cell lysates and ATP without (untreated) or with phosphatase inhibitors (phosphatase inhibitor treated). Proteins were visualized with Pro-Q diamond phosphoprotein stain. (B) Reactions of HeLa cell lysates and ATP-biotin without

    Journal: Chembiochem : a European journal of chemical biology

    Article Title: Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: Implications for phosphoproteomics

    doi: 10.1002/cbic.201200626

    Figure Lengend Snippet: (A) Reactions of HeLa cell lysates and ATP without (untreated) or with phosphatase inhibitors (phosphatase inhibitor treated). Proteins were visualized with Pro-Q diamond phosphoprotein stain. (B) Reactions of HeLa cell lysates and ATP-biotin without

    Article Snippet: For the reactions consisting of lysates, HeLa cell lysates (100 μg) were incubated with either ATP or ATP-biotin (2 mM) in the presence kinase inhibitor (Staurosporine, 0.10 mM) or phosphatase inhibitors (sodium fluoride and sodium orthovanadate, 0.10 mM each) in the manufacturer-provided buffer for PKA (1×, NEB) for 2 hours at 30 °C.

    Techniques: Staining

    RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Journal: mBio

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    doi: 10.1128/mBio.00833-16

    Figure Lengend Snippet: RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.

    Article Snippet: The reaction volume was 500 µl and included 1 mg to 2 mg total lysate protein, RNA (33 nM to 1 µM), and 2 mM AMP-PNP (Roche) or ATP (New England Biolabs).

    Techniques: Western Blot, Incubation, RNA Binding Assay, Protease Inhibitor

    RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Journal: mBio

    Article Title: RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

    doi: 10.1128/mBio.00833-16

    Figure Lengend Snippet: RNA mod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.

    Article Snippet: The reaction volume was 500 µl and included 1 mg to 2 mg total lysate protein, RNA (33 nM to 1 µM), and 2 mM AMP-PNP (Roche) or ATP (New England Biolabs).

    Techniques: Western Blot

    MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

    Journal: Cancers

    Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    doi: 10.3390/cancers6031464

    Figure Lengend Snippet: MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

    Article Snippet: The purified probe (100 ng) was then 5' labeled with [32 P-γ] ATP with T4 Polynucleotide Kinase (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions.

    Techniques: Sequencing, Clone Assay, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Labeling, Western Blot, Purification, Mutagenesis, Affinity Purification, Negative Control

    PELP1 is a novel substrate of GSK3β. Hippocampal lysates collected 24 h after GCI reperfusion were subjected to immunoprecipitation with PELP1 or IgG antibody. Immunoprecipitates were pulled down by adding dynabeads protein A. Immunoprecipitates were subjected to SDS/PAGE, followed by mass spectrometry analysis. ( A ) List of unique PELP1-interacting proteins. ( B ) Forebrain lysates were subjected to immunoprecipitation or GST pull-down assay, and PELP1 interaction with GSK3β was confirmed by Western blotting. ( C ) Forebrain lysates were used in pull-down assays using GST or various deletion fragments of PELP1, and interaction of PELP1 deletions with GSK3β was analyzed by Western blotting. ( D ) GST-tagged PELP1 deletions and full-length GST-PELP1 were incubated with purified GSK3β enzyme in kinase buffer with [ γ32 -P] ATP and 100 μM cold ATP for 30 min at 30 °C, and the phosphorylation of PELP1 fragments was analyzed by PhosphorImager.

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

    Article Title: Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

    doi: 10.1073/pnas.1516729112

    Figure Lengend Snippet: PELP1 is a novel substrate of GSK3β. Hippocampal lysates collected 24 h after GCI reperfusion were subjected to immunoprecipitation with PELP1 or IgG antibody. Immunoprecipitates were pulled down by adding dynabeads protein A. Immunoprecipitates were subjected to SDS/PAGE, followed by mass spectrometry analysis. ( A ) List of unique PELP1-interacting proteins. ( B ) Forebrain lysates were subjected to immunoprecipitation or GST pull-down assay, and PELP1 interaction with GSK3β was confirmed by Western blotting. ( C ) Forebrain lysates were used in pull-down assays using GST or various deletion fragments of PELP1, and interaction of PELP1 deletions with GSK3β was analyzed by Western blotting. ( D ) GST-tagged PELP1 deletions and full-length GST-PELP1 were incubated with purified GSK3β enzyme in kinase buffer with [ γ32 -P] ATP and 100 μM cold ATP for 30 min at 30 °C, and the phosphorylation of PELP1 fragments was analyzed by PhosphorImager.

    Article Snippet: In vitro kinase assay was performed using the kinase buffer (60 mM Hepes⋅NaOH at pH 7.5, 3 mM MgCl2 , 3 mM MnCl2 , 3 μM Na-orthovanadate, 1.2 mM DTT), 10 μCi [γ32 -P] ATP, 100 μM cold ATP, and 100 ng GSK3 β enzyme (New England Biolabs).

    Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry, Pull Down Assay, Western Blot, Incubation, Purification