in vitro phosphorylation assays in vitro radioactive assays  (PerkinElmer)

 
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    Structured Review

    PerkinElmer in vitro phosphorylation assays in vitro radioactive assays
    <t>In</t> <t>vitro</t> kinase assay. (A) Coomassie gel showing the purified proteins used in the in vitro kinase activity <t>assays.</t> (B) Recombinant ATG4B or ATG4B S34A was incubated with recombinant AKT2 and ATP γ- 32 P and incorporation of labeled γ- 32 P was measured by auto-radiography. The upper band corresponds to AKT2 <t>auto-phosphorylation</t> and the lower band corresponds to ATG4B. (C) Recombinant ATG4B wild-type (WT), S121A or S262A were incubated with (+) or without (–) recombinant AKT2 and incorporation of labeled γ- 32 P was measured by auto-radiography. On the left side, ULK1 mediated phosphorylation of ATG4B was included as control. On the right side, CLK2 (CLK2 catalytic domain with GST-tagged ( Prak et al., 2016 )) was included as another protein control to show that in the absence of ATG4B, AKT2 resulted in auto-phosphorylation.
    In Vitro Phosphorylation Assays In Vitro Radioactive Assays, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 91/100, based on 380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

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

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

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2018.00148

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

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

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

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

    Journal: Molecules

    doi: 10.3390/molecules21060766

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

    Techniques Used: Labeling, Incubation

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

    Techniques Used: Activity Assay, Labeling, Incubation

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

    Techniques Used: Labeling

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

    Techniques Used: Labeling, Incubation

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.772426

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

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

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064316

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

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

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

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

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

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

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003769

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

    Techniques Used: Polymerase Chain Reaction, Amplification

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

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

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

    Techniques Used: Northern Blot

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

    Techniques Used:

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

    Techniques Used: Over Expression

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

    Techniques Used: Northern Blot

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

    Techniques Used: Produced, Microarray

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

    Techniques Used:

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

    Techniques Used:

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

    Techniques Used: Binding Assay

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038246

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp525

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

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

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

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

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

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

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

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

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

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

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

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

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

    Techniques Used: Activation Assay, Transfection

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

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

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

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

    8) Product Images from "Activation of Arterial Wall Dendritic Cells and Breakdown of Self-tolerance in Giant Cell Arteritis"

    Article Title: Activation of Arterial Wall Dendritic Cells and Breakdown of Self-tolerance in Giant Cell Arteritis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20030850

    Functional characteristics of arterial wall DCs in normal arteries. Temporal arteries were collected from patients with neither GCA nor PMR. Tissue extracts from fresh shock-frozen samples were analyzed for TLR2- and TLR4-specific sequences by PCR. All negative arteries (marked as 1–6) contained mRNA transcripts for TRL2 and TLR4 (A). To test the responsiveness of arterial DCs to triggering with blood-born TNF-α or TLR ligands, we implanted pieces of arteries into SCID mice. 7–10 d after implantation, the SCID mouse chimeras were injected with 2 μg i.v. TNF-α, 10 μg i.v. LPS, or 100 μl i.p. CFA, and the arterial grafts were harvested 48 h later. Tissue extracts from the explanted grafts were analyzed for the mRNA transcripts of β-actin, CD83, IL-18, and the chemokines CCL18, CCL19, and CCL21. After stimulation with blood-born triggers, arterial wall DCs expressed CD83 + and began to produce an array of chemokines. The effect of TNF-α was limited to the induction of CCL21, whereas LPS induced the full spectrum of chemokines. One experiment representative of three is shown (B). Immunohistochemistry confirmed that arterial DCs from LPS-treated (left), but not control (right), arteries expressed CD83 (blue) and produced CCL21 (red) (C). Original magnification, 200 (except LPS-treated CD83 and CCL21 images, which were 600×). P, positive PCR control; N, untreated mouse control; and W, negative PCR control.
    Figure Legend Snippet: Functional characteristics of arterial wall DCs in normal arteries. Temporal arteries were collected from patients with neither GCA nor PMR. Tissue extracts from fresh shock-frozen samples were analyzed for TLR2- and TLR4-specific sequences by PCR. All negative arteries (marked as 1–6) contained mRNA transcripts for TRL2 and TLR4 (A). To test the responsiveness of arterial DCs to triggering with blood-born TNF-α or TLR ligands, we implanted pieces of arteries into SCID mice. 7–10 d after implantation, the SCID mouse chimeras were injected with 2 μg i.v. TNF-α, 10 μg i.v. LPS, or 100 μl i.p. CFA, and the arterial grafts were harvested 48 h later. Tissue extracts from the explanted grafts were analyzed for the mRNA transcripts of β-actin, CD83, IL-18, and the chemokines CCL18, CCL19, and CCL21. After stimulation with blood-born triggers, arterial wall DCs expressed CD83 + and began to produce an array of chemokines. The effect of TNF-α was limited to the induction of CCL21, whereas LPS induced the full spectrum of chemokines. One experiment representative of three is shown (B). Immunohistochemistry confirmed that arterial DCs from LPS-treated (left), but not control (right), arteries expressed CD83 (blue) and produced CCL21 (red) (C). Original magnification, 200 (except LPS-treated CD83 and CCL21 images, which were 600×). P, positive PCR control; N, untreated mouse control; and W, negative PCR control.

    Techniques Used: Functional Assay, Polymerase Chain Reaction, Mouse Assay, Injection, Immunohistochemistry, Produced

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004799

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

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

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

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

    10) Product Images from "Interleukin 12-mediated Prevention of Spontaneous Mammary Adenocarcinomas in Two Lines of Her-2/neu Transgenic Mice "

    Article Title: Interleukin 12-mediated Prevention of Spontaneous Mammary Adenocarcinomas in Two Lines of Her-2/neu Transgenic Mice

    Journal: The Journal of Experimental Medicine

    doi:

    Cryostat sections of invading lobular carcinomas in MSA control ( a and c ) and IL-12–treated ( b and d ) FVB–NeuN mice. TNF-α ( b ) and IFN-γ ( d ) are evident in the tumor growth area in IL-12–treated mice, whereas both are absent ( a and c ) in the MSA control, as revealed by staining with anti–TNF-α and –IFN-γ mAb in tumor masses of 49-wk-old mice. Original magnification: ×630.
    Figure Legend Snippet: Cryostat sections of invading lobular carcinomas in MSA control ( a and c ) and IL-12–treated ( b and d ) FVB–NeuN mice. TNF-α ( b ) and IFN-γ ( d ) are evident in the tumor growth area in IL-12–treated mice, whereas both are absent ( a and c ) in the MSA control, as revealed by staining with anti–TNF-α and –IFN-γ mAb in tumor masses of 49-wk-old mice. Original magnification: ×630.

    Techniques Used: Mouse Assay, Staining

    11) Product Images from "CspA regulation of Staphylococcus aureus carotenoid levels and σB activity is controlled by YjbH and Spx."

    Article Title: CspA regulation of Staphylococcus aureus carotenoid levels and σB activity is controlled by YjbH and Spx.

    Journal: Molecular microbiology

    doi: 10.1111/mmi.14273

    Spx and αCTD interact with the cspA promoter. EMSA analysis of αCTD and Spx binding to the cspA promoter probe in reactions containing Spx or mixtures of Spx with αCTD under various conditions. Bands corresponding to the cspA promoter/αCTD and cspA promoter/Spx/αCTD complexes are marked with arrows. The 32P cspA promoter probe was generated by labeling a PCR product of the cspA promoter (−300 to +1) with γ−32P dATP using T4 polynucleotide kinase. An internal fragment of sarX was used as the non-specific cold challenge DNA, while unlabeled cspA promoter (−300 to +1) was used as the specific cold challenge DNA.
    Figure Legend Snippet: Spx and αCTD interact with the cspA promoter. EMSA analysis of αCTD and Spx binding to the cspA promoter probe in reactions containing Spx or mixtures of Spx with αCTD under various conditions. Bands corresponding to the cspA promoter/αCTD and cspA promoter/Spx/αCTD complexes are marked with arrows. The 32P cspA promoter probe was generated by labeling a PCR product of the cspA promoter (−300 to +1) with γ−32P dATP using T4 polynucleotide kinase. An internal fragment of sarX was used as the non-specific cold challenge DNA, while unlabeled cspA promoter (−300 to +1) was used as the specific cold challenge DNA.

    Techniques Used: Binding Assay, Generated, Labeling, Polymerase Chain Reaction

    Transcriptional activity of the hbo-yjbH promoter is affected by cspA . (A) yjbH transcription in SH1000 wildtype, Δ cspA and Δ yjbH strains. RNA obtained from SH1000 wildtype, Δ cspA and Δ yjbH cells at various optical densities (15 μg per lane) was resolved on a denaturing agarose gel, blotted to Hybond XL membrane and hybridized with a 200-bp 32P-radiolabeled yjbH DNA fragment. The relative densitometric units (RDU) of the 1.9k and 1.2k nt bands were calculated relative to 1.2k nt levels in the wildtype at OD 650 =0.7 (set at 100). The hybridization analysis was repeated twice with similar results. A representative blot was shown. (B) hbo-yjbH promoter activity in S. aureus SH1000 strains deleted for cspA or yjbH . Cell density-normalized GFP fluorescence of S. aureus SH1000 wildtype, ΔcspA and Δ yjbH strains carrying the hbo-yjbH promoter reporter plasmid (pALC8134) at 4 and 10 hours of growth in TSB was shown. Cell densities were simultaneously measured at OD 650 and fluorescence normalized by it. The value for each strain represents the mean of two biological replicates that were read in triplicate, and the experiments were repeated three times. A representative experiment is displayed. The asterisks indicate statistical significance between wildtype and mutant strains, determined using Student t-test (*, P
    Figure Legend Snippet: Transcriptional activity of the hbo-yjbH promoter is affected by cspA . (A) yjbH transcription in SH1000 wildtype, Δ cspA and Δ yjbH strains. RNA obtained from SH1000 wildtype, Δ cspA and Δ yjbH cells at various optical densities (15 μg per lane) was resolved on a denaturing agarose gel, blotted to Hybond XL membrane and hybridized with a 200-bp 32P-radiolabeled yjbH DNA fragment. The relative densitometric units (RDU) of the 1.9k and 1.2k nt bands were calculated relative to 1.2k nt levels in the wildtype at OD 650 =0.7 (set at 100). The hybridization analysis was repeated twice with similar results. A representative blot was shown. (B) hbo-yjbH promoter activity in S. aureus SH1000 strains deleted for cspA or yjbH . Cell density-normalized GFP fluorescence of S. aureus SH1000 wildtype, ΔcspA and Δ yjbH strains carrying the hbo-yjbH promoter reporter plasmid (pALC8134) at 4 and 10 hours of growth in TSB was shown. Cell densities were simultaneously measured at OD 650 and fluorescence normalized by it. The value for each strain represents the mean of two biological replicates that were read in triplicate, and the experiments were repeated three times. A representative experiment is displayed. The asterisks indicate statistical significance between wildtype and mutant strains, determined using Student t-test (*, P

    Techniques Used: Activity Assay, Agarose Gel Electrophoresis, Hybridization, Fluorescence, Plasmid Preparation, Mutagenesis

    12) Product Images from "DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance"

    Article Title: DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.01635

    Heatmap of log2-fold changes of differentially expressed genes in C. metallidurans NA4S versus NA4, NA4SΔ agrRS versus NA4S and NA4SΔ agrRS versus NA4 under non-selective growth conditions.
    Figure Legend Snippet: Heatmap of log2-fold changes of differentially expressed genes in C. metallidurans NA4S versus NA4, NA4SΔ agrRS versus NA4S and NA4SΔ agrRS versus NA4 under non-selective growth conditions.

    Techniques Used:

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku945

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

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

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep05970

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

    Techniques Used: Activity Assay, Recombinant, Marker

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep24278

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

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

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

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

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

    Techniques Used: Autoradiography, Incubation, Activity Assay

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

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

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

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

    Journal: Science Advances

    doi: 10.1126/sciadv.1400203

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

    Techniques Used: DNA Synthesis

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

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

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

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

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E13-06-0339

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

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

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002204

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

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

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

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

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.408450

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

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

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

    Techniques Used: Labeling, Immunoprecipitation, Binding Assay

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

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

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

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

    Journal: eLife

    doi: 10.7554/eLife.13740

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

    Techniques Used: In Vitro, Generated, Sequencing

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

    Techniques Used: Activity Assay, Sequencing

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

    Techniques Used: Activity Assay

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

    Techniques Used: Generated, Incubation

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm463

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

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

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

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

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

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

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

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

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

    Techniques Used: Polyacrylamide Gel Electrophoresis

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep42741

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

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

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

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

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

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

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

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04010-4

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

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

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

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

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

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

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

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

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

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

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

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

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdf562

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

    Techniques Used: Immunoprecipitation, Expressing, Mutagenesis, Western Blot

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

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

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

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

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

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

    25) Product Images from "Identification and biochemical characterization of a novel eukaryotic-like Ser/Thr kinase in E. coli"

    Article Title: Identification and biochemical characterization of a novel eukaryotic-like Ser/Thr kinase in E. coli

    Journal: bioRxiv

    doi: 10.1101/819920

    YegI is an active kinase (A) Kinase activity of YegI : Autophosphorylation reactions were carried out at 37 °C with 0.2 μM of YegI (WT or K39D or D141N) in kinase buffer (50 mM Tris pH 7.5, 50 mM KCl, 1 mM DTT, 10 mM MgCl 2 , 10 mM MnCl 2 , 200 μM cold ATP and 5 μCi γ -[ 32 P]ATP. Reactions were stopped at t=30 mins and run on 12 % SDS-PAGE followed by autoradiography. Molecular weights (kDa) are indicated on the right of the gel. (B) Sensitivity to staurosporine: Autophosphorylation reactions were carried out at 37 °C with 1 μM of YegI (WT) in kinase buffer without cold ATP. Different concentrations of staurosporine (μM) was added to the reaction at indicated concentrations. Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel.
    Figure Legend Snippet: YegI is an active kinase (A) Kinase activity of YegI : Autophosphorylation reactions were carried out at 37 °C with 0.2 μM of YegI (WT or K39D or D141N) in kinase buffer (50 mM Tris pH 7.5, 50 mM KCl, 1 mM DTT, 10 mM MgCl 2 , 10 mM MnCl 2 , 200 μM cold ATP and 5 μCi γ -[ 32 P]ATP. Reactions were stopped at t=30 mins and run on 12 % SDS-PAGE followed by autoradiography. Molecular weights (kDa) are indicated on the right of the gel. (B) Sensitivity to staurosporine: Autophosphorylation reactions were carried out at 37 °C with 1 μM of YegI (WT) in kinase buffer without cold ATP. Different concentrations of staurosporine (μM) was added to the reaction at indicated concentrations. Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel.

    Techniques Used: Activity Assay, SDS Page, Autoradiography

    YegI is a Mn 2+ dependent kinase (A) Requirement of Mg 2+ /Mn 2+ for kinase activity: Autophosphorylation reactions were carried out at 37 °C with 1 μM of YegI (WT) in kinase buffer with indicated concentrations of MgCl 2 / MnCl 2 . Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel. (B) Requirement of DFG motif : Autophosphorylation reactions were carried out at 37 °C with 0.2 μM of YegI (WT or S160FD161G or D161G) in kinase buffer with either 10 mM MgCl 2 or MnCl 2 as described in Supp. Figure 3
    Figure Legend Snippet: YegI is a Mn 2+ dependent kinase (A) Requirement of Mg 2+ /Mn 2+ for kinase activity: Autophosphorylation reactions were carried out at 37 °C with 1 μM of YegI (WT) in kinase buffer with indicated concentrations of MgCl 2 / MnCl 2 . Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel. (B) Requirement of DFG motif : Autophosphorylation reactions were carried out at 37 °C with 0.2 μM of YegI (WT or S160FD161G or D161G) in kinase buffer with either 10 mM MgCl 2 or MnCl 2 as described in Supp. Figure 3

    Techniques Used: Activity Assay, SDS Page, Autoradiography

    Requirement of bivalent cations. Autophosphorylation reactions were carried out at 37 °C with 1 µM of YegI (WT) in kinase buffer with either 10 mM of MgCl 2 / MnCl 2 , CaCl 2 / NiCl 2 . Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography
    Figure Legend Snippet: Requirement of bivalent cations. Autophosphorylation reactions were carried out at 37 °C with 1 µM of YegI (WT) in kinase buffer with either 10 mM of MgCl 2 / MnCl 2 , CaCl 2 / NiCl 2 . Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography

    Techniques Used: SDS Page, Autoradiography

    YegI undergoes autophosphorylation on serine residues in the kinase domain and the C-terminus (A) Graphical representation of YegI phosphorylation sites: Domain structure depicting phosphorylation sites following mass spectrometry analysis of autophosphorylation. Phosphorylated residues are indicated by red circles. (B) Sites of autophosphorylation on YegI : Reactions were carried out at 37 °C with 1 μM of YegI (WT or phosphoablative mutants) in kinase buffer. Reactions were stopped at t=30 mins and run on 12 % SDS-PAGE followed by autoradiography. Molecular weights (kda) are indicated on the right of the gel.
    Figure Legend Snippet: YegI undergoes autophosphorylation on serine residues in the kinase domain and the C-terminus (A) Graphical representation of YegI phosphorylation sites: Domain structure depicting phosphorylation sites following mass spectrometry analysis of autophosphorylation. Phosphorylated residues are indicated by red circles. (B) Sites of autophosphorylation on YegI : Reactions were carried out at 37 °C with 1 μM of YegI (WT or phosphoablative mutants) in kinase buffer. Reactions were stopped at t=30 mins and run on 12 % SDS-PAGE followed by autoradiography. Molecular weights (kda) are indicated on the right of the gel.

    Techniques Used: Mass Spectrometry, SDS Page, Autoradiography

    Kinetics of YegI autophosphorylation (A) Autophosphorylation of YegI : Autophosphorylation reactions were carried out at 37 °C with indicated concentrations of YegI (WT) in kinase buffer. Reactions were stopped after 30 min and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel. (B) Autophosphorylation of YegI follows second order kinetics : Kinetics of YegI autophosphorylation was assessed using different concentrations of YegI. % P32 incorporation was calculated relative to autophosphorylation in the presence of 2 μM of YegI from Fig 4(A) .
    Figure Legend Snippet: Kinetics of YegI autophosphorylation (A) Autophosphorylation of YegI : Autophosphorylation reactions were carried out at 37 °C with indicated concentrations of YegI (WT) in kinase buffer. Reactions were stopped after 30 min and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel. (B) Autophosphorylation of YegI follows second order kinetics : Kinetics of YegI autophosphorylation was assessed using different concentrations of YegI. % P32 incorporation was calculated relative to autophosphorylation in the presence of 2 μM of YegI from Fig 4(A) .

    Techniques Used: SDS Page, Autoradiography

    26) Product Images from "Identification and biochemical characterization of a novel eukaryotic-like Ser/Thr kinase in E. coli"

    Article Title: Identification and biochemical characterization of a novel eukaryotic-like Ser/Thr kinase in E. coli

    Journal: bioRxiv

    doi: 10.1101/819920

    YegI is an active kinase (A) Kinase activity of YegI : Autophosphorylation reactions were carried out at 37 °C with 0.2 μM of YegI (WT or K39D or D141N) in kinase buffer (50 mM Tris pH 7.5, 50 mM KCl, 1 mM DTT, 10 mM MgCl 2 , 10 mM MnCl 2 , 200 μM cold ATP and 5 μCi γ -[ 32 P]ATP. Reactions were stopped at t=30 mins and run on 12 % SDS-PAGE followed by autoradiography. Molecular weights (kDa) are indicated on the right of the gel. (B) Sensitivity to staurosporine: Autophosphorylation reactions were carried out at 37 °C with 1 μM of YegI (WT) in kinase buffer without cold ATP. Different concentrations of staurosporine (μM) was added to the reaction at indicated concentrations. Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel.
    Figure Legend Snippet: YegI is an active kinase (A) Kinase activity of YegI : Autophosphorylation reactions were carried out at 37 °C with 0.2 μM of YegI (WT or K39D or D141N) in kinase buffer (50 mM Tris pH 7.5, 50 mM KCl, 1 mM DTT, 10 mM MgCl 2 , 10 mM MnCl 2 , 200 μM cold ATP and 5 μCi γ -[ 32 P]ATP. Reactions were stopped at t=30 mins and run on 12 % SDS-PAGE followed by autoradiography. Molecular weights (kDa) are indicated on the right of the gel. (B) Sensitivity to staurosporine: Autophosphorylation reactions were carried out at 37 °C with 1 μM of YegI (WT) in kinase buffer without cold ATP. Different concentrations of staurosporine (μM) was added to the reaction at indicated concentrations. Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel.

    Techniques Used: Activity Assay, SDS Page, Autoradiography

    YegI is a Mn 2+ dependent kinase (A) Requirement of Mg 2+ /Mn 2+ for kinase activity: Autophosphorylation reactions were carried out at 37 °C with 1 μM of YegI (WT) in kinase buffer with indicated concentrations of MgCl 2 / MnCl 2 . Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel. (B) Requirement of DFG motif : Autophosphorylation reactions were carried out at 37 °C with 0.2 μM of YegI (WT or S160FD161G or D161G) in kinase buffer with either 10 mM MgCl 2 or MnCl 2 as described in Supp. Figure 3
    Figure Legend Snippet: YegI is a Mn 2+ dependent kinase (A) Requirement of Mg 2+ /Mn 2+ for kinase activity: Autophosphorylation reactions were carried out at 37 °C with 1 μM of YegI (WT) in kinase buffer with indicated concentrations of MgCl 2 / MnCl 2 . Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel. (B) Requirement of DFG motif : Autophosphorylation reactions were carried out at 37 °C with 0.2 μM of YegI (WT or S160FD161G or D161G) in kinase buffer with either 10 mM MgCl 2 or MnCl 2 as described in Supp. Figure 3

    Techniques Used: Activity Assay, SDS Page, Autoradiography

    Requirement of bivalent cations. Autophosphorylation reactions were carried out at 37 °C with 1 µM of YegI (WT) in kinase buffer with either 10 mM of MgCl 2 / MnCl 2 , CaCl 2 / NiCl 2 . Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography
    Figure Legend Snippet: Requirement of bivalent cations. Autophosphorylation reactions were carried out at 37 °C with 1 µM of YegI (WT) in kinase buffer with either 10 mM of MgCl 2 / MnCl 2 , CaCl 2 / NiCl 2 . Reactions were stopped at t=30 mins and run on 12% SDS-PAGE followed by autoradiography

    Techniques Used: SDS Page, Autoradiography

    YegI undergoes autophosphorylation on serine residues in the kinase domain and the C-terminus (A) Graphical representation of YegI phosphorylation sites: Domain structure depicting phosphorylation sites following mass spectrometry analysis of autophosphorylation. Phosphorylated residues are indicated by red circles. (B) Sites of autophosphorylation on YegI : Reactions were carried out at 37 °C with 1 μM of YegI (WT or phosphoablative mutants) in kinase buffer. Reactions were stopped at t=30 mins and run on 12 % SDS-PAGE followed by autoradiography. Molecular weights (kda) are indicated on the right of the gel.
    Figure Legend Snippet: YegI undergoes autophosphorylation on serine residues in the kinase domain and the C-terminus (A) Graphical representation of YegI phosphorylation sites: Domain structure depicting phosphorylation sites following mass spectrometry analysis of autophosphorylation. Phosphorylated residues are indicated by red circles. (B) Sites of autophosphorylation on YegI : Reactions were carried out at 37 °C with 1 μM of YegI (WT or phosphoablative mutants) in kinase buffer. Reactions were stopped at t=30 mins and run on 12 % SDS-PAGE followed by autoradiography. Molecular weights (kda) are indicated on the right of the gel.

    Techniques Used: Mass Spectrometry, SDS Page, Autoradiography

    Kinetics of YegI autophosphorylation (A) Autophosphorylation of YegI : Autophosphorylation reactions were carried out at 37 °C with indicated concentrations of YegI (WT) in kinase buffer. Reactions were stopped after 30 min and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel. (B) Autophosphorylation of YegI follows second order kinetics : Kinetics of YegI autophosphorylation was assessed using different concentrations of YegI. % P32 incorporation was calculated relative to autophosphorylation in the presence of 2 μM of YegI from Fig 4(A) .
    Figure Legend Snippet: Kinetics of YegI autophosphorylation (A) Autophosphorylation of YegI : Autophosphorylation reactions were carried out at 37 °C with indicated concentrations of YegI (WT) in kinase buffer. Reactions were stopped after 30 min and run on 12% SDS-PAGE followed by autoradiography. Molecular weights are indicated on the right of the gel. (B) Autophosphorylation of YegI follows second order kinetics : Kinetics of YegI autophosphorylation was assessed using different concentrations of YegI. % P32 incorporation was calculated relative to autophosphorylation in the presence of 2 μM of YegI from Fig 4(A) .

    Techniques Used: SDS Page, Autoradiography

    27) Product Images from "A divergent CheW confers plasticity to nucleoid-associated chemosensory arrays"

    Article Title: A divergent CheW confers plasticity to nucleoid-associated chemosensory arrays

    Journal: bioRxiv

    doi: 10.1101/239202

    Interaction of FrzB with FrzCD and FrzE. (A) GST affinity chromatography co-purification of the indicated pairs. A, B, CD and E stand for GST-FrzA, GST-FrzB, 6His-FrzCD and 6His-FrzE CheA , respectively. In the first gel, lanes are issued from the same gel and were grouped for simplicity. (B) In the left panel, BLi sensograms show the binding of different amounts of GST-FrzB with immobilized 6His-FrzCD. BlitZ ProTM was used to calculate the affinity constant (Kd = 0.7 μM), using a global 1:1 fit. The central panel shows the responses at 145 s (nm, x axis) (average of two biological replicates) of the binding of immobilized 6His-FrzCD with different concentrations of GST-FrzB (μM, y axis). Data were analyzed by GraphPad Prism 5.0, based on steady-state levels of the responses. In the right panel, BLi sensograms show the binding of different amounts of GST-FrzB with immobilized 6His-FrzCD or 6His-FrzE CheA for comparison. A reference subtraction (sensor reference and GST-FrzB at 10 µM) was applied to account for non-specific binding, background, and signal drift to minimize sensor variability. (C) The effect of the FrzB β4-β5 chimera on the FrzE phosphorylation. The FrzE kinase domain (FrzE CheA ) auto-phosphorylation was tested in vitro by incubation of FrzE CheA in the presence of FrzA, FrzB or FrzB β4-β5 , the indicated different forms of FrzCD and ATPγP 33 as a phosphate donor. (D) 3D models of the FrzE P4- P5:FrzA and FrzE P4-P5 :FrzB complexes based on the T. maritima CheA P4-P5 :CheW crystal structure (PDB 2ch4). The 20 amino acid region forming the β-strands 4 and 5 of subdomain 2 and corresponding to the CheW region important for the CheW:CheA P5 interaction are highlighted in red in the FrzA structure. These residues are missing in FrzB. (E) Protein structural alignments between the T. maritima CheW x-ray structure and M. xanthus FrzA and FrzB theoretical 3D models. CheW residues in atomic contact with CheA and MCP are shown as red stars and blue squares, respectively. Secondary structures determined from CheW x-ray structure are shown on top of the alignment.
    Figure Legend Snippet: Interaction of FrzB with FrzCD and FrzE. (A) GST affinity chromatography co-purification of the indicated pairs. A, B, CD and E stand for GST-FrzA, GST-FrzB, 6His-FrzCD and 6His-FrzE CheA , respectively. In the first gel, lanes are issued from the same gel and were grouped for simplicity. (B) In the left panel, BLi sensograms show the binding of different amounts of GST-FrzB with immobilized 6His-FrzCD. BlitZ ProTM was used to calculate the affinity constant (Kd = 0.7 μM), using a global 1:1 fit. The central panel shows the responses at 145 s (nm, x axis) (average of two biological replicates) of the binding of immobilized 6His-FrzCD with different concentrations of GST-FrzB (μM, y axis). Data were analyzed by GraphPad Prism 5.0, based on steady-state levels of the responses. In the right panel, BLi sensograms show the binding of different amounts of GST-FrzB with immobilized 6His-FrzCD or 6His-FrzE CheA for comparison. A reference subtraction (sensor reference and GST-FrzB at 10 µM) was applied to account for non-specific binding, background, and signal drift to minimize sensor variability. (C) The effect of the FrzB β4-β5 chimera on the FrzE phosphorylation. The FrzE kinase domain (FrzE CheA ) auto-phosphorylation was tested in vitro by incubation of FrzE CheA in the presence of FrzA, FrzB or FrzB β4-β5 , the indicated different forms of FrzCD and ATPγP 33 as a phosphate donor. (D) 3D models of the FrzE P4- P5:FrzA and FrzE P4-P5 :FrzB complexes based on the T. maritima CheA P4-P5 :CheW crystal structure (PDB 2ch4). The 20 amino acid region forming the β-strands 4 and 5 of subdomain 2 and corresponding to the CheW region important for the CheW:CheA P5 interaction are highlighted in red in the FrzA structure. These residues are missing in FrzB. (E) Protein structural alignments between the T. maritima CheW x-ray structure and M. xanthus FrzA and FrzB theoretical 3D models. CheW residues in atomic contact with CheA and MCP are shown as red stars and blue squares, respectively. Secondary structures determined from CheW x-ray structure are shown on top of the alignment.

    Techniques Used: Affinity Chromatography, Copurification, Binding Assay, In Vitro, Incubation

    Localization of FrzB-mCherry in M. xanthus cells. (A) (Left) Micrographs of mCherry-frzB cells stained with the DNA DAPI stain. (Middle) mCherry (red) and DAPI fluorescence (blue) profiles are shown with the fluorescence intensity (arbitrary units) represented on the y -axis and the cell length positions with −1 and +1 indicating the poles, on the x- axis. The cell boundaries were drawn manually from the phase-contrast images. (Right) Plot of correlation coefficients of DAPI and mCherry localization. R 2 values > 0.5 indicate significant correlations. The table compares the R 2 values between the indicated fluorescent fusion and DAPI staining. (B) (Left) Fluorescence micrographs of the indicated M. xanthus strains carrying FrzCD-gfp fusions. The cell boundaries were drawn manually from the phase-contrast images. (Right) For each indicated strain, more than 120 cells ( x axis) from at least two biological replicates are represented as lines and ordered according to their length (pixels) in demographs. The GFP fluorescence intensity along the cell body is represented as colored pixels at the corresponding cell position (from −1 to +1 on the y axis). “0” is the cell center. On the right, a scale indicates the fluorescence intensity and the corresponding colors.
    Figure Legend Snippet: Localization of FrzB-mCherry in M. xanthus cells. (A) (Left) Micrographs of mCherry-frzB cells stained with the DNA DAPI stain. (Middle) mCherry (red) and DAPI fluorescence (blue) profiles are shown with the fluorescence intensity (arbitrary units) represented on the y -axis and the cell length positions with −1 and +1 indicating the poles, on the x- axis. The cell boundaries were drawn manually from the phase-contrast images. (Right) Plot of correlation coefficients of DAPI and mCherry localization. R 2 values > 0.5 indicate significant correlations. The table compares the R 2 values between the indicated fluorescent fusion and DAPI staining. (B) (Left) Fluorescence micrographs of the indicated M. xanthus strains carrying FrzCD-gfp fusions. The cell boundaries were drawn manually from the phase-contrast images. (Right) For each indicated strain, more than 120 cells ( x axis) from at least two biological replicates are represented as lines and ordered according to their length (pixels) in demographs. The GFP fluorescence intensity along the cell body is represented as colored pixels at the corresponding cell position (from −1 to +1 on the y axis). “0” is the cell center. On the right, a scale indicates the fluorescence intensity and the corresponding colors.

    Techniques Used: Staining, Fluorescence

    Schematic representation of the supramolecular organization of Che proteins. (A) MCP form trimers of dimers (each dimer is shown as a green circle), which, in turn, form hexagons connected with rings composed of the CheA-P5 domain (dark blue bars) and CheW (white bars). The light blue circles represent the CheA-P4 domain and, the red circles the interface between the β-strands 3 and 4 of subdomain 1 of CheA-P5 and the β-strands 4 and 5 of subdomain 2 of CheW. Rings containing six CheW proteins (shown at the center of the array) might serve to modulate the stability and activation of the system. A signaling unit is represented in the red box (Adapted from ( 43 )). (B) FrzCD, FrzE CheA , FrzA and FrzB proteins organization depicted by homology with Che proteins. (C) Schematic representation of the frz operon.
    Figure Legend Snippet: Schematic representation of the supramolecular organization of Che proteins. (A) MCP form trimers of dimers (each dimer is shown as a green circle), which, in turn, form hexagons connected with rings composed of the CheA-P5 domain (dark blue bars) and CheW (white bars). The light blue circles represent the CheA-P4 domain and, the red circles the interface between the β-strands 3 and 4 of subdomain 1 of CheA-P5 and the β-strands 4 and 5 of subdomain 2 of CheW. Rings containing six CheW proteins (shown at the center of the array) might serve to modulate the stability and activation of the system. A signaling unit is represented in the red box (Adapted from ( 43 )). (B) FrzCD, FrzE CheA , FrzA and FrzB proteins organization depicted by homology with Che proteins. (C) Schematic representation of the frz operon.

    Techniques Used: Activation Assay

    28) Product Images from "The C-Terminus Tail Regulates ERK3 Kinase Activity and Its Ability in Promoting Cancer Cell Migration and Invasion"

    Article Title: The C-Terminus Tail Regulates ERK3 Kinase Activity and Its Ability in Promoting Cancer Cell Migration and Invasion

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21114044

    Truncation of the C-terminus extension decreases the in vitro kinase activity of ERK3 towards myelin basic protein (MBP) and steroid receptor co-activator 3 (SRC-3) substrates. ( A ) In vitro kinase assay for full length or deletion mutant ERK3 proteins purified from mammalian 293T cells using MBP as a substrate. The assay was performed by incubating 40 nM of each ERK3 protein with 1 μg of recombinant MBP in kinase assay buffer containing (γ- 32 P)-ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. Total level of MBP substrate in each reaction is shown by Coomassie staining (left panel). ERK3 proteins in the Coomassie stained gel are marked with arrowheads. Please note that ERK3 proteins are barely seen in the Coomassie-stained gels due to their small amounts. The substrate phosphorylation was detected by autoradiography and marked with arrow (right panel). ( B ) Quantification of MBP phosphorylation by full length or deletion mutant ERK3 proteins. For the purpose of comparison, the normalized phosphorylation level of MBP by ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± SE of three independent experiments. * p
    Figure Legend Snippet: Truncation of the C-terminus extension decreases the in vitro kinase activity of ERK3 towards myelin basic protein (MBP) and steroid receptor co-activator 3 (SRC-3) substrates. ( A ) In vitro kinase assay for full length or deletion mutant ERK3 proteins purified from mammalian 293T cells using MBP as a substrate. The assay was performed by incubating 40 nM of each ERK3 protein with 1 μg of recombinant MBP in kinase assay buffer containing (γ- 32 P)-ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. Total level of MBP substrate in each reaction is shown by Coomassie staining (left panel). ERK3 proteins in the Coomassie stained gel are marked with arrowheads. Please note that ERK3 proteins are barely seen in the Coomassie-stained gels due to their small amounts. The substrate phosphorylation was detected by autoradiography and marked with arrow (right panel). ( B ) Quantification of MBP phosphorylation by full length or deletion mutant ERK3 proteins. For the purpose of comparison, the normalized phosphorylation level of MBP by ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± SE of three independent experiments. * p

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

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

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15135-9

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

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

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

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

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

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

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

    Techniques Used: Staining, Transfection, Expressing, Mutagenesis

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

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

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

    Techniques Used: Immunoprecipitation, Western Blot, Binding Assay

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

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15135-9

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

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

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.802587

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

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

    32) Product Images from "Evaluation of D-isomers of 4-borono-2-18F-fluoro-phenylalanine and O-11C-methyl-tyrosine as brain tumor imaging agents: a comparative PET study with their L-isomers in rat brain glioma"

    Article Title: Evaluation of D-isomers of 4-borono-2-18F-fluoro-phenylalanine and O-11C-methyl-tyrosine as brain tumor imaging agents: a comparative PET study with their L-isomers in rat brain glioma

    Journal: EJNMMI Research

    doi: 10.1186/s13550-018-0404-6

    PET imaging using L- and D- 18 F-FBPA ( a , b ) and L- and D- 11 C-CMT ( c , d ) in C6 glioma-bearing rat brains. Rats anesthetized by isoflurane were positioned prone on a fixation plate. After a transmission scan for 15 min, 10 MBq of each PET probe was intravenously injected, and an emission scan was performed for 90 min with 18 F-FBPA or for 60 min with 11 C-CMT. Summation images from 60 to 90 min for 18 F-FBPA and from 30 to 60 min for 11 C-CMT were reconstructed, and SUV images were created
    Figure Legend Snippet: PET imaging using L- and D- 18 F-FBPA ( a , b ) and L- and D- 11 C-CMT ( c , d ) in C6 glioma-bearing rat brains. Rats anesthetized by isoflurane were positioned prone on a fixation plate. After a transmission scan for 15 min, 10 MBq of each PET probe was intravenously injected, and an emission scan was performed for 90 min with 18 F-FBPA or for 60 min with 11 C-CMT. Summation images from 60 to 90 min for 18 F-FBPA and from 30 to 60 min for 11 C-CMT were reconstructed, and SUV images were created

    Techniques Used: Positron Emission Tomography, Imaging, Transmission Assay, Injection

    33) Product Images from "VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis"

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21491-x

    Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ) NIH3T3 cells transfected with the indicated plasmids were harvested 24 h post transfection and subjected to western blot analysis using anti-γ-tubulin antibodies. Equal total protein amounts were loaded (see also supplementary Fig. S9 ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ) (see also supplementary Fig. S9 ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.
    Figure Legend Snippet: Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ) NIH3T3 cells transfected with the indicated plasmids were harvested 24 h post transfection and subjected to western blot analysis using anti-γ-tubulin antibodies. Equal total protein amounts were loaded (see also supplementary Fig. S9 ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ) (see also supplementary Fig. S9 ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.

    Techniques Used: Staining, Transfection, Construct, Cotransfection, Western Blot

    ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ) Percentage of cells exhibiting centrosome localization of the indicated proteins (corresponds to Fig. 2a and supplementary Fig. S2a ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.
    Figure Legend Snippet: ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ) Percentage of cells exhibiting centrosome localization of the indicated proteins (corresponds to Fig. 2a and supplementary Fig. S2a ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.

    Techniques Used: Transfection, Expressing, Microscopy, Cotransfection

    A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.
    Figure Legend Snippet: A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.

    Techniques Used: Inhibition, Mutagenesis

    Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ . Cells were then processed for electron microscopy as described in material and methods. Cells selected by fluorescence microscopy were located in the TEM and serial sections were collected to locate the centrosome (supplementary Fig. S3 ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.
    Figure Legend Snippet: Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ . Cells were then processed for electron microscopy as described in material and methods. Cells selected by fluorescence microscopy were located in the TEM and serial sections were collected to locate the centrosome (supplementary Fig. S3 ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.

    Techniques Used: Expressing, Staining, Electron Microscopy, Fluorescence, Microscopy, Transmission Electron Microscopy

    Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ . Shown are time composite of sequential frames acquired for EB1-GFP channel on a spinning-disk confocal microscope at 1 second intervals for 1.5 minutes from representative cells (entire movie series are provided in supplementary Movies 1 and 2 ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.
    Figure Legend Snippet: Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ . Shown are time composite of sequential frames acquired for EB1-GFP channel on a spinning-disk confocal microscope at 1 second intervals for 1.5 minutes from representative cells (entire movie series are provided in supplementary Movies 1 and 2 ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.

    Techniques Used: Activity Assay, Transfection, Binding Assay, Microscopy, Expressing, Marker, Staining

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

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

    Journal:

    doi: 10.1016/j.nbd.2008.05.011

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

    Techniques Used: Inhibition, Activity Assay

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

    Techniques Used:

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

    Techniques Used:

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

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

    Journal:

    doi: 10.1128/JB.00046-08

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

    Techniques Used: Negative Control, Binding Assay, Incubation

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

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

    Journal:

    doi: 10.1261/rna.67906

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

    Techniques Used: Sequencing

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

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

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.11.026

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

    Techniques Used: Western Blot

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

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

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

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

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

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

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.239343

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

    Techniques Used: Plasmid Preparation

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

    Techniques Used:

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

    Techniques Used:

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

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

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

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

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01168-12

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

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

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

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

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01153-10

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

    Techniques Used: Footprinting

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

    Techniques Used: Mutagenesis

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

    Techniques Used:

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

    Techniques Used: Footprinting

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    Transfection:

    Article Title: Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA
    Article Snippet: .. Analysis of in vitro transcribed and transfected U1-snRNA integrity U1-snRNA was synthesized in-vitro using T7 RNA polymerase (as described above) in the presence of [γ-32 P]UTP (800 Ci/mmol; Perkin Elmer, Rodgau, Germany). .. After SAP-treatment, A549 cells were transfected (as described above) with 32 P-labeled U1-snRNA.

    Amplification:

    Article Title: The Uve1 Endonuclease Is Regulated by the White Collar Complex to Protect Cryptococcus neoformans from UV Damage
    Article Snippet: .. About 600 ng of the amplified fragment from UVE1 promoter region and nonspecific probe was radiolabeled with [γ-32 P] dATP (PerkinElmer) using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA). .. Labeling conditions were 1X T4 polynucleotide buffer, 5 µl 6000 Ci/mmol γ-32 P ATP, 10 units T4 polynucleotide kinase in a 50 µl reaction mixture at 37°C for 30 min.

    Article Title: Activation of Arterial Wall Dendritic Cells and Breakdown of Self-tolerance in Giant Cell Arteritis
    Article Snippet: .. TLR2 and TLR4 sequences were amplified using a thermocycler (model 9600; PerkinElmer), and CD14, CD83, CCL18, IL-18, CCL19, CCL21, and TCR α-chain were amplified using an UNO II thermocycler (Biometra) using the following conditions: 30 cycles of denaturation at 95°C for 60 s, primer annealing at 55°C for 60 s, and primer extension at 72°C for 90 s. The reaction products were visualized on ethidium bromide–stained 1% LE or 3% MS agarose gels (both Roche Applied Sciences) and digitally documented for further analysis (GelDoc 2000; Bio-Rad Laboratories). .. The method to quantify tissue cytokine mRNA, including the sequences of the primers and probes, has been described in detail previously ( ). mRNA for the chemokines CCL19 and CCL21, the cytokines IFN-γ and IL-18, and the cell surface markers CD83 and CD40L was quantified using the LightCycler PCR (Roche Applied Sciences).

    In Vitro:

    Article Title: Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells
    Article Snippet: .. In vitro Phosphorylation Assays In vitro radioactive assays were performed by incubating 100 ng recombinant ATG4B diluted in assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 5 mM DTT, 20 μM cold ATP, 0.16 μM ATP [γ-32 P] Perkin-Elmer NEG502A100UC) in the presence of recombinant AKT2 (Sigma-Millipore) at 30°C for the indicated time. ..

    Article Title: Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA
    Article Snippet: .. Analysis of in vitro transcribed and transfected U1-snRNA integrity U1-snRNA was synthesized in-vitro using T7 RNA polymerase (as described above) in the presence of [γ-32 P]UTP (800 Ci/mmol; Perkin Elmer, Rodgau, Germany). .. After SAP-treatment, A549 cells were transfected (as described above) with 32 P-labeled U1-snRNA.

    Immunoprecipitation:

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress
    Article Snippet: .. Immunoprecipitated proteins were resuspended in 50 µl of kinase assay buffer, with or without ATM specific inhibitor KU55933 (1 µM), with 1 µl of [γ-32 P]ATP (10µCi, 37.107 mBq/mMol, Perkin Elmer) and with 1 µg of purified GST-p65 substrate. .. The reaction was stopped by adding SDS loading buffer and was subjected to SDS-PAGE.

    Synthesized:

    Article Title: Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA
    Article Snippet: .. Analysis of in vitro transcribed and transfected U1-snRNA integrity U1-snRNA was synthesized in-vitro using T7 RNA polymerase (as described above) in the presence of [γ-32 P]UTP (800 Ci/mmol; Perkin Elmer, Rodgau, Germany). .. After SAP-treatment, A549 cells were transfected (as described above) with 32 P-labeled U1-snRNA.

    Purification:

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress
    Article Snippet: .. Immunoprecipitated proteins were resuspended in 50 µl of kinase assay buffer, with or without ATM specific inhibitor KU55933 (1 µM), with 1 µl of [γ-32 P]ATP (10µCi, 37.107 mBq/mMol, Perkin Elmer) and with 1 µg of purified GST-p65 substrate. .. The reaction was stopped by adding SDS loading buffer and was subjected to SDS-PAGE.

    Concentration Assay:

    Article Title: Kinetic and structural analyses reveal residues in phosphoinositide 3-kinase α that are critical for catalysis and substrate recognition
    Article Snippet: .. The reaction mixture contained 20 m m HEPES, pH 7.5, 10 m m MgCl2 , 0.25 m m DTT, 1 μg of PI3Kα, and 1 μl of γ-32 P-labeled ATP (3000 Ci/mmol, 10 mCi/ml, PerkinElmer Life Sciences) at a final concentration of 10 μ m ATP in a total of 50 μl. ..

    other:

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease
    Article Snippet: General Procedure for the Biochemical Experiments The 5′-32 P-labeled oligonucleotides were prepared by treating the CTNA-oligonucleotides (100 nmol) with T4 polynucleotide kinase (10 units, Takara Bio) at 37 °C for 30 min, in 70 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl2 , 5 mM DTT and γ-32 P adenosine triphosphate (~400 kBq, Perkin Elmer, Waltham, MA, USA).

    Mass Spectrometry:

    Article Title: Activation of Arterial Wall Dendritic Cells and Breakdown of Self-tolerance in Giant Cell Arteritis
    Article Snippet: .. TLR2 and TLR4 sequences were amplified using a thermocycler (model 9600; PerkinElmer), and CD14, CD83, CCL18, IL-18, CCL19, CCL21, and TCR α-chain were amplified using an UNO II thermocycler (Biometra) using the following conditions: 30 cycles of denaturation at 95°C for 60 s, primer annealing at 55°C for 60 s, and primer extension at 72°C for 90 s. The reaction products were visualized on ethidium bromide–stained 1% LE or 3% MS agarose gels (both Roche Applied Sciences) and digitally documented for further analysis (GelDoc 2000; Bio-Rad Laboratories). .. The method to quantify tissue cytokine mRNA, including the sequences of the primers and probes, has been described in detail previously ( ). mRNA for the chemokines CCL19 and CCL21, the cytokines IFN-γ and IL-18, and the cell surface markers CD83 and CD40L was quantified using the LightCycler PCR (Roche Applied Sciences).

    Kinase Assay:

    Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress
    Article Snippet: .. Immunoprecipitated proteins were resuspended in 50 µl of kinase assay buffer, with or without ATM specific inhibitor KU55933 (1 µM), with 1 µl of [γ-32 P]ATP (10µCi, 37.107 mBq/mMol, Perkin Elmer) and with 1 µg of purified GST-p65 substrate. .. The reaction was stopped by adding SDS loading buffer and was subjected to SDS-PAGE.

    Recombinant:

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    Article Snippet: .. In vitro Phosphorylation Assays In vitro radioactive assays were performed by incubating 100 ng recombinant ATG4B diluted in assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 5 mM DTT, 20 μM cold ATP, 0.16 μM ATP [γ-32 P] Perkin-Elmer NEG502A100UC) in the presence of recombinant AKT2 (Sigma-Millipore) at 30°C for the indicated time. ..

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  • 93
    PerkinElmer gamma counter
    Deadtime-corrected <t>gamma</t> counter CPM data as a function of activity in a decaying 18 F sample. The solid line indicates a linear fit to low count rate data and represents an idealized response under the assumption of perfect deadtime correction.
    Gamma Counter, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gamma counter/product/PerkinElmer
    Average 93 stars, based on 13 article reviews
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    92
    PerkinElmer ifn γ
    Cryostat sections of invading lobular carcinomas in MSA control ( a and c ) and IL-12–treated ( b and d ) FVB–NeuN mice. TNF-α ( b ) and <t>IFN-γ</t> ( d ) are evident in the tumor growth area in IL-12–treated mice, whereas both are absent ( a and c ) in the MSA control, as revealed by staining with anti–TNF-α and –IFN-γ mAb in tumor masses of 49-wk-old mice. Original magnification: ×630.
    Ifn γ, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PerkinElmer γ 32 p gtp
    Membrane-inserted CFTR catalyzes phosphotransfer from [γ- 32 <t>P]GTP</t> to N 3 -AMP. A , Western blot ( WB ) probed with antibody 13-1. Letters label highly ( C ) and core glycosylated ( B ) CFTR. Each lane represents 30 μg of membrane protein. B , autoradiograph and Western blot (probed with antibody M3A7) of the same gel. Experiments were performed as illustrated in Fig. 1 . Experimental conditions are indicated below the lanes. N 3 -AMP concentration was 65 μ m . Comparing the autoradiograph and Western blot corroborated that the labeled band was CFTR. C , CFTR photolabeling with 8-N 3 -AMP and 2-N 3 -AMP. N 3 -AMP concentration was 65 μ m . To compare the results from different autoradiographs, data were normalized to CFTR radioactivity under conditions indicated below bar 4. Asterisks indicate p ≤ 0.001 when compared with bar 4, and double daggers indicate p ≤ 0.001 when compared with bar 3 (one-way analysis of variance followed by the Holm-Sidak method for multiple comparisons, n = 3).
    γ 32 P Gtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Deadtime-corrected gamma counter CPM data as a function of activity in a decaying 18 F sample. The solid line indicates a linear fit to low count rate data and represents an idealized response under the assumption of perfect deadtime correction.

    Journal: EJNMMI physics

    Article Title: Performance assessment of a NaI(Tl) gamma counter for PET applications with methods for improved quantitative accuracy and greater standardization

    doi: 10.1186/s40658-015-0114-3

    Figure Lengend Snippet: Deadtime-corrected gamma counter CPM data as a function of activity in a decaying 18 F sample. The solid line indicates a linear fit to low count rate data and represents an idealized response under the assumption of perfect deadtime correction.

    Article Snippet: Gamma counter The instrument under evaluation was a commercial well-type gamma counter (2480 Wizard2 , PerkinElmer, Waltham, MA, USA) that consisted of a single 75-mm-diameter, 80-mm-high NaI(Tl) crystal with a 33-mm-diameter, 60-mm-deep hole.

    Techniques: Activity Assay

    18 F energy spectra measured with the 2480 Wizard 2 gamma counter. When the source was located in the conventional position, inside the well (b) , a peak at 511 keV and a coincidence sum peak at 1,022 keV were seen (a) . When the source was positioned (for illustrative purposes) just outside the well (d) , it was not possible for corresponding annihilation photons to be simultaneously measured and, as a result, the 1,022-keV coincidence sum peak was absent from the spectrum (c) . Note that the photographs were taken with the shielding removed to show the two different positions of the (white) source holder.

    Journal: EJNMMI physics

    Article Title: Performance assessment of a NaI(Tl) gamma counter for PET applications with methods for improved quantitative accuracy and greater standardization

    doi: 10.1186/s40658-015-0114-3

    Figure Lengend Snippet: 18 F energy spectra measured with the 2480 Wizard 2 gamma counter. When the source was located in the conventional position, inside the well (b) , a peak at 511 keV and a coincidence sum peak at 1,022 keV were seen (a) . When the source was positioned (for illustrative purposes) just outside the well (d) , it was not possible for corresponding annihilation photons to be simultaneously measured and, as a result, the 1,022-keV coincidence sum peak was absent from the spectrum (c) . Note that the photographs were taken with the shielding removed to show the two different positions of the (white) source holder.

    Article Snippet: Gamma counter The instrument under evaluation was a commercial well-type gamma counter (2480 Wizard2 , PerkinElmer, Waltham, MA, USA) that consisted of a single 75-mm-diameter, 80-mm-high NaI(Tl) crystal with a 33-mm-diameter, 60-mm-deep hole.

    Techniques:

    In vivo GLV-1h153 gene expression and therapy. ( a ) Gamma emissions from excised PC3 xenografts treated with intratumoural injection of 1 × 10 6 PFU GLV-1h153 and 1 mCi of 131I 48 h later, alongside controls that received 131 I only. ( b ) Viral GFP expression in intratumourally treated xenografts. ( c ) Long-term therapeutic effect of treatment with GLV-1h153 and 131 I on PC3 xenografts. Kaplan–Meier plot significance is the result of log-rank (Mantel Cox) test. * P

    Journal: Gene Therapy

    Article Title: Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer

    doi: 10.1038/gt.2016.5

    Figure Lengend Snippet: In vivo GLV-1h153 gene expression and therapy. ( a ) Gamma emissions from excised PC3 xenografts treated with intratumoural injection of 1 × 10 6 PFU GLV-1h153 and 1 mCi of 131I 48 h later, alongside controls that received 131 I only. ( b ) Viral GFP expression in intratumourally treated xenografts. ( c ) Long-term therapeutic effect of treatment with GLV-1h153 and 131 I on PC3 xenografts. Kaplan–Meier plot significance is the result of log-rank (Mantel Cox) test. * P

    Article Snippet: All wells were washed with copious amounts of PBS before detaching cells with 100 μl 1 m sodium hydroxide (Sigma-Aldrich) for 20 min. Gamma emissions from each sample were measured with a Wallac 1470 Wizard automatic gamma counter (Perkin Elmer).

    Techniques: In Vivo, Expressing, Injection

    Cryostat sections of invading lobular carcinomas in MSA control ( a and c ) and IL-12–treated ( b and d ) FVB–NeuN mice. TNF-α ( b ) and IFN-γ ( d ) are evident in the tumor growth area in IL-12–treated mice, whereas both are absent ( a and c ) in the MSA control, as revealed by staining with anti–TNF-α and –IFN-γ mAb in tumor masses of 49-wk-old mice. Original magnification: ×630.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 12-mediated Prevention of Spontaneous Mammary Adenocarcinomas in Two Lines of Her-2/neu Transgenic Mice

    doi:

    Figure Lengend Snippet: Cryostat sections of invading lobular carcinomas in MSA control ( a and c ) and IL-12–treated ( b and d ) FVB–NeuN mice. TNF-α ( b ) and IFN-γ ( d ) are evident in the tumor growth area in IL-12–treated mice, whereas both are absent ( a and c ) in the MSA control, as revealed by staining with anti–TNF-α and –IFN-γ mAb in tumor masses of 49-wk-old mice. Original magnification: ×630.

    Article Snippet: The cDNA were tested for the presence of murine glucose 3-phosphate dehydrogenase, IL-1α, IL-6, IFN-γ, TNF-α, GM-CSF, monokine induced by γ-IFN (MIG), and IFN-γ–inducible protein (IP-10) sequences in PCR reactions (Gene Amp Kit; Perkin Elmer Cetus, Norwalk, CT) performed in 50 μl volumes, by using specific primer pairs prepared by us (IP-10 and MIG) or from Clontech (Palo Alto, CA).

    Techniques: Mouse Assay, Staining

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M112.408450

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

    Article Snippet: [γ-32 P]GTP, dissolved in 10 mm Tricine, pH 7.6, was from PerkinElmer Life Sciences.

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

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M112.408450

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

    Article Snippet: [γ-32 P]GTP, dissolved in 10 mm Tricine, pH 7.6, was from PerkinElmer Life Sciences.

    Techniques: Labeling, Immunoprecipitation, Binding Assay

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M112.408450

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

    Article Snippet: [γ-32 P]GTP, dissolved in 10 mm Tricine, pH 7.6, was from PerkinElmer Life Sciences.

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