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 256 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 "Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives"

    Article Title: Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    doi: 10.1080/14756366.2018.1452919

    2D structures of the coumarin derivatives. The 3-phenylcoumarin analogues 1–7 produce HSD1 inhibition at a varying degree, but 8 and 9 were found to be inactive ( Table 1 ). Compound 10 or 3-imidazolecoumarin inhibit aromatase instead of HSD1.
    Figure Legend Snippet: 2D structures of the coumarin derivatives. The 3-phenylcoumarin analogues 1–7 produce HSD1 inhibition at a varying degree, but 8 and 9 were found to be inactive ( Table 1 ). Compound 10 or 3-imidazolecoumarin inhibit aromatase instead of HSD1.

    Techniques Used: Inhibition

    The binding of coumarin derivatives with aromatase, monoamine oxidase B and CYP1A2. (A) With the MAO-B (yellow cartoon), the docked pose of 6 demonstrates the analogous hydrophobic packing characteristic of the 3-phenylcoumarin analogues with the inhibitor C18 (stick model with orange backbone; PDB: 2V61) 27 . Notably, the R6-positioned polar group, fluorine in particular, improves the inhibition by forming a halogen bond with the Leu164°. (B) The docked pose of 4 (ball-and-stick model with green backbone) at the active site of CYP1A2 (grey cartoon) mimics α-naphthoflavone (stick model with orange backbone; PDB: 2HI4) 28 . Additionally, the R1-chlorine packs against the haeme and the C2-carbonyl and R4-hydroxyl, respectively, H-bond with crystal water (wat) and the Thr118 OH . (C) Based on docking, 10 (ball-and-stick model with green backbone) aligns similarly on top of the haeme (CPK model with cyan carbon atoms) in the active site of aromatase (magenta cartoon) as the androstenedione (stick model with orange backbone). Unlike the 3-phenylcoumarins the compound 10 has an acceptor group or the N3’ in the imidazole ring capable of H-bonding with the neutral Asp309 (PDB: 3EQM) 26 and, thus, 3-imidazolecoumarin is a potent aromatase inhibitor. Alternatively, the N3’ of 10 could be coordinated with the haeme (not shown). (D) The coumarin ring of 10 is aligned in a way that its C2-carbonyl accepts an H-bond from the Thr124 OH . Moreover, the deprotonated and electronegative N3’ of 3-imidazole ring is likely coordinated with the positively charged iron in the haeme (CPK model with cyan carbon atoms). See Figure 3 for further details.
    Figure Legend Snippet: The binding of coumarin derivatives with aromatase, monoamine oxidase B and CYP1A2. (A) With the MAO-B (yellow cartoon), the docked pose of 6 demonstrates the analogous hydrophobic packing characteristic of the 3-phenylcoumarin analogues with the inhibitor C18 (stick model with orange backbone; PDB: 2V61) 27 . Notably, the R6-positioned polar group, fluorine in particular, improves the inhibition by forming a halogen bond with the Leu164°. (B) The docked pose of 4 (ball-and-stick model with green backbone) at the active site of CYP1A2 (grey cartoon) mimics α-naphthoflavone (stick model with orange backbone; PDB: 2HI4) 28 . Additionally, the R1-chlorine packs against the haeme and the C2-carbonyl and R4-hydroxyl, respectively, H-bond with crystal water (wat) and the Thr118 OH . (C) Based on docking, 10 (ball-and-stick model with green backbone) aligns similarly on top of the haeme (CPK model with cyan carbon atoms) in the active site of aromatase (magenta cartoon) as the androstenedione (stick model with orange backbone). Unlike the 3-phenylcoumarins the compound 10 has an acceptor group or the N3’ in the imidazole ring capable of H-bonding with the neutral Asp309 (PDB: 3EQM) 26 and, thus, 3-imidazolecoumarin is a potent aromatase inhibitor. Alternatively, the N3’ of 10 could be coordinated with the haeme (not shown). (D) The coumarin ring of 10 is aligned in a way that its C2-carbonyl accepts an H-bond from the Thr124 OH . Moreover, the deprotonated and electronegative N3’ of 3-imidazole ring is likely coordinated with the positively charged iron in the haeme (CPK model with cyan carbon atoms). See Figure 3 for further details.

    Techniques Used: Binding Assay, Inhibition

    3) Product Images from "Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination"

    Article Title: Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.12.084

    Recombinant ID2 Complex Preferentially Binds Single-Stranded and Guanine-Rich RNA (A) Purified ID2 heterodimer proteins run on SDS-PAGE and stained with Coomassie Blue. (B) EMSA showing binding of ssDNA and ssRNA by recombinant ID2. (C) EMSA showing binding of dsDNA, dsRNA, and RNA:DNA hybrids by recombinant ID2. (D) Quantification of shifted nucleic acid substrates in (B) and (C). (E) EMSA showing binding of ssDNA substrates with different guanine content (8%, 28%, or 48%) by recombinant ID2. (F) Quantification of shifted substrates in (E). (G) EMSA showing binding of ssRNA substrates with different guanine content (8%, 28%, or 48%) by recombinant ID2. (H) Quantification of shifted substrates in (G). For (D), (F), and (H), means were calculated from 3 replicates; error bars represent SD.
    Figure Legend Snippet: Recombinant ID2 Complex Preferentially Binds Single-Stranded and Guanine-Rich RNA (A) Purified ID2 heterodimer proteins run on SDS-PAGE and stained with Coomassie Blue. (B) EMSA showing binding of ssDNA and ssRNA by recombinant ID2. (C) EMSA showing binding of dsDNA, dsRNA, and RNA:DNA hybrids by recombinant ID2. (D) Quantification of shifted nucleic acid substrates in (B) and (C). (E) EMSA showing binding of ssDNA substrates with different guanine content (8%, 28%, or 48%) by recombinant ID2. (F) Quantification of shifted substrates in (E). (G) EMSA showing binding of ssRNA substrates with different guanine content (8%, 28%, or 48%) by recombinant ID2. (H) Quantification of shifted substrates in (G). For (D), (F), and (H), means were calculated from 3 replicates; error bars represent SD.

    Techniques Used: Recombinant, Purification, SDS Page, Staining, Binding Assay

    RNA and R-Loop Stimulate FANCD2 Monoubiquitination (A) In vitro ubiquitination reaction of recombinant ID2 with either ssDNA or ssRNA substrates. FANCD2 was ubiquitinated maximally in presence of random sequence of ssRNA or ssDNA and ubiquitination was abrogated by T or U-rich nucleic acids. (B) DNA and RNA binding mutant FANCI(KKEE)/D2 complex failed to be ubiquitinated. (C) Comparison of ID2 ubiquitination efficiency using various nucleic acids substrates with different binding affinity. ssRNA conferred the most ubiquitination. (D) Comparison of ID2 ubiquitination efficiency using nucleic acids substrates with different guanine content. Nucleic acid with the highest guanine content conferred the most ubiquitination. (E) Comparison of ID2 ubiquitination efficiency using two configurations of D-loops and R-loops. D- and R-loops with guanine-containing single-strand DNA (ssDNA) supported ID2 ubiquitination similarly. (F) Comparison of ID2 ubiquitination efficiency using different R-loop substrates. R-loops with guanine-containing ssDNA and ssRNA tail supported ID2 ubiquitination with the highest efficiency. For (C)–(F), percentage of ubiquitinated FANCD2 is calculated by the intensity of the Ub-D2 band over total intensity of the Ub-D2 and D2 bands. (G) Model of R-loop binding and its stimulated monoubiquitination of FANCI-FANCD2 in response to DNA damage-induced transcriptional perturbation.
    Figure Legend Snippet: RNA and R-Loop Stimulate FANCD2 Monoubiquitination (A) In vitro ubiquitination reaction of recombinant ID2 with either ssDNA or ssRNA substrates. FANCD2 was ubiquitinated maximally in presence of random sequence of ssRNA or ssDNA and ubiquitination was abrogated by T or U-rich nucleic acids. (B) DNA and RNA binding mutant FANCI(KKEE)/D2 complex failed to be ubiquitinated. (C) Comparison of ID2 ubiquitination efficiency using various nucleic acids substrates with different binding affinity. ssRNA conferred the most ubiquitination. (D) Comparison of ID2 ubiquitination efficiency using nucleic acids substrates with different guanine content. Nucleic acid with the highest guanine content conferred the most ubiquitination. (E) Comparison of ID2 ubiquitination efficiency using two configurations of D-loops and R-loops. D- and R-loops with guanine-containing single-strand DNA (ssDNA) supported ID2 ubiquitination similarly. (F) Comparison of ID2 ubiquitination efficiency using different R-loop substrates. R-loops with guanine-containing ssDNA and ssRNA tail supported ID2 ubiquitination with the highest efficiency. For (C)–(F), percentage of ubiquitinated FANCD2 is calculated by the intensity of the Ub-D2 band over total intensity of the Ub-D2 and D2 bands. (G) Model of R-loop binding and its stimulated monoubiquitination of FANCI-FANCD2 in response to DNA damage-induced transcriptional perturbation.

    Techniques Used: In Vitro, Recombinant, Sequencing, RNA Binding Assay, Mutagenesis, Binding Assay

    4) 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

    5) Product Images from "Free Polyethylenimine Enhances Substrate-Mediated Gene Delivery on Titanium Substrates Modified With RGD-Functionalized Poly(acrylic acid) Brushes"

    Article Title: Free Polyethylenimine Enhances Substrate-Mediated Gene Delivery on Titanium Substrates Modified With RGD-Functionalized Poly(acrylic acid) Brushes

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2019.00051

    DNA complexes immobilized to PAA brushes compared to Flat Ti. For complexes formed at an N/P ratio of 20, the amount of material immobilized onto substrates measured by (A) radiolabeled DNA via scintillation counting and (B) total mass (bPEI and DNA plasmid, free and complexed) by spectroscopic ellipsometry. Statistical analyses were completed using one-way ANOVA with Tukey's post-test. There were no significant differences in the amount of DNA immobilized as measured by radioactivity (A) , but there were statistically significant differences between the amount of total mass on PAA-RGD and PAA substrates compared to Flat Ti ( **** P ≤ 0.0001) (B) . A dotted line marks the expected mass of bPEI-DNA complexes immobilized to the substrate based on the N/P ratio and quantification of DNA by radioactivity (B) .
    Figure Legend Snippet: DNA complexes immobilized to PAA brushes compared to Flat Ti. For complexes formed at an N/P ratio of 20, the amount of material immobilized onto substrates measured by (A) radiolabeled DNA via scintillation counting and (B) total mass (bPEI and DNA plasmid, free and complexed) by spectroscopic ellipsometry. Statistical analyses were completed using one-way ANOVA with Tukey's post-test. There were no significant differences in the amount of DNA immobilized as measured by radioactivity (A) , but there were statistically significant differences between the amount of total mass on PAA-RGD and PAA substrates compared to Flat Ti ( **** P ≤ 0.0001) (B) . A dotted line marks the expected mass of bPEI-DNA complexes immobilized to the substrate based on the N/P ratio and quantification of DNA by radioactivity (B) .

    Techniques Used: Plasmid Preparation, Radioactivity

    DNA complexes released from PAA and PAA-RGD brush substrates, compared to Flat Ti. The amount of DNA released from the substrates with OptiMEM (A) , serum-containing growth media (B) , or conditioned DMEM media (C) at 37°C was measured by radiolabeled DNA via scintillation counting. Release experiments were analyzed using one-way ANOVA with Tukey's post-tests at the final timepoint, which showed a statistically significant difference between PAA-RGD and PAA ( * P ≤ 0.05) for release with growth media (B) , and a statistically significant difference between PAA-RGD compared to Flat Ti ( * P ≤ 0.05) for release with conditioned media (C) .
    Figure Legend Snippet: DNA complexes released from PAA and PAA-RGD brush substrates, compared to Flat Ti. The amount of DNA released from the substrates with OptiMEM (A) , serum-containing growth media (B) , or conditioned DMEM media (C) at 37°C was measured by radiolabeled DNA via scintillation counting. Release experiments were analyzed using one-way ANOVA with Tukey's post-tests at the final timepoint, which showed a statistically significant difference between PAA-RGD and PAA ( * P ≤ 0.05) for release with growth media (B) , and a statistically significant difference between PAA-RGD compared to Flat Ti ( * P ≤ 0.05) for release with conditioned media (C) .

    Techniques Used:

    6) Product Images from "Comparative Systems Biology Analysis To Study the Mode of Action of the Isothiocyanate Compound Iberin on Pseudomonas aeruginosa"

    Article Title: Comparative Systems Biology Analysis To Study the Mode of Action of the Isothiocyanate Compound Iberin on Pseudomonas aeruginosa

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.02620-13

    Growth and normalized gfp gene expression (RFU/OD 450 ) of the P. aeruginosa PAO1 biosensor strains carrying rsmY-gfp (A and C) and rsmZ-gfp (B and D) in ABTGC medium containing various concentrations of iberin. Every 30 min, growth and gfp fluorescence were measured using OD 450 values and relative fluorescence units (RFU), respectively. The experiments were performed in triplicate; only representative ones are shown.
    Figure Legend Snippet: Growth and normalized gfp gene expression (RFU/OD 450 ) of the P. aeruginosa PAO1 biosensor strains carrying rsmY-gfp (A and C) and rsmZ-gfp (B and D) in ABTGC medium containing various concentrations of iberin. Every 30 min, growth and gfp fluorescence were measured using OD 450 values and relative fluorescence units (RFU), respectively. The experiments were performed in triplicate; only representative ones are shown.

    Techniques Used: Expressing, Fluorescence

    7) 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

    8) Product Images from "Restored expression of the atypical heat shock protein H11/HspB8 inhibits the growth of genetically diverse melanoma tumors through activation of novel TAK1-dependent death pathways"

    Article Title: Restored expression of the atypical heat shock protein H11/HspB8 inhibits the growth of genetically diverse melanoma tumors through activation of novel TAK1-dependent death pathways

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.108

    Caspase-1 activation is through TAK1-dependent ASC upregulation. ( a ) Protein extracts from stably transfected A375 cells untreated or treated with Dox (5 μ g/ml; 3 days) in the absence or presence of the TAK1 dominant-negative mutant K63W or the empty vector were immunoblotted with antibodies to activated caspase-7 (casp7p20), caspase-3 (casp3p20) or actin. Blots were stripped between probings. ( b ) Stably transfected A2058 cells as in ( a ) were stained with antibody to caspase-1p20 (AlexaFluor 488-conjugated secondary antibody; green) and DAPI (total cell number; blue). ( c ) Results for Dox-treated cells in ( a ) (densitometric units) and ( b ) (% positive cells) were averaged for three independent experiments and the % inhibition calculated from the formula [1−(K63W-transfected cells/untransfected cells)] × 100. ( d ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml; 3 days) in the presence or absence of the p38MAPK-specific inhibitor SB203580 (10 μ M) were immunoblotted with casp3p20, antibody, sequentially stripped and re-probed with antibodies to activated caspase-1 (casp1p20) and actin. ( e ) Extracts from stably transfected A375 and A2058 cells treated as in ( a ) were immunoblotted with antibodies to ASC or actin. Data were quantified by densitometric scanning and the results are expressed as ASC/actin densitometric units±S.D. ( f ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml; 3 days) were immunoprecipitated (IP) with ASC antibody or preimmune IgG and immunoblotted with antibodies to caspase 1 (recognizes the pro- and activated (p20) forms) and ASC. ( g ) Extracts from stably transfected A2058 cells given Dox (5 μ g/ml; 3 days) in the absence or presence of ASC aODN or sODN (30 μ M) were immunoblotted with antibodies to caspase1p20, ASC and actin. Data were quantified by densitometric scanning and the results are expressed as ASC or Casp1/actin densitometric units±S.D.
    Figure Legend Snippet: Caspase-1 activation is through TAK1-dependent ASC upregulation. ( a ) Protein extracts from stably transfected A375 cells untreated or treated with Dox (5 μ g/ml; 3 days) in the absence or presence of the TAK1 dominant-negative mutant K63W or the empty vector were immunoblotted with antibodies to activated caspase-7 (casp7p20), caspase-3 (casp3p20) or actin. Blots were stripped between probings. ( b ) Stably transfected A2058 cells as in ( a ) were stained with antibody to caspase-1p20 (AlexaFluor 488-conjugated secondary antibody; green) and DAPI (total cell number; blue). ( c ) Results for Dox-treated cells in ( a ) (densitometric units) and ( b ) (% positive cells) were averaged for three independent experiments and the % inhibition calculated from the formula [1−(K63W-transfected cells/untransfected cells)] × 100. ( d ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml; 3 days) in the presence or absence of the p38MAPK-specific inhibitor SB203580 (10 μ M) were immunoblotted with casp3p20, antibody, sequentially stripped and re-probed with antibodies to activated caspase-1 (casp1p20) and actin. ( e ) Extracts from stably transfected A375 and A2058 cells treated as in ( a ) were immunoblotted with antibodies to ASC or actin. Data were quantified by densitometric scanning and the results are expressed as ASC/actin densitometric units±S.D. ( f ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml; 3 days) were immunoprecipitated (IP) with ASC antibody or preimmune IgG and immunoblotted with antibodies to caspase 1 (recognizes the pro- and activated (p20) forms) and ASC. ( g ) Extracts from stably transfected A2058 cells given Dox (5 μ g/ml; 3 days) in the absence or presence of ASC aODN or sODN (30 μ M) were immunoblotted with antibodies to caspase1p20, ASC and actin. Data were quantified by densitometric scanning and the results are expressed as ASC or Casp1/actin densitometric units±S.D.

    Techniques Used: Activation Assay, Stable Transfection, Transfection, Dominant Negative Mutation, Plasmid Preparation, Staining, Inhibition, Immunoprecipitation

    TAK1 is activated by wild-type H11/HspB8 but not its non-killing mutants. ( a ) Pull-down assays of untreated and Dox-treated (5 μ g/ml; 3 days) stably transfected A375 and A2058 cells with H11/HspB8 or TAK1 antibodies or preimmune (Pre) IgG. ( b ) Immunocomplex PK assay of cell extracts as in ( a ) using TAK1 antibody and the TAK1 substrate MKK6 (PK). Kinase precipitates were blotted with TAK1 antibody (IB). ( c ) TAK1 was not activated in reciprocal pull-down assay as in ( a ) and immunocomplex PK assay as in ( b ) but using protein extracts from MeWo and TAG51 cells that respectively express the non-killing H11/HspB8 mutants, P173H and W51C
    Figure Legend Snippet: TAK1 is activated by wild-type H11/HspB8 but not its non-killing mutants. ( a ) Pull-down assays of untreated and Dox-treated (5 μ g/ml; 3 days) stably transfected A375 and A2058 cells with H11/HspB8 or TAK1 antibodies or preimmune (Pre) IgG. ( b ) Immunocomplex PK assay of cell extracts as in ( a ) using TAK1 antibody and the TAK1 substrate MKK6 (PK). Kinase precipitates were blotted with TAK1 antibody (IB). ( c ) TAK1 was not activated in reciprocal pull-down assay as in ( a ) and immunocomplex PK assay as in ( b ) but using protein extracts from MeWo and TAG51 cells that respectively express the non-killing H11/HspB8 mutants, P173H and W51C

    Techniques Used: Stable Transfection, Transfection, PK Assay, Pull Down Assay

    Schematic representation of the H11/HspB8-induced death pathways in A2058 and A375 cells. H11/HspB8 activates TAK1 in both melanoma lines. In A375 cells, the TAK1/p38MAPK pathway activates caspase-3/7 to cause apoptosis. In A2058 cells, the TAK1/p38MAPK pathway activates caspase-3, but TAK1 also activates caspase-1 through ASC upregulation and upregulates Beclin-1 through mTOR phosphorylation at S2481 (pmTORS2481). Caspase-1 cleaves Beclin-1 to promote apoptosis, but Beclin-1 also contributes to cell death through still unknown tumor-suppressor functions
    Figure Legend Snippet: Schematic representation of the H11/HspB8-induced death pathways in A2058 and A375 cells. H11/HspB8 activates TAK1 in both melanoma lines. In A375 cells, the TAK1/p38MAPK pathway activates caspase-3/7 to cause apoptosis. In A2058 cells, the TAK1/p38MAPK pathway activates caspase-3, but TAK1 also activates caspase-1 through ASC upregulation and upregulates Beclin-1 through mTOR phosphorylation at S2481 (pmTORS2481). Caspase-1 cleaves Beclin-1 to promote apoptosis, but Beclin-1 also contributes to cell death through still unknown tumor-suppressor functions

    Techniques Used:

    H11/HspB8 upregulates Beclin-1 through TAK1/pmTORS2481. ( a ) Serial sections from A2058 and A375 xenografts as in Figure 1b were stained by TUNEL (TMR-red labeled dUTP) and with antibody to Beclin-1 (AlexaFluor 488-conjugated secondary antibody; green) and the percentage of cells with co-localized staining was calculated relative to the total cell number determined by DAPI staining. ( b ) Extracts from stably transfected A375 and A2058 cells given Dox (5 μ g/ml; 3 days) in the absence or presence of the TAK1 dominant-negative mutant K63W or empty vector were immunoblotted with antibodies to Beclin-1 or actin. Data were quantified by densitometric scanning and the results are expressed as Beclin-1/actin densitometric units±S.D. ( c ) Extracts from cell cultures that were untreated or Dox treated in the presence or absence of K63W were immunoblotted with antibodies to pmTORS2481, pmTORS2448 or actin. Blots were stripped between probings. Data were quantified by densitometric scanning and the results are expressed as p-mTOR/actin densitometric units±S.D. ( d ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml) in the presence or absence of rapamycin (20 nM) were immunoblotted with Beclin-1 followed by actin antibodies. Data were quantified by densitometric scanning and the results are expressed as Beclin-1/actin densitometric units±S.D.
    Figure Legend Snippet: H11/HspB8 upregulates Beclin-1 through TAK1/pmTORS2481. ( a ) Serial sections from A2058 and A375 xenografts as in Figure 1b were stained by TUNEL (TMR-red labeled dUTP) and with antibody to Beclin-1 (AlexaFluor 488-conjugated secondary antibody; green) and the percentage of cells with co-localized staining was calculated relative to the total cell number determined by DAPI staining. ( b ) Extracts from stably transfected A375 and A2058 cells given Dox (5 μ g/ml; 3 days) in the absence or presence of the TAK1 dominant-negative mutant K63W or empty vector were immunoblotted with antibodies to Beclin-1 or actin. Data were quantified by densitometric scanning and the results are expressed as Beclin-1/actin densitometric units±S.D. ( c ) Extracts from cell cultures that were untreated or Dox treated in the presence or absence of K63W were immunoblotted with antibodies to pmTORS2481, pmTORS2448 or actin. Blots were stripped between probings. Data were quantified by densitometric scanning and the results are expressed as p-mTOR/actin densitometric units±S.D. ( d ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml) in the presence or absence of rapamycin (20 nM) were immunoblotted with Beclin-1 followed by actin antibodies. Data were quantified by densitometric scanning and the results are expressed as Beclin-1/actin densitometric units±S.D.

    Techniques Used: Staining, TUNEL Assay, Labeling, Stable Transfection, Transfection, Dominant Negative Mutation, Plasmid Preparation

    9) Product Images from "Pharmacological brake-release of mRNA translation enhances cognitive memory"

    Article Title: Pharmacological brake-release of mRNA translation enhances cognitive memory

    Journal: eLife

    doi: 10.7554/eLife.00498

    Identification of ISRIB as a potent cell-based inhibitor of PERK signaling. ( A ) Structures of ISRIB isosteromers. ( B ) Inhibition of the ATF4 luciferase reporter in HEK293T cells by ISRIB stereoisomers. Inhibition is plotted in relation to the concentration of either the cis or trans isomer of ISRIB. Cells were treated with 2 µg/ml of tunicamycin to induce ER stress and different concentrations of the inhibitors for 7 hr (N = 2, mean ± SD). ( C ) Effect of ISRIB on production of endogenous ATF4, PERK phosphorylation, and XBP1s production. An immunoblot analysis of PERK, ATF4 and XBP1s in HEK293T cells treated with different ER stress inducers (2.5 µg/ml tunicamycin [Tm] or 100 nM thapsigargin [Tg]) with or without 200 nM ISRIB for 3 hr is shown. The arrowhead marks the XBP1s specific band. ( D ) Effect of ISRIB on XBP1 mRNA splicing. Taqman assays for XBP1unspliced (XBP1u) and XBP1spliced (XBP1s) on cDNA synthesized from total RNA extracted from U2OS cells treated with 2 µg/ml of tunicamycin in the presence or absence of 200 nM ISRIB for the indicated times are shown. Percent splicing was calculated as the ratio of XBP1s over total XBP1 mRNA (XBP1u + XBP1s) (mean ± SD). DOI: http://dx.doi.org/10.7554/eLife.00498.004
    Figure Legend Snippet: Identification of ISRIB as a potent cell-based inhibitor of PERK signaling. ( A ) Structures of ISRIB isosteromers. ( B ) Inhibition of the ATF4 luciferase reporter in HEK293T cells by ISRIB stereoisomers. Inhibition is plotted in relation to the concentration of either the cis or trans isomer of ISRIB. Cells were treated with 2 µg/ml of tunicamycin to induce ER stress and different concentrations of the inhibitors for 7 hr (N = 2, mean ± SD). ( C ) Effect of ISRIB on production of endogenous ATF4, PERK phosphorylation, and XBP1s production. An immunoblot analysis of PERK, ATF4 and XBP1s in HEK293T cells treated with different ER stress inducers (2.5 µg/ml tunicamycin [Tm] or 100 nM thapsigargin [Tg]) with or without 200 nM ISRIB for 3 hr is shown. The arrowhead marks the XBP1s specific band. ( D ) Effect of ISRIB on XBP1 mRNA splicing. Taqman assays for XBP1unspliced (XBP1u) and XBP1spliced (XBP1s) on cDNA synthesized from total RNA extracted from U2OS cells treated with 2 µg/ml of tunicamycin in the presence or absence of 200 nM ISRIB for the indicated times are shown. Percent splicing was calculated as the ratio of XBP1s over total XBP1 mRNA (XBP1u + XBP1s) (mean ± SD). DOI: http://dx.doi.org/10.7554/eLife.00498.004

    Techniques Used: Inhibition, Luciferase, Concentration Assay, Synthesized

    ISRIB does not inhibit eIF2α phosphorylation or XBP1s production. Western blot analysis of PERK, ATF4, XBP1s, phospho S51-eIF2α, total eIF2α, phospho S539-eIF2Bε and total eIF2Bε in HEK293T cells treated with or without 2 µg/ml of tunicamycin or 100 nM thapsigargin in the presence or absence of 200 nM ISRIB for the indicated times. DOI: http://dx.doi.org/10.7554/eLife.00498.006
    Figure Legend Snippet: ISRIB does not inhibit eIF2α phosphorylation or XBP1s production. Western blot analysis of PERK, ATF4, XBP1s, phospho S51-eIF2α, total eIF2α, phospho S539-eIF2Bε and total eIF2Bε in HEK293T cells treated with or without 2 µg/ml of tunicamycin or 100 nM thapsigargin in the presence or absence of 200 nM ISRIB for the indicated times. DOI: http://dx.doi.org/10.7554/eLife.00498.006

    Techniques Used: Western Blot

    ISRIB blocks translational attenuation upon ER stress. Autoradiogram (left) and total protein (right) obtained from HEK293T cells that were treated with 100 nM thapsigargin with or without 200 nM ISRIB for either 1 or 3 hr prior to a 20 min pulse with 35 S-methionine before lysis. Equal amounts of lysate were loaded on an SDS-PAGE gel. DOI: http://dx.doi.org/10.7554/eLife.00498.007
    Figure Legend Snippet: ISRIB blocks translational attenuation upon ER stress. Autoradiogram (left) and total protein (right) obtained from HEK293T cells that were treated with 100 nM thapsigargin with or without 200 nM ISRIB for either 1 or 3 hr prior to a 20 min pulse with 35 S-methionine before lysis. Equal amounts of lysate were loaded on an SDS-PAGE gel. DOI: http://dx.doi.org/10.7554/eLife.00498.007

    Techniques Used: Lysis, SDS Page

    ISRIB impairs induction of the transcriptional network controlled by ATF4. ( A ) ER-Stress dependent induction of CHOP and GADD34 mRNA is impaired in cells treated with ISRIB. qPCR analysis of total RNA extracted from U2OS cells treated with 2 µg/ml of tunicamycin in the presence or absence of 200 nM ISRIB for the indicated times. mRNA levels for each sample were normalized to GAPDH (N = 4, mean ± SD). p values are derived from a one-tail Student’s t-test for unpaired samples. Statistical significance: CHOP, *p=0.0006; GADD34, *p=0.0008. ( B ) ISRIB blocks CHOP production during ER stress. An immunofluorescence analysis of U2OS cells treated with 100 nM thapsigargin for 2 or 4 hr in the presence or absence of 200 nM ISRIB is shown. A secondary Alexa Dye 488 anti-mouse antibody and rhodamine-phalloidin were used to visualize CHOP and actin, respectively. DOI: http://dx.doi.org/10.7554/eLife.00498.013
    Figure Legend Snippet: ISRIB impairs induction of the transcriptional network controlled by ATF4. ( A ) ER-Stress dependent induction of CHOP and GADD34 mRNA is impaired in cells treated with ISRIB. qPCR analysis of total RNA extracted from U2OS cells treated with 2 µg/ml of tunicamycin in the presence or absence of 200 nM ISRIB for the indicated times. mRNA levels for each sample were normalized to GAPDH (N = 4, mean ± SD). p values are derived from a one-tail Student’s t-test for unpaired samples. Statistical significance: CHOP, *p=0.0006; GADD34, *p=0.0008. ( B ) ISRIB blocks CHOP production during ER stress. An immunofluorescence analysis of U2OS cells treated with 100 nM thapsigargin for 2 or 4 hr in the presence or absence of 200 nM ISRIB is shown. A secondary Alexa Dye 488 anti-mouse antibody and rhodamine-phalloidin were used to visualize CHOP and actin, respectively. DOI: http://dx.doi.org/10.7554/eLife.00498.013

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Immunofluorescence

    High-throughput cell-based screen for inhibitors of PERK signaling. ( A ) Schematic representation of the ATF4 luciferase reporter used in the primary screen. The 5′ UTR of human ATF4 containing the uORFs 1 and 2 was fused to firefly luciferase and inserted into a retroviral expression system. ( B ) Primary screen optimization. HEK293T stably expressing the ATF4 luciferase reporter were plated in 384-well plates and treated for 6 hr with 100 nM thapsigargin (Tg) or DMSO as a no ER stress control. Luciferase production was measured at the end point after 6 hr (mean ± SD). The Z′ was calculated as 1−(3 [σ Tg + σ DMSO]/[μ Tg–μ DMSO]). ( C ) Primary screen results. The ATF4 luciferase reporter cell line was treated for 6 hr with 100 nM thapsigargin and library compounds (10 µM). Inhibition of the luciferase activity reporter was calculated as the percent reduction in relative luminescence normalized to thapsigargin treatment (0% inhibition) and the no-ER stress control (100% inhibition). Compounds were considered hits if they lied beyond three standard deviations (SD) from the thapsigargin treatment mean (red line). DOI: http://dx.doi.org/10.7554/eLife.00498.003
    Figure Legend Snippet: High-throughput cell-based screen for inhibitors of PERK signaling. ( A ) Schematic representation of the ATF4 luciferase reporter used in the primary screen. The 5′ UTR of human ATF4 containing the uORFs 1 and 2 was fused to firefly luciferase and inserted into a retroviral expression system. ( B ) Primary screen optimization. HEK293T stably expressing the ATF4 luciferase reporter were plated in 384-well plates and treated for 6 hr with 100 nM thapsigargin (Tg) or DMSO as a no ER stress control. Luciferase production was measured at the end point after 6 hr (mean ± SD). The Z′ was calculated as 1−(3 [σ Tg + σ DMSO]/[μ Tg–μ DMSO]). ( C ) Primary screen results. The ATF4 luciferase reporter cell line was treated for 6 hr with 100 nM thapsigargin and library compounds (10 µM). Inhibition of the luciferase activity reporter was calculated as the percent reduction in relative luminescence normalized to thapsigargin treatment (0% inhibition) and the no-ER stress control (100% inhibition). Compounds were considered hits if they lied beyond three standard deviations (SD) from the thapsigargin treatment mean (red line). DOI: http://dx.doi.org/10.7554/eLife.00498.003

    Techniques Used: High Throughput Screening Assay, Luciferase, Expressing, Stable Transfection, Inhibition, Activity Assay

    10) 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

    11) Product Images from "A Tuneable Switch for Controlling Environmental Degradation of Bioplastics: Addition of Isothiazolinone to Polyhydroxyalkanoates"

    Article Title: A Tuneable Switch for Controlling Environmental Degradation of Bioplastics: Addition of Isothiazolinone to Polyhydroxyalkanoates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075817

    Weight loss rates after initiation of weight loss during burial in mature soil for PHB ( ) and P(HB- co -8HV) (○) films as a consequence of initial DCOI loadings.
    Figure Legend Snippet: Weight loss rates after initiation of weight loss during burial in mature soil for PHB ( ) and P(HB- co -8HV) (○) films as a consequence of initial DCOI loadings.

    Techniques Used:

    Residual weight of PHB-DCOI (a) and P(HB-co-8HV)-DCOI films buried in soil; ( ) 0% w/w, (▪) 2.5%, (○) 5% and (□) 10% (w/w) initial DCOI loadings.
    Figure Legend Snippet: Residual weight of PHB-DCOI (a) and P(HB-co-8HV)-DCOI films buried in soil; ( ) 0% w/w, (▪) 2.5%, (○) 5% and (□) 10% (w/w) initial DCOI loadings.

    Techniques Used:

    Differential Scanning Calorimetry curves of virgin PHB films and P(HB- co -8HV) films (black line) and films containing 10% DCOI (grey line) in the second post annealed heating (a,c) and cooling (b,d) runs respectively.
    Figure Legend Snippet: Differential Scanning Calorimetry curves of virgin PHB films and P(HB- co -8HV) films (black line) and films containing 10% DCOI (grey line) in the second post annealed heating (a,c) and cooling (b,d) runs respectively.

    Techniques Used:

    DCOI content in PHB ( ) and P(HB-co-8HV) (○ ) films with burial time, (a) 2.5%, (b) 5% and (c) 10% (w/w) initial DCOI loadings.
    Figure Legend Snippet: DCOI content in PHB ( ) and P(HB-co-8HV) (○ ) films with burial time, (a) 2.5%, (b) 5% and (c) 10% (w/w) initial DCOI loadings.

    Techniques Used:

    Average Surface Roughness ( R a ) for PHB ( ) and P(HB- co -8HV) (○) films with DCOI loadings.
    Figure Legend Snippet: Average Surface Roughness ( R a ) for PHB ( ) and P(HB- co -8HV) (○) films with DCOI loadings.

    Techniques Used:

    Microbial percent surface area of PHB (a) and P(HB- co -8HV) (b) films, with burial time; ( ) 0% w/w, (▪) 2.5%, (○) 5% and (□) 10% (w/w) DCOI loadings, ‘X’ material weight loss too great to accurately measure biofilm coverage, arrows indicate onset of material weight loss for scl- PHA-DCOI films.
    Figure Legend Snippet: Microbial percent surface area of PHB (a) and P(HB- co -8HV) (b) films, with burial time; ( ) 0% w/w, (▪) 2.5%, (○) 5% and (□) 10% (w/w) DCOI loadings, ‘X’ material weight loss too great to accurately measure biofilm coverage, arrows indicate onset of material weight loss for scl- PHA-DCOI films.

    Techniques Used:

    12) Product Images from "The Bacterial Response Regulator ArcA Uses a Diverse Binding Site Architecture to Regulate Carbon Oxidation Globally"

    Article Title: The Bacterial Response Regulator ArcA Uses a Diverse Binding Site Architecture to Regulate Carbon Oxidation Globally

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003839

    Analysis of predicted multiple DR elements by DNase I footprinting. DNase I footprinting data for a subset of ArcA regulated promoters. The regions protected by ArcA-P are indicated with vertical lines with predicted DR elements indicated by filled boxes with arrows indicating the directional orientation of DR elements. The numbers indicate the position relative to the previously determined transcription start site. Predicted DR elements not protected by ArcA-P are indicated by dashed grey boxes while dashed black boxes represent protected regions where no DR element greater than 0 bits was predicted. Samples were electrophoresed with Maxam–Gilbert ladders (A+G) made using the same DNA (lane 1). ArcA-P protein concentrations are given from left to right in terms of nM total protein. (A) Coding strand of the astC promoter, (D) acs promoter, (E) putP promoter and (G) phoH promoter. ArcA-P: 0, 50, 100, 200, 400, 600 nM. (B) Coding strand of the trxC promoter and (H) dctA promoter. ArcA-P: 0, 100, 200, 400, 600, 1000 nM. (C) Coding strand of the icdA promoter. ArcA-P: 0, 50, 150, 300, 600, 1000 nM. (F) Coding strand of the paaA promoter. ArcA-P: 0, 100, 200, 400, 600 nM.
    Figure Legend Snippet: Analysis of predicted multiple DR elements by DNase I footprinting. DNase I footprinting data for a subset of ArcA regulated promoters. The regions protected by ArcA-P are indicated with vertical lines with predicted DR elements indicated by filled boxes with arrows indicating the directional orientation of DR elements. The numbers indicate the position relative to the previously determined transcription start site. Predicted DR elements not protected by ArcA-P are indicated by dashed grey boxes while dashed black boxes represent protected regions where no DR element greater than 0 bits was predicted. Samples were electrophoresed with Maxam–Gilbert ladders (A+G) made using the same DNA (lane 1). ArcA-P protein concentrations are given from left to right in terms of nM total protein. (A) Coding strand of the astC promoter, (D) acs promoter, (E) putP promoter and (G) phoH promoter. ArcA-P: 0, 50, 100, 200, 400, 600 nM. (B) Coding strand of the trxC promoter and (H) dctA promoter. ArcA-P: 0, 100, 200, 400, 600, 1000 nM. (C) Coding strand of the icdA promoter. ArcA-P: 0, 50, 150, 300, 600, 1000 nM. (F) Coding strand of the paaA promoter. ArcA-P: 0, 100, 200, 400, 600 nM.

    Techniques Used: Footprinting

    13) Product Images from "Polycomb Protein SCML2 Regulates the Cell Cycle by Binding and Modulating CDK/CYCLIN/p21 Complexes"

    Article Title: Polycomb Protein SCML2 Regulates the Cell Cycle by Binding and Modulating CDK/CYCLIN/p21 Complexes

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001737

    Expression and phosphorylation of SCML2 through the cell cycle. (A) Western blot analysis of nuclear extracts derived from HeLa cells growing asynchronously or arrested in mitosis with nocodazole, and incubated in the absence (−) or presence (+) of Antarctic phosphatase (top panel). The blot was probed for SCML2, and RNA polymerase II was used as a control. A densitometric analysis of the results is shown, with the positions of the peaks without phosphatase treatment indicated by a black line, and those derived from Antarctic phosphatase treatment with a red line (bottom panel). (B) Schematic representation of all the phospho-residues identified in the mass spectrometry analysis of SCML2B phosphorylated by CDK2/CYCE or CDK1/CYCB (black sticks). The phospho-sites of SCML2B previously identified in two phospho-proteomics reports are also shown (red sticks). (C) Autoradiography of SCML2B phosphorylated in vitro with CDK2/CYCE, CDK1/CYCB, Aurora kinase A, or in the absence of kinase, employing 32 P-γATP. (D) Quantification of the phosphorylation of SCML2B peptides from nuclear extracts of 293 cells growing asynchronously (AS) or arrested in mitosis with nocodazole (NCZ) and in S phase with thymidine (THY). Peptides with a higher level of phosphorylation in mitosis are highlighted in red, those with a higher level of phosphorylation in S phase are highlighted in blue, and those with a constant level of phosphorylation are highlighted in yellow. A schematic of SCML2B showing the phosphorylated residues is shown below. (E) Quantification of the phosphorylation of SCML2B peptides from nuclear extracts of 293 cells treated with increasing concentrations of Roscovitine for 8 h. (F) SCML2B was phosphorylated with CDK2/CYCE complexes in the absence (left panel) or presence (right panel) of Pin1, and the reaction was carried out with or without ATP, as indicated on top. After removing the CDK2/CYCE complexes in Ni-NTA column, SCML2B was incubated with GST alone or GST-p21. The proteins were pulled down using Glutathione sepharose beads. Five percent of the input is shown (In) as control. The pull-downs were analyzed by Western blot using specific antibodies for SCML2, GST, or CDK2, as indicated. (G) Autoradiography of SCML2B phosphorylated in vitro with CDK2/CYCE in the presence of increasing concentrations of Pin1 or without Pin1 (−), employing 32 P-γATP.
    Figure Legend Snippet: Expression and phosphorylation of SCML2 through the cell cycle. (A) Western blot analysis of nuclear extracts derived from HeLa cells growing asynchronously or arrested in mitosis with nocodazole, and incubated in the absence (−) or presence (+) of Antarctic phosphatase (top panel). The blot was probed for SCML2, and RNA polymerase II was used as a control. A densitometric analysis of the results is shown, with the positions of the peaks without phosphatase treatment indicated by a black line, and those derived from Antarctic phosphatase treatment with a red line (bottom panel). (B) Schematic representation of all the phospho-residues identified in the mass spectrometry analysis of SCML2B phosphorylated by CDK2/CYCE or CDK1/CYCB (black sticks). The phospho-sites of SCML2B previously identified in two phospho-proteomics reports are also shown (red sticks). (C) Autoradiography of SCML2B phosphorylated in vitro with CDK2/CYCE, CDK1/CYCB, Aurora kinase A, or in the absence of kinase, employing 32 P-γATP. (D) Quantification of the phosphorylation of SCML2B peptides from nuclear extracts of 293 cells growing asynchronously (AS) or arrested in mitosis with nocodazole (NCZ) and in S phase with thymidine (THY). Peptides with a higher level of phosphorylation in mitosis are highlighted in red, those with a higher level of phosphorylation in S phase are highlighted in blue, and those with a constant level of phosphorylation are highlighted in yellow. A schematic of SCML2B showing the phosphorylated residues is shown below. (E) Quantification of the phosphorylation of SCML2B peptides from nuclear extracts of 293 cells treated with increasing concentrations of Roscovitine for 8 h. (F) SCML2B was phosphorylated with CDK2/CYCE complexes in the absence (left panel) or presence (right panel) of Pin1, and the reaction was carried out with or without ATP, as indicated on top. After removing the CDK2/CYCE complexes in Ni-NTA column, SCML2B was incubated with GST alone or GST-p21. The proteins were pulled down using Glutathione sepharose beads. Five percent of the input is shown (In) as control. The pull-downs were analyzed by Western blot using specific antibodies for SCML2, GST, or CDK2, as indicated. (G) Autoradiography of SCML2B phosphorylated in vitro with CDK2/CYCE in the presence of increasing concentrations of Pin1 or without Pin1 (−), employing 32 P-γATP.

    Techniques Used: Expressing, Western Blot, Derivative Assay, Incubation, Mass Spectrometry, Autoradiography, In Vitro

    ES cell differentiation modulates the interaction of SCML2 with CDK2. (A) Immunoprecipitation of CDK2 in nuclear extracts from control-treated H9 cells (C) or cells treated with 15 µM Nutlin for 3 d (Nut). The material pulled down was analyzed with specific antibodies for SCML2, CDK2, and p21. Immunoprecipitation with a nonspecific IgG is used as control, and 1% input is shown (In). (B) Western blot analysis of the levels of Nanog and p21 in nuclear extracts of control H9 cells (C) and cells treated with 15 µM Nutlin for 3 d (Nut). Ponceau staining is shown as loading control. (C) Model for the proposed mechanism of action of SCML2B on the regulation of G1 progression. SCML2B and p21 or p27 cooperatively bind and inhibit CDK2/CYCE complexes in early G1 (step I, left), preventing their premature activation. As p21 and p27 levels increase, their association with CDK2/CYCE becomes more stable and independent of SCML2B, and restricts progression into S phase (step II, middle). Over time, increasing amounts of CDK2/CYCE lead to the phosphorylation of p21 and p27 and promote their degradation to allow the entry into S phase (step III, middle). Our data show that in the absence of SCML2B step I is missing, which results in a less effective step II and an accelerated progression to step III.
    Figure Legend Snippet: ES cell differentiation modulates the interaction of SCML2 with CDK2. (A) Immunoprecipitation of CDK2 in nuclear extracts from control-treated H9 cells (C) or cells treated with 15 µM Nutlin for 3 d (Nut). The material pulled down was analyzed with specific antibodies for SCML2, CDK2, and p21. Immunoprecipitation with a nonspecific IgG is used as control, and 1% input is shown (In). (B) Western blot analysis of the levels of Nanog and p21 in nuclear extracts of control H9 cells (C) and cells treated with 15 µM Nutlin for 3 d (Nut). Ponceau staining is shown as loading control. (C) Model for the proposed mechanism of action of SCML2B on the regulation of G1 progression. SCML2B and p21 or p27 cooperatively bind and inhibit CDK2/CYCE complexes in early G1 (step I, left), preventing their premature activation. As p21 and p27 levels increase, their association with CDK2/CYCE becomes more stable and independent of SCML2B, and restricts progression into S phase (step II, middle). Over time, increasing amounts of CDK2/CYCE lead to the phosphorylation of p21 and p27 and promote their degradation to allow the entry into S phase (step III, middle). Our data show that in the absence of SCML2B step I is missing, which results in a less effective step II and an accelerated progression to step III.

    Techniques Used: Cell Differentiation, Immunoprecipitation, Western Blot, Staining, Activation Assay

    Effect of SCML2B on the activity of CDK2/CYCE/p21-p27 in vitro . (A–B) Kinetic analysis of the kinase activity of CDK2/CYCE/p27 or CDK2/CYCE/p27/SCML2B (A) and CDK2/CYCE/p21 or CDK2/CYCE/p21/SCML2B (B) complexes with increasing concentrations of histone H1e, with the products analyzed by Western blot using antibody specific to H1T146Ph (lower panel) and quantified by densitometric analysis of the bands from three different experiments (upper panel). The results were fitted to Michaelis–Menten kinetics, and the amount of histone H1e was in 2–20 molar excess of SCML2B.
    Figure Legend Snippet: Effect of SCML2B on the activity of CDK2/CYCE/p21-p27 in vitro . (A–B) Kinetic analysis of the kinase activity of CDK2/CYCE/p27 or CDK2/CYCE/p27/SCML2B (A) and CDK2/CYCE/p21 or CDK2/CYCE/p21/SCML2B (B) complexes with increasing concentrations of histone H1e, with the products analyzed by Western blot using antibody specific to H1T146Ph (lower panel) and quantified by densitometric analysis of the bands from three different experiments (upper panel). The results were fitted to Michaelis–Menten kinetics, and the amount of histone H1e was in 2–20 molar excess of SCML2B.

    Techniques Used: Activity Assay, In Vitro, Western Blot

    14) 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

    15) 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

    16) 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

    17) Product Images from "Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture"

    Article Title: Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001176

    The effect of Zn 2+ on initiation and elongation activity of purified EAV and SARS-CoV RdRps. ( A ) An EAV RdRp reaction was initiated in the presence of [α- 32 P]ATP under conditions that do not allow elongation, i.e. , low ATP concentration. After 20 min, the reaction was split into two equal volumes, and Zn 2+ was added to one of the tubes. A chase with 50 µM unlabeled ATP, which allows elongation, was performed on both reactions and samples were taken after 5 and 30 min. ( B ) EAV RdRp reaction products that accumulated in the presence and absence of Zn 2+ (indicated above the lanes) after a 5- and 30-min chase with unlabeled ATP. The length of the reaction products in nt is indicated next to the gel. ( C ) A SARS-CoV RdRp reaction was initiated in the presence of 0.17 µM [α- 32 P]ATP, which limits elongation. After 10 min, the reaction was split into two equal volumes, and Zn 2+ was added to one of the tubes. A chase with 50 µM unlabeled ATP was performed on both reactions and samples were taken after 5, 10, 15, and 30 min. ( D ) SARS-CoV RdRp reaction products formed at the chase times indicated above the lanes in the presence and absence of Zn 2+ . The length of the reaction products in nt is indicated next to the gel (p is the primer length).
    Figure Legend Snippet: The effect of Zn 2+ on initiation and elongation activity of purified EAV and SARS-CoV RdRps. ( A ) An EAV RdRp reaction was initiated in the presence of [α- 32 P]ATP under conditions that do not allow elongation, i.e. , low ATP concentration. After 20 min, the reaction was split into two equal volumes, and Zn 2+ was added to one of the tubes. A chase with 50 µM unlabeled ATP, which allows elongation, was performed on both reactions and samples were taken after 5 and 30 min. ( B ) EAV RdRp reaction products that accumulated in the presence and absence of Zn 2+ (indicated above the lanes) after a 5- and 30-min chase with unlabeled ATP. The length of the reaction products in nt is indicated next to the gel. ( C ) A SARS-CoV RdRp reaction was initiated in the presence of 0.17 µM [α- 32 P]ATP, which limits elongation. After 10 min, the reaction was split into two equal volumes, and Zn 2+ was added to one of the tubes. A chase with 50 µM unlabeled ATP was performed on both reactions and samples were taken after 5, 10, 15, and 30 min. ( D ) SARS-CoV RdRp reaction products formed at the chase times indicated above the lanes in the presence and absence of Zn 2+ . The length of the reaction products in nt is indicated next to the gel (p is the primer length).

    Techniques Used: Activity Assay, Purification, Concentration Assay

    EAV and SARS-CoV RdRp assays with wild-type enzyme and active-site mutants. ( A ) The EAV polymerase was incapable of primer extension and required a free 3′ end and poly(U) residues to initiate. Nucleotide incorporating activity of the wild-type enzyme and D445A mutant of nsp9 on an 18-mer poly(U) template confirmed the specificity of our assay. ( B ) SARS-CoV nsp12 RdRp assays were performed with an RNA duplex with a 5′ U 10 overhang as template. The bar graph shows the nucleotide incorporating activities of wild-type and D618A nsp12. Error bars represent standard error of the mean (n = 3).
    Figure Legend Snippet: EAV and SARS-CoV RdRp assays with wild-type enzyme and active-site mutants. ( A ) The EAV polymerase was incapable of primer extension and required a free 3′ end and poly(U) residues to initiate. Nucleotide incorporating activity of the wild-type enzyme and D445A mutant of nsp9 on an 18-mer poly(U) template confirmed the specificity of our assay. ( B ) SARS-CoV nsp12 RdRp assays were performed with an RNA duplex with a 5′ U 10 overhang as template. The bar graph shows the nucleotide incorporating activities of wild-type and D618A nsp12. Error bars represent standard error of the mean (n = 3).

    Techniques Used: Activity Assay, Mutagenesis

    The activity of the RdRps of EAV and SARS-CoV is reversibly inhibited by Zn 2+ . RdRp activity of purified EAV nsp9 ( A ) and SARS-CoV nsp12 ( B ) in the presence of various Zn 2+ concentrations, as indicated above the lanes. ( C ) Schematic representation of the experiment to test whether Zn 2+ -mediated inhibition of RdRp activity could be reversed with MgEDTA. RdRp reactions, either untreated controls (sample 1 and 2) or reactions containing 6 mM Zn 2+ (samples 3 and 4) were incubated for 30 min. Both Zn 2+ -containing and control samples were split into two aliquots and 6 mM MgEDTA was added to sample 2 and 4. All reactions were incubated for an additional 30 min and then terminated. Reaction products of the RdRp assays with EAV nsp9 and SARS-CoV nsp12 are shown in ( D ) and ( E ), respectively. Numbers above the lanes refer to the sample numbers described under (C).
    Figure Legend Snippet: The activity of the RdRps of EAV and SARS-CoV is reversibly inhibited by Zn 2+ . RdRp activity of purified EAV nsp9 ( A ) and SARS-CoV nsp12 ( B ) in the presence of various Zn 2+ concentrations, as indicated above the lanes. ( C ) Schematic representation of the experiment to test whether Zn 2+ -mediated inhibition of RdRp activity could be reversed with MgEDTA. RdRp reactions, either untreated controls (sample 1 and 2) or reactions containing 6 mM Zn 2+ (samples 3 and 4) were incubated for 30 min. Both Zn 2+ -containing and control samples were split into two aliquots and 6 mM MgEDTA was added to sample 2 and 4. All reactions were incubated for an additional 30 min and then terminated. Reaction products of the RdRp assays with EAV nsp9 and SARS-CoV nsp12 are shown in ( D ) and ( E ), respectively. Numbers above the lanes refer to the sample numbers described under (C).

    Techniques Used: Activity Assay, Purification, Inhibition, Incubation

    18) Product Images from "A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori"

    Article Title: A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005369

    Direct Comparison of sodB Regulation in H. pylori Strains G27 and 26695. WT and Δ fur strains of G27 and 26695 were grown to exponential (A) and stationary (B) phase in iron replete and iron-limited (growth) media (60 µM dpp). After growth overnight, one-half of the exponential phase, iron replete culture was removed for RNA isolation. 200 µM dpp (final concentration) was added to create an iron-depletion shock condition to the remaining half of the iron replete cultures, and those cultures were grown for an additional hour prior to RNA isolation. The same procedure was applied the following day to the iron replete, stationary phase culture. After overnight growth, one-half of the iron-limited growth culture was removed for RNA isolation in exponential phase while the remaining half was allowed to grow into stationary phase, and RNA was isolated the following day. RNase Protection Assays (RPAs) were performed on RNA isolated from these strains using sodB , pfr , and amiE riboprobes. Data for Exponential phase cultures are shown in Panel A, and data for Stationary phase cultures are shown in Panel B. Fold-changes are indicated below each pair and were calculated by comparing either the relative amount of protected riboprobe in the iron-depletion shock environment (S) or the relative amount of protected riboprobe in the iron limited growth environment (G) to the iron replete lane (N). These data are representative of multiple independent experiments.
    Figure Legend Snippet: Direct Comparison of sodB Regulation in H. pylori Strains G27 and 26695. WT and Δ fur strains of G27 and 26695 were grown to exponential (A) and stationary (B) phase in iron replete and iron-limited (growth) media (60 µM dpp). After growth overnight, one-half of the exponential phase, iron replete culture was removed for RNA isolation. 200 µM dpp (final concentration) was added to create an iron-depletion shock condition to the remaining half of the iron replete cultures, and those cultures were grown for an additional hour prior to RNA isolation. The same procedure was applied the following day to the iron replete, stationary phase culture. After overnight growth, one-half of the iron-limited growth culture was removed for RNA isolation in exponential phase while the remaining half was allowed to grow into stationary phase, and RNA was isolated the following day. RNase Protection Assays (RPAs) were performed on RNA isolated from these strains using sodB , pfr , and amiE riboprobes. Data for Exponential phase cultures are shown in Panel A, and data for Stationary phase cultures are shown in Panel B. Fold-changes are indicated below each pair and were calculated by comparing either the relative amount of protected riboprobe in the iron-depletion shock environment (S) or the relative amount of protected riboprobe in the iron limited growth environment (G) to the iron replete lane (N). These data are representative of multiple independent experiments.

    Techniques Used: Isolation, Concentration Assay

    Strain specific differences in sodB regulation. Various H. pylori strains were grown to exponential phase as described in the Materials and Methods , and RNA was isolated from iron replete and iron-depleted shock conditions. RPAs were performed using sodB , pfr , and fur riboprobes and results are displayed in Panels A, B, and D, respectively. Basal levels of fur expression relative to the level of expression in 26695 are depicted in Panel C. Fold decrease in expression for sodB and pfr , fold increase for fur , and relative levels of basal fur expression are plotted as single points for each strain with squares, diamonds, triangles, and circles. Each shape represents a biologically independent set of RNA. Median fold change is represented as a bar for each strain. The dotted-dashed line represents the 2-fold significance cut-off in Panels A, B, and D. In Panel A only, the triangles represent the average of two technical repeats on that independent set of RNA.
    Figure Legend Snippet: Strain specific differences in sodB regulation. Various H. pylori strains were grown to exponential phase as described in the Materials and Methods , and RNA was isolated from iron replete and iron-depleted shock conditions. RPAs were performed using sodB , pfr , and fur riboprobes and results are displayed in Panels A, B, and D, respectively. Basal levels of fur expression relative to the level of expression in 26695 are depicted in Panel C. Fold decrease in expression for sodB and pfr , fold increase for fur , and relative levels of basal fur expression are plotted as single points for each strain with squares, diamonds, triangles, and circles. Each shape represents a biologically independent set of RNA. Median fold change is represented as a bar for each strain. The dotted-dashed line represents the 2-fold significance cut-off in Panels A, B, and D. In Panel A only, the triangles represent the average of two technical repeats on that independent set of RNA.

    Techniques Used: Isolation, Expressing

    Role of the −5 bp in sodB regulation. WT G27, WT 26695, and the “−5 bp swap” strain were grown as described in the Materials and Methods , and RNA was isolated under iron replete and iron-depletion shock conditions. RPAs were performed on RNA isolated from 4 biologically independent experiments using sodB and pfr riboprobes. Data from sodB RPAs are presented in Panel A, and data from pfr RPAs are presented in Panel B. Each square, diamond, triangle, and circle represent the average fold decrease calculated from three technical repeats with each independent set of RNA for each strain and growth condition combination. Median fold decrease is represented as a bar for each combination, and the dotted-dashed line represents the 2-fold significance cut-off. * p-value of 0.0001. # p-value of 0.006.
    Figure Legend Snippet: Role of the −5 bp in sodB regulation. WT G27, WT 26695, and the “−5 bp swap” strain were grown as described in the Materials and Methods , and RNA was isolated under iron replete and iron-depletion shock conditions. RPAs were performed on RNA isolated from 4 biologically independent experiments using sodB and pfr riboprobes. Data from sodB RPAs are presented in Panel A, and data from pfr RPAs are presented in Panel B. Each square, diamond, triangle, and circle represent the average fold decrease calculated from three technical repeats with each independent set of RNA for each strain and growth condition combination. Median fold decrease is represented as a bar for each combination, and the dotted-dashed line represents the 2-fold significance cut-off. * p-value of 0.0001. # p-value of 0.006.

    Techniques Used: Isolation

    19) 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

    20) Product Images from "Application of pooled genotyping to scan candidate regions for association with HDL cholesterol levels"

    Article Title: Application of pooled genotyping to scan candidate regions for association with HDL cholesterol levels

    Journal: Human Genomics

    doi: 10.1186/1479-7364-1-6-421

    Haplotype block-fitting analysis . Starting from estimated allele frequency differences for each individual single nucleotide polymorphism (SNP) from pooled genotyping, we use linear regression to solve for frequency differences of the underlying common haplotype patterns. In this example, we show a hypothetical haplotype block consisting of six SNPs and three common haplotypes. Measured frequency differences are shown for the haplotype tagging alleles for each SNP, which are also indicated by boxes in the haplotype patterns. In this example, we are estimating two free parameters from six SNP measurements, since the three pattern differences are constrained to sum to 0. Thus, these pattern differences should have lower variance than the individual SNP measurements. From the pattern differences, we are able to estimate the true allele frequency differences for each SNP more accurately.
    Figure Legend Snippet: Haplotype block-fitting analysis . Starting from estimated allele frequency differences for each individual single nucleotide polymorphism (SNP) from pooled genotyping, we use linear regression to solve for frequency differences of the underlying common haplotype patterns. In this example, we show a hypothetical haplotype block consisting of six SNPs and three common haplotypes. Measured frequency differences are shown for the haplotype tagging alleles for each SNP, which are also indicated by boxes in the haplotype patterns. In this example, we are estimating two free parameters from six SNP measurements, since the three pattern differences are constrained to sum to 0. Thus, these pattern differences should have lower variance than the individual SNP measurements. From the pattern differences, we are able to estimate the true allele frequency differences for each SNP more accurately.

    Techniques Used: Blocking Assay

    Genotyping single nucleotide polymorphisms (SNPs) using high-density oligonucleotide arrays . Each SNP is queried by 80 25-mer oligonucleotides synthesised on a glass substrate. The ten oligonucleotides shown are perfect-match probes for the reference (R) and alternate (A) alleles at five offsets on the forward strand sequence relative to the SNP (-2, -1, 0, + 1, + 2). Not shown are additional mismatch probes where the middle positions of the probes shown are replaced by the three alternate nucleotides, and an equivalent set of probes for the reverse strand.
    Figure Legend Snippet: Genotyping single nucleotide polymorphisms (SNPs) using high-density oligonucleotide arrays . Each SNP is queried by 80 25-mer oligonucleotides synthesised on a glass substrate. The ten oligonucleotides shown are perfect-match probes for the reference (R) and alternate (A) alleles at five offsets on the forward strand sequence relative to the SNP (-2, -1, 0, + 1, + 2). Not shown are additional mismatch probes where the middle positions of the probes shown are replaced by the three alternate nucleotides, and an equivalent set of probes for the reverse strand.

    Techniques Used: Sequencing

    Relationship between pooled allele frequency estimates and allele frequencies determined by individual genotyping . The frequency estimates from pooled genotyping, p ^ , are linearly related to allele frequencies, p , determined by individual genotyping. ( A ) Across all SNPs that were individually genotyped, variation in slope and intercept partially obscures the strength of this relationship. Here, we show p ^ plotted against p averaged over the four high-density lipoprotein pools for 284 SNPs. ( B ) For each SNP, we have independent measurements of p and p ^ in four pools. We show representative data for four SNPs having (due to sampling variation or association) relatively large separation between the four values of p .
    Figure Legend Snippet: Relationship between pooled allele frequency estimates and allele frequencies determined by individual genotyping . The frequency estimates from pooled genotyping, p ^ , are linearly related to allele frequencies, p , determined by individual genotyping. ( A ) Across all SNPs that were individually genotyped, variation in slope and intercept partially obscures the strength of this relationship. Here, we show p ^ plotted against p averaged over the four high-density lipoprotein pools for 284 SNPs. ( B ) For each SNP, we have independent measurements of p and p ^ in four pools. We show representative data for four SNPs having (due to sampling variation or association) relatively large separation between the four values of p .

    Techniques Used: Sampling

    Correspondence between estimated pooled allele frequency differences and actual differences determined by individual genotyping . For this analysis, we included data for single nucleotide polymorphisms (SNPs) having call rates of at least 90 per cent. (A) The correlation between estimated and actual allele frequency differences for 267 SNPs is 0.82. (B) For 164 SNPs in haplotype blocks with fitted p
    Figure Legend Snippet: Correspondence between estimated pooled allele frequency differences and actual differences determined by individual genotyping . For this analysis, we included data for single nucleotide polymorphisms (SNPs) having call rates of at least 90 per cent. (A) The correlation between estimated and actual allele frequency differences for 267 SNPs is 0.82. (B) For 164 SNPs in haplotype blocks with fitted p

    Techniques Used:

    21) Product Images from "In Vitro Study of Surface Modified Poly(ethylene glycol)-Impregnated Sintered Bovine Bone Scaffolds on Human Fibroblast Cells"

    Article Title: In Vitro Study of Surface Modified Poly(ethylene glycol)-Impregnated Sintered Bovine Bone Scaffolds on Human Fibroblast Cells

    Journal: Scientific Reports

    doi: 10.1038/srep09806

    FTIR spectra of (A) BA scaffolds sintered at 900°C, (B) PEG-impregnated BA scaffolds sintered at 900°C (PEG-P900), (C) pristine PEG, (D) BA scaffolds sintered at 1000°C, and (E) PEG-impregnated BA scaffolds sintered at 1000°C (PEG-P1000). Note that most of the peaks of PEG and the untreated BA are present in PEG-P900 and PEG-P1000, indicating the strong adsorption of PEG in the scaffolds.
    Figure Legend Snippet: FTIR spectra of (A) BA scaffolds sintered at 900°C, (B) PEG-impregnated BA scaffolds sintered at 900°C (PEG-P900), (C) pristine PEG, (D) BA scaffolds sintered at 1000°C, and (E) PEG-impregnated BA scaffolds sintered at 1000°C (PEG-P1000). Note that most of the peaks of PEG and the untreated BA are present in PEG-P900 and PEG-P1000, indicating the strong adsorption of PEG in the scaffolds.

    Techniques Used: Adsorption

    A) Alamar Blue reduction (%) behavior of PEG-impregnated BA scaffolds (for distal – square, middle – round, proximal – triangular) sintered at 900 and 1000°C in AB assay using 570 nm and 600 nm wavelengths. AB Reduction increases with time and it is highest for PEG-P1000 scaffold at day 7. B) Cell population or concentration varies with the PEG-impregnated BA scaffolds (symbols used for distal – square, middle – round, and proximal – triangular) sintered at 900 and 1000°C.
    Figure Legend Snippet: A) Alamar Blue reduction (%) behavior of PEG-impregnated BA scaffolds (for distal – square, middle – round, proximal – triangular) sintered at 900 and 1000°C in AB assay using 570 nm and 600 nm wavelengths. AB Reduction increases with time and it is highest for PEG-P1000 scaffold at day 7. B) Cell population or concentration varies with the PEG-impregnated BA scaffolds (symbols used for distal – square, middle – round, and proximal – triangular) sintered at 900 and 1000°C.

    Techniques Used: Concentration Assay

    Optical micrographs of the live fibroblast cells at wells adjacent to the PEG-impregnated BA scaffolds sintered at 900 and 1000°C derived from the three different femoral sections at day-1 (A – PEG-D900, B – PEG-M900, C – PEG-P900, D – PEG-D1000, E – PEG-M1000, and F – PEG-D1000), day-4 (G – PEG-D900, H – PEG-M900, I – PEG-P900, J – PEG-D1000, K – PEG-M1000, and L – PEG-D1000), and day-7 (M – PEG-D900, N – PEG-D1000, O – PEG-M900, P – PEG-M1000, Q – PEG-P900, and R – PEG-P1000) just before the AB assay. Images of (S) negative control or blank (only medium in well, without cell and scaffold) and (T) positive control (only medium and cells in well, without scaffold) wells after day 7 are presented for comparison. All the scale bars are 50 µm. Amount of live cell concentration is higher in the wells for the PEG-treated scaffolds sintered at 1000°C compared to 900°C.
    Figure Legend Snippet: Optical micrographs of the live fibroblast cells at wells adjacent to the PEG-impregnated BA scaffolds sintered at 900 and 1000°C derived from the three different femoral sections at day-1 (A – PEG-D900, B – PEG-M900, C – PEG-P900, D – PEG-D1000, E – PEG-M1000, and F – PEG-D1000), day-4 (G – PEG-D900, H – PEG-M900, I – PEG-P900, J – PEG-D1000, K – PEG-M1000, and L – PEG-D1000), and day-7 (M – PEG-D900, N – PEG-D1000, O – PEG-M900, P – PEG-M1000, Q – PEG-P900, and R – PEG-P1000) just before the AB assay. Images of (S) negative control or blank (only medium in well, without cell and scaffold) and (T) positive control (only medium and cells in well, without scaffold) wells after day 7 are presented for comparison. All the scale bars are 50 µm. Amount of live cell concentration is higher in the wells for the PEG-treated scaffolds sintered at 1000°C compared to 900°C.

    Techniques Used: Derivative Assay, Negative Control, Positive Control, Concentration Assay

    XRD patterns of the PEG-impregnated bovine apatite (BA) scaffolds: (A) PEG-D900, (B) PEG-M9000, (C) PEG-P900, (D) PEG-D1000, (E) PEG-M1000, and (F) PEG-P1000; inset (f) XRD pattern of pristine PEG. Intensity ratio (I PEG /I BA ) of PEG (I PEG ) to BA (I BA ) correspond to their maximum intensities (semicrystalline peak of PEG ‘*’ and crystalline peak of BA‘#’) indicates the quantitative presence of PEG in BA.
    Figure Legend Snippet: XRD patterns of the PEG-impregnated bovine apatite (BA) scaffolds: (A) PEG-D900, (B) PEG-M9000, (C) PEG-P900, (D) PEG-D1000, (E) PEG-M1000, and (F) PEG-P1000; inset (f) XRD pattern of pristine PEG. Intensity ratio (I PEG /I BA ) of PEG (I PEG ) to BA (I BA ) correspond to their maximum intensities (semicrystalline peak of PEG ‘*’ and crystalline peak of BA‘#’) indicates the quantitative presence of PEG in BA.

    Techniques Used:

    Surface morphology of PEG-treated BA scaffolds (a – PEG-D900, b – PEG-M900, c – PEG-P900, d – PEG-D1000, e – PEG-M1000, and f – PEG-P1000) after degradation test for day-7 at 37°C in PBS. Presence of PEG in a–c compared to d–f, but size of deposited particles is large in d–f compared to a–c. PEG impregnated sintered bovine scaffolds on Days 1, 4, and 7.
    Figure Legend Snippet: Surface morphology of PEG-treated BA scaffolds (a – PEG-D900, b – PEG-M900, c – PEG-P900, d – PEG-D1000, e – PEG-M1000, and f – PEG-P1000) after degradation test for day-7 at 37°C in PBS. Presence of PEG in a–c compared to d–f, but size of deposited particles is large in d–f compared to a–c. PEG impregnated sintered bovine scaffolds on Days 1, 4, and 7.

    Techniques Used:

    Inverted SEM images of PEG treated BA scaffolds (A – PEG-D900, B – PEG-M900, C – PEG-P900, D – PEG-D1000, E – PEG-M1000, and F– PEG-P1000) after degradation test for day-7 at 37°C in PBS. Pore size distribution of these scaffolds using image J software are for (a) PEG-D900, (b) PEG-M900, (c) PEG-P900, (d) PEG-D1000, (e) PEG-M1000, and (f) PEG-P1000, respectively. Note: single mode pores are present in the PEG-treated scaffolds sintered at 900°C and multimode pores at 1000 ° C. Scaffolds from proximal part have wide range of pore size compared to distal or middle part of the bovine femur.
    Figure Legend Snippet: Inverted SEM images of PEG treated BA scaffolds (A – PEG-D900, B – PEG-M900, C – PEG-P900, D – PEG-D1000, E – PEG-M1000, and F– PEG-P1000) after degradation test for day-7 at 37°C in PBS. Pore size distribution of these scaffolds using image J software are for (a) PEG-D900, (b) PEG-M900, (c) PEG-P900, (d) PEG-D1000, (e) PEG-M1000, and (f) PEG-P1000, respectively. Note: single mode pores are present in the PEG-treated scaffolds sintered at 900°C and multimode pores at 1000 ° C. Scaffolds from proximal part have wide range of pore size compared to distal or middle part of the bovine femur.

    Techniques Used: Software

    SEM morphology of the broken surface of PEG-impregnated BA scaffolds sintered at 900 and 1000°C derived from three different femoral sections with cells and morphology of the fibroblast cells that adhered to the PEG-treated scaffolds (a – PEG-D900, b – PEG-M900, c – PEG-P900, d – PEG-D1000, e – PEG-M1000, and f – PEG-P1000) after day 7 of AB assay. Attachment of the cell or cell colony (green colured arrow) is found inside the scaffolds and it is very high for PEG-P1000. Sharp edged particles, pores, and cells are indicated by white, yellow, and green coloured arrows, respectively.
    Figure Legend Snippet: SEM morphology of the broken surface of PEG-impregnated BA scaffolds sintered at 900 and 1000°C derived from three different femoral sections with cells and morphology of the fibroblast cells that adhered to the PEG-treated scaffolds (a – PEG-D900, b – PEG-M900, c – PEG-P900, d – PEG-D1000, e – PEG-M1000, and f – PEG-P1000) after day 7 of AB assay. Attachment of the cell or cell colony (green colured arrow) is found inside the scaffolds and it is very high for PEG-P1000. Sharp edged particles, pores, and cells are indicated by white, yellow, and green coloured arrows, respectively.

    Techniques Used: Derivative Assay

    Alamar Blue cell absorbance properties of PEG-impregnated BA scaffolds (a – PEG-D900, b – PEG-D1000, c – PEG-M900, d – PEG-M1000, e – PEG-P900, and f – PEG-P1000), control and blank at wavelengths of (A) 570 nm and (B) 600 nm. Note that the absorbance values increases significantly (p
    Figure Legend Snippet: Alamar Blue cell absorbance properties of PEG-impregnated BA scaffolds (a – PEG-D900, b – PEG-D1000, c – PEG-M900, d – PEG-M1000, e – PEG-P900, and f – PEG-P1000), control and blank at wavelengths of (A) 570 nm and (B) 600 nm. Note that the absorbance values increases significantly (p

    Techniques Used:

    22) Product Images from "Synthesis and Characterization of CeO2 Nanoparticles via Solution Combustion Method for Photocatalytic and Antibacterial Activity Studies"

    Article Title: Synthesis and Characterization of CeO2 Nanoparticles via Solution Combustion Method for Photocatalytic and Antibacterial Activity Studies

    Journal: ChemistryOpen

    doi: 10.1002/open.201402046

    Reduction of CrVI to CrIII using CeO2 nanoparticles
    Figure Legend Snippet: Reduction of CrVI to CrIII using CeO2 nanoparticles

    Techniques Used:

    Reduction of CrVI to CrIII using CeO2 nanoparticles
    Figure Legend Snippet: Reduction of CrVI to CrIII using CeO2 nanoparticles

    Techniques Used:

    Reduction of CrVI to CrIII using CeO2 nanoparticles
    Figure Legend Snippet: Reduction of CrVI to CrIII using CeO2 nanoparticles

    Techniques Used:

    Reduction of CrVI to CrIII using CeO2 nanoparticles
    Figure Legend Snippet: Reduction of CrVI to CrIII using CeO2 nanoparticles

    Techniques Used:

    Reduction of CrVI to CrIII using CeO2 nanoparticles
    Figure Legend Snippet: Reduction of CrVI to CrIII using CeO2 nanoparticles

    Techniques Used:

    23) Product Images from "Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms"

    Article Title: Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051096

    IMPDH1 directly binds ATP. (A) Representative ATP binding experiment with His-IMPDH proteins, shows [ 32 P] ATP bound to His-IMPDH1 only in the presence of 5 µM cold ATP. Data represents mean counts ± SD. (B) Specific binding was determined by expressing the counts per reaction as a fold over the non-specific counts present in the BSA sample. Shown is the mean specific binding ± SEM (fold over non-specific binding) from three to six independent experiments (*** p
    Figure Legend Snippet: IMPDH1 directly binds ATP. (A) Representative ATP binding experiment with His-IMPDH proteins, shows [ 32 P] ATP bound to His-IMPDH1 only in the presence of 5 µM cold ATP. Data represents mean counts ± SD. (B) Specific binding was determined by expressing the counts per reaction as a fold over the non-specific counts present in the BSA sample. Shown is the mean specific binding ± SEM (fold over non-specific binding) from three to six independent experiments (*** p

    Techniques Used: Binding Assay, Expressing

    24) Product Images from "Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase"

    Article Title: Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879585

    Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed that the initial recognition of nascent transcript at the 5′ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5′ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3′-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.
    Figure Legend Snippet: Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed that the initial recognition of nascent transcript at the 5′ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5′ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3′-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.

    Techniques Used: Activation Assay, RNA Binding Assay, Sequencing, Binding Assay

    Cloning, expression and purification of the L. delbrueckii subsp. bulgaricus Csm complex in E. coli . ( a ) Schematic of the LdCsm system. LdCsm genes and the adjacent CRISPR assay are indicated with filled large arrows and small rectangles, respectively. Line with an arrowhead denotes the promoter of the csm gene cassette and the direction of transcription. ( b ) Strategy for reconstitution of the LdCsm effector in E. coli . LdCsm genes ( cas6 + csm1-5 genes) were cloned into p15AIE, yielding p15AIE-cas (Supplementary Fig. S2). A CRISPR array carrying 10 copies of S1 spacer was generated and inserted into pUCE, giving pUCE-S1 (Supplementary Fig. S2). LdCsm2 was cloned into pET30a, giving pET30a-Csm2 that yields the His-tagged Csm2 upon plasmid-born gene expression in the cell. All three plasmids were introduced into E. coli BL21(DE3) by electroporation. ( c ) UV spectrum of SEC purification of LdCsm effector complex. E. coli cell extracts were employed for Nickel-His tag affinity purification of LdCsm2 by which LdCsm effector complexes were copurified. The resulting protein samples were further purified by SEC. Blue: UV absorbance at 280 nm; red: UV absorbance at 254 nm. ( d ) SDS-PAGE analysis of SEC samples collected in the peak region. M: protein mass marker; Input: proteins purified by nickel Csm2-His affinity chromatography. ( e ) Denaturing gel electrophoresis of 5′-labeled RNAs from LdCsm samples. RNAs were extracted from the SEC-purified LdCsm samples. M: RNA size ladder.
    Figure Legend Snippet: Cloning, expression and purification of the L. delbrueckii subsp. bulgaricus Csm complex in E. coli . ( a ) Schematic of the LdCsm system. LdCsm genes and the adjacent CRISPR assay are indicated with filled large arrows and small rectangles, respectively. Line with an arrowhead denotes the promoter of the csm gene cassette and the direction of transcription. ( b ) Strategy for reconstitution of the LdCsm effector in E. coli . LdCsm genes ( cas6 + csm1-5 genes) were cloned into p15AIE, yielding p15AIE-cas (Supplementary Fig. S2). A CRISPR array carrying 10 copies of S1 spacer was generated and inserted into pUCE, giving pUCE-S1 (Supplementary Fig. S2). LdCsm2 was cloned into pET30a, giving pET30a-Csm2 that yields the His-tagged Csm2 upon plasmid-born gene expression in the cell. All three plasmids were introduced into E. coli BL21(DE3) by electroporation. ( c ) UV spectrum of SEC purification of LdCsm effector complex. E. coli cell extracts were employed for Nickel-His tag affinity purification of LdCsm2 by which LdCsm effector complexes were copurified. The resulting protein samples were further purified by SEC. Blue: UV absorbance at 280 nm; red: UV absorbance at 254 nm. ( d ) SDS-PAGE analysis of SEC samples collected in the peak region. M: protein mass marker; Input: proteins purified by nickel Csm2-His affinity chromatography. ( e ) Denaturing gel electrophoresis of 5′-labeled RNAs from LdCsm samples. RNAs were extracted from the SEC-purified LdCsm samples. M: RNA size ladder.

    Techniques Used: Clone Assay, Expressing, Purification, CRISPR, Generated, Plasmid Preparation, Electroporation, Affinity Purification, SDS Page, Marker, Affinity Chromatography, Nucleic Acid Electrophoresis, Labeling

    Effect of LdCsm1 mutations on ssDNA binding and cleavage by the LdCsm effector complex. ( a ) Domain architecture of the LdCsm1 protein. HD represents the HD-type nuclease domain; Palm 1 and Palm 2 denote the two cyclase domains; Linker is a domain that adjoins the Palm1 and Palm2 domains, consisting 4 cysteine residues, D4 is located in the C-terminus rich in α-helices. Amino acid residues selected for alanine substitution mutagenesis are indicated with their names and positions. ( b ) RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm1 mutants. 50 nM S10-60 ssDNA substrates were mixed with 50 nM mutated LdCsm carrying each of LdCsm1 mutant proteins and 500 nM CTR, and incubated for 10 min. Samples were analyzed by denaturing PAGE. ( c ) ssDNA binding by effectors carrying each of the constructed LdCsm1 mutants. 5 nM labeled S10-60 ssDNA were incubated with 100 nM of LdCsm effectors indicated in each experiment. 400 nM of non-homologous RNA (S10 RNA) or in the presence of 500 nM of one of the target RNAs, PTR or CTR or NTR for 3 min. Samples were analyzed by non-denaturing PAGE. Red arrowheads indicate the Csm-ssDNA complex. ( d ) Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm1 mutated derivatives. The relative ssDNA binding activities were estimated by image quantification of the non-denaturing PAGE in (c) by the accessory analysis tool in Typhoon FLA 7000, the ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays, bars represent the mean standard deviation (± SD).
    Figure Legend Snippet: Effect of LdCsm1 mutations on ssDNA binding and cleavage by the LdCsm effector complex. ( a ) Domain architecture of the LdCsm1 protein. HD represents the HD-type nuclease domain; Palm 1 and Palm 2 denote the two cyclase domains; Linker is a domain that adjoins the Palm1 and Palm2 domains, consisting 4 cysteine residues, D4 is located in the C-terminus rich in α-helices. Amino acid residues selected for alanine substitution mutagenesis are indicated with their names and positions. ( b ) RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm1 mutants. 50 nM S10-60 ssDNA substrates were mixed with 50 nM mutated LdCsm carrying each of LdCsm1 mutant proteins and 500 nM CTR, and incubated for 10 min. Samples were analyzed by denaturing PAGE. ( c ) ssDNA binding by effectors carrying each of the constructed LdCsm1 mutants. 5 nM labeled S10-60 ssDNA were incubated with 100 nM of LdCsm effectors indicated in each experiment. 400 nM of non-homologous RNA (S10 RNA) or in the presence of 500 nM of one of the target RNAs, PTR or CTR or NTR for 3 min. Samples were analyzed by non-denaturing PAGE. Red arrowheads indicate the Csm-ssDNA complex. ( d ) Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm1 mutated derivatives. The relative ssDNA binding activities were estimated by image quantification of the non-denaturing PAGE in (c) by the accessory analysis tool in Typhoon FLA 7000, the ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays, bars represent the mean standard deviation (± SD).

    Techniques Used: Binding Assay, Mutagenesis, Construct, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Activity Assay, Standard Deviation

    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 trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export"

    Article Title: A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

    Journal: bioRxiv

    doi: 10.1101/835686

    Rab7 activation by the MCC GEF controls NPC1-dependent lysosomal cholesterol export. ( A, B ) Lysosomal cholesterol export is abolished in NPC1 -, C18orf8 -, Ccz1- and Mon1A/B -deficient cells. ( A ) Wild-type, NPC1-, C18orf8-, Ccz1 and Mon1A/B-deficient cells were treated for 24 hours with the NPC1 inhibitor U18666A to increase lysosomal cholesterol (pulse, top panels), followed by a 24 hours chase in the presence of LPDS and mevastatin (lower panels). Lysosomal cholesterol accumulation was visualised using Filipin co-staining with the LE/Ly marker CD63. ( B ) Colocalisation of Filipin with CD63 was plotted as Pearson correlation, calculated from 3 independent experiments with 6 representative fields per experiment and > 8 cells per field. ** p
    Figure Legend Snippet: Rab7 activation by the MCC GEF controls NPC1-dependent lysosomal cholesterol export. ( A, B ) Lysosomal cholesterol export is abolished in NPC1 -, C18orf8 -, Ccz1- and Mon1A/B -deficient cells. ( A ) Wild-type, NPC1-, C18orf8-, Ccz1 and Mon1A/B-deficient cells were treated for 24 hours with the NPC1 inhibitor U18666A to increase lysosomal cholesterol (pulse, top panels), followed by a 24 hours chase in the presence of LPDS and mevastatin (lower panels). Lysosomal cholesterol accumulation was visualised using Filipin co-staining with the LE/Ly marker CD63. ( B ) Colocalisation of Filipin with CD63 was plotted as Pearson correlation, calculated from 3 independent experiments with 6 representative fields per experiment and > 8 cells per field. ** p

    Techniques Used: Activation Assay, Staining, Marker

    28) 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

    29) 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

    30) Product Images from "Impact of an invasive nitrogen-fixing tree on arbuscular mycorrhizal fungi and the development of native species"

    Article Title: Impact of an invasive nitrogen-fixing tree on arbuscular mycorrhizal fungi and the development of native species

    Journal: AoB Plants

    doi: 10.1093/aobpla/plw018

    Percentage of colonization in P. lanceolata roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.
    Figure Legend Snippet: Percentage of colonization in P. lanceolata roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.

    Techniques Used:

    31) 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

    32) 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

    33) 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

    34) Product Images from "Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *"

    Article Title: Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.227603

    Substrate specificity of IbpA-Fic2. A , the Fic domains of IbpA, PfhB2 and HYPE target Tyr-32 of Cdc42, whereas VopS targets Thr-35. Bacterially expressed GST-tagged IbpA-Fic2, IbpA-Fic1, PfhB2-Fic2, VopS, or HYPE-Fic was incubated with wild type ( W ), Y32F ( Y ) or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro adenylylation assay using [α- 32 P]ATP. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of Cdc42 on the gel is indicated by arrows . The Fic domains of IbpA, PfhB2, and HYPE adenylylate wild type Cdc42 and Cdc42-T35A but not Cdc42-Y32F, indicating their specificity for the switch 1 tyrosine. In contrast, VopS fails to adenylylate only Cdc42-T35A, indicating its specificity for the switch 1 threonine. B , IbpA-Fic2 targets both the active and the inactive forms of Rho GTPases. Bacterially expressed untagged Cdc42, Rac, and RhoA loaded with GDP or GMP-PNP (as described under “Experimental Procedures”) were incubated with IbpA-Fic2 in an in vitro adenylylation assay. The protein load was visualized by Coomassie Blue staining, and the amount of adenylylation was visualized by autoradiography. The nucleotide status of the GTPases was confirmed prior to adenylylation by incubation with GST-Pak (Cdc42 and Rac) or GST-Rhotekin (RhoA) followed by separation on SDS-PAGE and Western analysis using antibodies directed against the individual GTPases. C , IbpA-Fic2 is active against the Cdc42-RhoGDI complex. HA-tagged Cdc42 was expressed in HEK293A cells. Bacterially expressed His 6 -SUMO-RhoGDI bound to nickel-agarose beads was incubated with the HEK239A cell extract treated for GDP loading of Rho GTPases (as described under “Experimental Procedures.”) to allow Cdc42-RhoGDI complex formation. After washing, the beads were subjected to the in vitro adenylylation reaction in the presence or absence of GST-tagged IbpA-Fic2. The supernatant ( supe ) and bead eluate were separated on SDS-PAGE and visualized by autoradiography. The protein load was monitored by Ponceau S staining.
    Figure Legend Snippet: Substrate specificity of IbpA-Fic2. A , the Fic domains of IbpA, PfhB2 and HYPE target Tyr-32 of Cdc42, whereas VopS targets Thr-35. Bacterially expressed GST-tagged IbpA-Fic2, IbpA-Fic1, PfhB2-Fic2, VopS, or HYPE-Fic was incubated with wild type ( W ), Y32F ( Y ) or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro adenylylation assay using [α- 32 P]ATP. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of Cdc42 on the gel is indicated by arrows . The Fic domains of IbpA, PfhB2, and HYPE adenylylate wild type Cdc42 and Cdc42-T35A but not Cdc42-Y32F, indicating their specificity for the switch 1 tyrosine. In contrast, VopS fails to adenylylate only Cdc42-T35A, indicating its specificity for the switch 1 threonine. B , IbpA-Fic2 targets both the active and the inactive forms of Rho GTPases. Bacterially expressed untagged Cdc42, Rac, and RhoA loaded with GDP or GMP-PNP (as described under “Experimental Procedures”) were incubated with IbpA-Fic2 in an in vitro adenylylation assay. The protein load was visualized by Coomassie Blue staining, and the amount of adenylylation was visualized by autoradiography. The nucleotide status of the GTPases was confirmed prior to adenylylation by incubation with GST-Pak (Cdc42 and Rac) or GST-Rhotekin (RhoA) followed by separation on SDS-PAGE and Western analysis using antibodies directed against the individual GTPases. C , IbpA-Fic2 is active against the Cdc42-RhoGDI complex. HA-tagged Cdc42 was expressed in HEK293A cells. Bacterially expressed His 6 -SUMO-RhoGDI bound to nickel-agarose beads was incubated with the HEK239A cell extract treated for GDP loading of Rho GTPases (as described under “Experimental Procedures.”) to allow Cdc42-RhoGDI complex formation. After washing, the beads were subjected to the in vitro adenylylation reaction in the presence or absence of GST-tagged IbpA-Fic2. The supernatant ( supe ) and bead eluate were separated on SDS-PAGE and visualized by autoradiography. The protein load was monitored by Ponceau S staining.

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

    Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.
    Figure Legend Snippet: Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

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

    Apparent steady-state kinetic measurements for ATP and constitutively active Cdc42. A , initial velocity measurements for ATP were obtained using a constant concentration of Cdc42 1–179 Q61L of 500 μ m while varying the ATP concentrations from 100 to 10,000 μ m . B , initial velocity measurements for Cdc42 were obtained at 5 m m ATP while varying the concentration of Cdc42 1–179 Q61L between 100 and 2800 μ m . Assays were performed in triplicate with IbpA-Fic2 at 0.56 n m . The line represents the fit of this data using the Michaelis-Menten equation (“Experimental Procedures”). Error bars indicate S.E.
    Figure Legend Snippet: Apparent steady-state kinetic measurements for ATP and constitutively active Cdc42. A , initial velocity measurements for ATP were obtained using a constant concentration of Cdc42 1–179 Q61L of 500 μ m while varying the ATP concentrations from 100 to 10,000 μ m . B , initial velocity measurements for Cdc42 were obtained at 5 m m ATP while varying the concentration of Cdc42 1–179 Q61L between 100 and 2800 μ m . Assays were performed in triplicate with IbpA-Fic2 at 0.56 n m . The line represents the fit of this data using the Michaelis-Menten equation (“Experimental Procedures”). Error bars indicate S.E.

    Techniques Used: Concentration Assay

    Fic domains of IbpA, PfhB2, and VopS preferentially target the Rho subfamily of GTPases for adenylylation. A , survey of Ras family Rho GTPases as substrates for Fic-mediated adenylylation. The indicated GST-tagged Rho GTPases were bacterially expressed and purified and incubated with purified IbpA-Fic2 in an in vitro adenylylation reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of IbpA-Fic2 on the gel is indicated by an arrow . IbpA-Fic2 adenylylated only the Rho family members, RhoB, RhoC, RhoG, and TC10. B , the ability of Fic enzymes to adenylylate RhoG. GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, VopS, and HYPE-Fic were incubated with bacterially expressed and purified GST-RhoG in an in vitro adenylylation reaction. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of RhoG on the gel is indicated by an arrow . The Fic domains of IbpA, PfhB2, and VopS efficiently adenylylate RhoG, whereas the Fic domain of HYPE displays a weaker adenylylation activity. C , the ability of Fic enzymes to adenylylate TC10. GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, VopS, and HYPE-Fic were incubated with bacterially expressed and purified GST-TC10 in an in vitro adenylylation reaction. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of TC10 on the gel is indicated by an arrow . The Fic domains of IbpA and PfhB2 can efficiently adenylylate TC10, whereas VopS shows minimal activity toward it. HYPE did not adenylylate TC10 in vitro .
    Figure Legend Snippet: Fic domains of IbpA, PfhB2, and VopS preferentially target the Rho subfamily of GTPases for adenylylation. A , survey of Ras family Rho GTPases as substrates for Fic-mediated adenylylation. The indicated GST-tagged Rho GTPases were bacterially expressed and purified and incubated with purified IbpA-Fic2 in an in vitro adenylylation reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of IbpA-Fic2 on the gel is indicated by an arrow . IbpA-Fic2 adenylylated only the Rho family members, RhoB, RhoC, RhoG, and TC10. B , the ability of Fic enzymes to adenylylate RhoG. GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, VopS, and HYPE-Fic were incubated with bacterially expressed and purified GST-RhoG in an in vitro adenylylation reaction. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of RhoG on the gel is indicated by an arrow . The Fic domains of IbpA, PfhB2, and VopS efficiently adenylylate RhoG, whereas the Fic domain of HYPE displays a weaker adenylylation activity. C , the ability of Fic enzymes to adenylylate TC10. GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, VopS, and HYPE-Fic were incubated with bacterially expressed and purified GST-TC10 in an in vitro adenylylation reaction. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The position of TC10 on the gel is indicated by an arrow . The Fic domains of IbpA and PfhB2 can efficiently adenylylate TC10, whereas VopS shows minimal activity toward it. HYPE did not adenylylate TC10 in vitro .

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

    35) 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

    36) 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

    37) Product Images from "Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response"

    Article Title: Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2011.18

    Face-to-face kinase domain dimerisation of human Ire1α. ( A ) Human Ire1α forms a dimer in which the kinase active sites of the two monomers face each other. The activation segment of one monomer is directed towards the other, so that the target substrate residue, Ser724 would come into close proximity of the Mg 2+ -ATP bound in the opposite active site, and be phosphorylated by it. This arrangement of Ire1α molecules provides a straightforward mechanistic model for how dimerisation of Ire1 N-terminal domains in the lumen of the ER would facilitate association and transphosphorylation of their associated kinase domains on the cytoplasmic side of the membrane. ( B ) Autophosphorylation of dephosphorylated dimer interface mutants Q636A and F637A Ire1 as compared with wild type. Both wild-type and mutant proteins were incubated with 5 mM MgCl and 5 mM ATP at 37°C and samples were run at specific time points. Protein samples were visualised by western blot with generic Ire1α or the phospho-specific pS724-Ire1α.
    Figure Legend Snippet: Face-to-face kinase domain dimerisation of human Ire1α. ( A ) Human Ire1α forms a dimer in which the kinase active sites of the two monomers face each other. The activation segment of one monomer is directed towards the other, so that the target substrate residue, Ser724 would come into close proximity of the Mg 2+ -ATP bound in the opposite active site, and be phosphorylated by it. This arrangement of Ire1α molecules provides a straightforward mechanistic model for how dimerisation of Ire1 N-terminal domains in the lumen of the ER would facilitate association and transphosphorylation of their associated kinase domains on the cytoplasmic side of the membrane. ( B ) Autophosphorylation of dephosphorylated dimer interface mutants Q636A and F637A Ire1 as compared with wild type. Both wild-type and mutant proteins were incubated with 5 mM MgCl and 5 mM ATP at 37°C and samples were run at specific time points. Protein samples were visualised by western blot with generic Ire1α or the phospho-specific pS724-Ire1α.

    Techniques Used: Activation Assay, Mutagenesis, Incubation, Western Blot

    Xbp1 mRNA cleavage by human Ire1α. ( A ) Elution profiles from Agilent Bioanalyser of (left) untreated Xbp1 mRNA, (middle) Xbp1 mRNA incubated with dephosphorylated Ire1α, and (right) Xbp1 mRNA incubated with phosphorylated Ire1α. Reaction conditions are given in Materials and methods section. The peak eluting at a position corresponding to 800 nucleotides in the left and middle panels is the unspliced Xbp1 mRNA, whereas the pair of peaks eluting at 300 and 500 nucleotides, respectively, in the right panel, corresponds to the two cleaved products of Xbp1 mRNA. The peak at 25 nucleotides in all three panels is a calibration marker. ( B ) In vitro cleavage activity of Ire1α, wild-type, and Q636A mutant, on a fluorescently tagged oligonucleotide encapsulating the sequence and secondary structure of a known Ire1α cleavage site in human Xbp1 mRNA. Cleavage generates a fluorescent species with higher gel mobility. The Q636A mutation, which causes a defect in autophosphorylation, results in a similar decrease in RNase activity, confirming the dependence of RNase activation on autophosphorylation. ( C ) Sunitinib inhibits Xbp1 mRNA splicing in vivo . Myeloma cell lines (U266+H929) were treated with tunicamycin to induce ER stress, in the presence or absence of sunitinib, and levels of spliced (Xbp1s) and unspliced (Xbp1u) mRNAs in the treated cells were determined by quantitative real-time PCR using LUX primers (see Materials and methods), and plotted as relative Xbp1u:Xbp1s ratio. In both cell lines, the addition of the Ire1α kinase inhibitor sunitinib significantly inhibits the splicing of Xbp1 mRNA. ( D ) Similar results were obtained by RT–PCR amplification. ( E ) Western blot of protein extracts from cells treated as in ( C ), showing inhibition of Xbp1 protein production in ER-stressed cells treated with sunitinib. Actin is shown as a loading control.
    Figure Legend Snippet: Xbp1 mRNA cleavage by human Ire1α. ( A ) Elution profiles from Agilent Bioanalyser of (left) untreated Xbp1 mRNA, (middle) Xbp1 mRNA incubated with dephosphorylated Ire1α, and (right) Xbp1 mRNA incubated with phosphorylated Ire1α. Reaction conditions are given in Materials and methods section. The peak eluting at a position corresponding to 800 nucleotides in the left and middle panels is the unspliced Xbp1 mRNA, whereas the pair of peaks eluting at 300 and 500 nucleotides, respectively, in the right panel, corresponds to the two cleaved products of Xbp1 mRNA. The peak at 25 nucleotides in all three panels is a calibration marker. ( B ) In vitro cleavage activity of Ire1α, wild-type, and Q636A mutant, on a fluorescently tagged oligonucleotide encapsulating the sequence and secondary structure of a known Ire1α cleavage site in human Xbp1 mRNA. Cleavage generates a fluorescent species with higher gel mobility. The Q636A mutation, which causes a defect in autophosphorylation, results in a similar decrease in RNase activity, confirming the dependence of RNase activation on autophosphorylation. ( C ) Sunitinib inhibits Xbp1 mRNA splicing in vivo . Myeloma cell lines (U266+H929) were treated with tunicamycin to induce ER stress, in the presence or absence of sunitinib, and levels of spliced (Xbp1s) and unspliced (Xbp1u) mRNAs in the treated cells were determined by quantitative real-time PCR using LUX primers (see Materials and methods), and plotted as relative Xbp1u:Xbp1s ratio. In both cell lines, the addition of the Ire1α kinase inhibitor sunitinib significantly inhibits the splicing of Xbp1 mRNA. ( D ) Similar results were obtained by RT–PCR amplification. ( E ) Western blot of protein extracts from cells treated as in ( C ), showing inhibition of Xbp1 protein production in ER-stressed cells treated with sunitinib. Actin is shown as a loading control.

    Techniques Used: Incubation, Marker, In Vitro, Activity Assay, Mutagenesis, Sequencing, Activation Assay, In Vivo, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Inhibition

    Autophosphorylation and inhibition. ( A ) Autophosphorylation of dephosphorylated Ire1α monitored by α-pS724-Ire1α phospho-specific antibody. Ire1α was incubated with 5 mM ATP and 5 mM MgCl 2 at 37°C for the times indicated. Protein was visualised by SDS–PAGE with Coomassie Brilliant Blue (CBB) and by western blot with a generic Ire1α antibody or the phospho-specific α-pS724-Ire1α antibody. ( B ) Quantitative DELFIA assay measuring autophosphorylation of dephosphorylated Ire1α monitored by α-pS724-Ire1α phospho-specific antibody. Reaction used 700 nM Ire1α at 37°C in the presence of 0.5 mM ATP and 20 mM Mg 2+ . The non-linear kinetics are consistent with a transphosphorylation reaction. ( C ) Inhibition of Ire1α autophosphorylation by the broad-specificity kinase inhibitor staurosporine, the licensed anti-cancer drug sunitinib, and ADP. Autophosphorylation reaction was followed by DELFIA assay as in ( D ) after 2.5 h. ( D ) Inhibition of Ire1α autophosphorylation by ADP. Autophosphorylation of previously dephosphorylated Ire1α, indicated by α-pS724-Ire1α western blot, is progressively inhibited in the presence of increasing concentrations of ADP, and essentially blocked at equimolar ADP to ATP, consistent with the higher affinity of ADP for the nucleotide-binding site in the unphosphorylated kinase indicated by the thermal-shift analysis ( A ). Reactions were carried out in the presence of 5 mM MgCl 2 . ( E ) Hetero-phosphorylation of the HTRF biotinylated peptide S2 (200 nM) by phosphorylated Ire1α (○=no enzyme; ▪=50 nM; ▵=100 nM; •=200 nM; □=400 nM; and ▴=800 nM) in the presence of 30 μM Mg-ATP. ( F ) Concentration-dependent inhibition of Ire1α hetero-phosphorylation activity. Curves are shown for sunitinib (IC 50 =3.7 μM±1.2) and ADP (IC 50 =38 μM±17). Staurosporine (data not plotted) had an IC 50 =13 nM±8.4. Reactions used 200 nM Ire1α, 200 nM peptide S2, and 30 μM ATP. IC 50 values are averages of three experiments.
    Figure Legend Snippet: Autophosphorylation and inhibition. ( A ) Autophosphorylation of dephosphorylated Ire1α monitored by α-pS724-Ire1α phospho-specific antibody. Ire1α was incubated with 5 mM ATP and 5 mM MgCl 2 at 37°C for the times indicated. Protein was visualised by SDS–PAGE with Coomassie Brilliant Blue (CBB) and by western blot with a generic Ire1α antibody or the phospho-specific α-pS724-Ire1α antibody. ( B ) Quantitative DELFIA assay measuring autophosphorylation of dephosphorylated Ire1α monitored by α-pS724-Ire1α phospho-specific antibody. Reaction used 700 nM Ire1α at 37°C in the presence of 0.5 mM ATP and 20 mM Mg 2+ . The non-linear kinetics are consistent with a transphosphorylation reaction. ( C ) Inhibition of Ire1α autophosphorylation by the broad-specificity kinase inhibitor staurosporine, the licensed anti-cancer drug sunitinib, and ADP. Autophosphorylation reaction was followed by DELFIA assay as in ( D ) after 2.5 h. ( D ) Inhibition of Ire1α autophosphorylation by ADP. Autophosphorylation of previously dephosphorylated Ire1α, indicated by α-pS724-Ire1α western blot, is progressively inhibited in the presence of increasing concentrations of ADP, and essentially blocked at equimolar ADP to ATP, consistent with the higher affinity of ADP for the nucleotide-binding site in the unphosphorylated kinase indicated by the thermal-shift analysis ( A ). Reactions were carried out in the presence of 5 mM MgCl 2 . ( E ) Hetero-phosphorylation of the HTRF biotinylated peptide S2 (200 nM) by phosphorylated Ire1α (○=no enzyme; ▪=50 nM; ▵=100 nM; •=200 nM; □=400 nM; and ▴=800 nM) in the presence of 30 μM Mg-ATP. ( F ) Concentration-dependent inhibition of Ire1α hetero-phosphorylation activity. Curves are shown for sunitinib (IC 50 =3.7 μM±1.2) and ADP (IC 50 =38 μM±17). Staurosporine (data not plotted) had an IC 50 =13 nM±8.4. Reactions used 200 nM Ire1α, 200 nM peptide S2, and 30 μM ATP. IC 50 values are averages of three experiments.

    Techniques Used: Inhibition, Incubation, SDS Page, Western Blot, Binding Assay, Concentration Assay, Activity Assay

    Autophosphorylation-dependent activation of Ire1α RNase activity. Model of Ire1 dimer-induced mechanism of action, highlighting the role of the face-to-face ‘phosphoryl-transfer' competent state defined here that facilitates transphosphorylation and phosphorylation-dependent downstream activation of the RNase activity. Whether the putatively RNase active back-to-back interaction observed in yeast Ire1 structures also occurs in the mammalian system is yet to be determined—the main structural components, not being conserved—and the mechanism by which autophosphorylation mediates RNase activation is yet to be directly demonstrated. Nonetheless, that inhibition of autophosphorylation by an ATP-competitive inhibitor such as sunitinib blocks consequent RNase activity provides a means by which therapeutic manipulation of the Ire1 arm of the UPRs might be achieved.
    Figure Legend Snippet: Autophosphorylation-dependent activation of Ire1α RNase activity. Model of Ire1 dimer-induced mechanism of action, highlighting the role of the face-to-face ‘phosphoryl-transfer' competent state defined here that facilitates transphosphorylation and phosphorylation-dependent downstream activation of the RNase activity. Whether the putatively RNase active back-to-back interaction observed in yeast Ire1 structures also occurs in the mammalian system is yet to be determined—the main structural components, not being conserved—and the mechanism by which autophosphorylation mediates RNase activation is yet to be directly demonstrated. Nonetheless, that inhibition of autophosphorylation by an ATP-competitive inhibitor such as sunitinib blocks consequent RNase activity provides a means by which therapeutic manipulation of the Ire1 arm of the UPRs might be achieved.

    Techniques Used: Activation Assay, Activity Assay, Inhibition

    38) Product Images from "CTCF Prevents the Epigenetic Drift of EBV Latency Promoter Qp"

    Article Title: CTCF Prevents the Epigenetic Drift of EBV Latency Promoter Qp

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001048

    RNA expression and promoter utilization in Qp mutated bacmids. A) Schematic representation of the EBV latency genes and promoters. Promoters are indicated by arrows. The position of the six EBNAs ORFs are indicated. B) Schematic representation of different EBNA1 transcripts. Exons present at 5′ end of EBNA1 mRNA are indicated in red. C) Quantitative RT-PCR was used to measure the abundance of EBNA2, EBNA3A and EBNA3C mRNA relative to bacmid GFP for Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection, as indicated. D) Same as in C, except EBNA1-transcripts initiating from either Cp/Wp, Qp, or Fp were measured relative to GFP in Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection. E) RT-PCR was measured for Wt rescue or ΔCTCF bacmids in 293 cell pools at 8 weeks post-transfection, as well as for type I (Mutu I) or type III (Mutu-LCL) controls. RNA was analyzed for the junction specific transcripts QUK (Qp initiation), C 1 C 2 W 1 W 2  (Cp initiation), W 0 W 1 W 2  (Wp initiation), BFLF1 (lytic gene adjacent to Qp), UK (EBNA1 mRNA in both type I and type III), and control cellular GAPDH.
    Figure Legend Snippet: RNA expression and promoter utilization in Qp mutated bacmids. A) Schematic representation of the EBV latency genes and promoters. Promoters are indicated by arrows. The position of the six EBNAs ORFs are indicated. B) Schematic representation of different EBNA1 transcripts. Exons present at 5′ end of EBNA1 mRNA are indicated in red. C) Quantitative RT-PCR was used to measure the abundance of EBNA2, EBNA3A and EBNA3C mRNA relative to bacmid GFP for Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection, as indicated. D) Same as in C, except EBNA1-transcripts initiating from either Cp/Wp, Qp, or Fp were measured relative to GFP in Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection. E) RT-PCR was measured for Wt rescue or ΔCTCF bacmids in 293 cell pools at 8 weeks post-transfection, as well as for type I (Mutu I) or type III (Mutu-LCL) controls. RNA was analyzed for the junction specific transcripts QUK (Qp initiation), C 1 C 2 W 1 W 2 (Cp initiation), W 0 W 1 W 2 (Wp initiation), BFLF1 (lytic gene adjacent to Qp), UK (EBNA1 mRNA in both type I and type III), and control cellular GAPDH.

    Techniques Used: RNA Expression, Quantitative RT-PCR, Transfection, Reverse Transcription Polymerase Chain Reaction

    39) Product Images from "Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln"

    Article Title: Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr619

    Pre-steady state measurements of Glu-tRNA Gln  formation by GluRS2. Aminoacylation reactions were performed at 4°C with 10 µM tRNA Gln , and 1 µM GluRS2, taking 10 µl aliquots to measure product formation. No burst indicative of a limiting product-release step was observed.
    Figure Legend Snippet: Pre-steady state measurements of Glu-tRNA Gln formation by GluRS2. Aminoacylation reactions were performed at 4°C with 10 µM tRNA Gln , and 1 µM GluRS2, taking 10 µl aliquots to measure product formation. No burst indicative of a limiting product-release step was observed.

    Techniques Used:

    A dynamic system for Gln-tRNA Gln  synthesis, which limits free Glu-tRNA Gln  in  H. pylori . The constants shown are the  k cat / K M  values calculated from the data in   Table 2 , for GluRS2 and GatCAB as free enzymes, and for the stoechiometric mix forming the Gln-transamidosome. Starting with tRNA Gln , the Gln-transamidosome pathway is favoured, with a  k cat / K M  value greater than that of the GluRS2 pathway. Glu-tRNA Gln  which is formed outside the Gln-transamidosome is susceptible to deacylation by GluRS2, or to an efficient transamidation by free GatCAB. The entire system therefore regulates against the presence of free Glu-tRNA Gln .
    Figure Legend Snippet: A dynamic system for Gln-tRNA Gln synthesis, which limits free Glu-tRNA Gln in H. pylori . The constants shown are the k cat / K M values calculated from the data in Table 2 , for GluRS2 and GatCAB as free enzymes, and for the stoechiometric mix forming the Gln-transamidosome. Starting with tRNA Gln , the Gln-transamidosome pathway is favoured, with a k cat / K M value greater than that of the GluRS2 pathway. Glu-tRNA Gln which is formed outside the Gln-transamidosome is susceptible to deacylation by GluRS2, or to an efficient transamidation by free GatCAB. The entire system therefore regulates against the presence of free Glu-tRNA Gln .

    Techniques Used:

    Binding experiments with tRNA Gln , GluRS2 and GatCAB as shown by size-exclusion chromatography. ( A ) Comparison of isolated GluRS2 and GluRS2/tRNA Gln  complex profiles. The association enabled us to estimate a  K D  value for GluRS2 and tRNA Gln  binding (see ‘Materials and Methods’ section). ( B ) Comparison between free and tRNA Gln -complexed GatCAB. ( C ) Gel-filtration of a solution containing the three partners. ( D ) The same experiment as described in (C) was performed with preformed Glu-tRNA Gln  (grey line) or after incubation of the three partners within an aminoacylation medium containing free Glu and ATP, to provide endogenous Glu-tRNA Gln  (dotted line). Proteins and tRNA Gln  were added at concentrations of 20 µM each for the experiments shown in all panels.
    Figure Legend Snippet: Binding experiments with tRNA Gln , GluRS2 and GatCAB as shown by size-exclusion chromatography. ( A ) Comparison of isolated GluRS2 and GluRS2/tRNA Gln complex profiles. The association enabled us to estimate a K D value for GluRS2 and tRNA Gln binding (see ‘Materials and Methods’ section). ( B ) Comparison between free and tRNA Gln -complexed GatCAB. ( C ) Gel-filtration of a solution containing the three partners. ( D ) The same experiment as described in (C) was performed with preformed Glu-tRNA Gln (grey line) or after incubation of the three partners within an aminoacylation medium containing free Glu and ATP, to provide endogenous Glu-tRNA Gln (dotted line). Proteins and tRNA Gln were added at concentrations of 20 µM each for the experiments shown in all panels.

    Techniques Used: Binding Assay, Size-exclusion Chromatography, Isolation, Filtration, Incubation

    Stoichiometry of the GluRS2/tRNA Gln /GatCAB complex. ( A ) Titration curve of a 1/1 mix of GluRS2 and GatCAB (20 µM each) with tRNA Gln  in molar ratios of 0.2–2.7. ( B ) Titration of a 1/1 mix of GluRS2 and tRNA Gln  (20 µM each) with free GatCAB in molar ratios of 0.3–2.0.
    Figure Legend Snippet: Stoichiometry of the GluRS2/tRNA Gln /GatCAB complex. ( A ) Titration curve of a 1/1 mix of GluRS2 and GatCAB (20 µM each) with tRNA Gln in molar ratios of 0.2–2.7. ( B ) Titration of a 1/1 mix of GluRS2 and tRNA Gln (20 µM each) with free GatCAB in molar ratios of 0.3–2.0.

    Techniques Used: Titration

    40) 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

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    In Vitro:

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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. ..

    Concentration Assay:

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    other:

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

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    Staining:

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    Recombinant:

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    Immunofluorescence:

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    Plasmid Preparation:

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    Software:

    Article Title: Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination
    Article Snippet: .. For staining of RNA:DNA hybrids in FA-D2 and derivative cells, Volocity software (Perkin Elmer) was used to quantify S9.6 immunofluorescence. ..

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    PerkinElmer p lanceolata
    Percentage of colonization in P. <t>lanceolata</t> roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.
    P Lanceolata, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 2 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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    PerkinElmer p nipam maa gma
    Ion transportation properties of the ICC membrane with different <t>NIPAM</t> and <t>MAA</t> content at different temperatures and pH values measured through conductivity variation in the downstream.
    P Nipam Maa Gma, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer γ 32 p radioactivity
    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
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    Image Search Results


    Percentage of colonization in P. lanceolata roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.

    Journal: AoB Plants

    Article Title: Impact of an invasive nitrogen-fixing tree on arbuscular mycorrhizal fungi and the development of native species

    doi: 10.1093/aobpla/plw018

    Figure Lengend Snippet: Percentage of colonization in P. lanceolata roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.

    Article Snippet: Due to the key role of AMF in P uptake, we also measured the content of P in leaves and roots of P. lanceolata using ICP-OES (Perkin Elmer Optima 4300 DV).

    Techniques:

    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

    Ion transportation properties of the ICC membrane with different NIPAM and MAA content at different temperatures and pH values measured through conductivity variation in the downstream.

    Journal: Scientific Reports

    Article Title: A smart thermo- and pH-responsive microfiltration membrane based on three-dimensional inverse colloidal crystals

    doi: 10.1038/s41598-017-12426-z

    Figure Lengend Snippet: Ion transportation properties of the ICC membrane with different NIPAM and MAA content at different temperatures and pH values measured through conductivity variation in the downstream.

    Article Snippet: The functional groups of PGMA, P(NIPAM-MAA-GMA)−30, P(NIPAM-MAA-GMA)−50 were determined by infrared spectroscopy (Spectrum 100, PerkinElmer).

    Techniques: Immunocytochemistry

    IR spectra of the co-polymers P(NIPAM-MAA-GMA) contains 50% ( a ), 30% ( b ) and 0% ( c ) of NIPAM and MAA.

    Journal: Scientific Reports

    Article Title: A smart thermo- and pH-responsive microfiltration membrane based on three-dimensional inverse colloidal crystals

    doi: 10.1038/s41598-017-12426-z

    Figure Lengend Snippet: IR spectra of the co-polymers P(NIPAM-MAA-GMA) contains 50% ( a ), 30% ( b ) and 0% ( c ) of NIPAM and MAA.

    Article Snippet: The functional groups of PGMA, P(NIPAM-MAA-GMA)−30, P(NIPAM-MAA-GMA)−50 were determined by infrared spectroscopy (Spectrum 100, PerkinElmer).

    Techniques:

    ( a ) Schematically illustration of the preparation of thermo-and pH-responsive ICC macroporous column; ( b ) Chemical structures of MAA and NIPAM.

    Journal: Scientific Reports

    Article Title: A smart thermo- and pH-responsive microfiltration membrane based on three-dimensional inverse colloidal crystals

    doi: 10.1038/s41598-017-12426-z

    Figure Lengend Snippet: ( a ) Schematically illustration of the preparation of thermo-and pH-responsive ICC macroporous column; ( b ) Chemical structures of MAA and NIPAM.

    Article Snippet: The functional groups of PGMA, P(NIPAM-MAA-GMA)−30, P(NIPAM-MAA-GMA)−50 were determined by infrared spectroscopy (Spectrum 100, PerkinElmer).

    Techniques: Immunocytochemistry

    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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M117.802587

    Figure Lengend 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

    Article Snippet: For liquid scintillation counting, either GST-HEF1 WT or GST-HEF1 S780A/T804A mutant band was excised from dried SDS-polyacrylamide gel band, and then gel pieces were dissolved in 30% H2 O2 (Sigma) at 50 °C for 4 h. After heating at 37 °C for an additional 1 h to drive off residual O2 , a scintillation mixture (ULTIMA GOLD, PerkinElmer Life Sciences) was added to sample, and the [γ-32 P]radioactivity was measured using a liquid scintillation analyzer (Tri-Carb 2910 TR, PerkinElmer Life Sciences).

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