γ 32 p atp  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92

    Structured Review

    New England Biolabs γ 32 p atp
    Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 <t>P]ATP,</t> followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.
    γ 32 P Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/New England Biolabs
    Average 92 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "Plk1 Phosphorylation of Mre11 Antagonizes the DNA Damage Response"

    Article Title: Plk1 Phosphorylation of Mre11 Antagonizes the DNA Damage Response

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-16-2787

    Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 P]ATP, followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.
    Figure Legend Snippet: Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 P]ATP, followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.

    Techniques Used: Incubation, Immunoprecipitation, Avidin-Biotin Assay, Lysis, Purification, Autoradiography, In Vitro, SDS Page, Staining, In Vivo, Transfection, Construct

    2) Product Images from "ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation"

    Article Title: ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003895

    FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo . A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser ( http://genome.ucsc.edu/ ) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ- 32 P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t -tests were performed. * P ≤0.05, ** P ≤0.01.
    Figure Legend Snippet: FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo . A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser ( http://genome.ucsc.edu/ ) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ- 32 P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t -tests were performed. * P ≤0.05, ** P ≤0.01.

    Techniques Used: In Vivo, Cross-linking Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Immunoprecipitation, Variant Assay, Labeling, Polymerase Chain Reaction

    FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels. A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ- 32 P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t -tests. Bar graphs represent mean ± SEM (n = 3). * P ≤0.05, *** P ≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t -tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P ≤0.05.
    Figure Legend Snippet: FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels. A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ- 32 P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t -tests. Bar graphs represent mean ± SEM (n = 3). * P ≤0.05, *** P ≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t -tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P ≤0.05.

    Techniques Used: Labeling, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Transfection, Western Blot, RNA Binding Assay, Expressing

    3) Product Images from "Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site"

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm031

    In vitro self-splicing ( A ) of B.c .I4 wild-type and mutant constructs and subsequent RT-PCR ( B ). In A , lane M shows the marker, γ [32-P] ATP 5′-end-labeled RNA Century-Plus Marker (Ambion). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 , and 100 mM MgCl 2 at 45°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. Schematic drawings are shown next to the bands corresponding to the different splicing products. The light grey box represents the extra 56-nt element. In B , RT-PCR with I4B_right and 5p_left_BamH1 primers ( Table 1 ) using in vitro splicing products as templates, confirming the size of the ligated exons. Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 1% agarose gel.
    Figure Legend Snippet: In vitro self-splicing ( A ) of B.c .I4 wild-type and mutant constructs and subsequent RT-PCR ( B ). In A , lane M shows the marker, γ [32-P] ATP 5′-end-labeled RNA Century-Plus Marker (Ambion). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 , and 100 mM MgCl 2 at 45°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. Schematic drawings are shown next to the bands corresponding to the different splicing products. The light grey box represents the extra 56-nt element. In B , RT-PCR with I4B_right and 5p_left_BamH1 primers ( Table 1 ) using in vitro splicing products as templates, confirming the size of the ligated exons. Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 1% agarose gel.

    Techniques Used: In Vitro, Mutagenesis, Construct, Reverse Transcription Polymerase Chain Reaction, Marker, Labeling, Agarose Gel Electrophoresis

    RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.
    Figure Legend Snippet: RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Labeling, Marker

    4) Product Images from "Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function"

    Article Title: Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0629-3

    a Schematic representation of HpMutS2 variants used in this study. Walker-A and Walker-B motifs of HpMutS2 were identified by multiple sequence alignment of MutS2 proteins from different bacteria using Clustal Omega ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). The mutations in the walker-A and Walker-B motifs were introduced using site directed mutagenesis. The mutated amino acids are highlighted in red. All the proteins were C-terminally His 6 -tagged. The LDLK motif and Smr domain are conserved nuclease sites of HpMutS2. b Effect of DNA substrates on ATPase activity of HpMutS2. HpMutS2 (45 nM) was incubated with increasing concentrations of ATP [0, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, and 1000 (μM)]. DNA substrates (1 μM) were added separately to the reaction mixtures. After incubation at 37 °C for 30 min the reactions were stopped by EDTA (50 mM) and the products were separated by TLC. ATP [γ- 32 P] was used as tracer to monitor the product formation. Reaction velocities were calculated by quantifying the proportion of products formed to un-reacted substrate divided by incubation time
    Figure Legend Snippet: a Schematic representation of HpMutS2 variants used in this study. Walker-A and Walker-B motifs of HpMutS2 were identified by multiple sequence alignment of MutS2 proteins from different bacteria using Clustal Omega ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). The mutations in the walker-A and Walker-B motifs were introduced using site directed mutagenesis. The mutated amino acids are highlighted in red. All the proteins were C-terminally His 6 -tagged. The LDLK motif and Smr domain are conserved nuclease sites of HpMutS2. b Effect of DNA substrates on ATPase activity of HpMutS2. HpMutS2 (45 nM) was incubated with increasing concentrations of ATP [0, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, and 1000 (μM)]. DNA substrates (1 μM) were added separately to the reaction mixtures. After incubation at 37 °C for 30 min the reactions were stopped by EDTA (50 mM) and the products were separated by TLC. ATP [γ- 32 P] was used as tracer to monitor the product formation. Reaction velocities were calculated by quantifying the proportion of products formed to un-reacted substrate divided by incubation time

    Techniques Used: Sequencing, Mutagenesis, Activity Assay, Incubation, Thin Layer Chromatography

    5) Product Images from "G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A"

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A

    Journal: Journal of Molecular Signaling

    doi: 10.1186/1750-2187-6-10

    PKA phosphorylates GPKOW at S27 and T316 in vitro . A . Recombinant GPKOW proteins were expressed in bacteria and purified on a HisTrap column followed by an ion exchange column. GPKOW wt (lane 1), GPKOW S27A (lane 2), GPKOW T316A (lane 3) and GPKOW S27A+T316A (lane 4) were all recognized by anti-GPKOW B01 after separation on SDS-PAGE and immunoblotting. One representative immunoblot is shown. B . Purified GPKOW wt (lanes 1 and 2), single- (lanes 3-6) or double-mutated GPKOW (lanes 7 and 8) were incubated with active (+) or heat inactivated (-) PKA Cα1 (7.4 ng) and γ- 32 P-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE and autoradiography. The CBB staining in the lower panel shows the amount of the different proteins. C . Unsaturated images from Syngene G-box and Typhoon 9400 phosphoimager were quantified by Genetools (Syngene) and the statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value
    Figure Legend Snippet: PKA phosphorylates GPKOW at S27 and T316 in vitro . A . Recombinant GPKOW proteins were expressed in bacteria and purified on a HisTrap column followed by an ion exchange column. GPKOW wt (lane 1), GPKOW S27A (lane 2), GPKOW T316A (lane 3) and GPKOW S27A+T316A (lane 4) were all recognized by anti-GPKOW B01 after separation on SDS-PAGE and immunoblotting. One representative immunoblot is shown. B . Purified GPKOW wt (lanes 1 and 2), single- (lanes 3-6) or double-mutated GPKOW (lanes 7 and 8) were incubated with active (+) or heat inactivated (-) PKA Cα1 (7.4 ng) and γ- 32 P-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE and autoradiography. The CBB staining in the lower panel shows the amount of the different proteins. C . Unsaturated images from Syngene G-box and Typhoon 9400 phosphoimager were quantified by Genetools (Syngene) and the statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value

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

    GPKOW binds RNA in vivo . A . 293T cells were harvested and exposed to UV irradiation (254 nm/200 mJ). The cells were lysed and treated with T1 RNase (0.048 U/μl) for 8 min followed by immunoprecipitation with Dynabeads conjugated with anti-GPKOW1894 (+ anti-GPKOW, lane 2) or rabbit IgG (+ IgG, lane 1). Immunoprecipitated samples were dephosphorylated with calf intestinal phosphatase (0.038 U/μl). The RNA was labeled with γ 32 -P ATP by PNK (0.5 U/μl) phosphorylation. All samples were separated on a denaturating (7 M urea) 6% PAA gel and subjected to autoradiography. The arrows indicate specific RNA species. The lower panel shows immunoreactive GPKOW from the cell lysate used (one representative lane is shown). B . Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop. The lanes were manually defined with identical frames. The statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value
    Figure Legend Snippet: GPKOW binds RNA in vivo . A . 293T cells were harvested and exposed to UV irradiation (254 nm/200 mJ). The cells were lysed and treated with T1 RNase (0.048 U/μl) for 8 min followed by immunoprecipitation with Dynabeads conjugated with anti-GPKOW1894 (+ anti-GPKOW, lane 2) or rabbit IgG (+ IgG, lane 1). Immunoprecipitated samples were dephosphorylated with calf intestinal phosphatase (0.038 U/μl). The RNA was labeled with γ 32 -P ATP by PNK (0.5 U/μl) phosphorylation. All samples were separated on a denaturating (7 M urea) 6% PAA gel and subjected to autoradiography. The arrows indicate specific RNA species. The lower panel shows immunoreactive GPKOW from the cell lysate used (one representative lane is shown). B . Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop. The lanes were manually defined with identical frames. The statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value

    Techniques Used: In Vivo, Irradiation, Immunoprecipitation, Labeling, Autoradiography

    6) Product Images from "pTAR-Encoded Proteins in Plasmid Partitioning"

    Article Title: pTAR-Encoded Proteins in Plasmid Partitioning

    Journal: Journal of Bacteriology

    doi:

    Stimulation of the ATPase activity of ParA by plasmid DNA. ATPase activity was measured as the release of 32 PO 4 from [γ- 32 P]ATP by thin-layer chromatography as described in Materials and Methods. Shown are results with pUCD550 (containing parS ). Identical results (data not shown) were obtained with the vector pUC4.
    Figure Legend Snippet: Stimulation of the ATPase activity of ParA by plasmid DNA. ATPase activity was measured as the release of 32 PO 4 from [γ- 32 P]ATP by thin-layer chromatography as described in Materials and Methods. Shown are results with pUCD550 (containing parS ). Identical results (data not shown) were obtained with the vector pUC4.

    Techniques Used: Activity Assay, Plasmid Preparation, Thin Layer Chromatography

    7) Product Images from "Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator"

    Article Title: Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator

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

    doi: 10.1073/pnas.1416652111

    CK1 promotes phosphorylation of Ci -PKA and inhibits recruitment of HIB. ( A and B ) Overexpression of CK1 inhibited HIB/Ci association. S2 cells were transfected with Myc-Ci -PKA with or without the indicated HIB and CK1 expression constructs. After treatment with MG132 for 4 h, cell lysates were subjected to immunoprecipitation and Western blot analysis using the indicated antibodies. Of note, both CK1α and CK1ε are tagged by a Flag epitope, and CK1ε overlaps with a nonspecific band (asterisk) detected by the anti-Flag antibody. ( C ) CK1 RNAi enhanced HIB/Ci association in response to Hh stimulation. S2 cells were treated with the control (luciferase) or CK1 α/ε dsRNA before transfection with Myc-Ci -PKA and Flag-MATH-CC. The transfected cells were treated with or without Hh-conditioned medium, followed by immunoprecipitation and Western blot analysis using the indicated antibodies. ( D ) CK1 induced phosphorylation of Ci -PKA but not Cim1-6. S2 cells were cotransfected with the indicated constructs and were treated with MG132. Cell lysates were separated on Phos-tag–conjugated SDS/PAGE, followed by Western blot analysis with an anti-Myc antibody. ( E ) Hh stimulated phosphorylation of Myc-Ci -PKA but not Myc-Cim1-6 through CK1. S2 cells treated with luciferase or CK1α/ε dsRNA were cotransfected with the indicated Ci constructs and were treated with or without Hh-conditioned medium. After treatment with MG132 for 4 h, cell lysates were prepared and separated on Phos-tag–conjugated SDS/PAGE, followed by Western blot analysis with an anti-Myc antibody. ( F ) A diagram of full-length Ci with six HIB-binding sites (S1–S6) indicated by individual bars and the sequences of individual sites shown underneath. The S/T-rich sequences are underlined. ( G ) In vitro kinase assay using a recombinant CK1 and GST-Ci fusion proteins containing the indicated wild-type or mutated HIB-binding sites in the presence of γ-[ 32 P]ATP. ( Left ) Short ( Top ) or long ( Middle ) exposure of the autoradiograph is shown. ( H ) GST-Ci fusion proteins containing wild-type or mutated S4 or S6 were incubated with cell extracts derived from HIB-N–expressing S2 cells. Input and bound HIB-N proteins were analyzed by Western blot using an anti-HA antibody. ( I ) S2 cells were transfected with Flag-MATH-CC alone or together with Myc-Ci -PKA or Myc-Ci -PKAS4D6D , followed by immunoprecipitation and Western blot analysis using the indicated antibodies. Myc-Ci -PKAS4D6D pulled down less Flag-MATH-CC than Myc-Ci -PKA .
    Figure Legend Snippet: CK1 promotes phosphorylation of Ci -PKA and inhibits recruitment of HIB. ( A and B ) Overexpression of CK1 inhibited HIB/Ci association. S2 cells were transfected with Myc-Ci -PKA with or without the indicated HIB and CK1 expression constructs. After treatment with MG132 for 4 h, cell lysates were subjected to immunoprecipitation and Western blot analysis using the indicated antibodies. Of note, both CK1α and CK1ε are tagged by a Flag epitope, and CK1ε overlaps with a nonspecific band (asterisk) detected by the anti-Flag antibody. ( C ) CK1 RNAi enhanced HIB/Ci association in response to Hh stimulation. S2 cells were treated with the control (luciferase) or CK1 α/ε dsRNA before transfection with Myc-Ci -PKA and Flag-MATH-CC. The transfected cells were treated with or without Hh-conditioned medium, followed by immunoprecipitation and Western blot analysis using the indicated antibodies. ( D ) CK1 induced phosphorylation of Ci -PKA but not Cim1-6. S2 cells were cotransfected with the indicated constructs and were treated with MG132. Cell lysates were separated on Phos-tag–conjugated SDS/PAGE, followed by Western blot analysis with an anti-Myc antibody. ( E ) Hh stimulated phosphorylation of Myc-Ci -PKA but not Myc-Cim1-6 through CK1. S2 cells treated with luciferase or CK1α/ε dsRNA were cotransfected with the indicated Ci constructs and were treated with or without Hh-conditioned medium. After treatment with MG132 for 4 h, cell lysates were prepared and separated on Phos-tag–conjugated SDS/PAGE, followed by Western blot analysis with an anti-Myc antibody. ( F ) A diagram of full-length Ci with six HIB-binding sites (S1–S6) indicated by individual bars and the sequences of individual sites shown underneath. The S/T-rich sequences are underlined. ( G ) In vitro kinase assay using a recombinant CK1 and GST-Ci fusion proteins containing the indicated wild-type or mutated HIB-binding sites in the presence of γ-[ 32 P]ATP. ( Left ) Short ( Top ) or long ( Middle ) exposure of the autoradiograph is shown. ( H ) GST-Ci fusion proteins containing wild-type or mutated S4 or S6 were incubated with cell extracts derived from HIB-N–expressing S2 cells. Input and bound HIB-N proteins were analyzed by Western blot using an anti-HA antibody. ( I ) S2 cells were transfected with Flag-MATH-CC alone or together with Myc-Ci -PKA or Myc-Ci -PKAS4D6D , followed by immunoprecipitation and Western blot analysis using the indicated antibodies. Myc-Ci -PKAS4D6D pulled down less Flag-MATH-CC than Myc-Ci -PKA .

    Techniques Used: Over Expression, Transfection, Expressing, Construct, Immunoprecipitation, Western Blot, FLAG-tag, Luciferase, SDS Page, Binding Assay, In Vitro, Kinase Assay, Recombinant, Autoradiography, Incubation, Derivative Assay

    8) Product Images from "The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion"

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00025-18

    TIN2L phosphorylation is dependent upon an intact CK2 consensus site. (A) Evolutionary trace analysis and summary of known interaction regions in TIN2. The residues comprising the putative CK2 phosphorylation sites are displayed. The protein interaction domains, the region where all DC-causing mutations cluster (DC), the position of R282 (*), and the putative TBM, all of which are present in both the TIN2L and TIN2S isoforms, and the C-terminal domain unique to TIN2L (TIN2L CTD) are indicated. (B) Characterization of TIN2S and TIN2L phosphorylation. Flag-tagged TIN2S or TIN2L was analyzed by SDS-PAGE in the presence of Phos-tag reagent. Phosphorylated TIN2L (TIN2L P ) is visible as a supershift compared to the λ phosphatase-treated TIN2L. (C) Characterization of TIN2L phosphorylation at S396 as in panel B. (D) Recombinant CK2 (NEB) analyzed by SDS-PAGE and silver staining. M, protein molecular mass marker; 1, CK2. (E) In vitro phosphorylation of TIN2L by CK2. Recombinant TIN2L purified from E. coli was incubated with CK2 and [γ- 32 P]ATP in the absence or presence of a CK2 inhibitor (TBB). (F) In vitro phosphorylation of partially purified TIN2L and TIN2L-S396A by CK2. Flag-tagged TIN2L or TIN2L-S396A from transiently transfected HEK 293T cells was dephosphorylated by incubation with λ phosphatase, purified via immunoprecipitation, and incubated with CK2. Phosphorylation status was assessed by SDS-PAGE in the presence of the Phos-tag reagent and compared to that of non-λ phosphatase-treated controls.
    Figure Legend Snippet: TIN2L phosphorylation is dependent upon an intact CK2 consensus site. (A) Evolutionary trace analysis and summary of known interaction regions in TIN2. The residues comprising the putative CK2 phosphorylation sites are displayed. The protein interaction domains, the region where all DC-causing mutations cluster (DC), the position of R282 (*), and the putative TBM, all of which are present in both the TIN2L and TIN2S isoforms, and the C-terminal domain unique to TIN2L (TIN2L CTD) are indicated. (B) Characterization of TIN2S and TIN2L phosphorylation. Flag-tagged TIN2S or TIN2L was analyzed by SDS-PAGE in the presence of Phos-tag reagent. Phosphorylated TIN2L (TIN2L P ) is visible as a supershift compared to the λ phosphatase-treated TIN2L. (C) Characterization of TIN2L phosphorylation at S396 as in panel B. (D) Recombinant CK2 (NEB) analyzed by SDS-PAGE and silver staining. M, protein molecular mass marker; 1, CK2. (E) In vitro phosphorylation of TIN2L by CK2. Recombinant TIN2L purified from E. coli was incubated with CK2 and [γ- 32 P]ATP in the absence or presence of a CK2 inhibitor (TBB). (F) In vitro phosphorylation of partially purified TIN2L and TIN2L-S396A by CK2. Flag-tagged TIN2L or TIN2L-S396A from transiently transfected HEK 293T cells was dephosphorylated by incubation with λ phosphatase, purified via immunoprecipitation, and incubated with CK2. Phosphorylation status was assessed by SDS-PAGE in the presence of the Phos-tag reagent and compared to that of non-λ phosphatase-treated controls.

    Techniques Used: SDS Page, Recombinant, Silver Staining, Marker, In Vitro, Purification, Incubation, Transfection, Immunoprecipitation

    9) Product Images from "Regulation of SAP102 Synaptic Targeting by Phosphorylation"

    Article Title: Regulation of SAP102 Synaptic Targeting by Phosphorylation

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-017-0836-4

    SAP102 is phosphorylated on Ser632 by CK2 in vitro a , Schematic diagram of SAP102 including an alignment of SAP102 (amino acids 625 – 638) and SAP97 (amino acids 680 – 693). SAP102 Ser632 is indicated with an arrowhead. b , GST-SAP102, GST-SAP102 ΔI2, and GST-SAP102 S632A were phosphorylated in vitro using [γ- 32 P] ATP with CK2 and analyzed by autoradiography. c , GST-SAP102, GST-SAP102 ΔI2, and GST-SAP102 S632A were phosphorylated in vitro with CK2 and analyzed by immunoblotting with SAP102 Ser632 phosphorylation state-specific antibody. The experiment was repeated three times and quantified using ImageQuant LAS TL software. Data represent means ± S.E. (*, p
    Figure Legend Snippet: SAP102 is phosphorylated on Ser632 by CK2 in vitro a , Schematic diagram of SAP102 including an alignment of SAP102 (amino acids 625 – 638) and SAP97 (amino acids 680 – 693). SAP102 Ser632 is indicated with an arrowhead. b , GST-SAP102, GST-SAP102 ΔI2, and GST-SAP102 S632A were phosphorylated in vitro using [γ- 32 P] ATP with CK2 and analyzed by autoradiography. c , GST-SAP102, GST-SAP102 ΔI2, and GST-SAP102 S632A were phosphorylated in vitro with CK2 and analyzed by immunoblotting with SAP102 Ser632 phosphorylation state-specific antibody. The experiment was repeated three times and quantified using ImageQuant LAS TL software. Data represent means ± S.E. (*, p

    Techniques Used: In Vitro, Autoradiography, Software

    10) Product Images from "In Vitro Selection of Cell-Internalizing DNA Aptamers in a Model System of Inflammatory Kidney Disease"

    Article Title: In Vitro Selection of Cell-Internalizing DNA Aptamers in a Model System of Inflammatory Kidney Disease

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.06.018

    Binding Activities of Selected DNA Aptamers to Cytokine-Stimulated Cells (A) Sequences of individual ssDNA aptamer candidates. G-quartet or G-rich motifs (underlined) within the random sequences (n = 43) of the individual aptamer are indicated. (B) Analysis of the consensus sequences of selected aptamers (A) employing WebLogo software. Results represent stacks of nucleic acid symbols based on a multiple alignment. The height of the symbols within the stack denotes the relative frequency of the nucleic acid, while the overall height of the stack represents the sequence conservation at that position. (C) Relative binding of full-length aptamers (A) labeled with [γ- 32 P]-ATP was analyzed by radioactive binding assay, employing cytokine-unstimulated (CK − ) or stimulated (CK + ) cells. Results are presented as the mean ± SD from three independent experiments.
    Figure Legend Snippet: Binding Activities of Selected DNA Aptamers to Cytokine-Stimulated Cells (A) Sequences of individual ssDNA aptamer candidates. G-quartet or G-rich motifs (underlined) within the random sequences (n = 43) of the individual aptamer are indicated. (B) Analysis of the consensus sequences of selected aptamers (A) employing WebLogo software. Results represent stacks of nucleic acid symbols based on a multiple alignment. The height of the symbols within the stack denotes the relative frequency of the nucleic acid, while the overall height of the stack represents the sequence conservation at that position. (C) Relative binding of full-length aptamers (A) labeled with [γ- 32 P]-ATP was analyzed by radioactive binding assay, employing cytokine-unstimulated (CK − ) or stimulated (CK + ) cells. Results are presented as the mean ± SD from three independent experiments.

    Techniques Used: Binding Assay, Software, Sequencing, Labeling

    11) Product Images from "Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period"

    Article Title: Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks779

    Top1 binds to intermediate region between ROREs in Bmal1 promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30 and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.
    Figure Legend Snippet: Top1 binds to intermediate region between ROREs in Bmal1 promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30 and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.

    Techniques Used: Cleavage Assay, Labeling, Purification, Cycling Probe Technology

    12) Product Images from "ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity"

    Article Title: ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2011.137

    ERK2 directly phosphorylates residue serine 74 and serine 77 in Bmf. ( a ) Alignment of Bmf protein sequences surrounding serine 74 and serine 77 among different species. Conserved serine 74 and serine 77 residues are shaded. The amino-acid positions relative to the start codon are shown in parentheses. ( b ) WM793TR cell lines expressing wild type, S74A, S77A, S74A/S77A (AA), S74D, S77D, S74D/S77D (DD) HA-Bmf were induced with with or without 100 ng/ml doxycycline for 7 h. Cells lysates were analyzed by western blotting using antibodies against the HA-tag and actin. ( c ) 1205LuTR/HA-Bmf (WT, S77A and AA) cells and A375TR/HA-Bmf (WT, S77A and AA) cells were treated with or without 100 ng/ml doxycycline for 7 h. Cell lysates were analyzed as in b . ( d ) WM793TR/HA Bmf cells were treated with 100 ng/ml doxycycline for 1.5 h and 1 μ M okadaic acid was added for another 30 min. Cells were then lysed for western blot analysis. Indicated are the percentages of Bmf that is slow migrating, as determined by quantitation of band intensity. ( e ) WM793TR/HA-Bmf S74A and WM793TR/HA-Bmf S77A cells were transfected with control, ERK1, ERK2 and p38 α siRNA. Seventy-two hours post transfection, cells were treated with 100 ng/ml doxycycline and with or without 10 μ M U0126 for 7 h before lysis for western blot analysis on indicated proteins. ( f ) Activated ERK2 was incubated with bacterially expressed GST-Bmf WT, GST-Bmf S74A, GST-Bmf S77A and GST-Bmf AA in the presence of [ γ - 32 P]ATP. Phosphorylation of GST-Bmf variants was examined by SDS-PAGE followed by autoradiography. Coomassie staining of the same gel is shown below
    Figure Legend Snippet: ERK2 directly phosphorylates residue serine 74 and serine 77 in Bmf. ( a ) Alignment of Bmf protein sequences surrounding serine 74 and serine 77 among different species. Conserved serine 74 and serine 77 residues are shaded. The amino-acid positions relative to the start codon are shown in parentheses. ( b ) WM793TR cell lines expressing wild type, S74A, S77A, S74A/S77A (AA), S74D, S77D, S74D/S77D (DD) HA-Bmf were induced with with or without 100 ng/ml doxycycline for 7 h. Cells lysates were analyzed by western blotting using antibodies against the HA-tag and actin. ( c ) 1205LuTR/HA-Bmf (WT, S77A and AA) cells and A375TR/HA-Bmf (WT, S77A and AA) cells were treated with or without 100 ng/ml doxycycline for 7 h. Cell lysates were analyzed as in b . ( d ) WM793TR/HA Bmf cells were treated with 100 ng/ml doxycycline for 1.5 h and 1 μ M okadaic acid was added for another 30 min. Cells were then lysed for western blot analysis. Indicated are the percentages of Bmf that is slow migrating, as determined by quantitation of band intensity. ( e ) WM793TR/HA-Bmf S74A and WM793TR/HA-Bmf S77A cells were transfected with control, ERK1, ERK2 and p38 α siRNA. Seventy-two hours post transfection, cells were treated with 100 ng/ml doxycycline and with or without 10 μ M U0126 for 7 h before lysis for western blot analysis on indicated proteins. ( f ) Activated ERK2 was incubated with bacterially expressed GST-Bmf WT, GST-Bmf S74A, GST-Bmf S77A and GST-Bmf AA in the presence of [ γ - 32 P]ATP. Phosphorylation of GST-Bmf variants was examined by SDS-PAGE followed by autoradiography. Coomassie staining of the same gel is shown below

    Techniques Used: Expressing, Western Blot, Quantitation Assay, Transfection, Lysis, Incubation, SDS Page, Autoradiography, Staining

    13) Product Images from "Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome"

    Article Title: Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv428

    Effects of hypomethylation and methylmimic on subcellular localization and RNA binding capacity of DRBD18. ( A ) Biochemical fractionation and western blot analysis of MHT-DRBD18(WT), MHT-DRBD18(R→K) and MHT-DRBD18(R→F) expressing cell lines. Cells were harvested two days post-induction and 1 × 10 6 cellular equivalents of cytoplasmic and nuclear fractions were separated via 10% SDS-PAGE. HSP70 and NOG are cytoplasmic and nuclear markers, respectively. MHT-DRBD18 constructs were visualized using anti-myc antibody. W, whole cell; C, cytoplasmic fraction; N, nuclear fraction. ( B ) Indirect immunofluorescence of uninduced MHT-DRBD18(WT) expressing cells and MHT-DRBD18(WT), MHT-DRBD18(R→K), and MHT-DRBD18(R→F) expressing cell lines 2 days post-induction with tet using anti-myc antibody. Nuclei and kinetoplasts were stained with DAPI, and merged signals (myc/DAPI) are shown on the right. DIC, differential interference contrast. Inset shows higher magnification image of perinuclear labeling. ( C ) CLIP analysis of cells expressing MHT-DRBD18 variants. Cells expressing MHT-DRBD18 variants were UV crosslinked at 254 nm and proteins were precipitated using anti-myc conjugated resin. Cell lysate pooled from all three samples and incubated with uncoated beads served as a negative control sample (mock). Left panel shows anti-myc western blot analysis of input samples (7.5 × 10 5 cell equivalents). Also shown are immunoprecipitated (bound) MHT-DRDB18 variant proteins following in vivo crosslinking and immunoprecipitation and just prior to labeling with γ 32 P-ATP by polynucleotide kinase. These bound samples were used for the analyses shown in the right panel. I, input, B, bound samples. Right panel shows phosphorimage analysis of bound MHT-DRBD18 variant RNPs. Values below the image represent the average signals from three replicate experiments.
    Figure Legend Snippet: Effects of hypomethylation and methylmimic on subcellular localization and RNA binding capacity of DRBD18. ( A ) Biochemical fractionation and western blot analysis of MHT-DRBD18(WT), MHT-DRBD18(R→K) and MHT-DRBD18(R→F) expressing cell lines. Cells were harvested two days post-induction and 1 × 10 6 cellular equivalents of cytoplasmic and nuclear fractions were separated via 10% SDS-PAGE. HSP70 and NOG are cytoplasmic and nuclear markers, respectively. MHT-DRBD18 constructs were visualized using anti-myc antibody. W, whole cell; C, cytoplasmic fraction; N, nuclear fraction. ( B ) Indirect immunofluorescence of uninduced MHT-DRBD18(WT) expressing cells and MHT-DRBD18(WT), MHT-DRBD18(R→K), and MHT-DRBD18(R→F) expressing cell lines 2 days post-induction with tet using anti-myc antibody. Nuclei and kinetoplasts were stained with DAPI, and merged signals (myc/DAPI) are shown on the right. DIC, differential interference contrast. Inset shows higher magnification image of perinuclear labeling. ( C ) CLIP analysis of cells expressing MHT-DRBD18 variants. Cells expressing MHT-DRBD18 variants were UV crosslinked at 254 nm and proteins were precipitated using anti-myc conjugated resin. Cell lysate pooled from all three samples and incubated with uncoated beads served as a negative control sample (mock). Left panel shows anti-myc western blot analysis of input samples (7.5 × 10 5 cell equivalents). Also shown are immunoprecipitated (bound) MHT-DRDB18 variant proteins following in vivo crosslinking and immunoprecipitation and just prior to labeling with γ 32 P-ATP by polynucleotide kinase. These bound samples were used for the analyses shown in the right panel. I, input, B, bound samples. Right panel shows phosphorimage analysis of bound MHT-DRBD18 variant RNPs. Values below the image represent the average signals from three replicate experiments.

    Techniques Used: RNA Binding Assay, Fractionation, Western Blot, Expressing, SDS Page, Construct, Immunofluorescence, Staining, Labeling, Cross-linking Immunoprecipitation, Incubation, Negative Control, Immunoprecipitation, Variant Assay, In Vivo

    14) Product Images from "Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period"

    Article Title: Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks779

    Top1 binds to intermediate region between ROREs in Bmal1 promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30 and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.
    Figure Legend Snippet: Top1 binds to intermediate region between ROREs in Bmal1 promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30 and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.

    Techniques Used: Cleavage Assay, Labeling, Purification, Cycling Probe Technology

    15) Product Images from "Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A"

    Article Title: Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr195

    SC35 promotes tau exon 10 inclusion through SC35-like enhancer. ( A ) SC35 was immunopurified by anti-HA. SC35 tagged with HA was overexpressed in HEK-293T cells and immunopurified with anti-HA-crosslinked protein G beads. Different elution fractions (left panel) were subjected to western blot analysis with anti-HA. Immunoprecipitated SC35 without elution was dephosphorylated with alkaline phosphatase and determined by anti-HA and 1H4, an antibody to phosphorylated SR proteins. E1, elution fraction 1; E2, elution fraction 2; E3, elution fraction 3; ALP, alkaline phosphatase. ( B ) SC35 bound to RNA of tau exon 10. Immunopurified SC35 (E1 and E2) was incubated with tau pre-mRNA (tau-RNA) or SC35-like enhancer deleted tau pre-mRNA (tau-RNA ΔSC35 like ) pre-labeled with γ- 32 P ATP. The incubation products were subjected to native-gel electrophoresis. After drying, the gel was analyzed with phosphoimaging device (BAS-1500, Fujifilm). ( C ) Schematic of mutations of mini-tau-gene on SC35-like enhancer of tau exon 10. ( D ) Mutations of SC35-like enhancer affected SC35 promoted tau exon 10 inclusion. Different mutants of mini-tau-gene, pCI/SI9–SI10, at SC35 like enhancer were transfected alone or together with pCEP4/SC35. RT–PCR was carried out to measure tau exon 10 splicing after 36 h transfection.
    Figure Legend Snippet: SC35 promotes tau exon 10 inclusion through SC35-like enhancer. ( A ) SC35 was immunopurified by anti-HA. SC35 tagged with HA was overexpressed in HEK-293T cells and immunopurified with anti-HA-crosslinked protein G beads. Different elution fractions (left panel) were subjected to western blot analysis with anti-HA. Immunoprecipitated SC35 without elution was dephosphorylated with alkaline phosphatase and determined by anti-HA and 1H4, an antibody to phosphorylated SR proteins. E1, elution fraction 1; E2, elution fraction 2; E3, elution fraction 3; ALP, alkaline phosphatase. ( B ) SC35 bound to RNA of tau exon 10. Immunopurified SC35 (E1 and E2) was incubated with tau pre-mRNA (tau-RNA) or SC35-like enhancer deleted tau pre-mRNA (tau-RNA ΔSC35 like ) pre-labeled with γ- 32 P ATP. The incubation products were subjected to native-gel electrophoresis. After drying, the gel was analyzed with phosphoimaging device (BAS-1500, Fujifilm). ( C ) Schematic of mutations of mini-tau-gene on SC35-like enhancer of tau exon 10. ( D ) Mutations of SC35-like enhancer affected SC35 promoted tau exon 10 inclusion. Different mutants of mini-tau-gene, pCI/SI9–SI10, at SC35 like enhancer were transfected alone or together with pCEP4/SC35. RT–PCR was carried out to measure tau exon 10 splicing after 36 h transfection.

    Techniques Used: Western Blot, Immunoprecipitation, ALP Assay, Incubation, Labeling, Nucleic Acid Electrophoresis, Transfection, Reverse Transcription Polymerase Chain Reaction

    16) Product Images from "The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion"

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00025-18

    TIN2L phosphorylation is dependent upon an intact CK2 consensus site. (A) Evolutionary trace analysis and summary of known interaction regions in TIN2. The residues comprising the putative CK2 phosphorylation sites are displayed. The protein interaction domains, the region where all DC-causing mutations cluster (DC), the position of R282 (*), and the putative TBM, all of which are present in both the TIN2L and TIN2S isoforms, and the C-terminal domain unique to TIN2L (TIN2L CTD) are indicated. (B) Characterization of TIN2S and TIN2L phosphorylation. Flag-tagged TIN2S or TIN2L was analyzed by SDS-PAGE in the presence of Phos-tag reagent. Phosphorylated TIN2L (TIN2L P ) is visible as a supershift compared to the λ phosphatase-treated TIN2L. (C) Characterization of TIN2L phosphorylation at S396 as in panel B. (D) Recombinant CK2 (NEB) analyzed by SDS-PAGE and silver staining. M, protein molecular mass marker; 1, CK2. (E) In vitro phosphorylation of TIN2L by CK2. Recombinant TIN2L purified from E. coli was incubated with CK2 and [γ- 32 P]ATP in the absence or presence of a CK2 inhibitor (TBB). (F) In vitro phosphorylation of partially purified TIN2L and TIN2L-S396A by CK2. Flag-tagged TIN2L or TIN2L-S396A from transiently transfected HEK 293T cells was dephosphorylated by incubation with λ phosphatase, purified via immunoprecipitation, and incubated with CK2. Phosphorylation status was assessed by SDS-PAGE in the presence of the Phos-tag reagent and compared to that of non-λ phosphatase-treated controls.
    Figure Legend Snippet: TIN2L phosphorylation is dependent upon an intact CK2 consensus site. (A) Evolutionary trace analysis and summary of known interaction regions in TIN2. The residues comprising the putative CK2 phosphorylation sites are displayed. The protein interaction domains, the region where all DC-causing mutations cluster (DC), the position of R282 (*), and the putative TBM, all of which are present in both the TIN2L and TIN2S isoforms, and the C-terminal domain unique to TIN2L (TIN2L CTD) are indicated. (B) Characterization of TIN2S and TIN2L phosphorylation. Flag-tagged TIN2S or TIN2L was analyzed by SDS-PAGE in the presence of Phos-tag reagent. Phosphorylated TIN2L (TIN2L P ) is visible as a supershift compared to the λ phosphatase-treated TIN2L. (C) Characterization of TIN2L phosphorylation at S396 as in panel B. (D) Recombinant CK2 (NEB) analyzed by SDS-PAGE and silver staining. M, protein molecular mass marker; 1, CK2. (E) In vitro phosphorylation of TIN2L by CK2. Recombinant TIN2L purified from E. coli was incubated with CK2 and [γ- 32 P]ATP in the absence or presence of a CK2 inhibitor (TBB). (F) In vitro phosphorylation of partially purified TIN2L and TIN2L-S396A by CK2. Flag-tagged TIN2L or TIN2L-S396A from transiently transfected HEK 293T cells was dephosphorylated by incubation with λ phosphatase, purified via immunoprecipitation, and incubated with CK2. Phosphorylation status was assessed by SDS-PAGE in the presence of the Phos-tag reagent and compared to that of non-λ phosphatase-treated controls.

    Techniques Used: SDS Page, Recombinant, Silver Staining, Marker, In Vitro, Purification, Incubation, Transfection, Immunoprecipitation

    17) Product Images from "Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress"

    Article Title: Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006133

    VgrR-VgrS is a two-component signaling system. (A) VgrS has autokinase activity. Inverted membrane vesicles of full-length VgrS and VgrS H186A recombinant proteins (5 μM) were incubated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. (B) VgrS transferred phosphoryl group to VgrR. VgrS (5 μM) was autophosphorylated as in (A) for 2 min. Twenty μM VgrR or 80 μM VgrR D51A proteins were added into the reaction, respectively. In both (A) and (B), the reaction was stopped by loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins (lower panels). Each experiment was repeated three times. (C) vgrR and vgrS constitute a bicistronic operon. RT-PCR was used to amplify cDNA. RT: amplification using cDNA transcribed from total RNA as template.–RT: negative control lacking reverse transcriptase during cDNA synthesis. DNA: amplification using bacterial DNA as template. Amplification of vgrS cDNA was used as a positive control. Location of the primers is shown in Fig 1A (upper panel). The assay was repeated three times. (D) Mapping the transcription initiation site (TIS) of the vgrR-vgrS operon. Primer extension using total RNA of X . campestris pv. campestris as the template. The G-A-T-C lanes show the dideoxy chain termination sequencing reaction in the same promoter region (note that the top of the ladder is blur for high GC content of the template). 1 and 2: vgrR mRNA was reverse transcribed at 42°C and 52°C, respectively, 3: △vgrR mRNA template (with primer binding site being deleted) was reverse transcribed at 42°C as negative control. TIS sites (P1 and P2) are shown as asterisks. NS: non-specific bands. The experiment was repeated independently twice. (E) Nucleotide sequence of the 5′ region upstream of vgrR . (F) GUS activity assay of promoters. GUS activity assays were conducted among all the recombinant bacterial strains with transcriptional fusions of P1-GUS, P2-GUS and P1+P2-GUS, respectively. Bacterial strains were induced in iron depleted (MMX) or iron replete (MMX plus Fe 3+ ) conditions. Vertical bars represent the standard deviations (n = 3).
    Figure Legend Snippet: VgrR-VgrS is a two-component signaling system. (A) VgrS has autokinase activity. Inverted membrane vesicles of full-length VgrS and VgrS H186A recombinant proteins (5 μM) were incubated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. (B) VgrS transferred phosphoryl group to VgrR. VgrS (5 μM) was autophosphorylated as in (A) for 2 min. Twenty μM VgrR or 80 μM VgrR D51A proteins were added into the reaction, respectively. In both (A) and (B), the reaction was stopped by loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins (lower panels). Each experiment was repeated three times. (C) vgrR and vgrS constitute a bicistronic operon. RT-PCR was used to amplify cDNA. RT: amplification using cDNA transcribed from total RNA as template.–RT: negative control lacking reverse transcriptase during cDNA synthesis. DNA: amplification using bacterial DNA as template. Amplification of vgrS cDNA was used as a positive control. Location of the primers is shown in Fig 1A (upper panel). The assay was repeated three times. (D) Mapping the transcription initiation site (TIS) of the vgrR-vgrS operon. Primer extension using total RNA of X . campestris pv. campestris as the template. The G-A-T-C lanes show the dideoxy chain termination sequencing reaction in the same promoter region (note that the top of the ladder is blur for high GC content of the template). 1 and 2: vgrR mRNA was reverse transcribed at 42°C and 52°C, respectively, 3: △vgrR mRNA template (with primer binding site being deleted) was reverse transcribed at 42°C as negative control. TIS sites (P1 and P2) are shown as asterisks. NS: non-specific bands. The experiment was repeated independently twice. (E) Nucleotide sequence of the 5′ region upstream of vgrR . (F) GUS activity assay of promoters. GUS activity assays were conducted among all the recombinant bacterial strains with transcriptional fusions of P1-GUS, P2-GUS and P1+P2-GUS, respectively. Bacterial strains were induced in iron depleted (MMX) or iron replete (MMX plus Fe 3+ ) conditions. Vertical bars represent the standard deviations (n = 3).

    Techniques Used: Activity Assay, Recombinant, Incubation, SDS Page, Autoradiography, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Sequencing, Binding Assay

    VgrS senses Fe 3+ depletion by its sensor region. (A) Fe 3+ inhibits the autophosphorylation of full-length VgrS. Upper panels: Inverted membrane vesicles containing full-length VgrS were phosphorylated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. Fe 3+ was added at different concentrations. Lower panels: Soluble, truncated VgrS is not stimulated by Fe 3+ . MBP-VgrS without the input and transmembrane domains was used in the autophosphorylation assay. The experiment was repeated three times. (B) Iron excess decreased the phosphotransfer level from VgrS to VgrR. Full-length VgrS membrane was phosphorylated as described in (A) for 2 min in the presence of Fe 3+ , 15.0 μM VgrR was added into the mixture for 20 sec before stopping the reaction. (C) VgrS sensor directly binds Fe 3+ . 2 μM VgrS sensor, truncated VgrS (MBP-VgrS) and VgrS E43A sensor were used in a microscale thermophoresis (MST) assay. The titer of Fe 3+ ranged from 0.061 to 250 μM. The experiment was repeated three times. (D) Substitution of residues in the ExxE motif of VgrS sensor eliminated VgrS’s sensing of Fe 3+ . Inverted membrane vesicles with full-length VgrS E43A , VgrS P44A , VgrS Q45A and VgrS E46A were used in the autokinase assay as in (A). Fe 3+ was added into the reaction mixtures. The experiment was repeated twice. In (A, B, and D), the reaction was stopped by adding loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins.
    Figure Legend Snippet: VgrS senses Fe 3+ depletion by its sensor region. (A) Fe 3+ inhibits the autophosphorylation of full-length VgrS. Upper panels: Inverted membrane vesicles containing full-length VgrS were phosphorylated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. Fe 3+ was added at different concentrations. Lower panels: Soluble, truncated VgrS is not stimulated by Fe 3+ . MBP-VgrS without the input and transmembrane domains was used in the autophosphorylation assay. The experiment was repeated three times. (B) Iron excess decreased the phosphotransfer level from VgrS to VgrR. Full-length VgrS membrane was phosphorylated as described in (A) for 2 min in the presence of Fe 3+ , 15.0 μM VgrR was added into the mixture for 20 sec before stopping the reaction. (C) VgrS sensor directly binds Fe 3+ . 2 μM VgrS sensor, truncated VgrS (MBP-VgrS) and VgrS E43A sensor were used in a microscale thermophoresis (MST) assay. The titer of Fe 3+ ranged from 0.061 to 250 μM. The experiment was repeated three times. (D) Substitution of residues in the ExxE motif of VgrS sensor eliminated VgrS’s sensing of Fe 3+ . Inverted membrane vesicles with full-length VgrS E43A , VgrS P44A , VgrS Q45A and VgrS E46A were used in the autokinase assay as in (A). Fe 3+ was added into the reaction mixtures. The experiment was repeated twice. In (A, B, and D), the reaction was stopped by adding loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins.

    Techniques Used: Size-exclusion Chromatography, Microscale Thermophoresis, SDS Page, Autoradiography, Staining

    Dissection of the VgrR binding consensus motif. (A) Venn diagram showing the number of VgrR-regulated genes identified by ChIP-seq. (B) Functional categories of the VgrR-regulated genes identified by ChIP-seq. Details of the genes are listed in S6 and S7 Tables. (C) Deduced consensus VgrR-binding DNA motif based on ChIP-seq data. Weblogo was used to show the nucleotide composition. (D) Mapping the VgrR protected DNA region in the 5′ upstream sequence of XC1241 ( tdvA ) by DNase I footprinting. The amounts of VgrR protein used in the reactions were 1: zero; 2: 0.08 μM; 3: 0.8 μM; 4: 3.2 μM; and 5: 8.0 μM. The DNA regions protected by VgrR are shown on the right of the footprinting results, with the three possible VgrR-binding motifs shown in red, green, and black, respectively. A-T-C-G lanes are the DNA ladders obtained by a dideoxy-mediated chain-termination method using the same DNA sequence as the template. (E) Electrophoretic mobility shift assay verified the DNA motif of the XC1241 promoter bound by VgrR. The DNA probes were chemically synthesized according to those shown in (D). Sequences of the promoter region of XC1241 are listed above each panel. Each DNA probe was labeled by [γ- 32 P]ATP. Triangles indicate the VgrR-DNA complexes. All experiments were repeated three times.
    Figure Legend Snippet: Dissection of the VgrR binding consensus motif. (A) Venn diagram showing the number of VgrR-regulated genes identified by ChIP-seq. (B) Functional categories of the VgrR-regulated genes identified by ChIP-seq. Details of the genes are listed in S6 and S7 Tables. (C) Deduced consensus VgrR-binding DNA motif based on ChIP-seq data. Weblogo was used to show the nucleotide composition. (D) Mapping the VgrR protected DNA region in the 5′ upstream sequence of XC1241 ( tdvA ) by DNase I footprinting. The amounts of VgrR protein used in the reactions were 1: zero; 2: 0.08 μM; 3: 0.8 μM; 4: 3.2 μM; and 5: 8.0 μM. The DNA regions protected by VgrR are shown on the right of the footprinting results, with the three possible VgrR-binding motifs shown in red, green, and black, respectively. A-T-C-G lanes are the DNA ladders obtained by a dideoxy-mediated chain-termination method using the same DNA sequence as the template. (E) Electrophoretic mobility shift assay verified the DNA motif of the XC1241 promoter bound by VgrR. The DNA probes were chemically synthesized according to those shown in (D). Sequences of the promoter region of XC1241 are listed above each panel. Each DNA probe was labeled by [γ- 32 P]ATP. Triangles indicate the VgrR-DNA complexes. All experiments were repeated three times.

    Techniques Used: Dissection, Binding Assay, Chromatin Immunoprecipitation, Functional Assay, Sequencing, Footprinting, Electrophoretic Mobility Shift Assay, Synthesized, Labeling

    18) Product Images from "Systematic Mutational Analysis of Histidine Kinase Genes in the Nosocomial Pathogen Stenotrophomonas maltophilia Identifies BfmAK System Control of Biofilm Development"

    Article Title: Systematic Mutational Analysis of Histidine Kinase Genes in the Nosocomial Pathogen Stenotrophomonas maltophilia Identifies BfmAK System Control of Biofilm Development

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03951-15

    Operon organization of the Smlt4208-Smlt4209 locus and its role in biofilm development. (A) Genomic localization of the Smlt4204-Smlt4209 locus. Arrows indicate genes and their transcriptional directions. Gene names are listed, and the primers (P1 to P12) used for RT-PCR are indicated around the arrows. (B) Dissection of the operon organization of the Smlt4204-Smlt4209 locus by RT-PCR. cDNA was reverse transcribed with random primers using total RNA from S. maltophilia ATCC 13637 grown overnight in NYG medium at 28°C. RT denotes PCR amplification using cDNA transcribed from RNA as the template; DNA denotes the positive control, using bacterial total DNA as the PCR template; −RT denotes the negative control, for which reverse transcriptase was absent during cDNA synthesis. (C) In vitro phosphorylation assay of the BfmA-BfmK system. BfmK inverted membrane vesicles (50 μg) and soluble BfmA protein (20 μM) were used. [γ- 32 P]ATP was added to the reaction mix for the times indicated. The reactions were stopped with SDS loading buffer, and the samples were separated by 12% SDS-PAGE. Int., band intensity that was estimated by Quantity One software. (D) bfmK ( Smlt4208 ) and bfmA ( Smlt4209 ) control biofilm formation in S. maltophilia ATCC 13637. Bacterial biofilms were quantified by OD 590 measurements using the crystal violet staining method. Bars represent standard deviations ( n = 8). *, P
    Figure Legend Snippet: Operon organization of the Smlt4208-Smlt4209 locus and its role in biofilm development. (A) Genomic localization of the Smlt4204-Smlt4209 locus. Arrows indicate genes and their transcriptional directions. Gene names are listed, and the primers (P1 to P12) used for RT-PCR are indicated around the arrows. (B) Dissection of the operon organization of the Smlt4204-Smlt4209 locus by RT-PCR. cDNA was reverse transcribed with random primers using total RNA from S. maltophilia ATCC 13637 grown overnight in NYG medium at 28°C. RT denotes PCR amplification using cDNA transcribed from RNA as the template; DNA denotes the positive control, using bacterial total DNA as the PCR template; −RT denotes the negative control, for which reverse transcriptase was absent during cDNA synthesis. (C) In vitro phosphorylation assay of the BfmA-BfmK system. BfmK inverted membrane vesicles (50 μg) and soluble BfmA protein (20 μM) were used. [γ- 32 P]ATP was added to the reaction mix for the times indicated. The reactions were stopped with SDS loading buffer, and the samples were separated by 12% SDS-PAGE. Int., band intensity that was estimated by Quantity One software. (D) bfmK ( Smlt4208 ) and bfmA ( Smlt4209 ) control biofilm formation in S. maltophilia ATCC 13637. Bacterial biofilms were quantified by OD 590 measurements using the crystal violet staining method. Bars represent standard deviations ( n = 8). *, P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Dissection, Polymerase Chain Reaction, Amplification, Positive Control, Negative Control, In Vitro, Phosphorylation Assay, SDS Page, Software, Staining

    BfmA directly binds to the promoter region of bfmA and acoT and regulates biofilm formation. EMSAs reveal that BfmA binds directly to the promoter region of bfmA itself (A) and acoT (B). A total of 4 fmol of [γ- 32 P]ATP-labeled DNA sequences corresponding to the bfmA and acoT promoter regions was used in the assay. Increasing amounts of unlabeled probes were used as competitors (10× to 1,000×) for binding to BfmA protein. The black triangle on the right side of each panel represents the BfmA-DNA binding complex. (C) bfmA is epistatic to acoT , which contributes to biofilm formation. Biofilm quantity was measured in different bacterial strains. IFD-acoT, acoT in-frame deletion mutant; IFD-acoT-acoT and IFD-bfmA-acoT, acoT and bfmA mutants containing a full-length acoT gene subjected to the control of the P lac promoter. All of the strains, including the wild-type strain, contained a corresponding blank or recombinant pBBRMCS2 vector. Bars represent standard deviations. *, significant difference ( P
    Figure Legend Snippet: BfmA directly binds to the promoter region of bfmA and acoT and regulates biofilm formation. EMSAs reveal that BfmA binds directly to the promoter region of bfmA itself (A) and acoT (B). A total of 4 fmol of [γ- 32 P]ATP-labeled DNA sequences corresponding to the bfmA and acoT promoter regions was used in the assay. Increasing amounts of unlabeled probes were used as competitors (10× to 1,000×) for binding to BfmA protein. The black triangle on the right side of each panel represents the BfmA-DNA binding complex. (C) bfmA is epistatic to acoT , which contributes to biofilm formation. Biofilm quantity was measured in different bacterial strains. IFD-acoT, acoT in-frame deletion mutant; IFD-acoT-acoT and IFD-bfmA-acoT, acoT and bfmA mutants containing a full-length acoT gene subjected to the control of the P lac promoter. All of the strains, including the wild-type strain, contained a corresponding blank or recombinant pBBRMCS2 vector. Bars represent standard deviations. *, significant difference ( P

    Techniques Used: Labeling, Binding Assay, Mutagenesis, Recombinant, Plasmid Preparation

    19) Product Images from "Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity"

    Article Title: Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

    Journal: Molecular Biology of the Cell

    doi:

    EMSA of DNA–protein complexes formed with the PKC/cAMP- and growth factor-responsive FGF-2 gene promoter regions. (A) EMSA of DNA–protein complexes formed with the cAMP/PKC-responsive region of the FGF-2 gene promoter. Nuclear proteins (5 μg) were used from subconfluent serum-free astrocytes that were untreated (lane 1) or treated with forskolin (10 μM, 24 h; lane 2) or PMA (100 nM, 24 h; lane 3). A double-stranded oligonucleotide corresponding to −624/−555-bp FGF-2 promoter sequences was used as a target DNA. (a–d) DNA–protein complexes; fp, free probe. (B) Binding to PKC/cAMP-responsive promoter region (−624/−555 bp): competition with unlabeled target DNA and consensus oligonucleotides for several transcription factors. Reactions contained 5 μg of nuclear proteins from PMA-treated astrocytes and a 150-fold molar excess of unlabeled competitor. A 50-fold excess of unlabeled target DNA was sufficient to completely abolish binding. The numbers are for the following competitors: (1) OCT1; (2) CRE; (3) AP1; (4) AP2; (5) NFκB; (6) STAT1/2; (7) STAT3/4; (8) unlabeled −624/−555-bp FGF-2 promoter fragment; (9) no competitor; and (10) no protein extract. (C) Binding to −555/−500-bp promoter region containing GFRE. Nuclear proteins (5 μg) from subconfluent astrocytic cultures treated with 5.0 × 10 −9 M EGF for 24 h were used in binding reactions with the −555/−500-bp fragment of the FGF-2 promoter. The target DNA was labeled with [γ- 32 P]ATP and T4 kinase, and 3000 cpm were used for each reaction as described in MATERIALS AND METHODS. The arrows indicate three DNA–protein complexes; fp, free probe. Binding reactions were carried out with or without unlabeled target DNA or various consensus oligonucleotides for known transcription factors. The numbers are for the following competitors: (1 and 2) no competitor; (3) unlabeled −555/−500 bp FGF-2 promoter fragment; (4) STAT 3/4; (5) STAT Ω; (6) NFκB; (7) AP1; (8) AP2; (9) CRE; and (10) OCT 1. Competitors were used in a 150-fold molar excess. A 50-fold excess of unlabeled target DNA was sufficient to completely abolish binding.
    Figure Legend Snippet: EMSA of DNA–protein complexes formed with the PKC/cAMP- and growth factor-responsive FGF-2 gene promoter regions. (A) EMSA of DNA–protein complexes formed with the cAMP/PKC-responsive region of the FGF-2 gene promoter. Nuclear proteins (5 μg) were used from subconfluent serum-free astrocytes that were untreated (lane 1) or treated with forskolin (10 μM, 24 h; lane 2) or PMA (100 nM, 24 h; lane 3). A double-stranded oligonucleotide corresponding to −624/−555-bp FGF-2 promoter sequences was used as a target DNA. (a–d) DNA–protein complexes; fp, free probe. (B) Binding to PKC/cAMP-responsive promoter region (−624/−555 bp): competition with unlabeled target DNA and consensus oligonucleotides for several transcription factors. Reactions contained 5 μg of nuclear proteins from PMA-treated astrocytes and a 150-fold molar excess of unlabeled competitor. A 50-fold excess of unlabeled target DNA was sufficient to completely abolish binding. The numbers are for the following competitors: (1) OCT1; (2) CRE; (3) AP1; (4) AP2; (5) NFκB; (6) STAT1/2; (7) STAT3/4; (8) unlabeled −624/−555-bp FGF-2 promoter fragment; (9) no competitor; and (10) no protein extract. (C) Binding to −555/−500-bp promoter region containing GFRE. Nuclear proteins (5 μg) from subconfluent astrocytic cultures treated with 5.0 × 10 −9 M EGF for 24 h were used in binding reactions with the −555/−500-bp fragment of the FGF-2 promoter. The target DNA was labeled with [γ- 32 P]ATP and T4 kinase, and 3000 cpm were used for each reaction as described in MATERIALS AND METHODS. The arrows indicate three DNA–protein complexes; fp, free probe. Binding reactions were carried out with or without unlabeled target DNA or various consensus oligonucleotides for known transcription factors. The numbers are for the following competitors: (1 and 2) no competitor; (3) unlabeled −555/−500 bp FGF-2 promoter fragment; (4) STAT 3/4; (5) STAT Ω; (6) NFκB; (7) AP1; (8) AP2; (9) CRE; and (10) OCT 1. Competitors were used in a 150-fold molar excess. A 50-fold excess of unlabeled target DNA was sufficient to completely abolish binding.

    Techniques Used: Binding Assay, Labeling

    20) Product Images from "Human Nudel and NudE as Regulators of Cytoplasmic Dynein in Poleward Protein Transport along the Mitotic Spindle"

    Article Title: Human Nudel and NudE as Regulators of Cytoplasmic Dynein in Poleward Protein Transport along the Mitotic Spindle

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.4.1239-1250.2003

    Phosphorylation by Cdc2 and Erk2. (A) Phosphorylation by Cdc2 in vitro. FLAG-tagged NudE, Nudel, and Nudel mt5 were immunoprecipitated with anti-FLAG beads and were treated with purified Cdc2 in the presence of [γ- 32 P]ATP. (B) Phosphorylation by Erk2 in vitro. Similar kinase assays were performed with or without purified Erk2 as indicated. (C) Phosphorylation sites in Nudel by Cdc2. FLAG-tagged Nudel S1 to Nudel S5 , each of which contained only one distinct S/TP motif, were immunoprecipitated and subjected to similar kinase assays. (D) Phosphorylation sites by Erk2. (E) Phosphorylation of Nudel by mitotic cell extracts. Histone H1 served as a control for the quality of the extracts. Kinase assays were performed in the presence or absence of olomoucine, a Cdc2/Erk inhibitor, as indicated.
    Figure Legend Snippet: Phosphorylation by Cdc2 and Erk2. (A) Phosphorylation by Cdc2 in vitro. FLAG-tagged NudE, Nudel, and Nudel mt5 were immunoprecipitated with anti-FLAG beads and were treated with purified Cdc2 in the presence of [γ- 32 P]ATP. (B) Phosphorylation by Erk2 in vitro. Similar kinase assays were performed with or without purified Erk2 as indicated. (C) Phosphorylation sites in Nudel by Cdc2. FLAG-tagged Nudel S1 to Nudel S5 , each of which contained only one distinct S/TP motif, were immunoprecipitated and subjected to similar kinase assays. (D) Phosphorylation sites by Erk2. (E) Phosphorylation of Nudel by mitotic cell extracts. Histone H1 served as a control for the quality of the extracts. Kinase assays were performed in the presence or absence of olomoucine, a Cdc2/Erk inhibitor, as indicated.

    Techniques Used: In Vitro, Immunoprecipitation, Purification

    21) Product Images from "Characterization of the Functional Domains of the SloR Metalloregulatory Protein in Streptococcus mutans"

    Article Title: Characterization of the Functional Domains of the SloR Metalloregulatory Protein in Streptococcus mutans

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01648-12

    Binding of wild-type (w/t) SloR and its metal ion-binding site 2 mutant variants to the sloABC promoter in EMSA. EMSAs were performed with a [γ- 32 P]ATP end-labeled 364-bp sloABC promoter-containing amplicon (the equivalent of 38 picograms) and
    Figure Legend Snippet: Binding of wild-type (w/t) SloR and its metal ion-binding site 2 mutant variants to the sloABC promoter in EMSA. EMSAs were performed with a [γ- 32 P]ATP end-labeled 364-bp sloABC promoter-containing amplicon (the equivalent of 38 picograms) and

    Techniques Used: Binding Assay, Mutagenesis, Labeling, Amplification

    Binding of wild-type SloR and its C-terminally truncated mutant variants to the sloABC promoter in EMSA. EMSAs were performed with a [γ- 32 P]ATP end-labeled 364-bp sloABC promoter-containing amplicon (the equivalent of 38 picograms) and whole-cell
    Figure Legend Snippet: Binding of wild-type SloR and its C-terminally truncated mutant variants to the sloABC promoter in EMSA. EMSAs were performed with a [γ- 32 P]ATP end-labeled 364-bp sloABC promoter-containing amplicon (the equivalent of 38 picograms) and whole-cell

    Techniques Used: Binding Assay, Mutagenesis, Labeling, Amplification

    22) Product Images from "Bacillus subtilis RecN binds and protects 3?-single-stranded DNA extensions in the presence of ATP"

    Article Title: Bacillus subtilis RecN binds and protects 3?-single-stranded DNA extensions in the presence of ATP

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki533

    DNA binding by RecN. ( A ) [ 32 P]Labelled ssDNA 60 (1 nM), at the 5′-terminus, was incubated with RecN (0.4, 0.8, 1.7, 3.7, 15, 30 and 60 nM) for 30 min at 37°C in buffer B in the absence (lanes 1–9) or presence of 1 mM ADP (lanes 10–18) or 1 mM ATP (lanes 19–27). RecN was not added to the controls in lane 1, 10 and 19. In lanes 10 and 19, (+) denotes that identical results were observed in the presence or absence of a nucleotide cofactor. ( B ) [γ- 32 P]Labelled ssDNA 60 (1 nM) and RecN (10 nM) were incubated and then ADP (0.01, 0.05, 0.1, 0.5 and 1 mM, lanes 3–7), ATP (0.01–1 mM, lanes 8–12) or AMP-PNP (0.01–1 mM, lanes 13–16) was added; incubation continued for 30 min at 37°C in buffer B. RecN was not added in lane 1. Complexes formed were separated by 10% ndPAGE and visualized by autoradiography. FD, free DNA; CI, complex I; CII, complex II; CIII, complex III; + and −, the presence or absence of the indicated factor.
    Figure Legend Snippet: DNA binding by RecN. ( A ) [ 32 P]Labelled ssDNA 60 (1 nM), at the 5′-terminus, was incubated with RecN (0.4, 0.8, 1.7, 3.7, 15, 30 and 60 nM) for 30 min at 37°C in buffer B in the absence (lanes 1–9) or presence of 1 mM ADP (lanes 10–18) or 1 mM ATP (lanes 19–27). RecN was not added to the controls in lane 1, 10 and 19. In lanes 10 and 19, (+) denotes that identical results were observed in the presence or absence of a nucleotide cofactor. ( B ) [γ- 32 P]Labelled ssDNA 60 (1 nM) and RecN (10 nM) were incubated and then ADP (0.01, 0.05, 0.1, 0.5 and 1 mM, lanes 3–7), ATP (0.01–1 mM, lanes 8–12) or AMP-PNP (0.01–1 mM, lanes 13–16) was added; incubation continued for 30 min at 37°C in buffer B. RecN was not added in lane 1. Complexes formed were separated by 10% ndPAGE and visualized by autoradiography. FD, free DNA; CI, complex I; CII, complex II; CIII, complex III; + and −, the presence or absence of the indicated factor.

    Techniques Used: Binding Assay, Incubation, Autoradiography

    23) Product Images from "DNA binding specificity of ATAF2, a NAC domain transcription factor targeted for degradation by Tobacco mosaic virus"

    Article Title: DNA binding specificity of ATAF2, a NAC domain transcription factor targeted for degradation by Tobacco mosaic virus

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-12-157

    ATAF2 binds DNA sequences from the genomic pull-down assay. ( A ) Schematic representation showing ATAF2 and ∆ ATAF2 deletion constructs. NAC domains C and D cover the DNA binding domain. ( B ) EMSA assay confirming ATAF2 and not ∆ ATAF2 binds to DNA clones identified in genomic pull-down assays. DNA probes were prepared by PCR-amplification followed by end-labeling with [γ- 32 P]ATP. The asterisk indicates the shifted band after ATAF2 binding. The four clones tested (C7, C34, C52, and C113) were all located within 1000 bp upstream of the coding sequences of At1G08540, At1G68907, At3G26540, and At3G11700 respectively.
    Figure Legend Snippet: ATAF2 binds DNA sequences from the genomic pull-down assay. ( A ) Schematic representation showing ATAF2 and ∆ ATAF2 deletion constructs. NAC domains C and D cover the DNA binding domain. ( B ) EMSA assay confirming ATAF2 and not ∆ ATAF2 binds to DNA clones identified in genomic pull-down assays. DNA probes were prepared by PCR-amplification followed by end-labeling with [γ- 32 P]ATP. The asterisk indicates the shifted band after ATAF2 binding. The four clones tested (C7, C34, C52, and C113) were all located within 1000 bp upstream of the coding sequences of At1G08540, At1G68907, At3G26540, and At3G11700 respectively.

    Techniques Used: Pull Down Assay, Construct, Binding Assay, Clone Assay, Polymerase Chain Reaction, Amplification, End Labeling

    24) Product Images from "The miR9863 Family Regulates Distinct Mla Alleles in Barley to Attenuate NLR Receptor-Triggered Disease Resistance and Cell-Death Signaling"

    Article Title: The miR9863 Family Regulates Distinct Mla Alleles in Barley to Attenuate NLR Receptor-Triggered Disease Resistance and Cell-Death Signaling

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004755

    22-nt miR9863s trigger the production of phasiRNAs with Mla1 transcripts. ( A ) Alignment of Mla1 sequence with 21-nt phased phasiRNAs. Raw reads of barley 21-nt phasiRNAs (y axis) obtained from deep sequencing were mapped to the sense strand (blue lines above the x axis) or antisense strand (red lines below the x axis) of the Mla1 sequence. Mla1 sequence adjacent to the miR9863 cleavage site was shown below the plot, and horizontal brackets below the sequence indicate 21-nt phasiRNAs, of which two most abundant ones (phasiRNAI and phasiRNAII) are indicated in the plot and in the sequence (in red). ( B ) Length of Mla1 phasiRNAs (x axis) was plotted to their raw reads value (y axis) obtained by sRNA deep sequencing in barley. ( C ) 5′-terminal nucleotide bias (x axis) of Mla1 phasiRNAs determined by raw reads (y axis) obtained by sRNA deep sequencing in barley. ( D ) Predicted secondary structure of precursors for expressing natural 22-nt miRNAs ( MIR9863b , MIR9863a ) and mutated 21-nt miRNAs ( MIR9863b 21 and MIR9863a 21 ). ( E ) miR9863b.1/b.2 and miR9863a of 22-nt regulate Mla1 with higher efficiency than the mutated miRNAs of 21-nt. The mature miRNAs were detected by RNA gel blotting (panel a) with the mixture of [γ- 32 P]ATP labeled probes for miR9863a and miR9863b.1 (see supplemental Table 2 online), and 5S rRNA was employed as a loading control. MLA1 and actin protein levels were determined by immunoblotting (panel b), and rubisco is shown as loading control. ( F ) to ( G ) miR9863b.1/b.2 and miR9863a of 22-nt trigger the biogenesis of phasiRNAs with Mla1 . Mla1 was co-expressed with EV or indicated miR9863 precursor in N. benthamiana , and the relative levels of phasiRNAI (F) and phasiRNAII (G) were measured by stem-loop quantitative RT-PCR (qRT-PCR) and normalized to U6 level. Letters above the bars (a-e) represent groups with significant differences [ p
    Figure Legend Snippet: 22-nt miR9863s trigger the production of phasiRNAs with Mla1 transcripts. ( A ) Alignment of Mla1 sequence with 21-nt phased phasiRNAs. Raw reads of barley 21-nt phasiRNAs (y axis) obtained from deep sequencing were mapped to the sense strand (blue lines above the x axis) or antisense strand (red lines below the x axis) of the Mla1 sequence. Mla1 sequence adjacent to the miR9863 cleavage site was shown below the plot, and horizontal brackets below the sequence indicate 21-nt phasiRNAs, of which two most abundant ones (phasiRNAI and phasiRNAII) are indicated in the plot and in the sequence (in red). ( B ) Length of Mla1 phasiRNAs (x axis) was plotted to their raw reads value (y axis) obtained by sRNA deep sequencing in barley. ( C ) 5′-terminal nucleotide bias (x axis) of Mla1 phasiRNAs determined by raw reads (y axis) obtained by sRNA deep sequencing in barley. ( D ) Predicted secondary structure of precursors for expressing natural 22-nt miRNAs ( MIR9863b , MIR9863a ) and mutated 21-nt miRNAs ( MIR9863b 21 and MIR9863a 21 ). ( E ) miR9863b.1/b.2 and miR9863a of 22-nt regulate Mla1 with higher efficiency than the mutated miRNAs of 21-nt. The mature miRNAs were detected by RNA gel blotting (panel a) with the mixture of [γ- 32 P]ATP labeled probes for miR9863a and miR9863b.1 (see supplemental Table 2 online), and 5S rRNA was employed as a loading control. MLA1 and actin protein levels were determined by immunoblotting (panel b), and rubisco is shown as loading control. ( F ) to ( G ) miR9863b.1/b.2 and miR9863a of 22-nt trigger the biogenesis of phasiRNAs with Mla1 . Mla1 was co-expressed with EV or indicated miR9863 precursor in N. benthamiana , and the relative levels of phasiRNAI (F) and phasiRNAII (G) were measured by stem-loop quantitative RT-PCR (qRT-PCR) and normalized to U6 level. Letters above the bars (a-e) represent groups with significant differences [ p

    Techniques Used: Sequencing, Expressing, Labeling, Quantitative RT-PCR

    25) Product Images from "Chloroplast Transcription at Different Light Intensities. Glutathione-Mediated Phosphorylation of the Major RNA Polymerase Involved in Redox-Regulated Organellar Gene Expression 1"

    Article Title: Chloroplast Transcription at Different Light Intensities. Glutathione-Mediated Phosphorylation of the Major RNA Polymerase Involved in Redox-Regulated Organellar Gene Expression 1

    Journal: Plant Physiology

    doi:

    Phosphorylation of chloroplast RNA polymerase-associated proteins from seedlings illuminated under HL and GL conditions. The enzyme complex was partially purified by heparin-Sepharose, followed by phosphocellulose chromatography. A, Polypeptides of chloroplast RNA polymerase preparations isolated from GL and HL seedlings, as revealed by SDS-PAGE and silver staining. B and C, In vitro phosphorylation assays in the presence of γ- 32 P-ATP. Polymerase-associated polypeptides from GL or HL plants were subjected to phosphorylation reactions using either the endogenous PTK activity (autophosphorylation; B), or by adding exogenous CK2 from rat liver (CK2-phosphorylation; C). The kinase-treated polypeptides were separated thereafter by SDS-PAGE and the dried gel was exposed to x-ray film. The horizontal arrows indicate the band in the 72- to 76-kD region, which is visible in A through C. Asterisks denote bands that differ in their intensity and dots denote bands with similar intensity in the GL and HL lanes in C.
    Figure Legend Snippet: Phosphorylation of chloroplast RNA polymerase-associated proteins from seedlings illuminated under HL and GL conditions. The enzyme complex was partially purified by heparin-Sepharose, followed by phosphocellulose chromatography. A, Polypeptides of chloroplast RNA polymerase preparations isolated from GL and HL seedlings, as revealed by SDS-PAGE and silver staining. B and C, In vitro phosphorylation assays in the presence of γ- 32 P-ATP. Polymerase-associated polypeptides from GL or HL plants were subjected to phosphorylation reactions using either the endogenous PTK activity (autophosphorylation; B), or by adding exogenous CK2 from rat liver (CK2-phosphorylation; C). The kinase-treated polypeptides were separated thereafter by SDS-PAGE and the dried gel was exposed to x-ray film. The horizontal arrows indicate the band in the 72- to 76-kD region, which is visible in A through C. Asterisks denote bands that differ in their intensity and dots denote bands with similar intensity in the GL and HL lanes in C.

    Techniques Used: Purification, Chromatography, Isolation, SDS Page, Silver Staining, In Vitro, Activity Assay

    26) 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

    27) Product Images from "Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans"

    Article Title: Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-69

    Purification of a putative PNKP from D. radiodurans and analysis of its 5' kinase and 3' phosphatase activities . A. Scheme of PNKP from D. radiodurans and H. sapiens . Protein domains are depicted according to predictions based on sequence similarities [36]. Schemes are not drawn to scale. HD, HD domain, kinase, polynucleotide kinase domain, HisB, histidinol phosphatase and related phosphatases domain. B. 3 μg of either D. radiodurans PNKP wt or PNKP R371K mutant were loaded onto a 10% SDS-PAGE and subsequently stained with Coomassie Blue R250. Both proteins were purified over 3 columns. Details are described in Methods. C. Titration of the D. radiodurans PNKP wt and PNKP R371K mutant to compare their polynucleotide kinase activity on a 5'OH 25 mer deoxyribose oligonucleotide. Different amounts of enzyme were incubated with the DNA substrate and γ-[ 32 P]-ATP as described in Methods. [ 32 P]-labelled 25 mer was detected by autoradiography.
    Figure Legend Snippet: Purification of a putative PNKP from D. radiodurans and analysis of its 5' kinase and 3' phosphatase activities . A. Scheme of PNKP from D. radiodurans and H. sapiens . Protein domains are depicted according to predictions based on sequence similarities [36]. Schemes are not drawn to scale. HD, HD domain, kinase, polynucleotide kinase domain, HisB, histidinol phosphatase and related phosphatases domain. B. 3 μg of either D. radiodurans PNKP wt or PNKP R371K mutant were loaded onto a 10% SDS-PAGE and subsequently stained with Coomassie Blue R250. Both proteins were purified over 3 columns. Details are described in Methods. C. Titration of the D. radiodurans PNKP wt and PNKP R371K mutant to compare their polynucleotide kinase activity on a 5'OH 25 mer deoxyribose oligonucleotide. Different amounts of enzyme were incubated with the DNA substrate and γ-[ 32 P]-ATP as described in Methods. [ 32 P]-labelled 25 mer was detected by autoradiography.

    Techniques Used: Purification, Sequencing, Mutagenesis, SDS Page, Staining, Titration, Activity Assay, Incubation, Autoradiography

    28) Product Images from "Identification of Toxoplasma gondii cAMP Dependent Protein Kinase and Its Role in the Tachyzoite Growth"

    Article Title: Identification of Toxoplasma gondii cAMP Dependent Protein Kinase and Its Role in the Tachyzoite Growth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022492

    Interaction of GST-TgPKA-C and MBP-TgPKA-R. A , Purified MBP-β-gal (lane 1) or MBP-TgPKA-R (lane 2) was incubated with GST-TgPKA-C in kinase buffer containing [γ- 32 P]ATP, separated on a denaturing gel, and Coomassie stained. B , Autoradiograph of the gel shown in ( A ). Arrows indicate the migration of GST-TgPKA-C, MBP-β-gal, or MBP-TgPKA-R. Molecular masses (kDa) are shown on the left.
    Figure Legend Snippet: Interaction of GST-TgPKA-C and MBP-TgPKA-R. A , Purified MBP-β-gal (lane 1) or MBP-TgPKA-R (lane 2) was incubated with GST-TgPKA-C in kinase buffer containing [γ- 32 P]ATP, separated on a denaturing gel, and Coomassie stained. B , Autoradiograph of the gel shown in ( A ). Arrows indicate the migration of GST-TgPKA-C, MBP-β-gal, or MBP-TgPKA-R. Molecular masses (kDa) are shown on the left.

    Techniques Used: Purification, Incubation, Staining, Autoradiography, Migration

    Expression, purification and kinase activity of TgPKA-C. A , A silver-stained ge and an immunoblot of purified GST-GFP or GST-TgPKA-C generated in the wheat germ cell-free protein synthesis system using pEU-GST-GFP (lanes 1 and 2) or pEU-GST-TgPKA-C (lanes 3 and 4). Total wheat germ extracts (lanes 1 and 3) were subjected to affinity chromatography on glutathione-Sepharose beads (lanes 2 and 4). The proteins were separated on denaturing gels and subjected to silver staining or transferred onto a nitrocellulose sheet and reacted with the anti-GST antibody. Molecular masses (kDa) are shown on the left. B , Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer containing [γ- 32 P]ATP, separated on a denaturing gel, and Coomassie stained (left panel). Autoradiograph of the gel (right panel). Arrows indicate the migration of GST-TgPKA-C and GST-GFP. C , Purified GST-TgPKA-C was incubated in kinase buffer (lane 1). The labeled protein was treated with λ-protein phosphatase (lane 2). Reaction mixtures were then subjected to resolution on an 8% SDS-PAGE gel, followed by Coomassie staining (left panel). Autoradiograph of the gel ( C ). D , Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer with Histone II AS and separated on a 15% denaturing gel followed by Coomassie staining (left panel). Autoradiograph of the gel (right panel). Molecular masses (kDa) are shown on the left.
    Figure Legend Snippet: Expression, purification and kinase activity of TgPKA-C. A , A silver-stained ge and an immunoblot of purified GST-GFP or GST-TgPKA-C generated in the wheat germ cell-free protein synthesis system using pEU-GST-GFP (lanes 1 and 2) or pEU-GST-TgPKA-C (lanes 3 and 4). Total wheat germ extracts (lanes 1 and 3) were subjected to affinity chromatography on glutathione-Sepharose beads (lanes 2 and 4). The proteins were separated on denaturing gels and subjected to silver staining or transferred onto a nitrocellulose sheet and reacted with the anti-GST antibody. Molecular masses (kDa) are shown on the left. B , Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer containing [γ- 32 P]ATP, separated on a denaturing gel, and Coomassie stained (left panel). Autoradiograph of the gel (right panel). Arrows indicate the migration of GST-TgPKA-C and GST-GFP. C , Purified GST-TgPKA-C was incubated in kinase buffer (lane 1). The labeled protein was treated with λ-protein phosphatase (lane 2). Reaction mixtures were then subjected to resolution on an 8% SDS-PAGE gel, followed by Coomassie staining (left panel). Autoradiograph of the gel ( C ). D , Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer with Histone II AS and separated on a 15% denaturing gel followed by Coomassie staining (left panel). Autoradiograph of the gel (right panel). Molecular masses (kDa) are shown on the left.

    Techniques Used: Expressing, Purification, Activity Assay, Staining, Generated, Affinity Chromatography, Silver Staining, Incubation, Autoradiography, Migration, Labeling, SDS Page

    29) Product Images from "The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action"

    Article Title: The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr271

    Mycobacterium tuberculosis UvrA is a DNA-dependent ATPase. Reactions mixtures contained 100 nM of the indicated DNA co-factor, 50 μM [γ- 32 P]ATP in the absence (lane 1) or presence of 10, 25, 50, 75, 100, 150, 200 and 250 nM Mt UvrA (lanes 2–9), respectively. Panels: ( a ), Mt UvrA alone; ( b ), Mt UvrA in the presence of single-stranded DNA (ODN8); ( c ) Mt UvrA in the presence of non-damaged duplex DNA (ODN1 annealed to ODN6); ( d ) Mt UvrA in presence of damaged duplex DNA (ODN7 annealed to ODN8) (ODN sequences are listed in Supplementary Table S1 ). The positions of [γ- 32 P]ATP and 32 Pi is indicated on the right-hand side. Panel ( e ): graphical representation of ATPase activity of Mt UvrA as a function of protein concentration. The percentage of [γ- 32 P]ATP hydrolyzed, in panels a–d, is plotted against the indicated amounts of Mt UvrA. (filled square), Mt UvrA in absence of DNA; (filled circle), Mt UvrA in the presence of single-stranded DNA; (filled triangle), Mt UvrA in the presence of non-damaged duplex DNA; (inverted filled triangle), Mt UvrA in the presence of damaged duplex DNA. Each point on the curves represents the mean of three separate experiments. Non-linear regression analysis (Michaelis-Menten) was applied to the data sets, using GraphPad PRISM ver5.00, for obtaining the best-fit curve.
    Figure Legend Snippet: Mycobacterium tuberculosis UvrA is a DNA-dependent ATPase. Reactions mixtures contained 100 nM of the indicated DNA co-factor, 50 μM [γ- 32 P]ATP in the absence (lane 1) or presence of 10, 25, 50, 75, 100, 150, 200 and 250 nM Mt UvrA (lanes 2–9), respectively. Panels: ( a ), Mt UvrA alone; ( b ), Mt UvrA in the presence of single-stranded DNA (ODN8); ( c ) Mt UvrA in the presence of non-damaged duplex DNA (ODN1 annealed to ODN6); ( d ) Mt UvrA in presence of damaged duplex DNA (ODN7 annealed to ODN8) (ODN sequences are listed in Supplementary Table S1 ). The positions of [γ- 32 P]ATP and 32 Pi is indicated on the right-hand side. Panel ( e ): graphical representation of ATPase activity of Mt UvrA as a function of protein concentration. The percentage of [γ- 32 P]ATP hydrolyzed, in panels a–d, is plotted against the indicated amounts of Mt UvrA. (filled square), Mt UvrA in absence of DNA; (filled circle), Mt UvrA in the presence of single-stranded DNA; (filled triangle), Mt UvrA in the presence of non-damaged duplex DNA; (inverted filled triangle), Mt UvrA in the presence of damaged duplex DNA. Each point on the curves represents the mean of three separate experiments. Non-linear regression analysis (Michaelis-Menten) was applied to the data sets, using GraphPad PRISM ver5.00, for obtaining the best-fit curve.

    Techniques Used: Activity Assay, Protein Concentration

    30) Product Images from "Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress"

    Article Title: Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006133

    VgrR-VgrS is a two-component signaling system. (A) VgrS has autokinase activity. Inverted membrane vesicles of full-length VgrS and VgrS H186A recombinant proteins (5 μM) were incubated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. (B) VgrS transferred phosphoryl group to VgrR. VgrS (5 μM) was autophosphorylated as in (A) for 2 min. Twenty μM VgrR or 80 μM VgrR D51A proteins were added into the reaction, respectively. In both (A) and (B), the reaction was stopped by loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins (lower panels). Each experiment was repeated three times. (C) vgrR and vgrS constitute a bicistronic operon. RT-PCR was used to amplify cDNA. RT: amplification using cDNA transcribed from total RNA as template.–RT: negative control lacking reverse transcriptase during cDNA synthesis. DNA: amplification using bacterial DNA as template. Amplification of vgrS cDNA was used as a positive control. Location of the primers is shown in Fig 1A (upper panel). The assay was repeated three times. (D) Mapping the transcription initiation site (TIS) of the vgrR-vgrS operon. Primer extension using total RNA of X . campestris pv. campestris as the template. The G-A-T-C lanes show the dideoxy chain termination sequencing reaction in the same promoter region (note that the top of the ladder is blur for high GC content of the template). 1 and 2: vgrR mRNA was reverse transcribed at 42°C and 52°C, respectively, 3: △vgrR mRNA template (with primer binding site being deleted) was reverse transcribed at 42°C as negative control. TIS sites (P1 and P2) are shown as asterisks. NS: non-specific bands. The experiment was repeated independently twice. (E) Nucleotide sequence of the 5′ region upstream of vgrR . (F) GUS activity assay of promoters. GUS activity assays were conducted among all the recombinant bacterial strains with transcriptional fusions of P1-GUS, P2-GUS and P1+P2-GUS, respectively. Bacterial strains were induced in iron depleted (MMX) or iron replete (MMX plus Fe 3+ ) conditions. Vertical bars represent the standard deviations (n = 3).
    Figure Legend Snippet: VgrR-VgrS is a two-component signaling system. (A) VgrS has autokinase activity. Inverted membrane vesicles of full-length VgrS and VgrS H186A recombinant proteins (5 μM) were incubated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. (B) VgrS transferred phosphoryl group to VgrR. VgrS (5 μM) was autophosphorylated as in (A) for 2 min. Twenty μM VgrR or 80 μM VgrR D51A proteins were added into the reaction, respectively. In both (A) and (B), the reaction was stopped by loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins (lower panels). Each experiment was repeated three times. (C) vgrR and vgrS constitute a bicistronic operon. RT-PCR was used to amplify cDNA. RT: amplification using cDNA transcribed from total RNA as template.–RT: negative control lacking reverse transcriptase during cDNA synthesis. DNA: amplification using bacterial DNA as template. Amplification of vgrS cDNA was used as a positive control. Location of the primers is shown in Fig 1A (upper panel). The assay was repeated three times. (D) Mapping the transcription initiation site (TIS) of the vgrR-vgrS operon. Primer extension using total RNA of X . campestris pv. campestris as the template. The G-A-T-C lanes show the dideoxy chain termination sequencing reaction in the same promoter region (note that the top of the ladder is blur for high GC content of the template). 1 and 2: vgrR mRNA was reverse transcribed at 42°C and 52°C, respectively, 3: △vgrR mRNA template (with primer binding site being deleted) was reverse transcribed at 42°C as negative control. TIS sites (P1 and P2) are shown as asterisks. NS: non-specific bands. The experiment was repeated independently twice. (E) Nucleotide sequence of the 5′ region upstream of vgrR . (F) GUS activity assay of promoters. GUS activity assays were conducted among all the recombinant bacterial strains with transcriptional fusions of P1-GUS, P2-GUS and P1+P2-GUS, respectively. Bacterial strains were induced in iron depleted (MMX) or iron replete (MMX plus Fe 3+ ) conditions. Vertical bars represent the standard deviations (n = 3).

    Techniques Used: Activity Assay, Recombinant, Incubation, SDS Page, Autoradiography, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Sequencing, Binding Assay

    VgrS senses Fe 3+ depletion by its sensor region. (A) Fe 3+ inhibits the autophosphorylation of full-length VgrS. Upper panels: Inverted membrane vesicles containing full-length VgrS were phosphorylated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. Fe 3+ was added at different concentrations. Lower panels: Soluble, truncated VgrS is not stimulated by Fe 3+ . MBP-VgrS without the input and transmembrane domains was used in the autophosphorylation assay. The experiment was repeated three times. (B) Iron excess decreased the phosphotransfer level from VgrS to VgrR. Full-length VgrS membrane was phosphorylated as described in (A) for 2 min in the presence of Fe 3+ , 15.0 μM VgrR was added into the mixture for 20 sec before stopping the reaction. (C) VgrS sensor directly binds Fe 3+ . 2 μM VgrS sensor, truncated VgrS (MBP-VgrS) and VgrS E43A sensor were used in a microscale thermophoresis (MST) assay. The titer of Fe 3+ ranged from 0.061 to 250 μM. The experiment was repeated three times. (D) Substitution of residues in the ExxE motif of VgrS sensor eliminated VgrS’s sensing of Fe 3+ . Inverted membrane vesicles with full-length VgrS E43A , VgrS P44A , VgrS Q45A and VgrS E46A were used in the autokinase assay as in (A). Fe 3+ was added into the reaction mixtures. The experiment was repeated twice. In (A, B, and D), the reaction was stopped by adding loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins.
    Figure Legend Snippet: VgrS senses Fe 3+ depletion by its sensor region. (A) Fe 3+ inhibits the autophosphorylation of full-length VgrS. Upper panels: Inverted membrane vesicles containing full-length VgrS were phosphorylated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. Fe 3+ was added at different concentrations. Lower panels: Soluble, truncated VgrS is not stimulated by Fe 3+ . MBP-VgrS without the input and transmembrane domains was used in the autophosphorylation assay. The experiment was repeated three times. (B) Iron excess decreased the phosphotransfer level from VgrS to VgrR. Full-length VgrS membrane was phosphorylated as described in (A) for 2 min in the presence of Fe 3+ , 15.0 μM VgrR was added into the mixture for 20 sec before stopping the reaction. (C) VgrS sensor directly binds Fe 3+ . 2 μM VgrS sensor, truncated VgrS (MBP-VgrS) and VgrS E43A sensor were used in a microscale thermophoresis (MST) assay. The titer of Fe 3+ ranged from 0.061 to 250 μM. The experiment was repeated three times. (D) Substitution of residues in the ExxE motif of VgrS sensor eliminated VgrS’s sensing of Fe 3+ . Inverted membrane vesicles with full-length VgrS E43A , VgrS P44A , VgrS Q45A and VgrS E46A were used in the autokinase assay as in (A). Fe 3+ was added into the reaction mixtures. The experiment was repeated twice. In (A, B, and D), the reaction was stopped by adding loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins.

    Techniques Used: Size-exclusion Chromatography, Microscale Thermophoresis, SDS Page, Autoradiography, Staining

    Dissection of the VgrR binding consensus motif. (A) Venn diagram showing the number of VgrR-regulated genes identified by ChIP-seq. (B) Functional categories of the VgrR-regulated genes identified by ChIP-seq. Details of the genes are listed in S6 and S7 Tables. (C) Deduced consensus VgrR-binding DNA motif based on ChIP-seq data. Weblogo was used to show the nucleotide composition. (D) Mapping the VgrR protected DNA region in the 5′ upstream sequence of XC1241 ( tdvA ) by DNase I footprinting. The amounts of VgrR protein used in the reactions were 1: zero; 2: 0.08 μM; 3: 0.8 μM; 4: 3.2 μM; and 5: 8.0 μM. The DNA regions protected by VgrR are shown on the right of the footprinting results, with the three possible VgrR-binding motifs shown in red, green, and black, respectively. A-T-C-G lanes are the DNA ladders obtained by a dideoxy-mediated chain-termination method using the same DNA sequence as the template. (E) Electrophoretic mobility shift assay verified the DNA motif of the XC1241 promoter bound by VgrR. The DNA probes were chemically synthesized according to those shown in (D). Sequences of the promoter region of XC1241 are listed above each panel. Each DNA probe was labeled by [γ- 32 P]ATP. Triangles indicate the VgrR-DNA complexes. All experiments were repeated three times.
    Figure Legend Snippet: Dissection of the VgrR binding consensus motif. (A) Venn diagram showing the number of VgrR-regulated genes identified by ChIP-seq. (B) Functional categories of the VgrR-regulated genes identified by ChIP-seq. Details of the genes are listed in S6 and S7 Tables. (C) Deduced consensus VgrR-binding DNA motif based on ChIP-seq data. Weblogo was used to show the nucleotide composition. (D) Mapping the VgrR protected DNA region in the 5′ upstream sequence of XC1241 ( tdvA ) by DNase I footprinting. The amounts of VgrR protein used in the reactions were 1: zero; 2: 0.08 μM; 3: 0.8 μM; 4: 3.2 μM; and 5: 8.0 μM. The DNA regions protected by VgrR are shown on the right of the footprinting results, with the three possible VgrR-binding motifs shown in red, green, and black, respectively. A-T-C-G lanes are the DNA ladders obtained by a dideoxy-mediated chain-termination method using the same DNA sequence as the template. (E) Electrophoretic mobility shift assay verified the DNA motif of the XC1241 promoter bound by VgrR. The DNA probes were chemically synthesized according to those shown in (D). Sequences of the promoter region of XC1241 are listed above each panel. Each DNA probe was labeled by [γ- 32 P]ATP. Triangles indicate the VgrR-DNA complexes. All experiments were repeated three times.

    Techniques Used: Dissection, Binding Assay, Chromatin Immunoprecipitation, Functional Assay, Sequencing, Footprinting, Electrophoretic Mobility Shift Assay, Synthesized, Labeling

    31) Product Images from "Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain"

    Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.8.2716-2727.2002

    m-Calpain phosphorylation at ST369/370 by PKA limits proteolytic activity. Both His-tagged wild-type- and ST369AA hCANP-expressing cells were grown and made quiescent for 24 h. Cells were treated with dexamethasone (2 μM) for 18 h to induce expression of exogenous hCANP constructs. To determine whether m-calpain was a target of PKA, hCANP was purified by Ni-NTA affinity chromatography and dialyzed with 50 mM Tris-HCl (pH 7.4) for 24 h. (A) Purified hCANP was treated with constitutively active cAMP-dependent PKA catalytic subunit and [γ- 32 P]ATP for 15 min. The samples were analyzed by separation through an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was used for autoradiography and subsequently blotted with anti-m-calpain antibody (Santa Cruz). The results shown are representative of two independent experiments. (B) Purified hCANP was incubated with recombinant Tau protein with or without calcium (40 μM) in the presence or absence of ERK (300 U) for 10 min. Samples were analyzed by SDS-10% polyacrylamide gel electrophoresis and transferred to a nylon membrane (Immobilon-P). The transferred membrane was blotted with anti-Tau antibody (Zymed). Reduction in the size of Tau indicates calpain activity. The results shown are representative of two independent experiments. (C) Purified hCANP was incubated with MAP2 in the presence or absence of ERK (300 U) and PKA catalytic subunit (25 U) for 20 min. Samples were analyzed on an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was blotted with anti-MAP2 antibody (Sigma). Loss of cleavage products of 75 to 120 kDa indicated activity. The results shown are representative of two independent experiments.
    Figure Legend Snippet: m-Calpain phosphorylation at ST369/370 by PKA limits proteolytic activity. Both His-tagged wild-type- and ST369AA hCANP-expressing cells were grown and made quiescent for 24 h. Cells were treated with dexamethasone (2 μM) for 18 h to induce expression of exogenous hCANP constructs. To determine whether m-calpain was a target of PKA, hCANP was purified by Ni-NTA affinity chromatography and dialyzed with 50 mM Tris-HCl (pH 7.4) for 24 h. (A) Purified hCANP was treated with constitutively active cAMP-dependent PKA catalytic subunit and [γ- 32 P]ATP for 15 min. The samples were analyzed by separation through an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was used for autoradiography and subsequently blotted with anti-m-calpain antibody (Santa Cruz). The results shown are representative of two independent experiments. (B) Purified hCANP was incubated with recombinant Tau protein with or without calcium (40 μM) in the presence or absence of ERK (300 U) for 10 min. Samples were analyzed by SDS-10% polyacrylamide gel electrophoresis and transferred to a nylon membrane (Immobilon-P). The transferred membrane was blotted with anti-Tau antibody (Zymed). Reduction in the size of Tau indicates calpain activity. The results shown are representative of two independent experiments. (C) Purified hCANP was incubated with MAP2 in the presence or absence of ERK (300 U) and PKA catalytic subunit (25 U) for 20 min. Samples were analyzed on an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was blotted with anti-MAP2 antibody (Sigma). Loss of cleavage products of 75 to 120 kDa indicated activity. The results shown are representative of two independent experiments.

    Techniques Used: Activity Assay, Expressing, Construct, Purification, Affinity Chromatography, Autoradiography, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis

    32) Product Images from "The Carboxyl Terminus of Brca2 Links the Disassembly of Rad51 Complexes to Mitotic Entry"

    Article Title: The Carboxyl Terminus of Brca2 Links the Disassembly of Rad51 Complexes to Mitotic Entry

    Journal: Current Biology

    doi: 10.1016/j.cub.2009.05.057

    The C-Terminal Motif of Gg Brca2 Is Functionally Cognate with Its Human Counterpart in CDK-Regulated Rad51 Binding (A) Protein sequence alignment showing the conserved cyclin-dependent kinase (CDK)-phosphorylated residues flanking Ser3239 of Gg Brca 2 and Ser3291 of Hs BRCA2. Gallus gallus residues used in this study are boxed, and residue numbers are indicated below. Asterisks indicate identical residues, double dots indicate conserved substitutions, and single dots indicate residues that are semiconserved. (B) Western blot analysis of Gg Rad51 and the myc epitope following immunoprecipitation of the myc-tagged construct encoding residues 3152–3398 of Gg Brca2 ( Gg B2 3152 aa–3398 aa ) transfected into DT40 cells. Roscovitine treatment for 30–120 min in asynchronous cells had little effect on Gg Rad51 binding (lanes 2 and 3). However, roscovitine effectively reversed the reduction in binding induced by nocodazole (compare lanes 5 and 6 with lane 4). (C and D) Western blot analysis of Gg Rad51 and the myc epitope following immunoprecipitation comparing the wild-type (WT) with S3239A/E or P3240L (C) or T3232A (D) variants of Gg B2 3152 aa–3398 aa transfected into DT40 cells. The S3239A/E and P3240L mutations in the conserved Ser-Pro consensus for CDK phosphorylation abrogated binding to Gg Rad51 in asynchronous and nocodazole-arrested mitotic cell extracts, whereas the T3232A mutation caused enhanced binding to Gg Rad51 under the same conditions. (E) Thr3232 and Ser3239 can be phosphorylated in vitro by CDK1. A wild-type Gg Brca2 peptide fused with GST (WT, sequence at top) or mutant forms in which Thr3232 (T3232A), Ser3239 (S3239A), or both (T3232A/S3239A) were substituted with Ala were subjected to an in vitro kinase assay in the presence of [γ- 32 P]ATP. Reaction products were cleaved with thrombin to separate the Brca2 peptides from GST. Silver staining (middle panel) measures the loading of the peptides in each reaction ( ∗ ). The bottom panel shows that CDK1 catalyzes the transfer of 32 P radiolabel from [γ- 32 P]ATP to the T3232A or S3239A peptides, but not to the double-mutant T3232A/S3239A peptide. Numbers at bottom indicate relative phosphorylation normalized to the amount of peptide present in each sample.
    Figure Legend Snippet: The C-Terminal Motif of Gg Brca2 Is Functionally Cognate with Its Human Counterpart in CDK-Regulated Rad51 Binding (A) Protein sequence alignment showing the conserved cyclin-dependent kinase (CDK)-phosphorylated residues flanking Ser3239 of Gg Brca 2 and Ser3291 of Hs BRCA2. Gallus gallus residues used in this study are boxed, and residue numbers are indicated below. Asterisks indicate identical residues, double dots indicate conserved substitutions, and single dots indicate residues that are semiconserved. (B) Western blot analysis of Gg Rad51 and the myc epitope following immunoprecipitation of the myc-tagged construct encoding residues 3152–3398 of Gg Brca2 ( Gg B2 3152 aa–3398 aa ) transfected into DT40 cells. Roscovitine treatment for 30–120 min in asynchronous cells had little effect on Gg Rad51 binding (lanes 2 and 3). However, roscovitine effectively reversed the reduction in binding induced by nocodazole (compare lanes 5 and 6 with lane 4). (C and D) Western blot analysis of Gg Rad51 and the myc epitope following immunoprecipitation comparing the wild-type (WT) with S3239A/E or P3240L (C) or T3232A (D) variants of Gg B2 3152 aa–3398 aa transfected into DT40 cells. The S3239A/E and P3240L mutations in the conserved Ser-Pro consensus for CDK phosphorylation abrogated binding to Gg Rad51 in asynchronous and nocodazole-arrested mitotic cell extracts, whereas the T3232A mutation caused enhanced binding to Gg Rad51 under the same conditions. (E) Thr3232 and Ser3239 can be phosphorylated in vitro by CDK1. A wild-type Gg Brca2 peptide fused with GST (WT, sequence at top) or mutant forms in which Thr3232 (T3232A), Ser3239 (S3239A), or both (T3232A/S3239A) were substituted with Ala were subjected to an in vitro kinase assay in the presence of [γ- 32 P]ATP. Reaction products were cleaved with thrombin to separate the Brca2 peptides from GST. Silver staining (middle panel) measures the loading of the peptides in each reaction ( ∗ ). The bottom panel shows that CDK1 catalyzes the transfer of 32 P radiolabel from [γ- 32 P]ATP to the T3232A or S3239A peptides, but not to the double-mutant T3232A/S3239A peptide. Numbers at bottom indicate relative phosphorylation normalized to the amount of peptide present in each sample.

    Techniques Used: Binding Assay, Sequencing, Western Blot, Immunoprecipitation, Construct, Transfection, Mutagenesis, In Vitro, Kinase Assay, Silver Staining

    33) Product Images from "Mycobacterium tuberculosis Rho Is an NTPase with Distinct Kinetic Properties and a Novel RNA-Binding Subdomain"

    Article Title: Mycobacterium tuberculosis Rho Is an NTPase with Distinct Kinetic Properties and a Novel RNA-Binding Subdomain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107474

    ATPase activity of MtbRho. (A) MtbRho hydrolyses ATP in presence of increasing concentrations of homopolymeric RNA – polyA (lanes1–3), polyC (lanes4–6) and polyU (lanes 7–9). No hydrolysis was observed in absence of RNA (lane 10). (B) MtbRho hydrolyzes ATP in presence of mycobacterial RNA. No hydrolysis was observed in absence of RNA (lane 1); 2 and 1 µg of M. smegmatis RNA stimulated ATP hydrolysis (lanes 2,3); the reaction is inhibited by Bicyclomycin (lane 4). ATPase assay was carried out as described in Methods . [1 mM unlabeled ATP was used as substrate, along with 100 nCi of α- 32 P-ATP (Panel A) or 100 nCi of γ- 32 P-ATP(Panel B), as tracer. Hydrolysis resulted in formation of α- 32 P-ADP (Panel A) or 32 Pi (Panel B) which were visualized using phosphorimager].
    Figure Legend Snippet: ATPase activity of MtbRho. (A) MtbRho hydrolyses ATP in presence of increasing concentrations of homopolymeric RNA – polyA (lanes1–3), polyC (lanes4–6) and polyU (lanes 7–9). No hydrolysis was observed in absence of RNA (lane 10). (B) MtbRho hydrolyzes ATP in presence of mycobacterial RNA. No hydrolysis was observed in absence of RNA (lane 1); 2 and 1 µg of M. smegmatis RNA stimulated ATP hydrolysis (lanes 2,3); the reaction is inhibited by Bicyclomycin (lane 4). ATPase assay was carried out as described in Methods . [1 mM unlabeled ATP was used as substrate, along with 100 nCi of α- 32 P-ATP (Panel A) or 100 nCi of γ- 32 P-ATP(Panel B), as tracer. Hydrolysis resulted in formation of α- 32 P-ADP (Panel A) or 32 Pi (Panel B) which were visualized using phosphorimager].

    Techniques Used: Activity Assay, ATPase Assay

    RNA preference by MtbRho. (A) Rates of ATPase activity of MtbRho and EcRho in presence of different RNAs. MtbRho is a weaker ATPase when polyC)is used(grey bars). But, in presence of mycobacterial RNA (brown bars), they have comparable rates of hydrolysis, while only MtbRho can hydrolyse ATP in presence of sdaA RNA(red bars). (B) Differential NTP hydrolysis by MtbRho. MtbRho can hydrolyze ATP and GTP in presence of the mycobacterial total RNA and sdaA RNA. Colorimetric assay was carried out as described in Methods (C) Differential ability of MtbRho to bind ATP and CTP. γ- 32 P-ATP alone can be UV-crosslinked to MtbRho (lane 1), and visualized on 8% SDS-PAGE. Addition of unlabeled ATP to the reaction competes out the γ- 32 P-ATP (lanes 2–4), but unlabeled CTP fails to do so (lanes 5–8). The reaction conditions were similar to ATPase assay, but without the addition of RNA.
    Figure Legend Snippet: RNA preference by MtbRho. (A) Rates of ATPase activity of MtbRho and EcRho in presence of different RNAs. MtbRho is a weaker ATPase when polyC)is used(grey bars). But, in presence of mycobacterial RNA (brown bars), they have comparable rates of hydrolysis, while only MtbRho can hydrolyse ATP in presence of sdaA RNA(red bars). (B) Differential NTP hydrolysis by MtbRho. MtbRho can hydrolyze ATP and GTP in presence of the mycobacterial total RNA and sdaA RNA. Colorimetric assay was carried out as described in Methods (C) Differential ability of MtbRho to bind ATP and CTP. γ- 32 P-ATP alone can be UV-crosslinked to MtbRho (lane 1), and visualized on 8% SDS-PAGE. Addition of unlabeled ATP to the reaction competes out the γ- 32 P-ATP (lanes 2–4), but unlabeled CTP fails to do so (lanes 5–8). The reaction conditions were similar to ATPase assay, but without the addition of RNA.

    Techniques Used: Activity Assay, Colorimetric Assay, SDS Page, ATPase Assay

    34) Product Images from "Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair"

    Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.16.5919-5927.2003

    Binding of DNA ligase III and PARP-1 to DNA single-strand interruptions. Effects of NAD on DNA binding by PARP-1 are shown. (A) Analysis of the binding of DNA ligase III to a DNA single-strand break by surface plasmon resonance. (Top panel) Schematic representation of the nicked hairpin oligonucleotide immobilized on the chip surface. (Bottom panel) Representative sensorgram showing the binding and release of untagged full-length DNA ligase III (LigIII), injected at the concentrations indicated, from the nicked hairpin oligonucleotide immobilized on the chip surface. (B) Visualization of DNA ligase III bound to a DNA single-strand break by DNase I footprinting. Intact DNA ligase III (LigIII; 20 nM) was preincubated with the labeled, nicked DNA substrate (16 nM) indicated and then incubated with DNase I as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled oligonucleotides were detected by autoradiography. −, no enzyme. The vertical line indicates the region protected from DNase I activity. (C) Effect of DNA strand break binding by DNA ligase III and PARP-1 on T4 polynucleotide kinase activity. Intact DNA ligase III (150 nM) or PARP-1 (150 nM) was preincubated with the indicated DNA substrate containing a single-nucleotide (1 nt) gap in the presence (+) or absence (−) of NAD as indicated prior to incubation with polynucleotide kinase and [γ- 32 P]ATP as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled 29-mer oligonucleotide was detected and quantitated by phosphorimager analysis. Lane C, no PARP-1 or DNA ligase III.
    Figure Legend Snippet: Binding of DNA ligase III and PARP-1 to DNA single-strand interruptions. Effects of NAD on DNA binding by PARP-1 are shown. (A) Analysis of the binding of DNA ligase III to a DNA single-strand break by surface plasmon resonance. (Top panel) Schematic representation of the nicked hairpin oligonucleotide immobilized on the chip surface. (Bottom panel) Representative sensorgram showing the binding and release of untagged full-length DNA ligase III (LigIII), injected at the concentrations indicated, from the nicked hairpin oligonucleotide immobilized on the chip surface. (B) Visualization of DNA ligase III bound to a DNA single-strand break by DNase I footprinting. Intact DNA ligase III (LigIII; 20 nM) was preincubated with the labeled, nicked DNA substrate (16 nM) indicated and then incubated with DNase I as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled oligonucleotides were detected by autoradiography. −, no enzyme. The vertical line indicates the region protected from DNase I activity. (C) Effect of DNA strand break binding by DNA ligase III and PARP-1 on T4 polynucleotide kinase activity. Intact DNA ligase III (150 nM) or PARP-1 (150 nM) was preincubated with the indicated DNA substrate containing a single-nucleotide (1 nt) gap in the presence (+) or absence (−) of NAD as indicated prior to incubation with polynucleotide kinase and [γ- 32 P]ATP as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled 29-mer oligonucleotide was detected and quantitated by phosphorimager analysis. Lane C, no PARP-1 or DNA ligase III.

    Techniques Used: Binding Assay, SPR Assay, Chromatin Immunoprecipitation, Injection, Footprinting, Labeling, Incubation, Nucleic Acid Electrophoresis, Autoradiography, Activity Assay

    35) Product Images from "Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6"

    Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.21.11347-11356.2003

    ATPase activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of wild-type VP6 or each VP6 mutant was incubated at 37°C for 15 min in a reaction mixture (20 μl) containing 30 mM Tris-HCl (pH 7.5), 3 mM MgCl 2 , 1 mM MnCl 2 , 10 mM DTT, 50 mg of bovine serum albumin per ml, 2 μg of poly(U), and 1 μCi of [γ- 32 P]ATP. The reaction product was analyzed by spotting 2-μl samples onto polyethyleneimine-cellulose plates, and the chromatograms were developed in 0.75 M potassium phosphate (pH 3.5). The plates were subsequently dried and autoradiographed. The amount of radioactivity at each position was estimated by phosphorimager, and the efficiencies of the ATPase activities of the three mutants were compared, with that of the wild type arbitrarily assigned a value of 100%. The control (C) represents a reaction mixture without VP6. The figure shows the mean values of the results of three different experiments.
    Figure Legend Snippet: ATPase activities of the wild-type (WT) and mutant VP6 proteins. One-half microgram of wild-type VP6 or each VP6 mutant was incubated at 37°C for 15 min in a reaction mixture (20 μl) containing 30 mM Tris-HCl (pH 7.5), 3 mM MgCl 2 , 1 mM MnCl 2 , 10 mM DTT, 50 mg of bovine serum albumin per ml, 2 μg of poly(U), and 1 μCi of [γ- 32 P]ATP. The reaction product was analyzed by spotting 2-μl samples onto polyethyleneimine-cellulose plates, and the chromatograms were developed in 0.75 M potassium phosphate (pH 3.5). The plates were subsequently dried and autoradiographed. The amount of radioactivity at each position was estimated by phosphorimager, and the efficiencies of the ATPase activities of the three mutants were compared, with that of the wild type arbitrarily assigned a value of 100%. The control (C) represents a reaction mixture without VP6. The figure shows the mean values of the results of three different experiments.

    Techniques Used: Mutagenesis, Incubation, Radioactivity

    36) Product Images from "HrpA, an RNA Helicase Involved in RNA Processing, Is Required for Mouse Infectivity and Tick Transmission of the Lyme Disease Spirochete"

    Article Title: HrpA, an RNA Helicase Involved in RNA Processing, Is Required for Mouse Infectivity and Tick Transmission of the Lyme Disease Spirochete

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003841

    RNA helicase assay. A ) Structure of the dsRNA substrate. Preparation of the helicase substrate is described in Materials and Methods . The long strand was prepared by in vitro transcription of pGEMX-1 that had been cleaved with NheI. The short strand was chemically synthesized and labeled with [γ- 32 P]ATP and polynucleotide kinase at the 5′ free end. The length of the double strand portion of the substrate is given in base pairs (bp), the single stranded and the overhang portions are given in nucleotides. B ) Autoradiograph of a polyacrylamide gel used to assay RNA helicase activity. The helicase assay was carried out for the wild-type and 5 different B. burgdorferi HrpA mutant proteins as described in the Materials and Methods . The enzymatic reaction products were resolved by 12% native PAGE followed by autoradiography. The autoradiograph shows wild-type and the S158A mutant HrpA as an example. Lane 1, No enzyme control, lane 2, ds RNA boiled at 95°C for 5 min to generate a marker for the single-stranded (SS) product. Lanes 3–5 contain wild-type HrpA and lines 6–8, the S158A HrpA mutant, with the indicated amounts of purified proteins. C ) Quantification of Helicase Activity. Helicase assays were performed in duplicate for three different concentrations of each purified HrpA. The results are plotted as the percentage of double strand RNA substrate converted to single stand as a function of the amount of enzyme per microliter of reaction. Errors bars represent the standard deviation.
    Figure Legend Snippet: RNA helicase assay. A ) Structure of the dsRNA substrate. Preparation of the helicase substrate is described in Materials and Methods . The long strand was prepared by in vitro transcription of pGEMX-1 that had been cleaved with NheI. The short strand was chemically synthesized and labeled with [γ- 32 P]ATP and polynucleotide kinase at the 5′ free end. The length of the double strand portion of the substrate is given in base pairs (bp), the single stranded and the overhang portions are given in nucleotides. B ) Autoradiograph of a polyacrylamide gel used to assay RNA helicase activity. The helicase assay was carried out for the wild-type and 5 different B. burgdorferi HrpA mutant proteins as described in the Materials and Methods . The enzymatic reaction products were resolved by 12% native PAGE followed by autoradiography. The autoradiograph shows wild-type and the S158A mutant HrpA as an example. Lane 1, No enzyme control, lane 2, ds RNA boiled at 95°C for 5 min to generate a marker for the single-stranded (SS) product. Lanes 3–5 contain wild-type HrpA and lines 6–8, the S158A HrpA mutant, with the indicated amounts of purified proteins. C ) Quantification of Helicase Activity. Helicase assays were performed in duplicate for three different concentrations of each purified HrpA. The results are plotted as the percentage of double strand RNA substrate converted to single stand as a function of the amount of enzyme per microliter of reaction. Errors bars represent the standard deviation.

    Techniques Used: Helicase Assay, In Vitro, Synthesized, Labeling, Autoradiography, Activity Assay, Mutagenesis, Clear Native PAGE, Marker, Purification, Standard Deviation

    ATP Hydrolysis by wild-type and mutant HrpA proteins. ATPase activity was assayed as described in Materials and Methods . ATPase activity of wild-type HrpA and five different point mutants was measured in the presence and absence of poly adenosine (RNA). The percentage of ATP hydrolyzed was determined by the release of 32 Pi from 1 mM of [γ- 32 P]ATP after incubation with 500 fmol purified HrpA per microliter of reaction for 1 h at 37°C with and without 1.1 nmol polyadenosine mononucleotide. Experiments were run in duplicate and the standard deviations are represented with error bars. Motifs and assigned functions are shown for the studied mutants.
    Figure Legend Snippet: ATP Hydrolysis by wild-type and mutant HrpA proteins. ATPase activity was assayed as described in Materials and Methods . ATPase activity of wild-type HrpA and five different point mutants was measured in the presence and absence of poly adenosine (RNA). The percentage of ATP hydrolyzed was determined by the release of 32 Pi from 1 mM of [γ- 32 P]ATP after incubation with 500 fmol purified HrpA per microliter of reaction for 1 h at 37°C with and without 1.1 nmol polyadenosine mononucleotide. Experiments were run in duplicate and the standard deviations are represented with error bars. Motifs and assigned functions are shown for the studied mutants.

    Techniques Used: Mutagenesis, Activity Assay, Incubation, Purification

    37) 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

    38) Product Images from "Biochemical analysis of human Dna2"

    Article Title: Biochemical analysis of human Dna2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl070

    ATPase activity of recombinant hDna2 proteins. ( A ) DNA-dependent ATPase activity of human DNA2. ATPase activity of recombinant hDna2 wild type (WT), D294A (DA) or K671E (KE) were measured using reaction mixtures containing [γ- 32 P]ATP in the presence (+) and absence (−) of single-stranded DNA as described in Materials And Methods. Reactions contained 600 fmol of recombinant protein and 0.5 mM [γ- 32 P]ATP. After incubation for 20 min at 37°C, the reaction products were separated by thin layer chromatography and detected using PhosphoImager. ATP indicates the position of uncleaved ATP. Pi indicates position of released inorganic phosphate. As a control, protein was omitted from the reaction (Buffer). ( B ) Extent of ATPase. The ATPase activity of wild-type hDna2 was measured using reaction mixtures containing various concentrations of [γ- 32 P]ATP, and extents of ATP hydrolysis were plotted against ATP concentration. To normalize background, ATPase activity was measured in the absence of single-stranded DNA and subtracted.
    Figure Legend Snippet: ATPase activity of recombinant hDna2 proteins. ( A ) DNA-dependent ATPase activity of human DNA2. ATPase activity of recombinant hDna2 wild type (WT), D294A (DA) or K671E (KE) were measured using reaction mixtures containing [γ- 32 P]ATP in the presence (+) and absence (−) of single-stranded DNA as described in Materials And Methods. Reactions contained 600 fmol of recombinant protein and 0.5 mM [γ- 32 P]ATP. After incubation for 20 min at 37°C, the reaction products were separated by thin layer chromatography and detected using PhosphoImager. ATP indicates the position of uncleaved ATP. Pi indicates position of released inorganic phosphate. As a control, protein was omitted from the reaction (Buffer). ( B ) Extent of ATPase. The ATPase activity of wild-type hDna2 was measured using reaction mixtures containing various concentrations of [γ- 32 P]ATP, and extents of ATP hydrolysis were plotted against ATP concentration. To normalize background, ATPase activity was measured in the absence of single-stranded DNA and subtracted.

    Techniques Used: Activity Assay, Recombinant, Incubation, Thin Layer Chromatography, Concentration Assay

    39) Product Images from "Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species"

    Article Title: Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-12-253

    Characterization of the binding reaction of Rep1A protein with iteron DNA by EMSA and footprinting. ( a ). Iteron of pWTY27. Possible iteron sequences from 338 to 606 bp on pWTY27 and AT-rich regions are shown. DR: direct repeat; IR: inverted repeat. The RepA binding sequences determined by DNA footprinting are boxed. The binding sequences of RepA protein are indicated by shading. ( b ). Detection of the binding activity of RepA protein with the iteron by EMSA. The DNA probe for each lane was 2 ng and the unlabeled probe was also used as specific competitor. The DNA-protein complex is indicated. ( c ). Determination of the binding sequence by DNA footprinting. The γ[ 32 p]ATP-radiolabelled primer was sequenced and electrophoresed (lanes G, A, T and C) as a control. The amounts of RepA protein used in lanes 1–5 were 0.17, 0.43, 0.85, 2.6 and 0 μg, respectively. Two sequences protected by RepA from digestion with DNaseI are shown and the RepA unbound sequences are underlined.
    Figure Legend Snippet: Characterization of the binding reaction of Rep1A protein with iteron DNA by EMSA and footprinting. ( a ). Iteron of pWTY27. Possible iteron sequences from 338 to 606 bp on pWTY27 and AT-rich regions are shown. DR: direct repeat; IR: inverted repeat. The RepA binding sequences determined by DNA footprinting are boxed. The binding sequences of RepA protein are indicated by shading. ( b ). Detection of the binding activity of RepA protein with the iteron by EMSA. The DNA probe for each lane was 2 ng and the unlabeled probe was also used as specific competitor. The DNA-protein complex is indicated. ( c ). Determination of the binding sequence by DNA footprinting. The γ[ 32 p]ATP-radiolabelled primer was sequenced and electrophoresed (lanes G, A, T and C) as a control. The amounts of RepA protein used in lanes 1–5 were 0.17, 0.43, 0.85, 2.6 and 0 μg, respectively. Two sequences protected by RepA from digestion with DNaseI are shown and the RepA unbound sequences are underlined.

    Techniques Used: Binding Assay, Footprinting, DNA Footprinting, Activity Assay, Sequencing

    40) Product Images from "siRNA function in RNAi: A chemical modification analysis"

    Article Title: siRNA function in RNAi: A chemical modification analysis

    Journal: RNA

    doi: 10.1261/rna.5103703

    Extending the half-life of siRNA duplexes prolongs the persistence of RNA interference in vivo. ( A ) Comparing the stability of unmodified siRNAs with siRNAs containing 2′-fluoro-uridine and 2′-fluoro-cytidine (2′-FU, 2′-FC) modifications ( a ) and thioate linkage (P–S) modifications ( b ). Unmodified or modified EGFP antisense strand siRNAs (AS) were 5′-labeled with [γ- 32 P]ATP by T4 polynucleotide kinases. Duplex siRNAs were formed by annealing equal molar ratios of sense-strand (SS) siRNAs with the 5′- 32 P-labeled antisense strand. To analyze siRNA stability in HeLa cell extract, 50 pmole of siRNA was incubated with 500 μg of HeLa cell extract in 50 μL of reaction mixture containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10 mM NaCl, 2 mM MgCl 2 , and 10% glycerol. At various time points, siRNAs were extracted and analyzed on 20% polyacrylamide gels containing 7 M urea followed by phosphorimage analysis (Fugi). ( B ) Kinetics of RNAi effects of duplex siRNA with 2′-fluoro-uridine and 2′-fluoro-cytidine modification in HeLa cells over a 144-h time course. The fluorescence intensity ratio of target (GFP) to control (RFP) protein was determined in the presence of unmodified dsRNA (blue bars) and duplex siRNA with 2′-fluoro-uridine and -cytidine modifications (DS-2′-FU, 2′-FC, cyan bar) and normalized to the ratio observed in the presence of mock-treated cells (red bars). Normalized ratios at
    Figure Legend Snippet: Extending the half-life of siRNA duplexes prolongs the persistence of RNA interference in vivo. ( A ) Comparing the stability of unmodified siRNAs with siRNAs containing 2′-fluoro-uridine and 2′-fluoro-cytidine (2′-FU, 2′-FC) modifications ( a ) and thioate linkage (P–S) modifications ( b ). Unmodified or modified EGFP antisense strand siRNAs (AS) were 5′-labeled with [γ- 32 P]ATP by T4 polynucleotide kinases. Duplex siRNAs were formed by annealing equal molar ratios of sense-strand (SS) siRNAs with the 5′- 32 P-labeled antisense strand. To analyze siRNA stability in HeLa cell extract, 50 pmole of siRNA was incubated with 500 μg of HeLa cell extract in 50 μL of reaction mixture containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10 mM NaCl, 2 mM MgCl 2 , and 10% glycerol. At various time points, siRNAs were extracted and analyzed on 20% polyacrylamide gels containing 7 M urea followed by phosphorimage analysis (Fugi). ( B ) Kinetics of RNAi effects of duplex siRNA with 2′-fluoro-uridine and 2′-fluoro-cytidine modification in HeLa cells over a 144-h time course. The fluorescence intensity ratio of target (GFP) to control (RFP) protein was determined in the presence of unmodified dsRNA (blue bars) and duplex siRNA with 2′-fluoro-uridine and -cytidine modifications (DS-2′-FU, 2′-FC, cyan bar) and normalized to the ratio observed in the presence of mock-treated cells (red bars). Normalized ratios at

    Techniques Used: In Vivo, Modification, Labeling, Incubation, Fluorescence

    Related Articles

    In Vitro:

    Article Title: Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator
    Article Snippet: .. For the in vitro kinase assay, individual GST-fusion proteins bound to glutathione beads were mixed with 0.1 mM ATP containing 10 mCi of γ-32 p-ATP and recombinant CK1 kinase (CK1δ; New England Biolabs) and were incubated at 30 °C for 1.5 h in reaction buffer [20 mM Tris⋅HCl (pH 8.0), 2 mM EDTA, 10 mM MgCl2 , 1 mM DTT]. ..

    Protease Inhibitor:

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A
    Article Snippet: .. Immunoprecipitated samples were dephosphorylated (0.038 U/μl calf intestinal phosphatase for 10 min, Roche 10713023001) and washed three times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2, 0,5% NP-40, 1 × protease inhibitor cocktail (Sigma-Aldrich P8340)), followed by labeling with γ-32 P ATP (2.1 μCi, Nerliens NEG502H) by polynucleotide kinase (PNK, 0.5 U/μl NEB M0201L) phosphorylation and further washed three times with Buffer C. All samples were resolved in FA dye and separated by electrophoresis on a denaturing (7 M urea) 6% polyacrylamide (PAA) gel. .. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop.

    Labeling:

    Article Title: ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation
    Article Snippet: .. The reverse primer was labeled with [γ-32 P] ATP using T4 PNK (NEB). .. PCR products were resolved on a 6% polyacrylamide/8M urea denaturing gel.

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A
    Article Snippet: .. Immunoprecipitated samples were dephosphorylated (0.038 U/μl calf intestinal phosphatase for 10 min, Roche 10713023001) and washed three times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2, 0,5% NP-40, 1 × protease inhibitor cocktail (Sigma-Aldrich P8340)), followed by labeling with γ-32 P ATP (2.1 μCi, Nerliens NEG502H) by polynucleotide kinase (PNK, 0.5 U/μl NEB M0201L) phosphorylation and further washed three times with Buffer C. All samples were resolved in FA dye and separated by electrophoresis on a denaturing (7 M urea) 6% polyacrylamide (PAA) gel. .. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop.

    Purification:

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Three micrograms of recombinant TIN2L purified from E. coli was incubated with 10 U of CK2 (NEB) and 10 μCi of [γ-32 P]ATP in 1× CK2 reaction buffer (NEB) at 30°C for 30 min. As a control, 3 μg of bovine serum albumin (BSA; NEB) was incubated with CK2 under the same conditions. ..

    Electrophoresis:

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A
    Article Snippet: .. Immunoprecipitated samples were dephosphorylated (0.038 U/μl calf intestinal phosphatase for 10 min, Roche 10713023001) and washed three times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2, 0,5% NP-40, 1 × protease inhibitor cocktail (Sigma-Aldrich P8340)), followed by labeling with γ-32 P ATP (2.1 μCi, Nerliens NEG502H) by polynucleotide kinase (PNK, 0.5 U/μl NEB M0201L) phosphorylation and further washed three times with Buffer C. All samples were resolved in FA dye and separated by electrophoresis on a denaturing (7 M urea) 6% polyacrylamide (PAA) gel. .. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop.

    Immunoprecipitation:

    Article Title: G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A
    Article Snippet: .. Immunoprecipitated samples were dephosphorylated (0.038 U/μl calf intestinal phosphatase for 10 min, Roche 10713023001) and washed three times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2, 0,5% NP-40, 1 × protease inhibitor cocktail (Sigma-Aldrich P8340)), followed by labeling with γ-32 P ATP (2.1 μCi, Nerliens NEG502H) by polynucleotide kinase (PNK, 0.5 U/μl NEB M0201L) phosphorylation and further washed three times with Buffer C. All samples were resolved in FA dye and separated by electrophoresis on a denaturing (7 M urea) 6% polyacrylamide (PAA) gel. .. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop.

    Incubation:

    Article Title: Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator
    Article Snippet: .. For the in vitro kinase assay, individual GST-fusion proteins bound to glutathione beads were mixed with 0.1 mM ATP containing 10 mCi of γ-32 p-ATP and recombinant CK1 kinase (CK1δ; New England Biolabs) and were incubated at 30 °C for 1.5 h in reaction buffer [20 mM Tris⋅HCl (pH 8.0), 2 mM EDTA, 10 mM MgCl2 , 1 mM DTT]. ..

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Three micrograms of recombinant TIN2L purified from E. coli was incubated with 10 U of CK2 (NEB) and 10 μCi of [γ-32 P]ATP in 1× CK2 reaction buffer (NEB) at 30°C for 30 min. As a control, 3 μg of bovine serum albumin (BSA; NEB) was incubated with CK2 under the same conditions. ..

    Kinase Assay:

    Article Title: Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator
    Article Snippet: .. For the in vitro kinase assay, individual GST-fusion proteins bound to glutathione beads were mixed with 0.1 mM ATP containing 10 mCi of γ-32 p-ATP and recombinant CK1 kinase (CK1δ; New England Biolabs) and were incubated at 30 °C for 1.5 h in reaction buffer [20 mM Tris⋅HCl (pH 8.0), 2 mM EDTA, 10 mM MgCl2 , 1 mM DTT]. ..

    Recombinant:

    Article Title: Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator
    Article Snippet: .. For the in vitro kinase assay, individual GST-fusion proteins bound to glutathione beads were mixed with 0.1 mM ATP containing 10 mCi of γ-32 p-ATP and recombinant CK1 kinase (CK1δ; New England Biolabs) and were incubated at 30 °C for 1.5 h in reaction buffer [20 mM Tris⋅HCl (pH 8.0), 2 mM EDTA, 10 mM MgCl2 , 1 mM DTT]. ..

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Three micrograms of recombinant TIN2L purified from E. coli was incubated with 10 U of CK2 (NEB) and 10 μCi of [γ-32 P]ATP in 1× CK2 reaction buffer (NEB) at 30°C for 30 min. As a control, 3 μg of bovine serum albumin (BSA; NEB) was incubated with CK2 under the same conditions. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    New England Biolabs ck2 buffer
    Phosphorylation of PACSIN 1 at serine 358 by <t>CK2.</t> A , scheme of PACSIN 1 represents its domain structure and location of Ser 358 . B , recombinant wild-type PACSIN 1 ( P1 wt ) and PACSIN 1 S358A ( P1 S358A ) were purified as GST fusion proteins, and GST was removed by proteolytic cleavage. The proteins were incubated with CK2 and [γ- 32 P]ATP for 20 min and analyzed by SDS-PAGE. The SDS-polyacrylamide gel was stained with Coomassie Brilliant Blue, dried, and exposed to an x-ray film. C , recombinant proteins including GST and an unrelated mutant PACSIN 1 T25A ( P1 T25A ) as controls were incubated with (+) or without (−) CK2, resolved by native PAGE, and immunoblotted with antibodies specific for PACSIN 1 or PACSIN 1 pS358. D , for kinetic analysis wild-type PACSIN 1 (P1 wt) was incubated with CK2 for the indicated time periods, separated by native PAGE and immunoblotted ( WB ) for PACSIN 1.
    Ck2 Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ck2 buffer/product/New England Biolabs
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ck2 buffer - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    92
    New England Biolabs γ 32 p atp
    Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 <t>P]ATP,</t> followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.
    γ 32 P Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/New England Biolabs
    Average 92 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Phosphorylation of PACSIN 1 at serine 358 by CK2. A , scheme of PACSIN 1 represents its domain structure and location of Ser 358 . B , recombinant wild-type PACSIN 1 ( P1 wt ) and PACSIN 1 S358A ( P1 S358A ) were purified as GST fusion proteins, and GST was removed by proteolytic cleavage. The proteins were incubated with CK2 and [γ- 32 P]ATP for 20 min and analyzed by SDS-PAGE. The SDS-polyacrylamide gel was stained with Coomassie Brilliant Blue, dried, and exposed to an x-ray film. C , recombinant proteins including GST and an unrelated mutant PACSIN 1 T25A ( P1 T25A ) as controls were incubated with (+) or without (−) CK2, resolved by native PAGE, and immunoblotted with antibodies specific for PACSIN 1 or PACSIN 1 pS358. D , for kinetic analysis wild-type PACSIN 1 (P1 wt) was incubated with CK2 for the indicated time periods, separated by native PAGE and immunoblotted ( WB ) for PACSIN 1.

    Journal: The Journal of Biological Chemistry

    Article Title: Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation *

    doi: 10.1074/jbc.M113.461293

    Figure Lengend Snippet: Phosphorylation of PACSIN 1 at serine 358 by CK2. A , scheme of PACSIN 1 represents its domain structure and location of Ser 358 . B , recombinant wild-type PACSIN 1 ( P1 wt ) and PACSIN 1 S358A ( P1 S358A ) were purified as GST fusion proteins, and GST was removed by proteolytic cleavage. The proteins were incubated with CK2 and [γ- 32 P]ATP for 20 min and analyzed by SDS-PAGE. The SDS-polyacrylamide gel was stained with Coomassie Brilliant Blue, dried, and exposed to an x-ray film. C , recombinant proteins including GST and an unrelated mutant PACSIN 1 T25A ( P1 T25A ) as controls were incubated with (+) or without (−) CK2, resolved by native PAGE, and immunoblotted with antibodies specific for PACSIN 1 or PACSIN 1 pS358. D , for kinetic analysis wild-type PACSIN 1 (P1 wt) was incubated with CK2 for the indicated time periods, separated by native PAGE and immunoblotted ( WB ) for PACSIN 1.

    Article Snippet: Samples of recombinant proteins (0.5 μg) were incubated at 30 °C for the indicated time periods in CK2 buffer (20 m m Tris, pH 7.5, 50 m m KCl, 10 m m MgCl2 with 200 μ m ATP (or 125 μ m ATP + 0.2 megabecquerel of [γ-32 P]ATP)) and 250 units of CK2 (New England Biolabs).

    Techniques: Recombinant, Purification, Incubation, SDS Page, Staining, Mutagenesis, Clear Native PAGE, Western Blot

    Reduced spine formation in the presence of the PACSIN 1 S358A mutant or after CK2 inhibition. A , rat hippocampal neurons were transfected with either PACSIN 1 wt ( left ). the mutant protein S358A ( right ) or the mutant protein S358D (data not shown) in combination with EGFP to visualize spines ( upper panel ). Enlargements of three dendrite segments of neurons transfected with either PACSIN 1 wt ( left ) or S358A mutant ( right ) are shown. Fewer spines can be detected in neurons expressing PACSIN 1 S358A ( lower panel ). B , quantification of two independent experiments. For each group five dendrites were counted on each of 12 cells, n = 60 dendrites; **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation *

    doi: 10.1074/jbc.M113.461293

    Figure Lengend Snippet: Reduced spine formation in the presence of the PACSIN 1 S358A mutant or after CK2 inhibition. A , rat hippocampal neurons were transfected with either PACSIN 1 wt ( left ). the mutant protein S358A ( right ) or the mutant protein S358D (data not shown) in combination with EGFP to visualize spines ( upper panel ). Enlargements of three dendrite segments of neurons transfected with either PACSIN 1 wt ( left ) or S358A mutant ( right ) are shown. Fewer spines can be detected in neurons expressing PACSIN 1 S358A ( lower panel ). B , quantification of two independent experiments. For each group five dendrites were counted on each of 12 cells, n = 60 dendrites; **, p

    Article Snippet: Samples of recombinant proteins (0.5 μg) were incubated at 30 °C for the indicated time periods in CK2 buffer (20 m m Tris, pH 7.5, 50 m m KCl, 10 m m MgCl2 with 200 μ m ATP (or 125 μ m ATP + 0.2 megabecquerel of [γ-32 P]ATP)) and 250 units of CK2 (New England Biolabs).

    Techniques: Mutagenesis, Inhibition, Transfection, Expressing

    Model of the mechanism by which PACSIN 1 affects spine formation. A , in the absence of BDNF, overexpression of PACSIN 1 S358A or inhibition of CK2 supports a stable Rac1-NADRIN-PACSIN 1 complex which leads to Rac1 GTP hydrolysis. B , in the presence of BDNF higher levels of activated CK2 phosphorylate PACSIN 1 at Ser 358 which causes a destabilization of the protein complex. This leads to higher concentrations of Rac1 GTP which induces spine formation. TrkB , tropomyosin-related kinase B.

    Journal: The Journal of Biological Chemistry

    Article Title: Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation *

    doi: 10.1074/jbc.M113.461293

    Figure Lengend Snippet: Model of the mechanism by which PACSIN 1 affects spine formation. A , in the absence of BDNF, overexpression of PACSIN 1 S358A or inhibition of CK2 supports a stable Rac1-NADRIN-PACSIN 1 complex which leads to Rac1 GTP hydrolysis. B , in the presence of BDNF higher levels of activated CK2 phosphorylate PACSIN 1 at Ser 358 which causes a destabilization of the protein complex. This leads to higher concentrations of Rac1 GTP which induces spine formation. TrkB , tropomyosin-related kinase B.

    Article Snippet: Samples of recombinant proteins (0.5 μg) were incubated at 30 °C for the indicated time periods in CK2 buffer (20 m m Tris, pH 7.5, 50 m m KCl, 10 m m MgCl2 with 200 μ m ATP (or 125 μ m ATP + 0.2 megabecquerel of [γ-32 P]ATP)) and 250 units of CK2 (New England Biolabs).

    Techniques: Over Expression, Inhibition

    Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 P]ATP, followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.

    Journal: Cancer research

    Article Title: Plk1 Phosphorylation of Mre11 Antagonizes the DNA Damage Response

    doi: 10.1158/0008-5472.CAN-16-2787

    Figure Lengend Snippet: Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 P]ATP, followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.

    Article Snippet: After the 5′ end of TP423 was labelled with [γ-32 P]ATP and polynucleotide kinase (New England Biolabs), TP423 and TP424 oligonucleotides were annealed to generate the 3′ overhanging DNA duplexes.

    Techniques: Incubation, Immunoprecipitation, Avidin-Biotin Assay, Lysis, Purification, Autoradiography, In Vitro, SDS Page, Staining, In Vivo, Transfection, Construct

    FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo . A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser ( http://genome.ucsc.edu/ ) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ- 32 P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t -tests were performed. * P ≤0.05, ** P ≤0.01.

    Journal: PLoS Genetics

    Article Title: ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation

    doi: 10.1371/journal.pgen.1003895

    Figure Lengend Snippet: FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo . A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser ( http://genome.ucsc.edu/ ) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ- 32 P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t -tests were performed. * P ≤0.05, ** P ≤0.01.

    Article Snippet: The reverse primer was labeled with [γ-32 P] ATP using T4 PNK (NEB).

    Techniques: In Vivo, Cross-linking Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Immunoprecipitation, Variant Assay, Labeling, Polymerase Chain Reaction

    FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels. A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ- 32 P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t -tests. Bar graphs represent mean ± SEM (n = 3). * P ≤0.05, *** P ≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t -tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P ≤0.05.

    Journal: PLoS Genetics

    Article Title: ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation

    doi: 10.1371/journal.pgen.1003895

    Figure Lengend Snippet: FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels. A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ- 32 P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t -tests. Bar graphs represent mean ± SEM (n = 3). * P ≤0.05, *** P ≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t -tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P ≤0.05.

    Article Snippet: The reverse primer was labeled with [γ-32 P] ATP using T4 PNK (NEB).

    Techniques: Labeling, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Transfection, Western Blot, RNA Binding Assay, Expressing

    In vitro self-splicing ( A ) of B.c .I4 wild-type and mutant constructs and subsequent RT-PCR ( B ). In A , lane M shows the marker, γ [32-P] ATP 5′-end-labeled RNA Century-Plus Marker (Ambion). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 , and 100 mM MgCl 2 at 45°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. Schematic drawings are shown next to the bands corresponding to the different splicing products. The light grey box represents the extra 56-nt element. In B , RT-PCR with I4B_right and 5p_left_BamH1 primers ( Table 1 ) using in vitro splicing products as templates, confirming the size of the ligated exons. Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 1% agarose gel.

    Journal: Nucleic Acids Research

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    doi: 10.1093/nar/gkm031

    Figure Lengend Snippet: In vitro self-splicing ( A ) of B.c .I4 wild-type and mutant constructs and subsequent RT-PCR ( B ). In A , lane M shows the marker, γ [32-P] ATP 5′-end-labeled RNA Century-Plus Marker (Ambion). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 , and 100 mM MgCl 2 at 45°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. Schematic drawings are shown next to the bands corresponding to the different splicing products. The light grey box represents the extra 56-nt element. In B , RT-PCR with I4B_right and 5p_left_BamH1 primers ( Table 1 ) using in vitro splicing products as templates, confirming the size of the ligated exons. Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 1% agarose gel.

    Article Snippet: Primer I4B_right was 5′-end-labeled with γ[32-P] ATP (3000 Ci/mmol, 10 mCi/ml) using T4 kinase (New England Biolabs).

    Techniques: In Vitro, Mutagenesis, Construct, Reverse Transcription Polymerase Chain Reaction, Marker, Labeling, Agarose Gel Electrophoresis

    RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Journal: Nucleic Acids Research

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    doi: 10.1093/nar/gkm031

    Figure Lengend Snippet: RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Article Snippet: Primer I4B_right was 5′-end-labeled with γ[32-P] ATP (3000 Ci/mmol, 10 mCi/ml) using T4 kinase (New England Biolabs).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Labeling, Marker