Structured Review

Stratagene y123w c1bα mutant
Distribution of open and closed loop conformations in <t>C1Bα.</t> (a) Histogram of the distances between the loop tips for the wt (yellow bars) and <t>Y123W</t> C1Bα (empty bars) observed during the interval between 2 and 10 ns of the MD trajectories. The distance between the loop tips is measured every 200 fs. Wt C1Bα shows a broad bimodal distribution centered at 12.5 Å and 9.5 Å, respectively. Y123W C1Bα has two preferred conformations: the open and closed, which are centered at 12.5 Å and 5 Å. (b) Histogram of the distances between the loop tips for the Y123W C1Bα (empty bars) observed during the trajectory interval between 10 and 18 ns, after the opening of the binding loops. The distance between the loop tips is measured every 200 fs. For comparison, the histogram generated using the three original 8 ns long trajectories of wt C1Bα is shown on the same plot (yellow bars). Both wt and Y123W C1Bα sample open or partially open conformations that show a bimodal distribution. Frequent transitions between open and partially open conformations are observed along the trajectories. Snapshots of the structures with closed and open loop conformations are shown in (c) and (d), respectively. Loop regions are highlighted in purple.
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Images

1) Product Images from "Probing the Determinants of Diacylglycerol Binding Affinity in C1B domain of Protein Kinase C?"

Article Title: Probing the Determinants of Diacylglycerol Binding Affinity in C1B domain of Protein Kinase C?

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2011.03.020

Distribution of open and closed loop conformations in C1Bα. (a) Histogram of the distances between the loop tips for the wt (yellow bars) and Y123W C1Bα (empty bars) observed during the interval between 2 and 10 ns of the MD trajectories. The distance between the loop tips is measured every 200 fs. Wt C1Bα shows a broad bimodal distribution centered at 12.5 Å and 9.5 Å, respectively. Y123W C1Bα has two preferred conformations: the open and closed, which are centered at 12.5 Å and 5 Å. (b) Histogram of the distances between the loop tips for the Y123W C1Bα (empty bars) observed during the trajectory interval between 10 and 18 ns, after the opening of the binding loops. The distance between the loop tips is measured every 200 fs. For comparison, the histogram generated using the three original 8 ns long trajectories of wt C1Bα is shown on the same plot (yellow bars). Both wt and Y123W C1Bα sample open or partially open conformations that show a bimodal distribution. Frequent transitions between open and partially open conformations are observed along the trajectories. Snapshots of the structures with closed and open loop conformations are shown in (c) and (d), respectively. Loop regions are highlighted in purple.
Figure Legend Snippet: Distribution of open and closed loop conformations in C1Bα. (a) Histogram of the distances between the loop tips for the wt (yellow bars) and Y123W C1Bα (empty bars) observed during the interval between 2 and 10 ns of the MD trajectories. The distance between the loop tips is measured every 200 fs. Wt C1Bα shows a broad bimodal distribution centered at 12.5 Å and 9.5 Å, respectively. Y123W C1Bα has two preferred conformations: the open and closed, which are centered at 12.5 Å and 5 Å. (b) Histogram of the distances between the loop tips for the Y123W C1Bα (empty bars) observed during the trajectory interval between 10 and 18 ns, after the opening of the binding loops. The distance between the loop tips is measured every 200 fs. For comparison, the histogram generated using the three original 8 ns long trajectories of wt C1Bα is shown on the same plot (yellow bars). Both wt and Y123W C1Bα sample open or partially open conformations that show a bimodal distribution. Frequent transitions between open and partially open conformations are observed along the trajectories. Snapshots of the structures with closed and open loop conformations are shown in (c) and (d), respectively. Loop regions are highlighted in purple.

Techniques Used: Binding Assay, Generated

NMR-detected titration of the wt (a) and Y123W C1Bα (b) with DOG in the presence of DPC/DPS micelles. The binding process is intermediate-to-fast and slow-to-intermediate on the chemical-shift timescale for the wt and Y123W C1Bα, respectively. The insets show the differences in the binding regimes for the wt and mutant proteins using Leu122 as an example. Large chemical shift perturbations are observed in the ligand-bound versus apo-spectra.
Figure Legend Snippet: NMR-detected titration of the wt (a) and Y123W C1Bα (b) with DOG in the presence of DPC/DPS micelles. The binding process is intermediate-to-fast and slow-to-intermediate on the chemical-shift timescale for the wt and Y123W C1Bα, respectively. The insets show the differences in the binding regimes for the wt and mutant proteins using Leu122 as an example. Large chemical shift perturbations are observed in the ligand-bound versus apo-spectra.

Techniques Used: Nuclear Magnetic Resonance, Titration, Binding Assay, Mutagenesis

DOG binding curves for the wt and Y123W C1Bα detected by NMR and fluorescence spectroscopy. In (a) and (b), the absolute values of the 1 H and 15 N chemical shift changes, Δ 1 H and Δ 15 using the dissociation constant K d produced P 0 of 0.23 ± 0.07 μM and K d of 6.7 ± 16.4 nM. Large errors in K d indicate that, in this protein concentration range, the binding is still tight, and we can only put an upper limit of 0.23 μM onto the K d value.
Figure Legend Snippet: DOG binding curves for the wt and Y123W C1Bα detected by NMR and fluorescence spectroscopy. In (a) and (b), the absolute values of the 1 H and 15 N chemical shift changes, Δ 1 H and Δ 15 using the dissociation constant K d produced P 0 of 0.23 ± 0.07 μM and K d of 6.7 ± 16.4 nM. Large errors in K d indicate that, in this protein concentration range, the binding is still tight, and we can only put an upper limit of 0.23 μM onto the K d value.

Techniques Used: Binding Assay, Nuclear Magnetic Resonance, Fluorescence, Spectroscopy, Produced, Protein Concentration

Assessment of structural differences between wt and Y123W C1Bα using chemical shift perturbation analysis and RDCs. Structural Zn 2+ ions are shown as black spheres in (a), (c), and (d). Prolines and residues that are missing from the 15 N- 1 The only significant perturbation is observed at the mutation site. (b) Comparison of the 1 D NH
Figure Legend Snippet: Assessment of structural differences between wt and Y123W C1Bα using chemical shift perturbation analysis and RDCs. Structural Zn 2+ ions are shown as black spheres in (a), (c), and (d). Prolines and residues that are missing from the 15 N- 1 The only significant perturbation is observed at the mutation site. (b) Comparison of the 1 D NH

Techniques Used: Mutagenesis

2) Product Images from "Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases"

Article Title: Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks381

Formation of modified nucleotides in a 24-mer RNA oligonucleotide incubated with the C/D sR47 RNP in the presence of cofactors AdoMet and SeAdoYn. The target sequence of the substrate oligonucleotide (top) contains a 33 P-labeled target nucleotide (shown in bold and an asterisk). Substrate oligonucleotide (1 μM) was incubated with 1 μM pre-assembled RNP and 50 μM AdoMet or 400 μM SeAdoYn for 40 min at 65°C. Samples were subjected to nuclease Bal31 digestion and TLC analysis of 33 P-labeled mononucleotides. Arrows point to target nucleotide products formed in the presence of cofactors AdoMet (lane 1, constitutes 45% of total nucleotide counts) or SeAdoYn (lane 2, constitutes 5% of total nucleotide counts). A control with SeAdoYn was carried in the absence of the Nop5p-aFib RNP core proteins (Lane 3).
Figure Legend Snippet: Formation of modified nucleotides in a 24-mer RNA oligonucleotide incubated with the C/D sR47 RNP in the presence of cofactors AdoMet and SeAdoYn. The target sequence of the substrate oligonucleotide (top) contains a 33 P-labeled target nucleotide (shown in bold and an asterisk). Substrate oligonucleotide (1 μM) was incubated with 1 μM pre-assembled RNP and 50 μM AdoMet or 400 μM SeAdoYn for 40 min at 65°C. Samples were subjected to nuclease Bal31 digestion and TLC analysis of 33 P-labeled mononucleotides. Arrows point to target nucleotide products formed in the presence of cofactors AdoMet (lane 1, constitutes 45% of total nucleotide counts) or SeAdoYn (lane 2, constitutes 5% of total nucleotide counts). A control with SeAdoYn was carried in the absence of the Nop5p-aFib RNP core proteins (Lane 3).

Techniques Used: Modification, Incubation, Sequencing, Labeling, Thin Layer Chromatography

Archaeal C/D RNP-directed sequence-specific modification and labeling of target RNA. ( A ) Schematic structure of a C/D RNP complex with substrate RNA. Core proteins L7Ae, Nop5p and aFib are bound at the C/D and C′/D′ sites of a guide RNA. One of the variable guide sequences is shown base-paired to a target sequence (green) of a substrate RNA. Modification occurs at a nucleotide complementary to the fifth position upstream from the D box. ( B ) C/D RNP-directed transfer of an activated side chain (red) from a cofactor S -adenosyl- l -methionine (AdoMet, X=S and R=methyl) or its analog SeAdoYn (X=Se and R=prop-2-ynyl) onto an RNA substrate. ( C ) Two-step ‘click’ labeling of target RNA via a C/D RNP-directed alkynylation, followed by Cu(I)-assisted 1,3-cycloaddition of a fluorogenic azide derivative (blue).
Figure Legend Snippet: Archaeal C/D RNP-directed sequence-specific modification and labeling of target RNA. ( A ) Schematic structure of a C/D RNP complex with substrate RNA. Core proteins L7Ae, Nop5p and aFib are bound at the C/D and C′/D′ sites of a guide RNA. One of the variable guide sequences is shown base-paired to a target sequence (green) of a substrate RNA. Modification occurs at a nucleotide complementary to the fifth position upstream from the D box. ( B ) C/D RNP-directed transfer of an activated side chain (red) from a cofactor S -adenosyl- l -methionine (AdoMet, X=S and R=methyl) or its analog SeAdoYn (X=Se and R=prop-2-ynyl) onto an RNA substrate. ( C ) Two-step ‘click’ labeling of target RNA via a C/D RNP-directed alkynylation, followed by Cu(I)-assisted 1,3-cycloaddition of a fluorogenic azide derivative (blue).

Techniques Used: Sequencing, Modification, Labeling

3) Product Images from "RECQL5 plays co-operative and complementary roles with WRN syndrome helicase"

Article Title: RECQL5 plays co-operative and complementary roles with WRN syndrome helicase

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1134

RECQL5 specifically stimulates the helicase activity of WRN but not BLM on fork duplexes. Helicase assays of ( A ) WRN (2 nM), ( B ) BLM (1 nM) and ( C ) GST fragment WRN H−R (2 nM) were performed in the presence of increasing concentrations of RECQL5 (2–20 nM). ( D ) Quantification of WRN unwinding activity in the presence of increasing concentrations of RECQL5. The plot represents mean of three independent experiments with error bars. ( E ) Helicase assays of K-WRN (2 nM) on a forked duplex with increasing concentrations of RECQL5. ( F ) Effect of increasing concentrations of both wt-RECQL5 and K58R RECQL5 on the helicase assays of WRN (2 nM) on a forked duplex. Helicase-dead K58R RECQL5 could not stimulate WRN as wt-RECQL5 under similar conditions. ( G ) Strand exchange of 2 nM WRN on a 3′-flap duplex with increasing concentrations of RECQL5 (2–20 nM). A synergistic increase in strand exchange of WRN was observed, indicating a functional co-operation of RECQL5 and WRN on synthetic stalled replication forks lacking the leading strand.
Figure Legend Snippet: RECQL5 specifically stimulates the helicase activity of WRN but not BLM on fork duplexes. Helicase assays of ( A ) WRN (2 nM), ( B ) BLM (1 nM) and ( C ) GST fragment WRN H−R (2 nM) were performed in the presence of increasing concentrations of RECQL5 (2–20 nM). ( D ) Quantification of WRN unwinding activity in the presence of increasing concentrations of RECQL5. The plot represents mean of three independent experiments with error bars. ( E ) Helicase assays of K-WRN (2 nM) on a forked duplex with increasing concentrations of RECQL5. ( F ) Effect of increasing concentrations of both wt-RECQL5 and K58R RECQL5 on the helicase assays of WRN (2 nM) on a forked duplex. Helicase-dead K58R RECQL5 could not stimulate WRN as wt-RECQL5 under similar conditions. ( G ) Strand exchange of 2 nM WRN on a 3′-flap duplex with increasing concentrations of RECQL5 (2–20 nM). A synergistic increase in strand exchange of WRN was observed, indicating a functional co-operation of RECQL5 and WRN on synthetic stalled replication forks lacking the leading strand.

Techniques Used: Activity Assay, Functional Assay

4) Product Images from "Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass"

Article Title: Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043828

Thermostability of CbCelA-TM1 in presence of CbHsp18 (A), MkHistone1 (B), and RNase A (C) and hydrolysis of Avicel with CbCelA-TM1 in presence of CbHsp18 (D), MkHistone1 (E), and RNase A (F). For the thermostability assay, 1 µM of CbCelA-TM1 was incubated with 8 µM, 16 µM, or 32 µM of CbHsp18, MkHistone1, or RNase A in a pH 6.0 citrate buffer. The mixtures were shaken end-over-end at 70°C for 24 hr. As a control, 1 µM of CbCelA-TM1 in the same buffer was incubated without shaking at 4°C. For hydrolysis of Avicel, 1 µM of CbCelA-TM1 was incubated with 20 mg/ml Avicel in the absence or presence of 8 µM, 16 µM, or 32 µM of CbHsp18, MkHistone1, or RNase A in a pH 6.0 citrate buffer. The reaction mixtures were shaken end-over-end at 70°C for 24 hr.
Figure Legend Snippet: Thermostability of CbCelA-TM1 in presence of CbHsp18 (A), MkHistone1 (B), and RNase A (C) and hydrolysis of Avicel with CbCelA-TM1 in presence of CbHsp18 (D), MkHistone1 (E), and RNase A (F). For the thermostability assay, 1 µM of CbCelA-TM1 was incubated with 8 µM, 16 µM, or 32 µM of CbHsp18, MkHistone1, or RNase A in a pH 6.0 citrate buffer. The mixtures were shaken end-over-end at 70°C for 24 hr. As a control, 1 µM of CbCelA-TM1 in the same buffer was incubated without shaking at 4°C. For hydrolysis of Avicel, 1 µM of CbCelA-TM1 was incubated with 20 mg/ml Avicel in the absence or presence of 8 µM, 16 µM, or 32 µM of CbHsp18, MkHistone1, or RNase A in a pH 6.0 citrate buffer. The reaction mixtures were shaken end-over-end at 70°C for 24 hr.

Techniques Used: Incubation

Thermostability of CbXyn10A (A, B, and C) and hydrolysis of Miscanthus by CbXyn10A (D, E, and F). A–C: Thermostability of CbXyn10A in the presence of CbHsp18 (A), MkHistone1 (B), and RNase A (C). One micromolar of CbXyn10A was incubated with 8 µM, 16 µM, or 32 µM of CbHsp18, MkHistone1, or RNase A in a pH 6.0 citrate buffer in a total volume of 500 µl. The mixtures were shaken end-over-end at 70°C for 24 hr. D–E: 1 µM of CbXyn10A was incubated with 20 mg/ml Miscanthus in the absence or presence of 8 µM, 16 µM, and 32 µM each of CbHsp18 (D), MkHistone1 (E), and RNase A (F) in a pH 6.0 citrate buffer in a total volume of 500 µl. The reaction mixtures were rotated end-over-end at 70°C for 24 hr.
Figure Legend Snippet: Thermostability of CbXyn10A (A, B, and C) and hydrolysis of Miscanthus by CbXyn10A (D, E, and F). A–C: Thermostability of CbXyn10A in the presence of CbHsp18 (A), MkHistone1 (B), and RNase A (C). One micromolar of CbXyn10A was incubated with 8 µM, 16 µM, or 32 µM of CbHsp18, MkHistone1, or RNase A in a pH 6.0 citrate buffer in a total volume of 500 µl. The mixtures were shaken end-over-end at 70°C for 24 hr. D–E: 1 µM of CbXyn10A was incubated with 20 mg/ml Miscanthus in the absence or presence of 8 µM, 16 µM, and 32 µM each of CbHsp18 (D), MkHistone1 (E), and RNase A (F) in a pH 6.0 citrate buffer in a total volume of 500 µl. The reaction mixtures were rotated end-over-end at 70°C for 24 hr.

Techniques Used: Incubation

Hydrolysis of Avicel by a binary mixture of CbCelA-TM1 and CbCdx1A. Avicel (20 mg/ml) was hydrolyzed with 1 µM each of CbCelA-TM1 and CbCdx1A in the absence or presence of 16 µM, 32 µM, and 64 µM of CbHsp18, MkHistone1, or RNase A in a pH 6.0 citrate buffer at a total volume of 500 µl. The reactions were carried out by shaking the mixtures end-over-end at 70°C for 24 hr. The reducing ends released in all samples were determined by the PAHBAH method.
Figure Legend Snippet: Hydrolysis of Avicel by a binary mixture of CbCelA-TM1 and CbCdx1A. Avicel (20 mg/ml) was hydrolyzed with 1 µM each of CbCelA-TM1 and CbCdx1A in the absence or presence of 16 µM, 32 µM, and 64 µM of CbHsp18, MkHistone1, or RNase A in a pH 6.0 citrate buffer at a total volume of 500 µl. The reactions were carried out by shaking the mixtures end-over-end at 70°C for 24 hr. The reducing ends released in all samples were determined by the PAHBAH method.

Techniques Used:

Schematic domain structures (A) and SDS-PAGE analysis (B) of the non-GH proteins and glycoside hydrolases. A: A schematic representation of the polypeptides of CbHsp18, MkHistone1, and the three glycoside hydrolases of C. bescii used in this study. GH9: Glycoside hydrolase family 9 domain; CBM3c: Carbohydrate binding module family 3 type C. B: SDS-PAGE analysis of purified recombinant non-GH proteins and the three glycoside hydrolases of C. bescii . Lane 1: molecular mass markers; lane 2: CbHsp18; lane 3: MkHistone1; lane 4: RNase A; lane 5: CbCelA-TM1; lane 6: CbCdx1A; lane 7: CbXyn10A. Two micrograms of each protein were resolved by 12% SDS-PAGE. RNase A was a commercial product (Roche).
Figure Legend Snippet: Schematic domain structures (A) and SDS-PAGE analysis (B) of the non-GH proteins and glycoside hydrolases. A: A schematic representation of the polypeptides of CbHsp18, MkHistone1, and the three glycoside hydrolases of C. bescii used in this study. GH9: Glycoside hydrolase family 9 domain; CBM3c: Carbohydrate binding module family 3 type C. B: SDS-PAGE analysis of purified recombinant non-GH proteins and the three glycoside hydrolases of C. bescii . Lane 1: molecular mass markers; lane 2: CbHsp18; lane 3: MkHistone1; lane 4: RNase A; lane 5: CbCelA-TM1; lane 6: CbCdx1A; lane 7: CbXyn10A. Two micrograms of each protein were resolved by 12% SDS-PAGE. RNase A was a commercial product (Roche).

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

5) Product Images from "A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein"

Article Title: A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002876

YLDV-IL18BP:IL18 interface. A). Key residues of YLDV-IL18BP at the interface. YLDV-IL18BP binds nearly identical surface of IL18 as previously observed in ECTV-IL18BP inhibitory complex. IL18 is shown as surface representation and colored grey. YLDV-IL18BP is drawn as a ribbon diagram with β-sheets colored in yellow. Binding sites A, B and C on IL18 surface are colored red, orange and cyan respectively. YLDV-IL18BP residues involved in binding IL18 are shown as stick representations. Each insert details the interactions involved in the respective binding site between YLDV-IL18BP and IL18. B). Unique interactions at binding site A. Carbon atoms of YLDV-IL18BP and IL18 are colored in yellow and pink, respectively. The secondary structures of YLDV-IL18BP and IL18 are colored in cyan and green, respectively. Red dashed lines indicate H-bonds. C). Unique interactions at binding site C. YLDV-IL18BP P116 is involved in favorable hydrophobic interactions with IL18. Polar interactions at site C involve S105 and D110 residues of IL18. The coloring scheme is the same as in B.
Figure Legend Snippet: YLDV-IL18BP:IL18 interface. A). Key residues of YLDV-IL18BP at the interface. YLDV-IL18BP binds nearly identical surface of IL18 as previously observed in ECTV-IL18BP inhibitory complex. IL18 is shown as surface representation and colored grey. YLDV-IL18BP is drawn as a ribbon diagram with β-sheets colored in yellow. Binding sites A, B and C on IL18 surface are colored red, orange and cyan respectively. YLDV-IL18BP residues involved in binding IL18 are shown as stick representations. Each insert details the interactions involved in the respective binding site between YLDV-IL18BP and IL18. B). Unique interactions at binding site A. Carbon atoms of YLDV-IL18BP and IL18 are colored in yellow and pink, respectively. The secondary structures of YLDV-IL18BP and IL18 are colored in cyan and green, respectively. Red dashed lines indicate H-bonds. C). Unique interactions at binding site C. YLDV-IL18BP P116 is involved in favorable hydrophobic interactions with IL18. Polar interactions at site C involve S105 and D110 residues of IL18. The coloring scheme is the same as in B.

Techniques Used: Binding Assay

Kinetic analyses of the binding of IL18 with YLDV-IL18BP. SPR analysis was performed as described in Figure 7 with YLDV-IL18BP at 5 different concentrations. The binding curves were globally fitted with BiaEvaluation software to a 1∶1 binding model. The colored and black lines are the actual responses in RU and globally fitted curves, respectively.
Figure Legend Snippet: Kinetic analyses of the binding of IL18 with YLDV-IL18BP. SPR analysis was performed as described in Figure 7 with YLDV-IL18BP at 5 different concentrations. The binding curves were globally fitted with BiaEvaluation software to a 1∶1 binding model. The colored and black lines are the actual responses in RU and globally fitted curves, respectively.

Techniques Used: Binding Assay, SPR Assay, Software

Biacore SPR analysis of IL18 mutants. Biotinylated YLDV-IL18BP and ECTV-IL18BP were captured on two different flow cells in a BIAcore streptavidin-coated CM5 chip, and their binding with IL18 was monitored simultaneously with a BIAcore 3000 sensor. The injection of IL18 started at ∼150 s and stopped at 900 s. The colored lines are the responses obtained with different IL18 mutants and normalized to a maximum of 100 RU (except for P57R, S105R for YLDV-IL18BP) for ease of comparison. YLDV-IL18BP and ECTV-IL18BP were expressed and secreted from mammalian cells and underwent in vitro biotinylation as described in Materials and Methods .
Figure Legend Snippet: Biacore SPR analysis of IL18 mutants. Biotinylated YLDV-IL18BP and ECTV-IL18BP were captured on two different flow cells in a BIAcore streptavidin-coated CM5 chip, and their binding with IL18 was monitored simultaneously with a BIAcore 3000 sensor. The injection of IL18 started at ∼150 s and stopped at 900 s. The colored lines are the responses obtained with different IL18 mutants and normalized to a maximum of 100 RU (except for P57R, S105R for YLDV-IL18BP) for ease of comparison. YLDV-IL18BP and ECTV-IL18BP were expressed and secreted from mammalian cells and underwent in vitro biotinylation as described in Materials and Methods .

Techniques Used: SPR Assay, Flow Cytometry, Chromatin Immunoprecipitation, Binding Assay, Injection, In Vitro

Overall structure of YLDV-IL18BP:IL18 complex. A). YLDV-IL18BP displays a homo-dimer (green and cyan), each adopting a β-sandwiched Ig-fold structure. Each protomer binds one molecular of IL18 (yellow and magenta), forming a 2∶2 complex. The intra-chain disulfide bonds (SS) in YLDV-IL18BPs are shown as yellow sticks. The inter-chain SS bond at the C-terminus of YLDV-IL18BPs is shown as orange spheres. B). Stereoview of superimposition of the monomer complexes of YLDV-IL18BP (green):IL18 (magenta) and ECTV-IL18BP (blue):IL18 (orange). The N- and C-terminus of YLDV-IL18BP are indicated. The intra-chain SS bonds are shown as in A. Residue C132 is shown as yellow spheres. The β-strands A, B, E and F in YLDV-IL18BP are labeled.
Figure Legend Snippet: Overall structure of YLDV-IL18BP:IL18 complex. A). YLDV-IL18BP displays a homo-dimer (green and cyan), each adopting a β-sandwiched Ig-fold structure. Each protomer binds one molecular of IL18 (yellow and magenta), forming a 2∶2 complex. The intra-chain disulfide bonds (SS) in YLDV-IL18BPs are shown as yellow sticks. The inter-chain SS bond at the C-terminus of YLDV-IL18BPs is shown as orange spheres. B). Stereoview of superimposition of the monomer complexes of YLDV-IL18BP (green):IL18 (magenta) and ECTV-IL18BP (blue):IL18 (orange). The N- and C-terminus of YLDV-IL18BP are indicated. The intra-chain SS bonds are shown as in A. Residue C132 is shown as yellow spheres. The β-strands A, B, E and F in YLDV-IL18BP are labeled.

Techniques Used: Labeling

YLDV-IL18BP forms a SS linked dimer in solution. SDS-PAGE analysis of purified YLDV-IL18BP proteins in the presence (+) or absence (−) of 2-mercaptoethanol (2-ME). A). Purified YLDV-IL18BP:IL18 protein complex that was expressed in E. coli . IL18 and YLDV-IL18BP are indicated by black and red arrows, respectively. B). Purified YLDV-IL18BP that was expressed and secreted from mammalian cells. The YLDV-IL18BP was engineered with a C-terminal site-specific biotinylation site, which allows its detection in Western blot with streptavidin. Two N-glycosylation sites were predicted for YLDV-IL18BP. Panel B also shows the SDS-PAGE of YLDV-IL18BP that was untreated (−) or treated (+) with endoglycosidase PNGase F.
Figure Legend Snippet: YLDV-IL18BP forms a SS linked dimer in solution. SDS-PAGE analysis of purified YLDV-IL18BP proteins in the presence (+) or absence (−) of 2-mercaptoethanol (2-ME). A). Purified YLDV-IL18BP:IL18 protein complex that was expressed in E. coli . IL18 and YLDV-IL18BP are indicated by black and red arrows, respectively. B). Purified YLDV-IL18BP that was expressed and secreted from mammalian cells. The YLDV-IL18BP was engineered with a C-terminal site-specific biotinylation site, which allows its detection in Western blot with streptavidin. Two N-glycosylation sites were predicted for YLDV-IL18BP. Panel B also shows the SDS-PAGE of YLDV-IL18BP that was untreated (−) or treated (+) with endoglycosidase PNGase F.

Techniques Used: SDS Page, Purification, Western Blot

Biacore SPR Analysis of YLDV-IL18BP mutants. Biotinylated IL18 was captured on a BIAcore streptavidin-coated CM5 chip, and its binding with IL18BP was monitored with a BIAcore 3000 sensor. All the YLDV-IL18BP proteins were expressed in E. coli as SUMO fusion and purified to near homogeneity. The IL18BP mutants were derived from the monomeric form of the protein with the HVEC mutation. The injection of IL18BP started at ∼150 s and stopped at 900 s. The colored lines are the responses obtained with different IL18BP mutants and normalized to a maximum of 100 resonance units (RU) (except for Y56A) for ease of comparison.
Figure Legend Snippet: Biacore SPR Analysis of YLDV-IL18BP mutants. Biotinylated IL18 was captured on a BIAcore streptavidin-coated CM5 chip, and its binding with IL18BP was monitored with a BIAcore 3000 sensor. All the YLDV-IL18BP proteins were expressed in E. coli as SUMO fusion and purified to near homogeneity. The IL18BP mutants were derived from the monomeric form of the protein with the HVEC mutation. The injection of IL18BP started at ∼150 s and stopped at 900 s. The colored lines are the responses obtained with different IL18BP mutants and normalized to a maximum of 100 resonance units (RU) (except for Y56A) for ease of comparison.

Techniques Used: SPR Assay, Chromatin Immunoprecipitation, Binding Assay, Purification, Derivative Assay, Mutagenesis, Injection

Structure based sequence alignment of IL18 binding proteins. Structure based sequence alignment of various IL18BPs was created using the crystal structure of YLDV-IL18BP as the template. Lettering and numbering above alignment correspond to YLDV-IL18BP topology and numbering scheme. Colored stars above residues indicate the three binding sites on IL18 with which they interact, red: site A, orange: site B, cyan: site C. Solid black circles above residues indicate resides involved in homo-dimerization. The two intra-chain disulfide bonds are indicated with the green letters. The cysteine residue forming the unique inter-chain disulfide bond is indicated with a pink triangle. The sequence alignment was performed with the FATCAT server [44] and ClustalX [45] , and the figure was created with ESPript [46] .
Figure Legend Snippet: Structure based sequence alignment of IL18 binding proteins. Structure based sequence alignment of various IL18BPs was created using the crystal structure of YLDV-IL18BP as the template. Lettering and numbering above alignment correspond to YLDV-IL18BP topology and numbering scheme. Colored stars above residues indicate the three binding sites on IL18 with which they interact, red: site A, orange: site B, cyan: site C. Solid black circles above residues indicate resides involved in homo-dimerization. The two intra-chain disulfide bonds are indicated with the green letters. The cysteine residue forming the unique inter-chain disulfide bond is indicated with a pink triangle. The sequence alignment was performed with the FATCAT server [44] and ClustalX [45] , and the figure was created with ESPript [46] .

Techniques Used: Sequencing, Binding Assay

Kinetic analyses of the binding of IL18 with YLDV-IL18BP or ECTV-IL18BP. SPR analysis was performed as described in Figure 9 with IL18 at 5 different concentrations. The binding curves were globally fitted with BiaEvaluation software to a 1∶1 binding model. The colored and black lines are the actual responses in RU and globally fitted curves, respectively.
Figure Legend Snippet: Kinetic analyses of the binding of IL18 with YLDV-IL18BP or ECTV-IL18BP. SPR analysis was performed as described in Figure 9 with IL18 at 5 different concentrations. The binding curves were globally fitted with BiaEvaluation software to a 1∶1 binding model. The colored and black lines are the actual responses in RU and globally fitted curves, respectively.

Techniques Used: Binding Assay, SPR Assay, Software

6) Product Images from "Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms"

Article Title: Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms

Journal: BMC Structural Biology

doi: 10.1186/1472-6807-8-8

Bleicher5.png . Average interaction energies involved in the binding of GC-1 to TRα and TRβ as computed from molecular dynamics simulations.
Figure Legend Snippet: Bleicher5.png . Average interaction energies involved in the binding of GC-1 to TRα and TRβ as computed from molecular dynamics simulations.

Techniques Used: Binding Assay

Bleicher3.png . Ligand-receptor interactions for thyroid receptors: (a) T 3 as bound to hTRα (green) and hTRβ (magenta). All interactions are maintained between the ligand and the binding site residues in both hTR isoforms. Both Ser277 and Asn331 interact with the amino group of T 3 through their amide nitrogen, leading to similar conformations of these residues. (b) GC1 bound to TRα: multiple conformations of the Arg228 are observed. In the productive conformation there is a strong interaction with the ligand (cyan), while in non-productive conformations this residue interacts with the side-chain of Ser277. The Arg228 double conformation is observed in the first crystal form of hTRα LBD+GC1 complex (Table 1). In the intermediate conformation Arg228 interacts both with the GC1 and the Ser277 amino group (white, second hTRα LBD+GC-1 crystal form, Table 1). (c) Comparison of GC1 bound to hTRα and hTRβ. For hTRβ (magenta) only a single productive conformation of the Arg282 side-chain was observed, which resembles the productive Arg228 (hTRα) conformation (green). Arg282 (hTRβ), is strongly interacting with the ligand and its productive conformation is locked in place by the interactions with the side-chain of Asn331.
Figure Legend Snippet: Bleicher3.png . Ligand-receptor interactions for thyroid receptors: (a) T 3 as bound to hTRα (green) and hTRβ (magenta). All interactions are maintained between the ligand and the binding site residues in both hTR isoforms. Both Ser277 and Asn331 interact with the amino group of T 3 through their amide nitrogen, leading to similar conformations of these residues. (b) GC1 bound to TRα: multiple conformations of the Arg228 are observed. In the productive conformation there is a strong interaction with the ligand (cyan), while in non-productive conformations this residue interacts with the side-chain of Ser277. The Arg228 double conformation is observed in the first crystal form of hTRα LBD+GC1 complex (Table 1). In the intermediate conformation Arg228 interacts both with the GC1 and the Ser277 amino group (white, second hTRα LBD+GC-1 crystal form, Table 1). (c) Comparison of GC1 bound to hTRα and hTRβ. For hTRβ (magenta) only a single productive conformation of the Arg282 side-chain was observed, which resembles the productive Arg228 (hTRα) conformation (green). Arg282 (hTRβ), is strongly interacting with the ligand and its productive conformation is locked in place by the interactions with the side-chain of Asn331.

Techniques Used: Binding Assay

Bleicher6.png . Superposition of the crystal structures of T 3 (blue and sky blue) and GC1 (red and orange) bound to two hTR isoforms highlights the conformational variability associated with the Ser277 and Asn331 residues. This variability is mostly a result of the lack of the amine group in GC-1, but its presence in T 3 .
Figure Legend Snippet: Bleicher6.png . Superposition of the crystal structures of T 3 (blue and sky blue) and GC1 (red and orange) bound to two hTR isoforms highlights the conformational variability associated with the Ser277 and Asn331 residues. This variability is mostly a result of the lack of the amine group in GC-1, but its presence in T 3 .

Techniques Used:

Bleicher1.png . Chemical formulas of GC-1 (a) and T 3 (b) .
Figure Legend Snippet: Bleicher1.png . Chemical formulas of GC-1 (a) and T 3 (b) .

Techniques Used:

7) Product Images from "The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi"

Article Title: The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200407088

Rud3p is a member of a family of coiled-coil proteins with a conserved COOH-terminal domain. (A) Schematic representation of S. cerevisiae Rud3p and its relatives from the indicated species. At, A. thaliana ; Ce, C. elegans ; Dm, D. melanogaster ; Hs, Homo sapiens ; Sc, S. cerevisiae ; Tb, Trypanosoma brucei . (B) Alignment of the COOH-terminal regions of the GRAB domain proteins with those of GRIP domain proteins golgin-245 and golgin-97, and the structure of the GRIP domain of human golgin-245. The two sets of sequences were independently aligned with CLUSTAL W and shaded where more than half the residues are related (gray) or identical (black). Hydrophobic residues conserved in each set are marked with filled circles and with a red circle for the critical tyrosine in the GRIP domain, and the leucine is in the equivalent position in Rud3p. In both alignments the tryptophans are shaded orange and cluster downstream of the conserved region. In the case of golgin-245, the tryptophan apparently stabilizes the interaction of the GRIP domain with Golgi membranes ( Panic et al., 2003a ). (C) A schematic representation of the GRAB domain proteins from metazoans and yeasts, along with a GRIP domain protein. All contain either a GRIP or GRAB domain, and the latter have a downstream GA1 motif. Metazoan GRAB proteins also have an extended, proline-rich, COOH-terminal region, whereas GRAB proteins from yeasts and filamentous fungi have an upstream region of sequence conservation (GA2, blue). (D) Fluorescent micrographs of rud3 Δ cells expressing GFP-tagged wild-type Rud3p or the mutant L410A as in Fig. 1 A.
Figure Legend Snippet: Rud3p is a member of a family of coiled-coil proteins with a conserved COOH-terminal domain. (A) Schematic representation of S. cerevisiae Rud3p and its relatives from the indicated species. At, A. thaliana ; Ce, C. elegans ; Dm, D. melanogaster ; Hs, Homo sapiens ; Sc, S. cerevisiae ; Tb, Trypanosoma brucei . (B) Alignment of the COOH-terminal regions of the GRAB domain proteins with those of GRIP domain proteins golgin-245 and golgin-97, and the structure of the GRIP domain of human golgin-245. The two sets of sequences were independently aligned with CLUSTAL W and shaded where more than half the residues are related (gray) or identical (black). Hydrophobic residues conserved in each set are marked with filled circles and with a red circle for the critical tyrosine in the GRIP domain, and the leucine is in the equivalent position in Rud3p. In both alignments the tryptophans are shaded orange and cluster downstream of the conserved region. In the case of golgin-245, the tryptophan apparently stabilizes the interaction of the GRIP domain with Golgi membranes ( Panic et al., 2003a ). (C) A schematic representation of the GRAB domain proteins from metazoans and yeasts, along with a GRIP domain protein. All contain either a GRIP or GRAB domain, and the latter have a downstream GA1 motif. Metazoan GRAB proteins also have an extended, proline-rich, COOH-terminal region, whereas GRAB proteins from yeasts and filamentous fungi have an upstream region of sequence conservation (GA2, blue). (D) Fluorescent micrographs of rud3 Δ cells expressing GFP-tagged wild-type Rud3p or the mutant L410A as in Fig. 1 A.

Techniques Used: Sequencing, Expressing, Mutagenesis

Erv14p is required for the Golgi localization of Rud3p. (A) Fluorescent micrographs of live yeast (BY4741) expressing GFP-Rud3p from plasmid pE1, with the indicated genes deleted. (B) Fluorescent micrographs of an erv14 Δ strain with the RUD3 ORF NH 2 -terminally GFP tagged in the genome and containing either an empty CEN plasmid or a similar plasmid encoding Erv14p-HA as indicated. (C) Fluorescent micrographs of a yeast strain lacking ERV14 and with USO1 COOH-terminally GFP tagged in the genome and containing a CEN plasmid expressing RFP-Rud3p as in Fig. 1 A. (D) Distribution of the Golgi markers Gos1p and Vrg4p expressed as GFP-fusions in either wild-type cells or a mutant lacking ERV14 as indicated. GFP-tagged proteins expressed as in A.
Figure Legend Snippet: Erv14p is required for the Golgi localization of Rud3p. (A) Fluorescent micrographs of live yeast (BY4741) expressing GFP-Rud3p from plasmid pE1, with the indicated genes deleted. (B) Fluorescent micrographs of an erv14 Δ strain with the RUD3 ORF NH 2 -terminally GFP tagged in the genome and containing either an empty CEN plasmid or a similar plasmid encoding Erv14p-HA as indicated. (C) Fluorescent micrographs of a yeast strain lacking ERV14 and with USO1 COOH-terminally GFP tagged in the genome and containing a CEN plasmid expressing RFP-Rud3p as in Fig. 1 A. (D) Distribution of the Golgi markers Gos1p and Vrg4p expressed as GFP-fusions in either wild-type cells or a mutant lacking ERV14 as indicated. GFP-tagged proteins expressed as in A.

Techniques Used: Expressing, Plasmid Preparation, Mutagenesis

Rud3p interacts with the small GTPase Arf1p. (A) Anti-HA protein blot of total cell lysate (Lys.) from a strain expressing Rud3p tagged in the genome with an NH 2 -terminal HA epitope tag (AGY10) and of proteins that bound to GST fusions of the GTP-locked versions of Arf1p, Arf3p, Ypt1p, Ypt6, Ypt31p, and Ypt32p. For the GTP-locked forms of Arf1p (Q71L) and Arf3p (Q71L), the first 14 amino acids that form an amphipathic helix were removed and replaced with an NH 2 -terminal GST tag. For Ypt1p (Q67L), Ypt6p (Q67L), Ypt31p (Q72L), and Ypt32p (Q72L), the COOH-terminal cysteine residues were replaced with a COOH-terminal GST tag. Lysate is 10% of material applied to beads. (B) Anti-HA protein blot of total cell lysate (Lys.; 10% of material loaded) and proteins that bound to GST fusions of wild-type Arf1p and Arf3p preloaded with GDP or a nonhydrolysable analogue of GTP, GTPγS. The blot was stripped and reprobed with a rabbit antibody against Imh1p. (C) As for GST-Arf1p in B, except cell lysates were prepared from a strain expressing either GFP-Rud3p or the mutant proteins L410A expressed under the control of a constitutively active PHO5 promoter from the CEN plasmid pRS416. (D) Binding of the COOH-terminal 126 amino acids of Rud3p to Arf1p (T31N) or Arf1p (Q71L). The indicated forms of GST-Arf1p were coexpressed in E. coli with the COOH terminus of Rud3p, and after cell lysis isolated on glutathione Sepharose beads. Bound proteins were analyzed by gel electrophoresis, and the indicated band was identified as the COOH terminus of Rud3p by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides ( Shevchenko et al., 1996 ).
Figure Legend Snippet: Rud3p interacts with the small GTPase Arf1p. (A) Anti-HA protein blot of total cell lysate (Lys.) from a strain expressing Rud3p tagged in the genome with an NH 2 -terminal HA epitope tag (AGY10) and of proteins that bound to GST fusions of the GTP-locked versions of Arf1p, Arf3p, Ypt1p, Ypt6, Ypt31p, and Ypt32p. For the GTP-locked forms of Arf1p (Q71L) and Arf3p (Q71L), the first 14 amino acids that form an amphipathic helix were removed and replaced with an NH 2 -terminal GST tag. For Ypt1p (Q67L), Ypt6p (Q67L), Ypt31p (Q72L), and Ypt32p (Q72L), the COOH-terminal cysteine residues were replaced with a COOH-terminal GST tag. Lysate is 10% of material applied to beads. (B) Anti-HA protein blot of total cell lysate (Lys.; 10% of material loaded) and proteins that bound to GST fusions of wild-type Arf1p and Arf3p preloaded with GDP or a nonhydrolysable analogue of GTP, GTPγS. The blot was stripped and reprobed with a rabbit antibody against Imh1p. (C) As for GST-Arf1p in B, except cell lysates were prepared from a strain expressing either GFP-Rud3p or the mutant proteins L410A expressed under the control of a constitutively active PHO5 promoter from the CEN plasmid pRS416. (D) Binding of the COOH-terminal 126 amino acids of Rud3p to Arf1p (T31N) or Arf1p (Q71L). The indicated forms of GST-Arf1p were coexpressed in E. coli with the COOH terminus of Rud3p, and after cell lysis isolated on glutathione Sepharose beads. Bound proteins were analyzed by gel electrophoresis, and the indicated band was identified as the COOH terminus of Rud3p by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides ( Shevchenko et al., 1996 ).

Techniques Used: Expressing, Mutagenesis, Plasmid Preparation, Binding Assay, Lysis, Isolation, Nucleic Acid Electrophoresis, Mass Spectrometry

COOH-terminal mutants of Rud3p are not functional. (A) Growth at the indicated temperatures of yeast lacking genomic copies of both RIC1 and RUD3 , but containing the either an empty plasmid or expressing wild-type or mutant GFP-Rud3p (indicated on the diagram) from a constitutive PHO5 promoter. Cells lacking RUD3 and with only copy of RIC1 on a CEN , URA3 plasmid were transformed with CEN , LEU2 plasmids expressing GFP-Rud3p or mutants, and the yeast plated onto plates containing 5-fluoroorotic acid (5-FOA) to remove the RIC1 containing URA3 plasmid. (B) A yeast strain expressing GFP-tagged Rud3p lacking the GA1 motif (463ΔC) was used in a binding assay as in Fig. 3 C with GST-Arf1p loaded with either GDP or GTP-γ-S. Also shown is a fluorescent micrograph of live yeast lacking genomic RUD3 and expressing GFP-Rud3p463ΔC. (C) Fluorescent micrographs of a rud3 Δ strain with the ARF1 ORF tagged in the genome with a COOH-terminal GFP tag (AGY26) and expressing RFP-Rud3p under the constitutive PHO5 promoter from the CEN plasmid pRS416. Some structures contain both RFP-Rud3p and Arf1p-GFP (arrows) and some contain just the latter (arrowheads). (D) Fluorescent micrographs of live BY4741 yeast with the indicated genes deleted and expressing GFP-Rud3p as in Fig. 1 C.
Figure Legend Snippet: COOH-terminal mutants of Rud3p are not functional. (A) Growth at the indicated temperatures of yeast lacking genomic copies of both RIC1 and RUD3 , but containing the either an empty plasmid or expressing wild-type or mutant GFP-Rud3p (indicated on the diagram) from a constitutive PHO5 promoter. Cells lacking RUD3 and with only copy of RIC1 on a CEN , URA3 plasmid were transformed with CEN , LEU2 plasmids expressing GFP-Rud3p or mutants, and the yeast plated onto plates containing 5-fluoroorotic acid (5-FOA) to remove the RIC1 containing URA3 plasmid. (B) A yeast strain expressing GFP-tagged Rud3p lacking the GA1 motif (463ΔC) was used in a binding assay as in Fig. 3 C with GST-Arf1p loaded with either GDP or GTP-γ-S. Also shown is a fluorescent micrograph of live yeast lacking genomic RUD3 and expressing GFP-Rud3p463ΔC. (C) Fluorescent micrographs of a rud3 Δ strain with the ARF1 ORF tagged in the genome with a COOH-terminal GFP tag (AGY26) and expressing RFP-Rud3p under the constitutive PHO5 promoter from the CEN plasmid pRS416. Some structures contain both RFP-Rud3p and Arf1p-GFP (arrows) and some contain just the latter (arrowheads). (D) Fluorescent micrographs of live BY4741 yeast with the indicated genes deleted and expressing GFP-Rud3p as in Fig. 1 C.

Techniques Used: Functional Assay, Plasmid Preparation, Expressing, Mutagenesis, Transformation Assay, Binding Assay

The COOH terminus of Rud3p mediates Golgi association. (A) Fluorescent micrographs of live yeast expressing the indicated fusion proteins. Uso1p was tagged with GFP at the COOH terminus in the genome of the wild-type strain BY4741, whereas RFP-Rud3p was expressed from a CEN plasmid under the control of a constitutive version of the PHO5 promoter. The two proteins were found to be localized to the same punctate structures, as illustrated by those marked with arrows. (B) Schematic diagram of truncations made in the RUD3 ORF in the yeast strain SEY6210 ( Robinson et al., 1988 ) by insertion by homologous recombination of a cassette encoding a PHO5 promoter and an NH 2 -terminal GFP tag. Also shown is a coiled-coil prediction for Rud3p ( Lupas, 1996 ). (C) Fluorescent micrographs of live yeast expressing the indicated GFP-Rud3p truncations as in B.
Figure Legend Snippet: The COOH terminus of Rud3p mediates Golgi association. (A) Fluorescent micrographs of live yeast expressing the indicated fusion proteins. Uso1p was tagged with GFP at the COOH terminus in the genome of the wild-type strain BY4741, whereas RFP-Rud3p was expressed from a CEN plasmid under the control of a constitutive version of the PHO5 promoter. The two proteins were found to be localized to the same punctate structures, as illustrated by those marked with arrows. (B) Schematic diagram of truncations made in the RUD3 ORF in the yeast strain SEY6210 ( Robinson et al., 1988 ) by insertion by homologous recombination of a cassette encoding a PHO5 promoter and an NH 2 -terminal GFP tag. Also shown is a coiled-coil prediction for Rud3p ( Lupas, 1996 ). (C) Fluorescent micrographs of live yeast expressing the indicated GFP-Rud3p truncations as in B.

Techniques Used: Expressing, Plasmid Preparation, Homologous Recombination

Erv14p and Rud3p in Golgi function. (A) Fluorescent micrographs of yeast (BY4741) expressing Arf1p-GFP from a CEN , URA3 plasmid, with the genomic copies of ARF1 and ERV14 deleted as indicated. (B) Anti-GFP protein blot of lysates (Lys.; 10% of material loaded) from wild-type BY4741 (WT) or erv14 Δ cells expressing GFP-Rud3p or of the proteins bound when the lysates were applied to immobilized GST-Arf1p loaded with the indicated nucleotides, as in Fig. 3 C. (C) Anti-GFP protein blot of total cellular proteins from BY4741 (WT) or the indicated strains expressing Axl2p-GFP from a CEN plasmid, with or without endoglycosidase H digestion. (D) As C, except that the cells were rud3 Δ and contained either an empty CEN vector or the same with the indicated forms of GFP-Rud3p.
Figure Legend Snippet: Erv14p and Rud3p in Golgi function. (A) Fluorescent micrographs of yeast (BY4741) expressing Arf1p-GFP from a CEN , URA3 plasmid, with the genomic copies of ARF1 and ERV14 deleted as indicated. (B) Anti-GFP protein blot of lysates (Lys.; 10% of material loaded) from wild-type BY4741 (WT) or erv14 Δ cells expressing GFP-Rud3p or of the proteins bound when the lysates were applied to immobilized GST-Arf1p loaded with the indicated nucleotides, as in Fig. 3 C. (C) Anti-GFP protein blot of total cellular proteins from BY4741 (WT) or the indicated strains expressing Axl2p-GFP from a CEN plasmid, with or without endoglycosidase H digestion. (D) As C, except that the cells were rud3 Δ and contained either an empty CEN vector or the same with the indicated forms of GFP-Rud3p.

Techniques Used: Expressing, Plasmid Preparation

8) Product Images from "New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats"

Article Title: New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr1228

p68 increases the binding of MBNL1 to 95 CUG repeats. ( A ) Plasmid (CUG)95 was in vitro transcribed in the presence of [α- 32 P]UTP. Labeled (CUG)95 RNA was incubated with a constant amount of recombinant MBNL1 protein (200 ng) and increasing amounts of recombinant p68ΔCt2 under splicing conditions with ATP. Proteins cross-linked to labeled RNA were separated on a 10% SDS–PAGE. Bottom image shows a silver stain of a gel run in parallel. Note that p68 ΔCt2 migrates as two bands that are recognized by anti-p68 antibodies (data not shown). ( B ) The cross-linked proteins shown in (A) were immunoprecipitated with anti-MBNL1 antibodies and separated by SDS-PAGE. ( C ) (CUG)95, (CAG)61 and (CCUG)62 RNAs were labeled with [α- 32 P]CTP and used for UV-cross-linking experiments. ( D ) The increase of cross-linked MBNL1 to CUG repeats is specific to p68. Labeled (CUG)95 was incubated with 200 ng of recombinant MBNL1 protein and 25 or 75 ng of recombinant p68ΔCt2, UAP56 or eIF4A3 proteins. Quantifications result from three independent experiments, with error bars indicating standard deviation. * P
Figure Legend Snippet: p68 increases the binding of MBNL1 to 95 CUG repeats. ( A ) Plasmid (CUG)95 was in vitro transcribed in the presence of [α- 32 P]UTP. Labeled (CUG)95 RNA was incubated with a constant amount of recombinant MBNL1 protein (200 ng) and increasing amounts of recombinant p68ΔCt2 under splicing conditions with ATP. Proteins cross-linked to labeled RNA were separated on a 10% SDS–PAGE. Bottom image shows a silver stain of a gel run in parallel. Note that p68 ΔCt2 migrates as two bands that are recognized by anti-p68 antibodies (data not shown). ( B ) The cross-linked proteins shown in (A) were immunoprecipitated with anti-MBNL1 antibodies and separated by SDS-PAGE. ( C ) (CUG)95, (CAG)61 and (CCUG)62 RNAs were labeled with [α- 32 P]CTP and used for UV-cross-linking experiments. ( D ) The increase of cross-linked MBNL1 to CUG repeats is specific to p68. Labeled (CUG)95 was incubated with 200 ng of recombinant MBNL1 protein and 25 or 75 ng of recombinant p68ΔCt2, UAP56 or eIF4A3 proteins. Quantifications result from three independent experiments, with error bars indicating standard deviation. * P

Techniques Used: Binding Assay, Plasmid Preparation, In Vitro, Labeling, Incubation, Recombinant, SDS Page, Silver Staining, Immunoprecipitation, Standard Deviation

9) Product Images from "RINL, Guanine Nucleotide Exchange Factor Rab5-Subfamily, Is Involved in the EphA8-Degradation Pathway with Odin"

Article Title: RINL, Guanine Nucleotide Exchange Factor Rab5-Subfamily, Is Involved in the EphA8-Degradation Pathway with Odin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0030575

GEF activity of RINL for Rab5 subfamily proteins. (A–D) HEK293T cells expressing myc-Rab5b (A), Rab21 (B), Rab22 (C), or Rab31 (D) and FLAG-mock, RINL, RIN3, or Rabex-5 were metabolically radiolabeled with 32 P i for 4 hours. Myc-Rab5 subfamily proteins were immunoprecipitated with an anti-myc monoclonal antibody, and nucleotides associating with each Rab protein were separated by thin-layer chromatography. The radioactivity of GTP and GDP was quantified, and the percentages (%) of each GTP-bound Rab are shown. Total lysates (bottom) and immunoprecipitated samples (middle) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-myc antibodies, respectively. *p
Figure Legend Snippet: GEF activity of RINL for Rab5 subfamily proteins. (A–D) HEK293T cells expressing myc-Rab5b (A), Rab21 (B), Rab22 (C), or Rab31 (D) and FLAG-mock, RINL, RIN3, or Rabex-5 were metabolically radiolabeled with 32 P i for 4 hours. Myc-Rab5 subfamily proteins were immunoprecipitated with an anti-myc monoclonal antibody, and nucleotides associating with each Rab protein were separated by thin-layer chromatography. The radioactivity of GTP and GDP was quantified, and the percentages (%) of each GTP-bound Rab are shown. Total lysates (bottom) and immunoprecipitated samples (middle) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-myc antibodies, respectively. *p

Techniques Used: Activity Assay, Expressing, Metabolic Labelling, Immunoprecipitation, Thin Layer Chromatography, Radioactivity, SDS Page

10) Product Images from "Structural Investigation of the Transmembrane Domain of KCNE1 in Proteoliposomes"

Article Title: Structural Investigation of the Transmembrane Domain of KCNE1 in Proteoliposomes

Journal: Biochemistry

doi: 10.1021/bi500943p

Four-pulse Q-band DEER data of KCNE1 mutant (Val50/Ser68) bearing two MTSL spin labels. Background-subtracted dipolar evolutions for the indicated mutants (left) and their corresponding distance probability distribution from Tikhonov regularization (right) are shown for (A) 1% LMPG micelles, (B) proteoliposomes (POPC/POPG = 3:1), and (C) lipodisq nanoparticles.
Figure Legend Snippet: Four-pulse Q-band DEER data of KCNE1 mutant (Val50/Ser68) bearing two MTSL spin labels. Background-subtracted dipolar evolutions for the indicated mutants (left) and their corresponding distance probability distribution from Tikhonov regularization (right) are shown for (A) 1% LMPG micelles, (B) proteoliposomes (POPC/POPG = 3:1), and (C) lipodisq nanoparticles.

Techniques Used: Mutagenesis

Overlay of distance probability distribution data obtained from Tikhonov regularization of DEER data for MTSL-labeled KCNE1 in micelles, proteoliposomes, and lipodisq nanoparticles. The black traces represent micelles, blue represents proteoliposomes, and red represents lipodisq nanoparticles. The label in each box indicates the dual-labeling sites. The Y -axis scale represents the probability of distance distribution in each plot.
Figure Legend Snippet: Overlay of distance probability distribution data obtained from Tikhonov regularization of DEER data for MTSL-labeled KCNE1 in micelles, proteoliposomes, and lipodisq nanoparticles. The black traces represent micelles, blue represents proteoliposomes, and red represents lipodisq nanoparticles. The label in each box indicates the dual-labeling sites. The Y -axis scale represents the probability of distance distribution in each plot.

Techniques Used: Labeling

Four-pulse Q-band DEER data of KCNE1 mutants (Tyr46–Val50/Arg67–Leu71) bearing two BSLs. Background-subtracted dipolar evolutions of the indicated mutants (left) and their corresponding distance probability distributions from Tikhonov regularization are shown (right) for conditions of (A) 1% LMPG micelles, (B) proteoliposomes (POPC/POPG = 3:1), and (C) lipodisq nanoparticles.
Figure Legend Snippet: Four-pulse Q-band DEER data of KCNE1 mutants (Tyr46–Val50/Arg67–Leu71) bearing two BSLs. Background-subtracted dipolar evolutions of the indicated mutants (left) and their corresponding distance probability distributions from Tikhonov regularization are shown (right) for conditions of (A) 1% LMPG micelles, (B) proteoliposomes (POPC/POPG = 3:1), and (C) lipodisq nanoparticles.

Techniques Used:

Schematic model of the overlay of the 10 lowest energy structures of KCNE1 TMD in a lipid bilayer.
Figure Legend Snippet: Schematic model of the overlay of the 10 lowest energy structures of KCNE1 TMD in a lipid bilayer.

Techniques Used:

Overlay of distance probability data obtained from Tikhonov regularization in DEER data analysis for BSL-labeled KCNE1 samples in micelles, proteoliposomes, and lipodisq nanoparticles. The black traces represent micelles, blue represents proteoliposomes, and red represents lipodisq nanoparticles. The label in each box indicates the dual-labeling sites. The Y -axis scale represents the probability of distance distribution in each plot.
Figure Legend Snippet: Overlay of distance probability data obtained from Tikhonov regularization in DEER data analysis for BSL-labeled KCNE1 samples in micelles, proteoliposomes, and lipodisq nanoparticles. The black traces represent micelles, blue represents proteoliposomes, and red represents lipodisq nanoparticles. The label in each box indicates the dual-labeling sites. The Y -axis scale represents the probability of distance distribution in each plot.

Techniques Used: Labeling

Schematic representation of spin-labeling probes and sites. (A) MTSL, (B) BSL, and (C) ribbon model of transmembrane domain of KCNE1 (PDB ID: 2k21) highlighting representative sites used in this study with spheres at their α-carbons. All spin-labeling sites are located inside the membrane. The dashed lines represent the lipid bilayer interfaces. Spin-labeling sites 45 and 71 are at the termini of the transmembrane domain that spans the membrane bilayers.
Figure Legend Snippet: Schematic representation of spin-labeling probes and sites. (A) MTSL, (B) BSL, and (C) ribbon model of transmembrane domain of KCNE1 (PDB ID: 2k21) highlighting representative sites used in this study with spheres at their α-carbons. All spin-labeling sites are located inside the membrane. The dashed lines represent the lipid bilayer interfaces. Spin-labeling sites 45 and 71 are at the termini of the transmembrane domain that spans the membrane bilayers.

Techniques Used: Labeling

Results of the structure refinement of the KCNE1 TMD in proteoliposomes incorporating MTSL DEER distance-restraint data using an Xplor-NIH simulated annealing molecular dynamics protocol. (A) Overlay of the 10 structures with lowest energy obtained from restrained simulated annealing calculations using amino acids 45–71 (transmembrane domain) of KCNE1. The final structures were generated by replacing the MTSL-labeled side chains by the native amino acid side chains with retention of the C β position in the label. (B) Ribbon representation of overlaid DEER structures. (C) Overlay of DEER structures and the previously determined NMR structure (blue cartoon represents micellar NMR structure, and purple cartoons represent DEER structures in lipid bilayers).
Figure Legend Snippet: Results of the structure refinement of the KCNE1 TMD in proteoliposomes incorporating MTSL DEER distance-restraint data using an Xplor-NIH simulated annealing molecular dynamics protocol. (A) Overlay of the 10 structures with lowest energy obtained from restrained simulated annealing calculations using amino acids 45–71 (transmembrane domain) of KCNE1. The final structures were generated by replacing the MTSL-labeled side chains by the native amino acid side chains with retention of the C β position in the label. (B) Ribbon representation of overlaid DEER structures. (C) Overlay of DEER structures and the previously determined NMR structure (blue cartoon represents micellar NMR structure, and purple cartoons represent DEER structures in lipid bilayers).

Techniques Used: Generated, Labeling, Nuclear Magnetic Resonance

11) Product Images from "Transcriptional control by two interacting regulatory proteins: identification of the PtxS binding site at PtxR"

Article Title: Transcriptional control by two interacting regulatory proteins: identification of the PtxS binding site at PtxR

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt773

In vitro transcription from P gad . Transcription assays were carried out as described in ‘Materials and Methods’ section. ( A ) The assay performed in the presence of 20 μM combined wild-type PtxS with wild type PtxR, PtxR(V173A) or PtxR(W269A) mutants. ( B ) In vitro transcription assays obtained by supplementation of increase concentration of 2-ketogluconate (0–150 µM). ( C ) The densitometric analysis of the in vitro transcription gels. Circles: WT PtxR protein, triangle: V173A PtxR protein and star: W269A.
Figure Legend Snippet: In vitro transcription from P gad . Transcription assays were carried out as described in ‘Materials and Methods’ section. ( A ) The assay performed in the presence of 20 μM combined wild-type PtxS with wild type PtxR, PtxR(V173A) or PtxR(W269A) mutants. ( B ) In vitro transcription assays obtained by supplementation of increase concentration of 2-ketogluconate (0–150 µM). ( C ) The densitometric analysis of the in vitro transcription gels. Circles: WT PtxR protein, triangle: V173A PtxR protein and star: W269A.

Techniques Used: In Vitro, Concentration Assay

12) Product Images from "The Actinobacillus pleuropneumoniae HMW1C-Like Glycosyltransferase Mediates N-Linked Glycosylation of the Haemophilus influenzae HMW1 Adhesin"

Article Title: The Actinobacillus pleuropneumoniae HMW1C-Like Glycosyltransferase Mediates N-Linked Glycosylation of the Haemophilus influenzae HMW1 Adhesin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0015888

Ability of ApHMW1C to complement a deficiency in HMW1C. Panel A shows Western immunoblots of whole cell sonicates of E. coli BL21(DE3)/pACYC-HMW1ΔC (lane 1), E. coli BL21(DE3)/pACYC-HMW1ΔC + pET45b-HMW1C (lane 2), and E. coli BL21(DE3)/pACYC-HMW1ΔC + pET45b-ApHMW1C (lane 3). Lane 1 contains twice as much protein as loaded in lanes 2 and 3 to increase the visibility of the non-glycosylated HMW1 species. The blot in the upper panel was performed with a guinea pig antiserum reactive with HMW1, and the blot in the lower panel was performed with a guinea pig antiserum reactive with H. influenzae HMW1C. The asterisk indicates the glycosylated HMW1 pro-protein, and the plus sign indicates the non-glycosylated HMW1 pro-protein. The diamond indicates the glycosylated HMW1 mature protein, and the circle indicates the non-glycosylated HMW1 mature protein. Panel B shows in vitro adherence results comparing adherence by E. coli BL21(DE3)/pACYC-HMW1ΔC ( hmw1AB ), E. coli BL21(DE3)/pACYC-HMW1ΔC + pET45b-HMW1C ( hmw1AB + hmw1C ), and E. coli BL21(DE3)/pACYC-HMW1ΔC + pET45b-ApHMW1C ( hmw1AB + Aphmw1C ) to Chang epithelial cells. Bars and error bars represent mean and standard error measurements from a representative assay with measurements performed in triplicate.
Figure Legend Snippet: Ability of ApHMW1C to complement a deficiency in HMW1C. Panel A shows Western immunoblots of whole cell sonicates of E. coli BL21(DE3)/pACYC-HMW1ΔC (lane 1), E. coli BL21(DE3)/pACYC-HMW1ΔC + pET45b-HMW1C (lane 2), and E. coli BL21(DE3)/pACYC-HMW1ΔC + pET45b-ApHMW1C (lane 3). Lane 1 contains twice as much protein as loaded in lanes 2 and 3 to increase the visibility of the non-glycosylated HMW1 species. The blot in the upper panel was performed with a guinea pig antiserum reactive with HMW1, and the blot in the lower panel was performed with a guinea pig antiserum reactive with H. influenzae HMW1C. The asterisk indicates the glycosylated HMW1 pro-protein, and the plus sign indicates the non-glycosylated HMW1 pro-protein. The diamond indicates the glycosylated HMW1 mature protein, and the circle indicates the non-glycosylated HMW1 mature protein. Panel B shows in vitro adherence results comparing adherence by E. coli BL21(DE3)/pACYC-HMW1ΔC ( hmw1AB ), E. coli BL21(DE3)/pACYC-HMW1ΔC + pET45b-HMW1C ( hmw1AB + hmw1C ), and E. coli BL21(DE3)/pACYC-HMW1ΔC + pET45b-ApHMW1C ( hmw1AB + Aphmw1C ) to Chang epithelial cells. Bars and error bars represent mean and standard error measurements from a representative assay with measurements performed in triplicate.

Techniques Used: Western Blot, In Vitro

Representative GT41 members, HMW1C sequences, and schematics of recombinant proteins used in this study. ( A ) Domain organization of three GT41 members, including the human OGT (HsOGT), the Xanthomonas campestris OGT (XcOGT), and Haemophilus influenzae HMW1C protein (HMW1C). The TPR and GT domains are indicated in blue and cyan, respectively. Based on the XcOGT structure [17] , [18] , domain boundaries of HsOGT were assigned (the nucleus localization signal in red). Fly and mammalian OGTs have a large insertion (in white) within the GT domain. In HMW1C, the N-terminal domain (in magenta) is different from the TPR domains in HsOGT and XcOGT. HMW1C residues 155 and 260 correspond to ApHMW1C residue 125 (limited proteolysis boundary) and XcOGT residue 203 (boundary for GT), respectively. ( B ) The sequence alignment of HMW1C (Hi) with its ortholog from A. pleuropneumoniae (Ap). The protease cleavage site is indicated with star. ( C ) Schematics of HMW1 and acceptor protein constructs. The known domain organization of HMW1 is shown: SP, the signal peptide (residues 1–68); HMW1-PP, the HMW1 pro-piece (residues 69–441) containing the secretion domain; and the mature adhesin (residues 442–1536). Several constructs representing different regions of HMW1 were generated as GST-fusion proteins to serve as acceptor proteins. Based on assessment of solubility and stability of each protein in solution, the best substrate was HMW1ct. For the substrate HMW1ct, a His-tagged version was also produced. The N-glycosylation sites, N1348, N1352, and N1366, within HMW1ct are indicated. ( D ) Schematics of enzyme constructs. ApHMW1C (GenBank: ABN74719.1) and its two sub-domains (P15 and P55) identified from the analysis of limited proteolysis were produced as His-tagged proteins. An analytical gel filtration profile of purified ApHMW1C (marked with star) revealed a calculated molecular weight of ∼70 kDa, consistent with a monomer. The peak positions of molecular standards are indicated as arrowheads (aldolase, 158 kDa; conalbumin, 75 kDa; and ovalbumin, 43 kDa).
Figure Legend Snippet: Representative GT41 members, HMW1C sequences, and schematics of recombinant proteins used in this study. ( A ) Domain organization of three GT41 members, including the human OGT (HsOGT), the Xanthomonas campestris OGT (XcOGT), and Haemophilus influenzae HMW1C protein (HMW1C). The TPR and GT domains are indicated in blue and cyan, respectively. Based on the XcOGT structure [17] , [18] , domain boundaries of HsOGT were assigned (the nucleus localization signal in red). Fly and mammalian OGTs have a large insertion (in white) within the GT domain. In HMW1C, the N-terminal domain (in magenta) is different from the TPR domains in HsOGT and XcOGT. HMW1C residues 155 and 260 correspond to ApHMW1C residue 125 (limited proteolysis boundary) and XcOGT residue 203 (boundary for GT), respectively. ( B ) The sequence alignment of HMW1C (Hi) with its ortholog from A. pleuropneumoniae (Ap). The protease cleavage site is indicated with star. ( C ) Schematics of HMW1 and acceptor protein constructs. The known domain organization of HMW1 is shown: SP, the signal peptide (residues 1–68); HMW1-PP, the HMW1 pro-piece (residues 69–441) containing the secretion domain; and the mature adhesin (residues 442–1536). Several constructs representing different regions of HMW1 were generated as GST-fusion proteins to serve as acceptor proteins. Based on assessment of solubility and stability of each protein in solution, the best substrate was HMW1ct. For the substrate HMW1ct, a His-tagged version was also produced. The N-glycosylation sites, N1348, N1352, and N1366, within HMW1ct are indicated. ( D ) Schematics of enzyme constructs. ApHMW1C (GenBank: ABN74719.1) and its two sub-domains (P15 and P55) identified from the analysis of limited proteolysis were produced as His-tagged proteins. An analytical gel filtration profile of purified ApHMW1C (marked with star) revealed a calculated molecular weight of ∼70 kDa, consistent with a monomer. The peak positions of molecular standards are indicated as arrowheads (aldolase, 158 kDa; conalbumin, 75 kDa; and ovalbumin, 43 kDa).

Techniques Used: Recombinant, Sequencing, Construct, Generated, Solubility, Produced, Filtration, Purification, Molecular Weight

13) Product Images from "Activation of a synapse weakening pathway by human Val66 but not Met66 pro-brain-derived neurotrophic factor (proBDNF)"

Article Title: Activation of a synapse weakening pathway by human Val66 but not Met66 pro-brain-derived neurotrophic factor (proBDNF)

Journal: Pharmacological Research

doi: 10.1016/j.phrs.2015.12.008

Characterization of proBDNF Val66Met FCR polymorphic variants. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (a) Wild-type (WT) proBDNF or furin cleavage-resistant (FCR) proBDNF Val66Met variants (50 ng) and BDNF (10 ng) resolved with SDS PAGE and western blotted using anti-BDNF-N20 1:3000. Val* denotes commercial proBDNF Val66-FCR (Alomone labs). ProBDNF variants have a similar molecular weight of ∼26 kDa (unglycosylated) with high purity. (b) ProBDNF (∼1 μg) resolved on acidic native PAGE gels stained with Coomassie Blue showed no laddering or unresolved proteins, confirming absence of oligomerization and soluble aggregates. Mature BDNF (∼14 kDa) used as standard. (c) ProBDNF variants (50 μg/ml) furin digested 1 h at 30 °C, resolved on 12% SDS PAGE and analyzed using western blotting. Protein bands were detected using anti-BDNF N20 at 1:3000. WT was the positive control, cleaved to mature BDNF. Cleavage-resistant proBDNF variants were intact following furin exposure. (d) Overlay of PONDR outputs of Val66 and Met66. Values are predicted and scaled 0–1. The thick black line (‘disorder bar’) denotes strength of disorder prediction. (e) ProBDNF variants (∼0.1 mg/ml) were measured by CD in the far-UV range. Spectra of both variants showed predominant β-structure with subtle differences in the 222 nm region. Lines show Val66 in light blue Met66 in dark blue. (f) and (g) E16 cortical neurons (DIV11) were serum starved for 2 h and treated with a range of concentrations (0–10 nM) of proBDNF variants for 18 h, after which cells were processed and measured at (f) 485 nm for LDH release (ProBDNF FCR Val66 in light blue, Met66 in dark blue) or (g) at excitation/emission 560/595 nm for JC-1 assay with relative fluorescence of J-aggregates measured (ProBDNF WT Val66 in light blue, Met66 in dark blue). The polymorphism was not associated ( P = 0.473) with the reduction in membrane potential (two-way ANOVA followed by Bonferroni’s post hoc test). In (f) and (g) changes are represented as column graphs with subsequent analysis using one-way ANOVA followed by Dunnett’s multiple comparison post hoc test for comparison within group. Throughout, error bars denote standard error of mean. Significance is denoted as * P
Figure Legend Snippet: Characterization of proBDNF Val66Met FCR polymorphic variants. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (a) Wild-type (WT) proBDNF or furin cleavage-resistant (FCR) proBDNF Val66Met variants (50 ng) and BDNF (10 ng) resolved with SDS PAGE and western blotted using anti-BDNF-N20 1:3000. Val* denotes commercial proBDNF Val66-FCR (Alomone labs). ProBDNF variants have a similar molecular weight of ∼26 kDa (unglycosylated) with high purity. (b) ProBDNF (∼1 μg) resolved on acidic native PAGE gels stained with Coomassie Blue showed no laddering or unresolved proteins, confirming absence of oligomerization and soluble aggregates. Mature BDNF (∼14 kDa) used as standard. (c) ProBDNF variants (50 μg/ml) furin digested 1 h at 30 °C, resolved on 12% SDS PAGE and analyzed using western blotting. Protein bands were detected using anti-BDNF N20 at 1:3000. WT was the positive control, cleaved to mature BDNF. Cleavage-resistant proBDNF variants were intact following furin exposure. (d) Overlay of PONDR outputs of Val66 and Met66. Values are predicted and scaled 0–1. The thick black line (‘disorder bar’) denotes strength of disorder prediction. (e) ProBDNF variants (∼0.1 mg/ml) were measured by CD in the far-UV range. Spectra of both variants showed predominant β-structure with subtle differences in the 222 nm region. Lines show Val66 in light blue Met66 in dark blue. (f) and (g) E16 cortical neurons (DIV11) were serum starved for 2 h and treated with a range of concentrations (0–10 nM) of proBDNF variants for 18 h, after which cells were processed and measured at (f) 485 nm for LDH release (ProBDNF FCR Val66 in light blue, Met66 in dark blue) or (g) at excitation/emission 560/595 nm for JC-1 assay with relative fluorescence of J-aggregates measured (ProBDNF WT Val66 in light blue, Met66 in dark blue). The polymorphism was not associated ( P = 0.473) with the reduction in membrane potential (two-way ANOVA followed by Bonferroni’s post hoc test). In (f) and (g) changes are represented as column graphs with subsequent analysis using one-way ANOVA followed by Dunnett’s multiple comparison post hoc test for comparison within group. Throughout, error bars denote standard error of mean. Significance is denoted as * P

Techniques Used: SDS Page, Western Blot, Molecular Weight, Clear Native PAGE, Staining, Positive Control, Fluorescence

ProBDNF variants are similar in their binding to TrkB, p75NTR, sortilin and SorCS2. Representative sensorgrams are shown for 0–20 nM proBDNF FCR Val66 (a, c, e, and g) and FCR Met66 (b, d, f, and h) binding to their isolated receptor binding domains TrkBIg 2 , p75NTR ECD, sortilin LD and SorCS2 LD. Data was double referenced (RU) and plotted as a function of time. All the kinetic data were fitted to 1:1 Langmuir mathematical model and kinetic constants were calculated. KD denotes mean equilibrium dissociation constant. Mean constants for both Val66 and Met66 variants were not statistically different. Full kinetic constants are given in Table 1 .
Figure Legend Snippet: ProBDNF variants are similar in their binding to TrkB, p75NTR, sortilin and SorCS2. Representative sensorgrams are shown for 0–20 nM proBDNF FCR Val66 (a, c, e, and g) and FCR Met66 (b, d, f, and h) binding to their isolated receptor binding domains TrkBIg 2 , p75NTR ECD, sortilin LD and SorCS2 LD. Data was double referenced (RU) and plotted as a function of time. All the kinetic data were fitted to 1:1 Langmuir mathematical model and kinetic constants were calculated. KD denotes mean equilibrium dissociation constant. Mean constants for both Val66 and Met66 variants were not statistically different. Full kinetic constants are given in Table 1 .

Techniques Used: Binding Assay, Isolation

ProBDNF Val66 activates GSK3β to mediate effects on synaptic plasticity. (a) LTP was induced by delivering 2 tetanic stimuli to the Schaffer collateral input of acute hippocampus slices. Prior to tetanus, slices were pre-incubated (90 min) with 1 nM proBDNF Val66 and Met66 FCR variants and perfused constantly with appropriate 1 nM variants. Statistical comparison of the mean fEPSPs values, calculated ∼45 min after application of tetanus, showed prominent differences between Val66 and Met66 in inhibiting LTP. (b) LTD was induced using standard LFS protocol of 1 Hz, 900 pulses for 15 min in rat (P24–26) hippocampal slices. Prior to LFS, slices were pre-incubated (90 min) with 1 nM proBDNF Val66 or Met66 and perfused constantly with 1 nM proBDNF variants as appropriate. Statistical comparison of mean fEPSPs values calculated approximately 45 min after application of LFS showed prominent differences between Val66 and Met66 in facilitating LTD. (c) Western blotting analysis of phosphorylated GSK3β at serine 9 and total levels of GSK3β after incubation with proBDNF variants for 90 min, n = 4. (d) Representative western blot showing PHF-1 phosphorylation levels normalized to tau-5 after treatment with proBDNF Val66 or Met66 (1 nM; 90 min). Bar-chart showed quantification of pooled data ( n = 4). (e) Quantification of LTP levels following 2 tetanic stimuli with proBDNF Val66 in the presence and absence of CT99021 (1 μM; n = 6). Significance ( * P
Figure Legend Snippet: ProBDNF Val66 activates GSK3β to mediate effects on synaptic plasticity. (a) LTP was induced by delivering 2 tetanic stimuli to the Schaffer collateral input of acute hippocampus slices. Prior to tetanus, slices were pre-incubated (90 min) with 1 nM proBDNF Val66 and Met66 FCR variants and perfused constantly with appropriate 1 nM variants. Statistical comparison of the mean fEPSPs values, calculated ∼45 min after application of tetanus, showed prominent differences between Val66 and Met66 in inhibiting LTP. (b) LTD was induced using standard LFS protocol of 1 Hz, 900 pulses for 15 min in rat (P24–26) hippocampal slices. Prior to LFS, slices were pre-incubated (90 min) with 1 nM proBDNF Val66 or Met66 and perfused constantly with 1 nM proBDNF variants as appropriate. Statistical comparison of mean fEPSPs values calculated approximately 45 min after application of LFS showed prominent differences between Val66 and Met66 in facilitating LTD. (c) Western blotting analysis of phosphorylated GSK3β at serine 9 and total levels of GSK3β after incubation with proBDNF variants for 90 min, n = 4. (d) Representative western blot showing PHF-1 phosphorylation levels normalized to tau-5 after treatment with proBDNF Val66 or Met66 (1 nM; 90 min). Bar-chart showed quantification of pooled data ( n = 4). (e) Quantification of LTP levels following 2 tetanic stimuli with proBDNF Val66 in the presence and absence of CT99021 (1 μM; n = 6). Significance ( * P

Techniques Used: Incubation, Western Blot

14) Product Images from "The inverted free energy landscape of an intrinsically disordered peptide by simulations and experiments"

Article Title: The inverted free energy landscape of an intrinsically disordered peptide by simulations and experiments

Journal: Scientific Reports

doi: 10.1038/srep15449

Secondary structure populations of the Aβ40 peptide at increasing values of the free energy. The lines indicate different secondary structure types: α-helical regions are shown in blue, β-sheet regions in red, and polyproline II regions in green. The different panels report the secondary structure populations (‘%SS’, which is given as a fraction of the total population, i.e.from 0 to 1) corresponding to different slices of the free energy landscape: ( a ) entire free energy landscape, ( b ) lower region of the free energy landscape (0–6 kJ/mol), ( c ) 6–12 kJ/mol region, ( d ) 12–18 kJ/mol region, ( e ) 18–24 kJ/mol region and ( f ) higher region (above 24 kJ/mol).
Figure Legend Snippet: Secondary structure populations of the Aβ40 peptide at increasing values of the free energy. The lines indicate different secondary structure types: α-helical regions are shown in blue, β-sheet regions in red, and polyproline II regions in green. The different panels report the secondary structure populations (‘%SS’, which is given as a fraction of the total population, i.e.from 0 to 1) corresponding to different slices of the free energy landscape: ( a ) entire free energy landscape, ( b ) lower region of the free energy landscape (0–6 kJ/mol), ( c ) 6–12 kJ/mol region, ( d ) 12–18 kJ/mol region, ( e ) 18–24 kJ/mol region and ( f ) higher region (above 24 kJ/mol).

Techniques Used:

Characterization of the high free energy structures of the Aβ40 peptide. Representative conformations sampled during the simulations are shown at increasing values of free energy with respect to the global disordered minimum. On the left we report the average values of radius of gyration and secondary structure population for the whole ensemble and for difference slices in the free energy with a width of 6 kJ/mol (see also Figs 3 and 4 ). For increasing free energies, Aβ40 becomes more structured by populating both α-helical and β-sheet conformations.
Figure Legend Snippet: Characterization of the high free energy structures of the Aβ40 peptide. Representative conformations sampled during the simulations are shown at increasing values of free energy with respect to the global disordered minimum. On the left we report the average values of radius of gyration and secondary structure population for the whole ensemble and for difference slices in the free energy with a width of 6 kJ/mol (see also Figs 3 and 4 ). For increasing free energies, Aβ40 becomes more structured by populating both α-helical and β-sheet conformations.

Techniques Used:

Temperature-induced compaction of the Aβ40 peptide in 20 mM phosphate buffer at pH 8. ( A ) Values of the compaction index (CI) relative to that at 5 °C, as calculated from the hydrodynamic radii determined by NMR diffusion experiments at a concentration of 200 μM. ( B ) Far UV-CD spectra recorded at a concentration of 20 μM. ( C ) CD spectra-based values showing the increase (MRE) and the red-shift (wavelength) of the minimum mean residue ellipticities with increasing temperature. ( D ) CD-derived secondary structure population showing the relative change of the α-helical, β-strand, unfolded and turn populations of Aβ40 between relative to those at 5 °C. ( E ) Quenching of tyrosine fluorescence intensity with increasing temperature. ( F ) Reversibility of the quenching of tyrosine fluorescence intensity followed at 305 nm between 5 and 100 °C.
Figure Legend Snippet: Temperature-induced compaction of the Aβ40 peptide in 20 mM phosphate buffer at pH 8. ( A ) Values of the compaction index (CI) relative to that at 5 °C, as calculated from the hydrodynamic radii determined by NMR diffusion experiments at a concentration of 200 μM. ( B ) Far UV-CD spectra recorded at a concentration of 20 μM. ( C ) CD spectra-based values showing the increase (MRE) and the red-shift (wavelength) of the minimum mean residue ellipticities with increasing temperature. ( D ) CD-derived secondary structure population showing the relative change of the α-helical, β-strand, unfolded and turn populations of Aβ40 between relative to those at 5 °C. ( E ) Quenching of tyrosine fluorescence intensity with increasing temperature. ( F ) Reversibility of the quenching of tyrosine fluorescence intensity followed at 305 nm between 5 and 100 °C.

Techniques Used: Nuclear Magnetic Resonance, Diffusion-based Assay, Concentration Assay, Derivative Assay, Fluorescence

Residue-based analysis of solvent accessible surface area (SASA) of Aβ40 at increasing values of the free energy. Black bars correspond to the SASA difference with the conformations in the global minimum for each residue averaged on the same free energy windows. The horizontal red line shows the average SASA value for the sequence, which is progressively increasing with the free energy. This effect is particularly evident for all the larger hydrophobic residues, highlighted by cyan bars in background.
Figure Legend Snippet: Residue-based analysis of solvent accessible surface area (SASA) of Aβ40 at increasing values of the free energy. Black bars correspond to the SASA difference with the conformations in the global minimum for each residue averaged on the same free energy windows. The horizontal red line shows the average SASA value for the sequence, which is progressively increasing with the free energy. This effect is particularly evident for all the larger hydrophobic residues, highlighted by cyan bars in background.

Techniques Used: Sequencing

Free energy landscape of the Aβ40 peptide. The free energy landscape is shown as a function of three collective variables used in the NMR-guided metadynamics simulations: anti-parallel β-sheet content (X-axis), α-helical content (Y-axis) and number of hydrophobic contacts (or compactness, Z-axis). Isosurfaces are shown at 5 (red), 10 (blue), 18 (yellow) and 25 kJ/mol (cyan); white regions are not visited as they have higher free energies. Representative structures sampled during the simulation are also shown.
Figure Legend Snippet: Free energy landscape of the Aβ40 peptide. The free energy landscape is shown as a function of three collective variables used in the NMR-guided metadynamics simulations: anti-parallel β-sheet content (X-axis), α-helical content (Y-axis) and number of hydrophobic contacts (or compactness, Z-axis). Isosurfaces are shown at 5 (red), 10 (blue), 18 (yellow) and 25 kJ/mol (cyan); white regions are not visited as they have higher free energies. Representative structures sampled during the simulation are also shown.

Techniques Used: Nuclear Magnetic Resonance

15) Product Images from "TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol"

Article Title: TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol

Journal: Nature Communications

doi: 10.1038/ncomms13296

Tth PrimPol is a DNA primase that can be coupled to processive elongation by Φ29DNApol. ( a ) Left panel: metal and sugar selectivity of Tth PrimPol primase activity. 3′-GTCC-5′ oligonucleotide (1 μM) was used as a preferred template. Labelled nucleotide [γ- 32 P] ATP or [α- 32 P] dATP nM) were alternatively used as 5′-nucleotide and either unlabelled GTP or dGTP (10 μM) were tested as 3′-nucleotide to form the initiating dimer. Primer synthesis mediated by Tth PrimPol (400 nM) was evaluated either with 1 mM MnCl 2 or 5 mM MgCl 2 at 55 °C during 60 min. Right panel: recognition of the priming site. The assay was as in the left panel, but using templates differing in the base preceding the primase initiation site (…X TC C…) and the indicated metal and nucleotides. ( b ) Tth PrimPol-mediated DNA primer synthesis at 30 °C occurs at multiple sites on a heterogeneous ssDNA template. Tth PrimPol (100 nM) was able to generate DNA primers on circular M13mp18 ssDNA (5 ng μl −1 ), when using four alternative combinations of dNTPs, implying that initiation occurred at multiple sites. In all cases, [α- 32 P] dGTP (16 nM) was provided to label the nascent primers, combined with either dATP, dCTP, dGTP or dTTP (1 μM), in the presence of 10 mM MgCl 2 at 30 °C during 20 min. ( c ) To evaluate the processivity of primer synthesis by Tth PrimPol, we used heparin as a competitor. Tth PrimPol (10 nM) was preincubated for 5 min on ice, either in the absence/presence of heparin (1 ng μl −1 ). Subsequently, the reaction was complemented with M13mp18 ssDNA (5 ng μl −1 ), dATP, dCTP and dTTP (10 μM each), [α- 32 P] dGTP (16 nM; 3,000 Ci mmol −1 ) and heparin (1 ng μl −1 ) when indicated and the incubation was maintained for 10 min at 30 °C, and processed as described. ( d ) Tth PrimPol-synthesized DNA primers are efficiently extended by Φ29DNApol. The contribution of each enzyme was assayed in two consecutive stages (pulse and chase), as indicated in the scheme. The pulse demonstrated the synthesis of primers with a mean size of 7–9 nt (left panel). During chase (right panel), Φ29DNApol generated high-molecular-weight primer-elongated products (detected at the top of the gel) and some degradation products (evidenced at the bottom).
Figure Legend Snippet: Tth PrimPol is a DNA primase that can be coupled to processive elongation by Φ29DNApol. ( a ) Left panel: metal and sugar selectivity of Tth PrimPol primase activity. 3′-GTCC-5′ oligonucleotide (1 μM) was used as a preferred template. Labelled nucleotide [γ- 32 P] ATP or [α- 32 P] dATP nM) were alternatively used as 5′-nucleotide and either unlabelled GTP or dGTP (10 μM) were tested as 3′-nucleotide to form the initiating dimer. Primer synthesis mediated by Tth PrimPol (400 nM) was evaluated either with 1 mM MnCl 2 or 5 mM MgCl 2 at 55 °C during 60 min. Right panel: recognition of the priming site. The assay was as in the left panel, but using templates differing in the base preceding the primase initiation site (…X TC C…) and the indicated metal and nucleotides. ( b ) Tth PrimPol-mediated DNA primer synthesis at 30 °C occurs at multiple sites on a heterogeneous ssDNA template. Tth PrimPol (100 nM) was able to generate DNA primers on circular M13mp18 ssDNA (5 ng μl −1 ), when using four alternative combinations of dNTPs, implying that initiation occurred at multiple sites. In all cases, [α- 32 P] dGTP (16 nM) was provided to label the nascent primers, combined with either dATP, dCTP, dGTP or dTTP (1 μM), in the presence of 10 mM MgCl 2 at 30 °C during 20 min. ( c ) To evaluate the processivity of primer synthesis by Tth PrimPol, we used heparin as a competitor. Tth PrimPol (10 nM) was preincubated for 5 min on ice, either in the absence/presence of heparin (1 ng μl −1 ). Subsequently, the reaction was complemented with M13mp18 ssDNA (5 ng μl −1 ), dATP, dCTP and dTTP (10 μM each), [α- 32 P] dGTP (16 nM; 3,000 Ci mmol −1 ) and heparin (1 ng μl −1 ) when indicated and the incubation was maintained for 10 min at 30 °C, and processed as described. ( d ) Tth PrimPol-synthesized DNA primers are efficiently extended by Φ29DNApol. The contribution of each enzyme was assayed in two consecutive stages (pulse and chase), as indicated in the scheme. The pulse demonstrated the synthesis of primers with a mean size of 7–9 nt (left panel). During chase (right panel), Φ29DNApol generated high-molecular-weight primer-elongated products (detected at the top of the gel) and some degradation products (evidenced at the bottom).

Techniques Used: Activity Assay, Incubation, Synthesized, Generated, Molecular Weight

Scheme of the TruePrime reaction. TruePrime reaction steps leading to amplification of (genomic) DNA. ( a ) Tth PrimPol binds to denatured DNA at different sites. ( b ) Tth PrimPol synthesizes short DNA primers. ( c ) DNA primers are recognized by Φ29DNApol, which replaces Tth PrimPol extending the primers. ( d ) DNA primer elongation coupled to strand displacement by Φ29DNApol leads to the exposure of new single-stranded template regions. ( e ) Tth PrimPol catalyses new rounds of priming on the displaced ssDNA. ( f ) New DNA primers trigger further rounds of strand-displacement synthesis, leading to exponential amplification of the target DNA.
Figure Legend Snippet: Scheme of the TruePrime reaction. TruePrime reaction steps leading to amplification of (genomic) DNA. ( a ) Tth PrimPol binds to denatured DNA at different sites. ( b ) Tth PrimPol synthesizes short DNA primers. ( c ) DNA primers are recognized by Φ29DNApol, which replaces Tth PrimPol extending the primers. ( d ) DNA primer elongation coupled to strand displacement by Φ29DNApol leads to the exposure of new single-stranded template regions. ( e ) Tth PrimPol catalyses new rounds of priming on the displaced ssDNA. ( f ) New DNA primers trigger further rounds of strand-displacement synthesis, leading to exponential amplification of the target DNA.

Techniques Used: Amplification

Multiple amino acid sequence alignment of the closest Tth PrimPol orthologues. The first block of sequences corresponds to Thermales ( Thermus and Meiothermus ) and the second block includes Si /pRN1 PrimPol and some other putative bifunctional primases/polymerases from bacteria, archaea and phage; in addition, plasmidic Rep (a potential PrimPol) and Bc MCM PrimPol were included. Numbers in parentheses indicate the number of amino acid residues not shown. Invariant or conserved residues among Thermales (first set of sequences) were labelled in red and blue letters, respectively. Identity matches in the second set of sequences were equally coloured. The alignment defines several conserved regions, including the highly conserved motifs A, B and C (boxed in yellow), characteristic of AEP-like primases. Experimentally determined secondary structure elements in Si /pRN1 PrimPol are indicated above pRN1 sequence (α-helices, lettered cylinders; green) and β-strands (numbered arrows; orange). Modelled secondary structure elements in Tth PrimPol are tentatively depicted above the Tth PrimPol sequence (see also Fig 1b). The corresponding aligned regions are boxed in the same colours to emphasize structural conservation between Tth PrimPol and the AEP core of pRN1. The C-terminal region of Tth PrimPol, conserved in other Thermales (boxed in grey), aligns with the PriCT-1 domain of Rep and Eph primases; this region is not yet crystallized in pRN1 and it has been described as pRN1_helical 29 . Dots indicate invariant residues acting either as metal (red), nucleotide (purple) or Zn (magenta) ligands. Selection of the closest Tth PrimPol homologues and multiple alignment of their amino acid sequence were initially performed with the BLAST programme and further adjusted manually to maximize similarities with the structured regions of pRN1 PrimPol. Nomenclature: YP_004631.1 T. thermophilus ( Tth ); YP_006971229.1 Thermus oshimai ( Tos ); WP_003046664.1 Thermus aquaticus ( Taq ); YP_004202830.1 Thermus scotoductus ( Tsc 1); YP_004202855.1 T. scotoductus ( Tsc 2); ETN89075.1 Thermus sp ( Tsp ); YP_003508539.1 Meiothermus ruber ( Mru ); YP_003684976.1 Meiothermus silvanus ( Msi 1); YP_003684747.1 M. silvanus ( Msi 2); AAC44111.1 plasmid pRN1 ORF904 from S. islandicus (pRN1); YP_502469.1 Methanospirillum hungatei ( Mhu ); YP_006262572.1 Deinococcus gobiensis ( Dgo ); YP_002829910.1 S. islandicus ( Sis ); YP_003357218.1 Methanocella paludícola ( Mpa ); AFO10831.1 Enterococcus phage EfaCPT1 (Eph); YP_009074444.1 Shigella sonnei Rep protein from plasmid ColE4-CT9 (Rep); WP_044797243 PrimPol-helicase from B. cereus ( Bc MCM).
Figure Legend Snippet: Multiple amino acid sequence alignment of the closest Tth PrimPol orthologues. The first block of sequences corresponds to Thermales ( Thermus and Meiothermus ) and the second block includes Si /pRN1 PrimPol and some other putative bifunctional primases/polymerases from bacteria, archaea and phage; in addition, plasmidic Rep (a potential PrimPol) and Bc MCM PrimPol were included. Numbers in parentheses indicate the number of amino acid residues not shown. Invariant or conserved residues among Thermales (first set of sequences) were labelled in red and blue letters, respectively. Identity matches in the second set of sequences were equally coloured. The alignment defines several conserved regions, including the highly conserved motifs A, B and C (boxed in yellow), characteristic of AEP-like primases. Experimentally determined secondary structure elements in Si /pRN1 PrimPol are indicated above pRN1 sequence (α-helices, lettered cylinders; green) and β-strands (numbered arrows; orange). Modelled secondary structure elements in Tth PrimPol are tentatively depicted above the Tth PrimPol sequence (see also Fig 1b). The corresponding aligned regions are boxed in the same colours to emphasize structural conservation between Tth PrimPol and the AEP core of pRN1. The C-terminal region of Tth PrimPol, conserved in other Thermales (boxed in grey), aligns with the PriCT-1 domain of Rep and Eph primases; this region is not yet crystallized in pRN1 and it has been described as pRN1_helical 29 . Dots indicate invariant residues acting either as metal (red), nucleotide (purple) or Zn (magenta) ligands. Selection of the closest Tth PrimPol homologues and multiple alignment of their amino acid sequence were initially performed with the BLAST programme and further adjusted manually to maximize similarities with the structured regions of pRN1 PrimPol. Nomenclature: YP_004631.1 T. thermophilus ( Tth ); YP_006971229.1 Thermus oshimai ( Tos ); WP_003046664.1 Thermus aquaticus ( Taq ); YP_004202830.1 Thermus scotoductus ( Tsc 1); YP_004202855.1 T. scotoductus ( Tsc 2); ETN89075.1 Thermus sp ( Tsp ); YP_003508539.1 Meiothermus ruber ( Mru ); YP_003684976.1 Meiothermus silvanus ( Msi 1); YP_003684747.1 M. silvanus ( Msi 2); AAC44111.1 plasmid pRN1 ORF904 from S. islandicus (pRN1); YP_502469.1 Methanospirillum hungatei ( Mhu ); YP_006262572.1 Deinococcus gobiensis ( Dgo ); YP_002829910.1 S. islandicus ( Sis ); YP_003357218.1 Methanocella paludícola ( Mpa ); AFO10831.1 Enterococcus phage EfaCPT1 (Eph); YP_009074444.1 Shigella sonnei Rep protein from plasmid ColE4-CT9 (Rep); WP_044797243 PrimPol-helicase from B. cereus ( Bc MCM).

Techniques Used: Sequencing, Blocking Assay, Selection, Plasmid Preparation

Tth PrimPol-mediated MDA (TruePrime) is able to efficiently amplify circular and linear DNA molecules. ( a ) Tth PrimPol can be used instead of RPs to trigger MDA by Φ29DNApol. Combination of both enzymes (TruePrime) is able to proficiently amplify single-stranded M13mp1 circular DNA (100 fg), unlike the combination of Φ29DNApol plus human PrimPol. Individual addition of Φ29DNApol or Tth PrimPol does not lead to amplification of input DNA. Non-template controls (NTC) are included, to ensure the lack of background amplification in the absence of input DNA. Error bars are s.d. ( b ) Amplification of human genomic DNA (6 pg; the equivalent amount contained in a single human cell) by combination of Tth PrimPol and Φ29DNApol. Hs PrimPol again failed to amplify DNA in cooperation with Φ29DNApol, highlighting the requirements of the specific primase features of Tth PrimPol. Error bars are s.d. ( c ) Agarose gel image showing the similar high size distribution of the amplified fragments obtained with TruePrime versus RPs in part B. ( d ) Superior sensitivity of Tth PrimPol-mediated MDA (TruePrime) for the amplification of human genomic DNA (efficient with a DNA input as low as 1 fg), having about 100-fold higher sensitivity than RP-mediated MDA. Error bars are s.d.
Figure Legend Snippet: Tth PrimPol-mediated MDA (TruePrime) is able to efficiently amplify circular and linear DNA molecules. ( a ) Tth PrimPol can be used instead of RPs to trigger MDA by Φ29DNApol. Combination of both enzymes (TruePrime) is able to proficiently amplify single-stranded M13mp1 circular DNA (100 fg), unlike the combination of Φ29DNApol plus human PrimPol. Individual addition of Φ29DNApol or Tth PrimPol does not lead to amplification of input DNA. Non-template controls (NTC) are included, to ensure the lack of background amplification in the absence of input DNA. Error bars are s.d. ( b ) Amplification of human genomic DNA (6 pg; the equivalent amount contained in a single human cell) by combination of Tth PrimPol and Φ29DNApol. Hs PrimPol again failed to amplify DNA in cooperation with Φ29DNApol, highlighting the requirements of the specific primase features of Tth PrimPol. Error bars are s.d. ( c ) Agarose gel image showing the similar high size distribution of the amplified fragments obtained with TruePrime versus RPs in part B. ( d ) Superior sensitivity of Tth PrimPol-mediated MDA (TruePrime) for the amplification of human genomic DNA (efficient with a DNA input as low as 1 fg), having about 100-fold higher sensitivity than RP-mediated MDA. Error bars are s.d.

Techniques Used: Multiple Displacement Amplification, Amplification, Agarose Gel Electrophoresis

A putative PrimPol in T. thermophilus . ( a ) Modular organization of various AEP-like enzymes. A conserved AEP domain (green bar) contains the three conserved regions A, B and C forming the primase active site. Unlike conventional primases as Hs Prim1, PrimPols frequently have a Zn-finger-containing region ( Hs PrimPol) or even a helicase domain ( Bc MCM PrimPol; Si /pRN1 PrimPol). A putative AEP-like enzyme in T. thermophilus lacks both Zn finger and helicase domain; however, its C-terminal domain contains a PriCT-1 domain characteristic of some prokaryotic primases, also shared by Bc MCM and Si /pRN1 PrimPols (see later in b ). Nomenclature: small catalytic subunit of the human RNA primase ( Hs Prim1); human PrimPol ( Hs PrimPol); PrimPol-helicase from Bacillus cereus ( Bc MCM); plasmid pRN1 ORF904 from Sulfolobus islandicus ( Si /pRN1 PrimPol); putative PrimPol from T. thermophilus ( Tth PrimPol). ( b ) 3D structure of Tth PrimPol. The computer-modelled crystal structure of Tth PrimPol (amino acids 4–208 modelled as described in Methods) is depicted in ribbon format by using the graphic program PyMol. α-Helices are green (lettered), β-strands are orange (numbered) and intervening loop regions are grey; metal ligands (Asp70, Asp72 and Asp123) are shown in red; dNTP ligand (His101) is shown in purple; DNA template (dark purple) and primer (blue) strands, activating metals (grey spheres) and incoming nucleotide (cyan) are derived from 3D structures of M. tuberculosis PolDom Ligase D (4MKY and 3PKY).
Figure Legend Snippet: A putative PrimPol in T. thermophilus . ( a ) Modular organization of various AEP-like enzymes. A conserved AEP domain (green bar) contains the three conserved regions A, B and C forming the primase active site. Unlike conventional primases as Hs Prim1, PrimPols frequently have a Zn-finger-containing region ( Hs PrimPol) or even a helicase domain ( Bc MCM PrimPol; Si /pRN1 PrimPol). A putative AEP-like enzyme in T. thermophilus lacks both Zn finger and helicase domain; however, its C-terminal domain contains a PriCT-1 domain characteristic of some prokaryotic primases, also shared by Bc MCM and Si /pRN1 PrimPols (see later in b ). Nomenclature: small catalytic subunit of the human RNA primase ( Hs Prim1); human PrimPol ( Hs PrimPol); PrimPol-helicase from Bacillus cereus ( Bc MCM); plasmid pRN1 ORF904 from Sulfolobus islandicus ( Si /pRN1 PrimPol); putative PrimPol from T. thermophilus ( Tth PrimPol). ( b ) 3D structure of Tth PrimPol. The computer-modelled crystal structure of Tth PrimPol (amino acids 4–208 modelled as described in Methods) is depicted in ribbon format by using the graphic program PyMol. α-Helices are green (lettered), β-strands are orange (numbered) and intervening loop regions are grey; metal ligands (Asp70, Asp72 and Asp123) are shown in red; dNTP ligand (His101) is shown in purple; DNA template (dark purple) and primer (blue) strands, activating metals (grey spheres) and incoming nucleotide (cyan) are derived from 3D structures of M. tuberculosis PolDom Ligase D (4MKY and 3PKY).

Techniques Used: Plasmid Preparation, Derivative Assay

16) Product Images from "TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol"

Article Title: TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol

Journal: Nature Communications

doi: 10.1038/ncomms13296

Tth PrimPol is a DNA primase that can be coupled to processive elongation by Φ29DNApol. ( a ) Left panel: metal and sugar selectivity of Tth PrimPol primase activity. 3′-GTCC-5′ oligonucleotide (1 μM) was used as a preferred template. Labelled nucleotide [γ- 32 P] ATP or [α- 32 P] dATP nM) were alternatively used as 5′-nucleotide and either unlabelled GTP or dGTP (10 μM) were tested as 3′-nucleotide to form the initiating dimer. Primer synthesis mediated by Tth PrimPol (400 nM) was evaluated either with 1 mM MnCl 2 or 5 mM MgCl 2 at 55 °C during 60 min. Right panel: recognition of the priming site. The assay was as in the left panel, but using templates differing in the base preceding the primase initiation site (…X TC C…) and the indicated metal and nucleotides. ( b ) Tth PrimPol-mediated DNA primer synthesis at 30 °C occurs at multiple sites on a heterogeneous ssDNA template. Tth PrimPol (100 nM) was able to generate DNA primers on circular M13mp18 ssDNA (5 ng μl −1 ), when using four alternative combinations of dNTPs, implying that initiation occurred at multiple sites. In all cases, [α- 32 P] dGTP (16 nM) was provided to label the nascent primers, combined with either dATP, dCTP, dGTP or dTTP (1 μM), in the presence of 10 mM MgCl 2 at 30 °C during 20 min. ( c ) To evaluate the processivity of primer synthesis by Tth PrimPol, we used heparin as a competitor. Tth PrimPol (10 nM) was preincubated for 5 min on ice, either in the absence/presence of heparin (1 ng μl −1 ). Subsequently, the reaction was complemented with M13mp18 ssDNA (5 ng μl −1 ), dATP, dCTP and dTTP (10 μM each), [α- 32 P] dGTP (16 nM; 3,000 Ci mmol −1 ) and heparin (1 ng μl −1 ) when indicated and the incubation was maintained for 10 min at 30 °C, and processed as described. ( d ) Tth PrimPol-synthesized DNA primers are efficiently extended by Φ29DNApol. The contribution of each enzyme was assayed in two consecutive stages (pulse and chase), as indicated in the scheme. The pulse demonstrated the synthesis of primers with a mean size of 7–9 nt (left panel). During chase (right panel), Φ29DNApol generated high-molecular-weight primer-elongated products (detected at the top of the gel) and some degradation products (evidenced at the bottom).
Figure Legend Snippet: Tth PrimPol is a DNA primase that can be coupled to processive elongation by Φ29DNApol. ( a ) Left panel: metal and sugar selectivity of Tth PrimPol primase activity. 3′-GTCC-5′ oligonucleotide (1 μM) was used as a preferred template. Labelled nucleotide [γ- 32 P] ATP or [α- 32 P] dATP nM) were alternatively used as 5′-nucleotide and either unlabelled GTP or dGTP (10 μM) were tested as 3′-nucleotide to form the initiating dimer. Primer synthesis mediated by Tth PrimPol (400 nM) was evaluated either with 1 mM MnCl 2 or 5 mM MgCl 2 at 55 °C during 60 min. Right panel: recognition of the priming site. The assay was as in the left panel, but using templates differing in the base preceding the primase initiation site (…X TC C…) and the indicated metal and nucleotides. ( b ) Tth PrimPol-mediated DNA primer synthesis at 30 °C occurs at multiple sites on a heterogeneous ssDNA template. Tth PrimPol (100 nM) was able to generate DNA primers on circular M13mp18 ssDNA (5 ng μl −1 ), when using four alternative combinations of dNTPs, implying that initiation occurred at multiple sites. In all cases, [α- 32 P] dGTP (16 nM) was provided to label the nascent primers, combined with either dATP, dCTP, dGTP or dTTP (1 μM), in the presence of 10 mM MgCl 2 at 30 °C during 20 min. ( c ) To evaluate the processivity of primer synthesis by Tth PrimPol, we used heparin as a competitor. Tth PrimPol (10 nM) was preincubated for 5 min on ice, either in the absence/presence of heparin (1 ng μl −1 ). Subsequently, the reaction was complemented with M13mp18 ssDNA (5 ng μl −1 ), dATP, dCTP and dTTP (10 μM each), [α- 32 P] dGTP (16 nM; 3,000 Ci mmol −1 ) and heparin (1 ng μl −1 ) when indicated and the incubation was maintained for 10 min at 30 °C, and processed as described. ( d ) Tth PrimPol-synthesized DNA primers are efficiently extended by Φ29DNApol. The contribution of each enzyme was assayed in two consecutive stages (pulse and chase), as indicated in the scheme. The pulse demonstrated the synthesis of primers with a mean size of 7–9 nt (left panel). During chase (right panel), Φ29DNApol generated high-molecular-weight primer-elongated products (detected at the top of the gel) and some degradation products (evidenced at the bottom).

Techniques Used: Activity Assay, Incubation, Synthesized, Generated, Molecular Weight

Scheme of the TruePrime reaction. TruePrime reaction steps leading to amplification of (genomic) DNA. ( a ) Tth PrimPol binds to denatured DNA at different sites. ( b ) Tth PrimPol synthesizes short DNA primers. ( c ) DNA primers are recognized by Φ29DNApol, which replaces Tth PrimPol extending the primers. ( d ) DNA primer elongation coupled to strand displacement by Φ29DNApol leads to the exposure of new single-stranded template regions. ( e ) Tth PrimPol catalyses new rounds of priming on the displaced ssDNA. ( f ) New DNA primers trigger further rounds of strand-displacement synthesis, leading to exponential amplification of the target DNA.
Figure Legend Snippet: Scheme of the TruePrime reaction. TruePrime reaction steps leading to amplification of (genomic) DNA. ( a ) Tth PrimPol binds to denatured DNA at different sites. ( b ) Tth PrimPol synthesizes short DNA primers. ( c ) DNA primers are recognized by Φ29DNApol, which replaces Tth PrimPol extending the primers. ( d ) DNA primer elongation coupled to strand displacement by Φ29DNApol leads to the exposure of new single-stranded template regions. ( e ) Tth PrimPol catalyses new rounds of priming on the displaced ssDNA. ( f ) New DNA primers trigger further rounds of strand-displacement synthesis, leading to exponential amplification of the target DNA.

Techniques Used: Amplification

Multiple amino acid sequence alignment of the closest Tth PrimPol orthologues. The first block of sequences corresponds to Thermales ( Thermus and Meiothermus ) and the second block includes Si /pRN1 PrimPol and some other putative bifunctional primases/polymerases from bacteria, archaea and phage; in addition, plasmidic Rep (a potential PrimPol) and Bc MCM PrimPol were included. Numbers in parentheses indicate the number of amino acid residues not shown. Invariant or conserved residues among Thermales (first set of sequences) were labelled in red and blue letters, respectively. Identity matches in the second set of sequences were equally coloured. The alignment defines several conserved regions, including the highly conserved motifs A, B and C (boxed in yellow), characteristic of AEP-like primases. Experimentally determined secondary structure elements in Si /pRN1 PrimPol are indicated above pRN1 sequence (α-helices, lettered cylinders; green) and β-strands (numbered arrows; orange). Modelled secondary structure elements in Tth PrimPol are tentatively depicted above the Tth PrimPol sequence (see also Fig 1b). The corresponding aligned regions are boxed in the same colours to emphasize structural conservation between Tth PrimPol and the AEP core of pRN1. The C-terminal region of Tth PrimPol, conserved in other Thermales (boxed in grey), aligns with the PriCT-1 domain of Rep and Eph primases; this region is not yet crystallized in pRN1 and it has been described as pRN1_helical 29 . Dots indicate invariant residues acting either as metal (red), nucleotide (purple) or Zn (magenta) ligands. Selection of the closest Tth PrimPol homologues and multiple alignment of their amino acid sequence were initially performed with the BLAST programme and further adjusted manually to maximize similarities with the structured regions of pRN1 PrimPol. Nomenclature: YP_004631.1 T. thermophilus ( Tth ); YP_006971229.1 Thermus oshimai ( Tos ); WP_003046664.1 Thermus aquaticus ( Taq ); YP_004202830.1 Thermus scotoductus ( Tsc 1); YP_004202855.1 T. scotoductus ( Tsc 2); ETN89075.1 Thermus sp ( Tsp ); YP_003508539.1 Meiothermus ruber ( Mru ); YP_003684976.1 Meiothermus silvanus ( Msi 1); YP_003684747.1 M. silvanus ( Msi 2); AAC44111.1 plasmid pRN1 ORF904 from S. islandicus (pRN1); YP_502469.1 Methanospirillum hungatei ( Mhu ); YP_006262572.1 Deinococcus gobiensis ( Dgo ); YP_002829910.1 S. islandicus ( Sis ); YP_003357218.1 Methanocella paludícola ( Mpa ); AFO10831.1 Enterococcus phage EfaCPT1 (Eph); YP_009074444.1 Shigella sonnei Rep protein from plasmid ColE4-CT9 (Rep); WP_044797243 PrimPol-helicase from B. cereus ( Bc MCM).
Figure Legend Snippet: Multiple amino acid sequence alignment of the closest Tth PrimPol orthologues. The first block of sequences corresponds to Thermales ( Thermus and Meiothermus ) and the second block includes Si /pRN1 PrimPol and some other putative bifunctional primases/polymerases from bacteria, archaea and phage; in addition, plasmidic Rep (a potential PrimPol) and Bc MCM PrimPol were included. Numbers in parentheses indicate the number of amino acid residues not shown. Invariant or conserved residues among Thermales (first set of sequences) were labelled in red and blue letters, respectively. Identity matches in the second set of sequences were equally coloured. The alignment defines several conserved regions, including the highly conserved motifs A, B and C (boxed in yellow), characteristic of AEP-like primases. Experimentally determined secondary structure elements in Si /pRN1 PrimPol are indicated above pRN1 sequence (α-helices, lettered cylinders; green) and β-strands (numbered arrows; orange). Modelled secondary structure elements in Tth PrimPol are tentatively depicted above the Tth PrimPol sequence (see also Fig 1b). The corresponding aligned regions are boxed in the same colours to emphasize structural conservation between Tth PrimPol and the AEP core of pRN1. The C-terminal region of Tth PrimPol, conserved in other Thermales (boxed in grey), aligns with the PriCT-1 domain of Rep and Eph primases; this region is not yet crystallized in pRN1 and it has been described as pRN1_helical 29 . Dots indicate invariant residues acting either as metal (red), nucleotide (purple) or Zn (magenta) ligands. Selection of the closest Tth PrimPol homologues and multiple alignment of their amino acid sequence were initially performed with the BLAST programme and further adjusted manually to maximize similarities with the structured regions of pRN1 PrimPol. Nomenclature: YP_004631.1 T. thermophilus ( Tth ); YP_006971229.1 Thermus oshimai ( Tos ); WP_003046664.1 Thermus aquaticus ( Taq ); YP_004202830.1 Thermus scotoductus ( Tsc 1); YP_004202855.1 T. scotoductus ( Tsc 2); ETN89075.1 Thermus sp ( Tsp ); YP_003508539.1 Meiothermus ruber ( Mru ); YP_003684976.1 Meiothermus silvanus ( Msi 1); YP_003684747.1 M. silvanus ( Msi 2); AAC44111.1 plasmid pRN1 ORF904 from S. islandicus (pRN1); YP_502469.1 Methanospirillum hungatei ( Mhu ); YP_006262572.1 Deinococcus gobiensis ( Dgo ); YP_002829910.1 S. islandicus ( Sis ); YP_003357218.1 Methanocella paludícola ( Mpa ); AFO10831.1 Enterococcus phage EfaCPT1 (Eph); YP_009074444.1 Shigella sonnei Rep protein from plasmid ColE4-CT9 (Rep); WP_044797243 PrimPol-helicase from B. cereus ( Bc MCM).

Techniques Used: Sequencing, Blocking Assay, Selection, Plasmid Preparation

Tth PrimPol-mediated MDA (TruePrime) is able to efficiently amplify circular and linear DNA molecules. ( a ) Tth PrimPol can be used instead of RPs to trigger MDA by Φ29DNApol. Combination of both enzymes (TruePrime) is able to proficiently amplify single-stranded M13mp1 circular DNA (100 fg), unlike the combination of Φ29DNApol plus human PrimPol. Individual addition of Φ29DNApol or Tth PrimPol does not lead to amplification of input DNA. Non-template controls (NTC) are included, to ensure the lack of background amplification in the absence of input DNA. Error bars are s.d. ( b ) Amplification of human genomic DNA (6 pg; the equivalent amount contained in a single human cell) by combination of Tth PrimPol and Φ29DNApol. Hs PrimPol again failed to amplify DNA in cooperation with Φ29DNApol, highlighting the requirements of the specific primase features of Tth PrimPol. Error bars are s.d. ( c ) Agarose gel image showing the similar high size distribution of the amplified fragments obtained with TruePrime versus RPs in part B. ( d ) Superior sensitivity of Tth PrimPol-mediated MDA (TruePrime) for the amplification of human genomic DNA (efficient with a DNA input as low as 1 fg), having about 100-fold higher sensitivity than RP-mediated MDA. Error bars are s.d.
Figure Legend Snippet: Tth PrimPol-mediated MDA (TruePrime) is able to efficiently amplify circular and linear DNA molecules. ( a ) Tth PrimPol can be used instead of RPs to trigger MDA by Φ29DNApol. Combination of both enzymes (TruePrime) is able to proficiently amplify single-stranded M13mp1 circular DNA (100 fg), unlike the combination of Φ29DNApol plus human PrimPol. Individual addition of Φ29DNApol or Tth PrimPol does not lead to amplification of input DNA. Non-template controls (NTC) are included, to ensure the lack of background amplification in the absence of input DNA. Error bars are s.d. ( b ) Amplification of human genomic DNA (6 pg; the equivalent amount contained in a single human cell) by combination of Tth PrimPol and Φ29DNApol. Hs PrimPol again failed to amplify DNA in cooperation with Φ29DNApol, highlighting the requirements of the specific primase features of Tth PrimPol. Error bars are s.d. ( c ) Agarose gel image showing the similar high size distribution of the amplified fragments obtained with TruePrime versus RPs in part B. ( d ) Superior sensitivity of Tth PrimPol-mediated MDA (TruePrime) for the amplification of human genomic DNA (efficient with a DNA input as low as 1 fg), having about 100-fold higher sensitivity than RP-mediated MDA. Error bars are s.d.

Techniques Used: Multiple Displacement Amplification, Amplification, Agarose Gel Electrophoresis

A putative PrimPol in T. thermophilus . ( a ) Modular organization of various AEP-like enzymes. A conserved AEP domain (green bar) contains the three conserved regions A, B and C forming the primase active site. Unlike conventional primases as Hs Prim1, PrimPols frequently have a Zn-finger-containing region ( Hs PrimPol) or even a helicase domain ( Bc MCM PrimPol; Si /pRN1 PrimPol). A putative AEP-like enzyme in T. thermophilus lacks both Zn finger and helicase domain; however, its C-terminal domain contains a PriCT-1 domain characteristic of some prokaryotic primases, also shared by Bc MCM and Si /pRN1 PrimPols (see later in b ). Nomenclature: small catalytic subunit of the human RNA primase ( Hs Prim1); human PrimPol ( Hs PrimPol); PrimPol-helicase from Bacillus cereus ( Bc MCM); plasmid pRN1 ORF904 from Sulfolobus islandicus ( Si /pRN1 PrimPol); putative PrimPol from T. thermophilus ( Tth PrimPol). ( b ) 3D structure of Tth PrimPol. The computer-modelled crystal structure of Tth PrimPol (amino acids 4–208 modelled as described in Methods) is depicted in ribbon format by using the graphic program PyMol. α-Helices are green (lettered), β-strands are orange (numbered) and intervening loop regions are grey; metal ligands (Asp70, Asp72 and Asp123) are shown in red; dNTP ligand (His101) is shown in purple; DNA template (dark purple) and primer (blue) strands, activating metals (grey spheres) and incoming nucleotide (cyan) are derived from 3D structures of M. tuberculosis PolDom Ligase D (4MKY and 3PKY).
Figure Legend Snippet: A putative PrimPol in T. thermophilus . ( a ) Modular organization of various AEP-like enzymes. A conserved AEP domain (green bar) contains the three conserved regions A, B and C forming the primase active site. Unlike conventional primases as Hs Prim1, PrimPols frequently have a Zn-finger-containing region ( Hs PrimPol) or even a helicase domain ( Bc MCM PrimPol; Si /pRN1 PrimPol). A putative AEP-like enzyme in T. thermophilus lacks both Zn finger and helicase domain; however, its C-terminal domain contains a PriCT-1 domain characteristic of some prokaryotic primases, also shared by Bc MCM and Si /pRN1 PrimPols (see later in b ). Nomenclature: small catalytic subunit of the human RNA primase ( Hs Prim1); human PrimPol ( Hs PrimPol); PrimPol-helicase from Bacillus cereus ( Bc MCM); plasmid pRN1 ORF904 from Sulfolobus islandicus ( Si /pRN1 PrimPol); putative PrimPol from T. thermophilus ( Tth PrimPol). ( b ) 3D structure of Tth PrimPol. The computer-modelled crystal structure of Tth PrimPol (amino acids 4–208 modelled as described in Methods) is depicted in ribbon format by using the graphic program PyMol. α-Helices are green (lettered), β-strands are orange (numbered) and intervening loop regions are grey; metal ligands (Asp70, Asp72 and Asp123) are shown in red; dNTP ligand (His101) is shown in purple; DNA template (dark purple) and primer (blue) strands, activating metals (grey spheres) and incoming nucleotide (cyan) are derived from 3D structures of M. tuberculosis PolDom Ligase D (4MKY and 3PKY).

Techniques Used: Plasmid Preparation, Derivative Assay

17) Product Images from "Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin"

Article Title: Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0160980

Fusion proteins enhanced cytotoxic outcome and delayed tumor development. (A) MDA-MB-231 and MCF-7 cells were treated with different concentrations of RL2, fusion proteins or saline (control) for 48 h and MTT analysis was performed. Tumor cell viability was determined relative to the viability of the control cells (incubated without the proteins). Data are presented as mean ± SD. (B) SCID mice with subcutaneously grafted MDA-MB-231 tumors, which reached an average (in the group) size of 20 ± 10 mm 3 , were subjected to intravenous injection of fusion proteins (RL2, T3-RL2, RL2-iRGD or RL-iRGD-His) at a dose of 40 mg/kg in saline three times every second day. The control group of mice received saline with the same mode of as administration the proteins. Tumors were excised and weighed on the 17th day after the last injection. Boxes represent the 25th, 50th and 75th percentiles. Squares with lines represent the median. Whiskers represent minimum/maximum. Data were statistically analyzed using one-way ANOVA with post hoc Fisher test; a p value
Figure Legend Snippet: Fusion proteins enhanced cytotoxic outcome and delayed tumor development. (A) MDA-MB-231 and MCF-7 cells were treated with different concentrations of RL2, fusion proteins or saline (control) for 48 h and MTT analysis was performed. Tumor cell viability was determined relative to the viability of the control cells (incubated without the proteins). Data are presented as mean ± SD. (B) SCID mice with subcutaneously grafted MDA-MB-231 tumors, which reached an average (in the group) size of 20 ± 10 mm 3 , were subjected to intravenous injection of fusion proteins (RL2, T3-RL2, RL2-iRGD or RL-iRGD-His) at a dose of 40 mg/kg in saline three times every second day. The control group of mice received saline with the same mode of as administration the proteins. Tumors were excised and weighed on the 17th day after the last injection. Boxes represent the 25th, 50th and 75th percentiles. Squares with lines represent the median. Whiskers represent minimum/maximum. Data were statistically analyzed using one-way ANOVA with post hoc Fisher test; a p value

Techniques Used: Multiple Displacement Amplification, MTT Assay, Incubation, Mouse Assay, Injection

18) Product Images from "Substrate Distortion and the Catalytic Reaction Mechanism of 5-Carboxyvanillate Decarboxylase"

Article Title: Substrate Distortion and the Catalytic Reaction Mechanism of 5-Carboxyvanillate Decarboxylase

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.5b08251

Geometric distortion of 5-NV in the active site of LigW from N. aromaticivorans (PDB id: 4QRN). (a) 5-NV structure shown as a stick model and the 2FoFc map (contour level σ = 3.0, red colored) is drawn around it. Carbon atoms are green, oxygen red and nitrogen blue. (b) Superposition of coordinates for 5-NV bound to LigW (atoms are colored) and the small molecule 5-NV 0.96 Å structure (all atoms are gray). (c) Distortion of enzyme-bound 5-NV, the planes are drawn through sets of atoms forming different planar groups: the ring plane (green; C1, C2, C6, carboxylate), the hydroxyl group plane (yellow; C3, C4, C5, methoxy group), and the nitro group plane (cyan; C5 and the nitro group).
Figure Legend Snippet: Geometric distortion of 5-NV in the active site of LigW from N. aromaticivorans (PDB id: 4QRN). (a) 5-NV structure shown as a stick model and the 2FoFc map (contour level σ = 3.0, red colored) is drawn around it. Carbon atoms are green, oxygen red and nitrogen blue. (b) Superposition of coordinates for 5-NV bound to LigW (atoms are colored) and the small molecule 5-NV 0.96 Å structure (all atoms are gray). (c) Distortion of enzyme-bound 5-NV, the planes are drawn through sets of atoms forming different planar groups: the ring plane (green; C1, C2, C6, carboxylate), the hydroxyl group plane (yellow; C3, C4, C5, methoxy group), and the nitro group plane (cyan; C5 and the nitro group).

Techniques Used:

(a) 2FoFc map at a contour level of σ = 3.0 is shown with 5-NV (the carbon atoms are colored in magenta), selected amino acid side chains, and Mn 2+ (the map contour level is σ = 5.0) in the active site of LigW from N. aromaticivorans . (b) Mn 2+ and 5-NV coordination in the active site. The distances are in Angstroms. (c) The “distorted” conformation of the inhibitor 5-NV (all atoms are colored magenta) represents a “perfect” fit in the LigW active site pocket (PDB id: 4QRN). The amino acid side chains are shown as stick models and the semitransparent van der Waals spheres. Oxygen atoms are red, nitrogen atoms are blue, manganese is drawn as a gray nonbonded sphere, metal coordination bonds are shown as dotted gray lines. Hydrogen atoms were omitted for clarity.
Figure Legend Snippet: (a) 2FoFc map at a contour level of σ = 3.0 is shown with 5-NV (the carbon atoms are colored in magenta), selected amino acid side chains, and Mn 2+ (the map contour level is σ = 5.0) in the active site of LigW from N. aromaticivorans . (b) Mn 2+ and 5-NV coordination in the active site. The distances are in Angstroms. (c) The “distorted” conformation of the inhibitor 5-NV (all atoms are colored magenta) represents a “perfect” fit in the LigW active site pocket (PDB id: 4QRN). The amino acid side chains are shown as stick models and the semitransparent van der Waals spheres. Oxygen atoms are red, nitrogen atoms are blue, manganese is drawn as a gray nonbonded sphere, metal coordination bonds are shown as dotted gray lines. Hydrogen atoms were omitted for clarity.

Techniques Used:

19) Product Images from "Identification of white campion (Silene latifolia) guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction"

Article Title: Identification of white campion (Silene latifolia) guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-12-158

Purification of SlGOMT1 and SlGOMT2. Both GOMT1 and GOMT2 were purified by Ni 2+ affinity chromatography and separated by SDS-PAGE. Lane 1 shows the soluble crude bacterial extract (~10 μg) and lane 2 shows the purified protein (~1 μg). Molecular weight markers are shown on the left.
Figure Legend Snippet: Purification of SlGOMT1 and SlGOMT2. Both GOMT1 and GOMT2 were purified by Ni 2+ affinity chromatography and separated by SDS-PAGE. Lane 1 shows the soluble crude bacterial extract (~10 μg) and lane 2 shows the purified protein (~1 μg). Molecular weight markers are shown on the left.

Techniques Used: Purification, Affinity Chromatography, SDS Page, Molecular Weight

A representative GC chromatogram showing the conversion of guaiacol to veratrole by SlGOMT1. A desalted crude extract from E. coli cells expressing SlGOMT1 was supplied with guaiacol and SAM. Volatile compounds were collected and analyzed as described in methods section.
Figure Legend Snippet: A representative GC chromatogram showing the conversion of guaiacol to veratrole by SlGOMT1. A desalted crude extract from E. coli cells expressing SlGOMT1 was supplied with guaiacol and SAM. Volatile compounds were collected and analyzed as described in methods section.

Techniques Used: Expressing

Velocity versus substrate curves for SlGOMT1 (closed circles) and SlGOMT2 (open circles) with increasing amounts of guaiacol.
Figure Legend Snippet: Velocity versus substrate curves for SlGOMT1 (closed circles) and SlGOMT2 (open circles) with increasing amounts of guaiacol.

Techniques Used:

Alignment of SlGOMT1 ( Silene latifolia ), SlGOMT2 ( Silene latifolia ), SdOMT1 ( Silene dioica ), and SdOMT2 ( Silene dioica ). White letters on black background show identical amino acids of at least five sequences. One amino acid (in yellow background) that is involved in the difference of catalytic efficiency is highlighted . Three conserved SAM-binding domains are also shown in red blocks. GenBank accession numbers are as follows: RhOOMT1 ( Rosa hybrida , NCBI: AF502433), RhOOMT2 ( Rosa hybrida , NCBI:AF502434), VvROMT ( Vitis vinifera , NCBI:CAQ76879), ObEOMT ( Ocimum basilicum , NCBI:AF435008), and CTOMT1 ( Solanum lycopersicum , sol genomics network:SGN-U582403).
Figure Legend Snippet: Alignment of SlGOMT1 ( Silene latifolia ), SlGOMT2 ( Silene latifolia ), SdOMT1 ( Silene dioica ), and SdOMT2 ( Silene dioica ). White letters on black background show identical amino acids of at least five sequences. One amino acid (in yellow background) that is involved in the difference of catalytic efficiency is highlighted . Three conserved SAM-binding domains are also shown in red blocks. GenBank accession numbers are as follows: RhOOMT1 ( Rosa hybrida , NCBI: AF502433), RhOOMT2 ( Rosa hybrida , NCBI:AF502434), VvROMT ( Vitis vinifera , NCBI:CAQ76879), ObEOMT ( Ocimum basilicum , NCBI:AF435008), and CTOMT1 ( Solanum lycopersicum , sol genomics network:SGN-U582403).

Techniques Used: Binding Assay

20) Product Images from "Identification of white campion (Silene latifolia) guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction"

Article Title: Identification of white campion (Silene latifolia) guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-12-158

Alignment of SlGOMT1 ( Silene latifolia ), SlGOMT2 ( Silene latifolia ), SdOMT1 ( Silene dioica ), and SdOMT2 ( Silene dioica ). White letters on black background show identical amino acids of at least five sequences. One amino acid (in yellow background) that is involved in the difference of catalytic efficiency is highlighted . Three conserved SAM-binding domains are also shown in red blocks. GenBank accession numbers are as follows: RhOOMT1 ( Rosa hybrida , NCBI: AF502433), RhOOMT2 ( Rosa hybrida , NCBI:AF502434), VvROMT ( Vitis vinifera , NCBI:CAQ76879), ObEOMT ( Ocimum basilicum , NCBI:AF435008), and CTOMT1 ( Solanum lycopersicum , sol genomics network:SGN-U582403).
Figure Legend Snippet: Alignment of SlGOMT1 ( Silene latifolia ), SlGOMT2 ( Silene latifolia ), SdOMT1 ( Silene dioica ), and SdOMT2 ( Silene dioica ). White letters on black background show identical amino acids of at least five sequences. One amino acid (in yellow background) that is involved in the difference of catalytic efficiency is highlighted . Three conserved SAM-binding domains are also shown in red blocks. GenBank accession numbers are as follows: RhOOMT1 ( Rosa hybrida , NCBI: AF502433), RhOOMT2 ( Rosa hybrida , NCBI:AF502434), VvROMT ( Vitis vinifera , NCBI:CAQ76879), ObEOMT ( Ocimum basilicum , NCBI:AF435008), and CTOMT1 ( Solanum lycopersicum , sol genomics network:SGN-U582403).

Techniques Used: Binding Assay

21) Product Images from "Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification"

Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2012.890

SDS-PAGE analysis of purified protein recombinant SllB in E. coli BL21 cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.
Figure Legend Snippet: SDS-PAGE analysis of purified protein recombinant SllB in E. coli BL21 cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.

Techniques Used: SDS Page, Purification, Recombinant, Marker

A transmission electron microscopic observation on the E. coli BL21 with recombinant protein. (A) Normal E. coli BL21 was treated as control. (B) The E. coli BL21 cells recombinant S-layer protein. (C) Crystal lattice structures on surface of the E. coli BL21 cells recombinant S-layer protein.
Figure Legend Snippet: A transmission electron microscopic observation on the E. coli BL21 with recombinant protein. (A) Normal E. coli BL21 was treated as control. (B) The E. coli BL21 cells recombinant S-layer protein. (C) Crystal lattice structures on surface of the E. coli BL21 cells recombinant S-layer protein.

Techniques Used: Transmission Assay, Recombinant

Expression of sllB in E. coli BL21 cells. (A) SDS-PAGE analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; lane M represent Takara Protein Marker (Broad). (B) Western blot analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; M, Takara Protein Marker (Broad). Lane M1, Precision plus protein standards; lane M2, perfect protein marker. Note the band pointed with arrows is the recombinant S-layer protein.
Figure Legend Snippet: Expression of sllB in E. coli BL21 cells. (A) SDS-PAGE analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; lane M represent Takara Protein Marker (Broad). (B) Western blot analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; M, Takara Protein Marker (Broad). Lane M1, Precision plus protein standards; lane M2, perfect protein marker. Note the band pointed with arrows is the recombinant S-layer protein.

Techniques Used: Expressing, SDS Page, Marker, Western Blot, Recombinant

22) Product Images from "Increasing and Decreasing the Ultrastability of Bacterial Chemotaxis Core Signaling Complexes by Modifying Protein−Protein Contacts"

Article Title: Increasing and Decreasing the Ultrastability of Bacterial Chemotaxis Core Signaling Complexes by Modifying Protein−Protein Contacts

Journal: Biochemistry

doi: 10.1021/bi500849p

Effects on ultrastability of a disulfide bond bridging a core unit interface. Decay time courses for reconstituted and washed core complexes are shown (see the legend of Figure 2 ). These decays demonstrate the effects of the reduced or oxidized kpD586C/apN50C Cys pair at kinase–adaptor interface 1 within the core unit; the kpD586C–apN50C disulfide covalently cross-links this interface. 16 Each plot summarizes the decay of intact, full-length kinase (Kinase Intactness, dashed line and empty symbols) and of kinase enzymatic function (Kinase Activity, solid line and filled symbols). Addition of attractant (Ser) is observed to fully inhibit kinase activity via native receptor-mediated kinase regulation. Table 2 summarizes the kinetic parameters. (A) A 72 h decay time course of control reconstituted Cysless complexes, showing that the redox treatments yield minimal perturbation of the decay kinetics (compare with Figure 2 A and Table 2 ). Both the reduced and oxidized complexes exhibit a quasi-stable component (τ = 17–34 and 19–32 h, respectively, for 60–70% of the population) and an ultrastable component (τ ≫ 72 h for 30–40% of the population). (B) A 72 h time course of the decay of reconstituted di-Cys complexes containing the reduced Cys pair or the oxidized disulfide linkage. The reduced di-Cys complexes exhibit both a quasi-stable component (τ = 21–36 h for 60–80% of the population) and an ultrastable component (τ ≫ 72 h for 20–40% of the population) as observed in panel A for native Cysless complexes. In contrast, the oxidized di-Cys complexes exhibit only the ultrastable component (τ > 300 h for 100% of the population). (C) A 432 h time course comparing the decay of Cysless complexes to that of oxidized di-Cys complexes containing the disulfide linkage. As usual, the Cysless complexes exhibit both a quasi-stable component (τ = 35–42 h for 50% of the population) and an ultrastable component (τ = 440–750 h for 50% of the population). In contrast, the oxidized di-Cys complexes exhibit only the ultrastable component (τ ≫ 1000 h for 100% of the population).
Figure Legend Snippet: Effects on ultrastability of a disulfide bond bridging a core unit interface. Decay time courses for reconstituted and washed core complexes are shown (see the legend of Figure 2 ). These decays demonstrate the effects of the reduced or oxidized kpD586C/apN50C Cys pair at kinase–adaptor interface 1 within the core unit; the kpD586C–apN50C disulfide covalently cross-links this interface. 16 Each plot summarizes the decay of intact, full-length kinase (Kinase Intactness, dashed line and empty symbols) and of kinase enzymatic function (Kinase Activity, solid line and filled symbols). Addition of attractant (Ser) is observed to fully inhibit kinase activity via native receptor-mediated kinase regulation. Table 2 summarizes the kinetic parameters. (A) A 72 h decay time course of control reconstituted Cysless complexes, showing that the redox treatments yield minimal perturbation of the decay kinetics (compare with Figure 2 A and Table 2 ). Both the reduced and oxidized complexes exhibit a quasi-stable component (τ = 17–34 and 19–32 h, respectively, for 60–70% of the population) and an ultrastable component (τ ≫ 72 h for 30–40% of the population). (B) A 72 h time course of the decay of reconstituted di-Cys complexes containing the reduced Cys pair or the oxidized disulfide linkage. The reduced di-Cys complexes exhibit both a quasi-stable component (τ = 21–36 h for 60–80% of the population) and an ultrastable component (τ ≫ 72 h for 20–40% of the population) as observed in panel A for native Cysless complexes. In contrast, the oxidized di-Cys complexes exhibit only the ultrastable component (τ > 300 h for 100% of the population). (C) A 432 h time course comparing the decay of Cysless complexes to that of oxidized di-Cys complexes containing the disulfide linkage. As usual, the Cysless complexes exhibit both a quasi-stable component (τ = 35–42 h for 50% of the population) and an ultrastable component (τ = 440–750 h for 50% of the population). In contrast, the oxidized di-Cys complexes exhibit only the ultrastable component (τ ≫ 1000 h for 100% of the population).

Techniques Used: Activity Assay

Effect of a disulfide bond bridging a core unit interface on susceptibility to proteolysis. A 24 h time course illustrating the effects of added trypsin on Cysless and di-Cys complexes possessing the kpD586C/apN50C Cys pair (see the legends of Figures 2 and 3 ). Each plot summarizes the decay of intact, full-length kinase (Kinase Intactness, dashed line and empty symbols) and of kinase enzyme activity (Kinase Activity, solid line and filled symbols). Addition of attractant (Ser) is observed to fully inhibit, within error, receptor-mediated stimulation of kinase activity. Table 3 summarizes the kinetic parameters. (A) Decay time courses of control, reconstituted Cysless complexes showing that in the absence of trypsin the decay exhibits the usual quasi-stable (τ = 35–42 h for 50% of the population) and ultrastable (τ ≫ 42 h for 50% of the population) components. Trypsin speeds the decay of most of the quasi-stable component (τ = 1.8–6.3 h for 30–40% of the population, and τ = 35–42 h for 10–20% of the population), whereas trypsin has no detectable effect on the ultrastable component (τ ≫ 48 h for 50% of the population). (B) Decay time course of reconstituted di-Cys complexes containing the reduced Cys pair or the oxidized disulfide linkage, both in the presence of trypsin. For reduced di-Cys complexes, as for Cysless complexes (see panel A), trypsin speeds the decay of most of the quasi-stable component (τ = 1.4–1.5 h for 20–40% of the population, and τ = 35–42 h for 10–30% of the population), whereas trypsin has no detectable effect on the ultrastable component (τ ≫ 48 h for 50% of the population). In striking contrast, the oxidized di-Cys complexes possess no detectable quasi-stable component, and the ultrastable component is fully resistant to trypsin on the examined time scale (τ ≫ 48 h for 100% of the population).
Figure Legend Snippet: Effect of a disulfide bond bridging a core unit interface on susceptibility to proteolysis. A 24 h time course illustrating the effects of added trypsin on Cysless and di-Cys complexes possessing the kpD586C/apN50C Cys pair (see the legends of Figures 2 and 3 ). Each plot summarizes the decay of intact, full-length kinase (Kinase Intactness, dashed line and empty symbols) and of kinase enzyme activity (Kinase Activity, solid line and filled symbols). Addition of attractant (Ser) is observed to fully inhibit, within error, receptor-mediated stimulation of kinase activity. Table 3 summarizes the kinetic parameters. (A) Decay time courses of control, reconstituted Cysless complexes showing that in the absence of trypsin the decay exhibits the usual quasi-stable (τ = 35–42 h for 50% of the population) and ultrastable (τ ≫ 42 h for 50% of the population) components. Trypsin speeds the decay of most of the quasi-stable component (τ = 1.8–6.3 h for 30–40% of the population, and τ = 35–42 h for 10–20% of the population), whereas trypsin has no detectable effect on the ultrastable component (τ ≫ 48 h for 50% of the population). (B) Decay time course of reconstituted di-Cys complexes containing the reduced Cys pair or the oxidized disulfide linkage, both in the presence of trypsin. For reduced di-Cys complexes, as for Cysless complexes (see panel A), trypsin speeds the decay of most of the quasi-stable component (τ = 1.4–1.5 h for 20–40% of the population, and τ = 35–42 h for 10–30% of the population), whereas trypsin has no detectable effect on the ultrastable component (τ ≫ 48 h for 50% of the population). In striking contrast, the oxidized di-Cys complexes possess no detectable quasi-stable component, and the ultrastable component is fully resistant to trypsin on the examined time scale (τ ≫ 48 h for 100% of the population).

Techniques Used: Activity Assay

Effect of bulky kpL545W and kpV634W Trp substitutions on the ultrastability of reconstituted core complexes. Shown are 72 h decay time courses for reconstituted, washed core complexes formed on isolated E. coli membranes. Serine receptor (Tsr), histidine kinase (CheA), and adaptor protein (CheW) were mixed and incubated to reconstitute core complexes, and then the resulting membrane-bound complexes were washed to remove unbound components. Each plot summarizes the decay of intact, full-length kinase (Kinase Intactness, dashed line and empty symbols) and of kinase enzymatic function (Kinase Activity, solid line and filled symbols). In each case, the addition of attractant (Ser) fully inhibited kinase activity via native, receptor-mediated kinase regulation. Table 2 summarizes the kinetic parameters for each time course. (A) Decay of control Cysless reconstituted complexes, exhibiting both a quasi-stable component (τ = 17–34 h for 60–70% of the population) and an ultrastable component (τ ≫ 72 h for the remaining 30–40% of the population). (B) Decay of reconstituted complexes containing the kinase protein kpL545W mutation to perturb the receptor–kinase interface within the core unit, exhibiting a quasi-stable (τ = 24–41 h for 100% of the population) but no ultrastable component. (C) Decay of reconstituted complexes containing the kinase protein kpV634W mutation to perturb kinase–adaptor protein interface 1, located within the core unit. The decay exhibits a quasi-stable (τ = 25–35 h for 100% of the population) but no ultrastable component.
Figure Legend Snippet: Effect of bulky kpL545W and kpV634W Trp substitutions on the ultrastability of reconstituted core complexes. Shown are 72 h decay time courses for reconstituted, washed core complexes formed on isolated E. coli membranes. Serine receptor (Tsr), histidine kinase (CheA), and adaptor protein (CheW) were mixed and incubated to reconstitute core complexes, and then the resulting membrane-bound complexes were washed to remove unbound components. Each plot summarizes the decay of intact, full-length kinase (Kinase Intactness, dashed line and empty symbols) and of kinase enzymatic function (Kinase Activity, solid line and filled symbols). In each case, the addition of attractant (Ser) fully inhibited kinase activity via native, receptor-mediated kinase regulation. Table 2 summarizes the kinetic parameters for each time course. (A) Decay of control Cysless reconstituted complexes, exhibiting both a quasi-stable component (τ = 17–34 h for 60–70% of the population) and an ultrastable component (τ ≫ 72 h for the remaining 30–40% of the population). (B) Decay of reconstituted complexes containing the kinase protein kpL545W mutation to perturb the receptor–kinase interface within the core unit, exhibiting a quasi-stable (τ = 24–41 h for 100% of the population) but no ultrastable component. (C) Decay of reconstituted complexes containing the kinase protein kpV634W mutation to perturb kinase–adaptor protein interface 1, located within the core unit. The decay exhibits a quasi-stable (τ = 25–35 h for 100% of the population) but no ultrastable component.

Techniques Used: Isolation, Incubation, Activity Assay, Mutagenesis

23) Product Images from "Kinetic and spectroscopic studies of hemin acquisition in the hemophore HasAp from Pseudomonas aeruginosa †"

Article Title: Kinetic and spectroscopic studies of hemin acquisition in the hemophore HasAp from Pseudomonas aeruginosa †

Journal: Biochemistry

doi: 10.1021/bi100692f

(A) Kinetics of hemin association measured at 408 nm for wt (black) and H32A (red) HasAp. (B) Observed rate constants plotted as a function of apo-HasAp concentration for wt (black) and H32A (red) HasAp as determined by single exponential fitting of the
Figure Legend Snippet: (A) Kinetics of hemin association measured at 408 nm for wt (black) and H32A (red) HasAp. (B) Observed rate constants plotted as a function of apo-HasAp concentration for wt (black) and H32A (red) HasAp as determined by single exponential fitting of the

Techniques Used: Concentration Assay

Low-frequency RR spectra of RFQ samples of the reaction of one equivalent hemin with (A) wt and (B) H32A apo-HasAp at 760 and 950 μM, respectively (λ exc = 413 nm, 20 mW; all spectra are normalized on the intensity of the ν 7 observed
Figure Legend Snippet: Low-frequency RR spectra of RFQ samples of the reaction of one equivalent hemin with (A) wt and (B) H32A apo-HasAp at 760 and 950 μM, respectively (λ exc = 413 nm, 20 mW; all spectra are normalized on the intensity of the ν 7 observed

Techniques Used:

Absorption spectra of human met-Hb (blue), wt holo-HasAp (black), and H32A holo-HasAp (red) normalized to 5.0 μM heme.
Figure Legend Snippet: Absorption spectra of human met-Hb (blue), wt holo-HasAp (black), and H32A holo-HasAp (red) normalized to 5.0 μM heme.

Techniques Used:

High-frequency RR spectra of RFQ samples of the reaction of one equivalent hemin with (A) wt and (B) H32A apo-HasAp at 760 and 950 μM, respectively (λ exc = 413 nm, 20 mW; all spectra are normalized on the intensity of the ν 4 observed
Figure Legend Snippet: High-frequency RR spectra of RFQ samples of the reaction of one equivalent hemin with (A) wt and (B) H32A apo-HasAp at 760 and 950 μM, respectively (λ exc = 413 nm, 20 mW; all spectra are normalized on the intensity of the ν 4 observed

Techniques Used:

Stopped-flow absorption spectra of the association of 4.45 μM hemin to (A) wt and (B) H32A apo-HasAp at 31 μM. Blue traces are hemin + buffer controls at the same times as those indicated for the proteins. (C) Comparison between the absorption
Figure Legend Snippet: Stopped-flow absorption spectra of the association of 4.45 μM hemin to (A) wt and (B) H32A apo-HasAp at 31 μM. Blue traces are hemin + buffer controls at the same times as those indicated for the proteins. (C) Comparison between the absorption

Techniques Used: Flow Cytometry

X-band EPR spectra of RFQ samples of the reaction of one equivalent hemin with (A) wt and (B) H32A apo-HasAp at 760 and 950 μM, respectively. Spectra are normalized according to double integration of EPR signals from known concentrations of holo-HasAp
Figure Legend Snippet: X-band EPR spectra of RFQ samples of the reaction of one equivalent hemin with (A) wt and (B) H32A apo-HasAp at 760 and 950 μM, respectively. Spectra are normalized according to double integration of EPR signals from known concentrations of holo-HasAp

Techniques Used: Electron Paramagnetic Resonance

24) Product Images from "Cloning and Sequencing of a Poly(dl-Lactic Acid) Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13 and Its Functional Expression in Escherichia coli"

Article Title: Cloning and Sequencing of a Poly(dl-Lactic Acid) Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13 and Its Functional Expression in Escherichia coli

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.69.5.2498-2504.2003

Restriction map of the 2.9-kbp Hin dIII fragment from pLA29, showing the positions of the Hin dIII (Hin), Sph I (Sph), Sma I (Sma), Eco RI (Eco), and Pst I (Pst) restriction sites. The location of the plaA gene is indicated by an arrow. E. coli XL10-Gold transformed with each plasmid was grown on an agar plate containing emulsified PLA05 or PBSA3020 at 37°C for 24 h.
Figure Legend Snippet: Restriction map of the 2.9-kbp Hin dIII fragment from pLA29, showing the positions of the Hin dIII (Hin), Sph I (Sph), Sma I (Sma), Eco RI (Eco), and Pst I (Pst) restriction sites. The location of the plaA gene is indicated by an arrow. E. coli XL10-Gold transformed with each plasmid was grown on an agar plate containing emulsified PLA05 or PBSA3020 at 37°C for 24 h.

Techniques Used: Transformation Assay, Plasmid Preparation

Clear-zone formation obtained with recombinant E. coli XL10-Gold on agar plates containing emulsified PLA05 (A) and PBSA3020 (B). The plates were incubated at 37°C for 48 h.
Figure Legend Snippet: Clear-zone formation obtained with recombinant E. coli XL10-Gold on agar plates containing emulsified PLA05 (A) and PBSA3020 (B). The plates were incubated at 37°C for 48 h.

Techniques Used: Recombinant, Incubation

25) Product Images from "Effects of the Fc-III tag on activity and stability of green fluorescent protein and human muscle creatine kinase"

Article Title: Effects of the Fc-III tag on activity and stability of green fluorescent protein and human muscle creatine kinase

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.2282

Purification of GFPs from E. coli . (A) Lane 1, the reduced PAGE of the Fc-III tagged GFP; Lane 2, the reduced PAGE of His-tagged GFP; Lane 3, the nonreduced PAGE of the Fc-III tagged GFP; Lane 4, the nonreduced PAGE of the His-tagged GFP. (B) The MS spectrum
Figure Legend Snippet: Purification of GFPs from E. coli . (A) Lane 1, the reduced PAGE of the Fc-III tagged GFP; Lane 2, the reduced PAGE of His-tagged GFP; Lane 3, the nonreduced PAGE of the Fc-III tagged GFP; Lane 4, the nonreduced PAGE of the His-tagged GFP. (B) The MS spectrum

Techniques Used: Purification, Polyacrylamide Gel Electrophoresis, Mass Spectrometry

26) Product Images from "Alpha-Synuclein is a Target of Fic-mediated Adenylylation/AMPylation: Implications for Parkinson’s Disease"

Article Title: Alpha-Synuclein is a Target of Fic-mediated Adenylylation/AMPylation: Implications for Parkinson’s Disease

Journal: bioRxiv

doi: 10.1101/525659

HYPE binds to and adenylylates αSyn. A ) Steady state binding affinity determination for E234G/H363A-HYPE incubated with His6-tagged αSyn immobilized on HIS1K sensors were obtained by Biolayer Interferometry. B ) In vitro adenylylation assay with purified HYPE and αSyn using α 32 P-ATP as a nucleotide source showing adenylylation of αSyn by enzymatically active E234G-HYPE but not by its catalytically dead mutant E234G/H363A-HYPE.
Figure Legend Snippet: HYPE binds to and adenylylates αSyn. A ) Steady state binding affinity determination for E234G/H363A-HYPE incubated with His6-tagged αSyn immobilized on HIS1K sensors were obtained by Biolayer Interferometry. B ) In vitro adenylylation assay with purified HYPE and αSyn using α 32 P-ATP as a nucleotide source showing adenylylation of αSyn by enzymatically active E234G-HYPE but not by its catalytically dead mutant E234G/H363A-HYPE.

Techniques Used: Binding Assay, Incubation, In Vitro, Purification, Mutagenesis

Adenylylation reduces αSyn’s ability to disrupt membranes. Calcein-loaded PG: PC (1:1) SUVs were incubated with adenylylated or non-adenylylated (control) αSyn and examined for an increase in calcein fluorescence (excitation and emission wavelengths, 485 nm and 515 nm). The data are presented as relative membrane permeabilization after 12 h, normalized with respect to non-adenylylated WT αSyn control. Mean ± SEM, n = 4 (each with 3 technical replicates).
Figure Legend Snippet: Adenylylation reduces αSyn’s ability to disrupt membranes. Calcein-loaded PG: PC (1:1) SUVs were incubated with adenylylated or non-adenylylated (control) αSyn and examined for an increase in calcein fluorescence (excitation and emission wavelengths, 485 nm and 515 nm). The data are presented as relative membrane permeabilization after 12 h, normalized with respect to non-adenylylated WT αSyn control. Mean ± SEM, n = 4 (each with 3 technical replicates).

Techniques Used: Incubation, Fluorescence

A ) Schematic representation of human HYPE. HYPE’s Fic motif with the catalytic H363 and its α-inh domain (yellow box) with E234 are indicated. SS/TM, signal sequence/transmembrane domain. TPR, tetratricopeptide domain. Fic, filamentation induced by cAMP domain. B ) Schematic representation of human alpha-Synuclein (αSyn). Familial mutants associated with Parkinson’s disease (A30P, E46K, H50Q, G51D, A53E, and A53T) are indicated. Sites of adenylylation (T33, T54, and T75) are indicated in red. αSyn’s N-terminal (N-term), central hydrophobic NAC (non-Abeta component of Alzheimer’s disease amyloid), and C-terminal (C-term) domains are shown. Schematic representations for HYPE and αSyn are not to scale.
Figure Legend Snippet: A ) Schematic representation of human HYPE. HYPE’s Fic motif with the catalytic H363 and its α-inh domain (yellow box) with E234 are indicated. SS/TM, signal sequence/transmembrane domain. TPR, tetratricopeptide domain. Fic, filamentation induced by cAMP domain. B ) Schematic representation of human alpha-Synuclein (αSyn). Familial mutants associated with Parkinson’s disease (A30P, E46K, H50Q, G51D, A53E, and A53T) are indicated. Sites of adenylylation (T33, T54, and T75) are indicated in red. αSyn’s N-terminal (N-term), central hydrophobic NAC (non-Abeta component of Alzheimer’s disease amyloid), and C-terminal (C-term) domains are shown. Schematic representations for HYPE and αSyn are not to scale.

Techniques Used: Sequencing

Kinetic traces of a fibrillation assay conducted with purified human WT αSyn adenylylated in the presence of E234G HYPE (green line) or left unmodified as αSyn alone (blue line) show a significant reduction in the yield of ThT fluorescence – potentially reflecting inhibition of fibril elongation – as a result of αSyn adenylylation. Control samples consist of αSyn incubated in the presence of WT-HYPE (red line) or catalytically dead E234G/H363A HYPE (purple line) prior to fibrillation. The background fluorescence for the ThT only control against which all samples were blanked is also shown (light blue line).
Figure Legend Snippet: Kinetic traces of a fibrillation assay conducted with purified human WT αSyn adenylylated in the presence of E234G HYPE (green line) or left unmodified as αSyn alone (blue line) show a significant reduction in the yield of ThT fluorescence – potentially reflecting inhibition of fibril elongation – as a result of αSyn adenylylation. Control samples consist of αSyn incubated in the presence of WT-HYPE (red line) or catalytically dead E234G/H363A HYPE (purple line) prior to fibrillation. The background fluorescence for the ThT only control against which all samples were blanked is also shown (light blue line).

Techniques Used: Purification, Fluorescence, Inhibition, Incubation

Adenylylation alters the morphology of αSyn fibrils. Transmission electron microscopy images of fibrils formed by human WT αSyn. The protein was untreated (A) or incubated with WT HYPE (B), its catalytically inactive E234G/H363A mutant, (C) or the constitutively adenylylation competent E234G mutant (D). Fibrils formed by αSyn largely consist of bundles of fibril pairs that are intertwined, with each twist occurring at regular pitch lengths (red arrows). However, significant levels of fibrils formed by adenylylated αSyn (incubated with E234G) do not display this twist and are instead structured as parallel fibril pairs (yellow arrows). Magnified images of fibrils showing one representative twist (A-C) or one parallel fibril polymorph (D) are shown beside each image. Scale 200nm.
Figure Legend Snippet: Adenylylation alters the morphology of αSyn fibrils. Transmission electron microscopy images of fibrils formed by human WT αSyn. The protein was untreated (A) or incubated with WT HYPE (B), its catalytically inactive E234G/H363A mutant, (C) or the constitutively adenylylation competent E234G mutant (D). Fibrils formed by αSyn largely consist of bundles of fibril pairs that are intertwined, with each twist occurring at regular pitch lengths (red arrows). However, significant levels of fibrils formed by adenylylated αSyn (incubated with E234G) do not display this twist and are instead structured as parallel fibril pairs (yellow arrows). Magnified images of fibrils showing one representative twist (A-C) or one parallel fibril polymorph (D) are shown beside each image. Scale 200nm.

Techniques Used: Transmission Assay, Electron Microscopy, Incubation, Mutagenesis

HYPE-mediated adenylylation of αSyn apparently inhibits fibril elongation without altering the lag time. A ) Representative kinetic traces of fibrillation assays conducted with purified αSyn adenylylated in the presence of E234G HYPE (green line) or left unmodified as αSyn alone (blue line) show a significant reduction in the yield of ThT fluorescence – potentially reflecting inhibition of fibril elongation – as a result of αSyn adenylylation. Control samples consist of αSyn incubated in the presence of WT HYPE (red line) or catalytically dead E234G/H363A HYPE (purple line) prior to fibrillation. B ) Quantification of endpoint ThT signal intensities obtained from N=3 fibrillation assays. Asterisk * indicates a p value = 0.02. C ) Endpoint fibril samples from (A) were ultracentrifuged, and soluble αSyn in the supernatant (S) fraction and insoluble/aggregated αSyn in the pellet (P) fraction were analyzed by SDS-PAGE. D ) Quantification of the bands corresponding to monomeric αSyn in the pellet fractions of the gel shown in (C). Adenylylation of αSyn following incubation with E234G-HYPE results in a decrease (74% vs. 100%) in the amount of αSyn seen in the pellet fraction, suggesting that more αSyn is soluble/not aggregated. E ) Normalized ThT values indicate that the lag time of fibrillation is unaltered by HYPE-mediated adenylylation, as the curves for WT αSyn and αSyn incubated with various HYPE samples overlap. In contrast, control samples of mouse WT αSyn (dark blue line) or human A53T αSyn mutant (orange line) exhibit a shorter lag time, indicating a much faster rate of nucleation.
Figure Legend Snippet: HYPE-mediated adenylylation of αSyn apparently inhibits fibril elongation without altering the lag time. A ) Representative kinetic traces of fibrillation assays conducted with purified αSyn adenylylated in the presence of E234G HYPE (green line) or left unmodified as αSyn alone (blue line) show a significant reduction in the yield of ThT fluorescence – potentially reflecting inhibition of fibril elongation – as a result of αSyn adenylylation. Control samples consist of αSyn incubated in the presence of WT HYPE (red line) or catalytically dead E234G/H363A HYPE (purple line) prior to fibrillation. B ) Quantification of endpoint ThT signal intensities obtained from N=3 fibrillation assays. Asterisk * indicates a p value = 0.02. C ) Endpoint fibril samples from (A) were ultracentrifuged, and soluble αSyn in the supernatant (S) fraction and insoluble/aggregated αSyn in the pellet (P) fraction were analyzed by SDS-PAGE. D ) Quantification of the bands corresponding to monomeric αSyn in the pellet fractions of the gel shown in (C). Adenylylation of αSyn following incubation with E234G-HYPE results in a decrease (74% vs. 100%) in the amount of αSyn seen in the pellet fraction, suggesting that more αSyn is soluble/not aggregated. E ) Normalized ThT values indicate that the lag time of fibrillation is unaltered by HYPE-mediated adenylylation, as the curves for WT αSyn and αSyn incubated with various HYPE samples overlap. In contrast, control samples of mouse WT αSyn (dark blue line) or human A53T αSyn mutant (orange line) exhibit a shorter lag time, indicating a much faster rate of nucleation.

Techniques Used: Purification, Fluorescence, Inhibition, Incubation, SDS Page, Mutagenesis

27) Product Images from "Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration"

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

Journal: bioRxiv

doi: 10.1101/2020.05.11.083030

Abi SH3 domain binds to two fragments of NHSL1 (A) Far western overlay with purified MBP-tagged full-length Abi1 (MBP-Abi1 full length) or an MBP fusion protein with Abi1 in which the SH3 domain had been deleted (MBP-Abi1-delta-SH3) and MBP as control on a blot of different purified GST-NHSL1 fusion proteins covering the entire length of NHSL1. Representative blots from three independent experiments. Fragments 4 and 5 contain three putative SH3 binding si tes. (B) Coomassie gel showing GST fragments covering the entire length of the NHSL1 amino acid sequence (see Fig. 1E for fragment sizes and location within NHSL1) and GST only as control which are used in the Far Western Blot in (A).
Figure Legend Snippet: Abi SH3 domain binds to two fragments of NHSL1 (A) Far western overlay with purified MBP-tagged full-length Abi1 (MBP-Abi1 full length) or an MBP fusion protein with Abi1 in which the SH3 domain had been deleted (MBP-Abi1-delta-SH3) and MBP as control on a blot of different purified GST-NHSL1 fusion proteins covering the entire length of NHSL1. Representative blots from three independent experiments. Fragments 4 and 5 contain three putative SH3 binding si tes. (B) Coomassie gel showing GST fragments covering the entire length of the NHSL1 amino acid sequence (see Fig. 1E for fragment sizes and location within NHSL1) and GST only as control which are used in the Far Western Blot in (A).

Techniques Used: Western Blot, Purification, Binding Assay, Sequencing, Far Western Blot

28) Product Images from "Screening for potential nuclear substrates for the plant cell death suppressor kinase Adi3 using peptide microarrays"

Article Title: Screening for potential nuclear substrates for the plant cell death suppressor kinase Adi3 using peptide microarrays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0234011

Confirmation of Adi3-mediated phosphorylation events on RPB2 as a potential substrate for Adi3. (A) BLAST results from the identification of RPB2 as a potential Adi3 substrate. RPB2 was identified by BLAST using the 48 th , 62 th , and 139 th peptide as queries. In the peptide sequence column, Ser and Thr residues highlighted in red or blue, respectively, indicate possible phosphorylation sites. Peptide # refers to the ranking of each indicated peptide used for BLAST within the top 63 peptides and the 139 th peptide phosphorylated by Adi3. In the BLAST results column, numbers represent amino acid positions in the peptide or RPB2 protein. (B) in vitro kinase activity of Adi3 toward RPB2. Three μg of each RPB2 domain protein was incubated with 1 μCi of [γ- 32 P]ATP in the presence or absence of 1 μg of Adi3 S212D/S595D . Red arrows indicate the expected position of RPB2 domain proteins. Top and bottom panels show the phosphorimage and Coomassie stained gel, respectively. Experiments were repeated three times with similar results. (C) Adi3 phosphorylates Thr675 and Thr676 of RPB2 D3. The indicated RPB2 D3 Thr or Ser residues were mutated to Ala individually or in combination and tested for Adi3-mediated phosphorylation using in vitro kinase assays. The assay was conducted as described in B. Quantification of the auto- and trans -phosphorylation activities of Adi3 were from three independent assays. Top and bottom panels indicate the phosphorimage and Coomassie stained gel, respectively. Asterisks indicate significantly decreased (*) auto- and trans -phosphorylation activity of Adi3 compared to RPB2 D3 WT (Student’ t test, P
Figure Legend Snippet: Confirmation of Adi3-mediated phosphorylation events on RPB2 as a potential substrate for Adi3. (A) BLAST results from the identification of RPB2 as a potential Adi3 substrate. RPB2 was identified by BLAST using the 48 th , 62 th , and 139 th peptide as queries. In the peptide sequence column, Ser and Thr residues highlighted in red or blue, respectively, indicate possible phosphorylation sites. Peptide # refers to the ranking of each indicated peptide used for BLAST within the top 63 peptides and the 139 th peptide phosphorylated by Adi3. In the BLAST results column, numbers represent amino acid positions in the peptide or RPB2 protein. (B) in vitro kinase activity of Adi3 toward RPB2. Three μg of each RPB2 domain protein was incubated with 1 μCi of [γ- 32 P]ATP in the presence or absence of 1 μg of Adi3 S212D/S595D . Red arrows indicate the expected position of RPB2 domain proteins. Top and bottom panels show the phosphorimage and Coomassie stained gel, respectively. Experiments were repeated three times with similar results. (C) Adi3 phosphorylates Thr675 and Thr676 of RPB2 D3. The indicated RPB2 D3 Thr or Ser residues were mutated to Ala individually or in combination and tested for Adi3-mediated phosphorylation using in vitro kinase assays. The assay was conducted as described in B. Quantification of the auto- and trans -phosphorylation activities of Adi3 were from three independent assays. Top and bottom panels indicate the phosphorimage and Coomassie stained gel, respectively. Asterisks indicate significantly decreased (*) auto- and trans -phosphorylation activity of Adi3 compared to RPB2 D3 WT (Student’ t test, P

Techniques Used: Sequencing, In Vitro, Activity Assay, Incubation, Staining

Identification of potential Adi3 nuclear substrates. (A) Bioinformatics and experimental steps followed to screen putative nuclear substrates for Adi3. (B) Categorization of 294 selected nuclear or nuclear event-related proteins identified by BLAST using the top 63 Ser peptides phosphorylated by Adi3. (C) Information for the final 11 tomato protein candidates as potential Adi3 substrates. See [ 31 – 33 ] for information on OX2.
Figure Legend Snippet: Identification of potential Adi3 nuclear substrates. (A) Bioinformatics and experimental steps followed to screen putative nuclear substrates for Adi3. (B) Categorization of 294 selected nuclear or nuclear event-related proteins identified by BLAST using the top 63 Ser peptides phosphorylated by Adi3. (C) Information for the final 11 tomato protein candidates as potential Adi3 substrates. See [ 31 – 33 ] for information on OX2.

Techniques Used:

Analysis of amino acid preferences in Adi3 phosphorylated peptides. (A) Distribution of amino acid composition of the top 63 peptides comparted to entire peptide microarray library. Over- and under-representation of amino acids results in a positive value and a negative value, respectively. (B) Stack-plots of position-dependent deviations of frequencies for single amino acids. The height of the bars indicates the extent (in %) of over- or under-represented individual amino acids in the top 63 peptides as compared to the composition of all peptides in the microarray library. In C and D, amino acid positional probability consensus with top 10 phosphorylated peptides from the (C) Ser- and (D) Thr-peptide microarrays using Sequence logo (WebLogo 3). The size of the amino acid code in the sequence logo represents the frequency of that amino acid at a particular position.
Figure Legend Snippet: Analysis of amino acid preferences in Adi3 phosphorylated peptides. (A) Distribution of amino acid composition of the top 63 peptides comparted to entire peptide microarray library. Over- and under-representation of amino acids results in a positive value and a negative value, respectively. (B) Stack-plots of position-dependent deviations of frequencies for single amino acids. The height of the bars indicates the extent (in %) of over- or under-represented individual amino acids in the top 63 peptides as compared to the composition of all peptides in the microarray library. In C and D, amino acid positional probability consensus with top 10 phosphorylated peptides from the (C) Ser- and (D) Thr-peptide microarrays using Sequence logo (WebLogo 3). The size of the amino acid code in the sequence logo represents the frequency of that amino acid at a particular position.

Techniques Used: Peptide Microarray, Microarray, Sequencing

The Adi3 phosphorylated peptide chips and comparison of kinase activity of the Adi3 S212D/S539D mutant on Ser and Thr residues. (A) Phosphorimage of the whole Ser-peptide microarray chip. Numbers represent each subarray region. Image shown is representative of 4 Ser-peptide chips phosphorylated by Adi3 S212D/S539D . (B) and (C) show one subarray region of Ser- and Thr-peptide microarray chips, respectively. (D) in vitro Adi3 phosphorylation of Gal83 and Gal83 S26T to analyze Adi3 kinase activity on both Ser and Thr residues. Adi3 was incubated with [γ- 32 P]ATP in the absence (lane 1, 2) and presence of Gal83 (lane 3 to 5) or Gal83 S26T mutant (lane 6 to 8). Top and bottom panels indicate the phosphorimage and Coomassie stained gel, respectively.
Figure Legend Snippet: The Adi3 phosphorylated peptide chips and comparison of kinase activity of the Adi3 S212D/S539D mutant on Ser and Thr residues. (A) Phosphorimage of the whole Ser-peptide microarray chip. Numbers represent each subarray region. Image shown is representative of 4 Ser-peptide chips phosphorylated by Adi3 S212D/S539D . (B) and (C) show one subarray region of Ser- and Thr-peptide microarray chips, respectively. (D) in vitro Adi3 phosphorylation of Gal83 and Gal83 S26T to analyze Adi3 kinase activity on both Ser and Thr residues. Adi3 was incubated with [γ- 32 P]ATP in the absence (lane 1, 2) and presence of Gal83 (lane 3 to 5) or Gal83 S26T mutant (lane 6 to 8). Top and bottom panels indicate the phosphorimage and Coomassie stained gel, respectively.

Techniques Used: Activity Assay, Mutagenesis, Peptide Microarray, Chromatin Immunoprecipitation, In Vitro, Incubation, Staining

29) Product Images from "Role of the disulfide bond in stabilizing and folding of the fimbrial protein DraE from uropathogenic Escherichia coli"

Article Title: Role of the disulfide bond in stabilizing and folding of the fimbrial protein DraE from uropathogenic Escherichia coli

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.785477

The free energy diagram of DraE-sc ( without asterisk , black lines ) and DraE-sc-ΔSS (with asterisk , gray lines ) folding/unfolding. N corresponds to native states of proteins, U corresponds to unfolded states, and ‡ corresponds to the transition state. Δ G N→ ‡ denotes the free energy barrier for unfolding, and Δ G U→‡ denotes the free energy barrier of folding. Inset , thermodynamic and kinetic parameters of DraE-sc and DraE-sc-ΔSS denaturation, determined or calculated for 25 °C. a , calculated on the basis of k unfold and k fold : Δ G N→U = − RTln ( k unfold / k fold ); b , value taken from Ref. 10 ; c , taken from Ref. 9 ; d , calculated on the basis of Δ G N→U and k fold : k unfold = k fold · exp (−Δ G N→U / RT ).
Figure Legend Snippet: The free energy diagram of DraE-sc ( without asterisk , black lines ) and DraE-sc-ΔSS (with asterisk , gray lines ) folding/unfolding. N corresponds to native states of proteins, U corresponds to unfolded states, and ‡ corresponds to the transition state. Δ G N→ ‡ denotes the free energy barrier for unfolding, and Δ G U→‡ denotes the free energy barrier of folding. Inset , thermodynamic and kinetic parameters of DraE-sc and DraE-sc-ΔSS denaturation, determined or calculated for 25 °C. a , calculated on the basis of k unfold and k fold : Δ G N→U = − RTln ( k unfold / k fold ); b , value taken from Ref. 10 ; c , taken from Ref. 9 ; d , calculated on the basis of Δ G N→U and k fold : k unfold = k fold · exp (−Δ G N→U / RT ).

Techniques Used:

Per-residue RMSD of atomic positions in thermal denaturation MD simulations of DraE-sc and DraE-sc-ΔSS. The top panel shows the temperature profile used to enforce thermal unfolding over the course of 300 ns. RMSD values, representing deviation from the native structure, were averaged over five independent simulation runs. Triangles indicate strand unfolding events in which strand A ( top panel ) or strand G ( bottom panel ) lose their secondary structure. These triangles are color-coded according to the presence or absence of the disulfide bond; e.g. the position of an orange top triangle corresponds to the simulation time at which strand A dissociated in the reduced protein form (without the disulfide bond).
Figure Legend Snippet: Per-residue RMSD of atomic positions in thermal denaturation MD simulations of DraE-sc and DraE-sc-ΔSS. The top panel shows the temperature profile used to enforce thermal unfolding over the course of 300 ns. RMSD values, representing deviation from the native structure, were averaged over five independent simulation runs. Triangles indicate strand unfolding events in which strand A ( top panel ) or strand G ( bottom panel ) lose their secondary structure. These triangles are color-coded according to the presence or absence of the disulfide bond; e.g. the position of an orange top triangle corresponds to the simulation time at which strand A dissociated in the reduced protein form (without the disulfide bond).

Techniques Used:

Kinetics of DraE-sc and DraE-sc-ΔSS refolding. A , far-UV CD spectra of both protein variants in their native (0 m ) and denatured (6 m GdmCl) state. B and C , protein refolding monitored at the respective time by the shape of far-UV CD spectra, initiated by 100-fold dilution of initial solutions of proteins in 6 m GdmCl. D and E , changes in relative ellipticity measured at 227 nm during the refolding experiments. Red lines denote the fitted first rate kinetic exponential function.
Figure Legend Snippet: Kinetics of DraE-sc and DraE-sc-ΔSS refolding. A , far-UV CD spectra of both protein variants in their native (0 m ) and denatured (6 m GdmCl) state. B and C , protein refolding monitored at the respective time by the shape of far-UV CD spectra, initiated by 100-fold dilution of initial solutions of proteins in 6 m GdmCl. D and E , changes in relative ellipticity measured at 227 nm during the refolding experiments. Red lines denote the fitted first rate kinetic exponential function.

Techniques Used:

Detection of surface located Dr fimbriae in the E. coli AAEC191 strains encoded in the pCC90, pCC90DraE-ΔSS, and pCC90D54stop (DraE negative mutant) plasmids, denoted as DraE , DraE -Δ SS , and DraE-stop , respectively. A–C , for Dr fimbriae, immunofluorescence examination with rabbit anti-DraE and goat TRITC-labeled anti-rabbit antibodies of Dr fimbriae at the surface of AAEC191A/pCC90 ( A ), AAEC191A/pCC90DraE-ΔSS ( B ), and control AAEC191A/pCC90D54stop ( C ) strains. The left and right halves of B and C represent fluorescence and white light views of the same bacterial sample. D–L , microscopic fluorescence detection of Dr fimbriae-mediated adherence of AAEC191A/pCC90/pGFP ( D , G , and J ), AAEC191A/pCC90DraE-ΔSS/pGFP ( E , H , and K ) and control AAEC191A/pCC90D54stop/pGFP ( F , I , and L ) strains to cancer T24 urinary bladder cells. Bacterial adherence were performed in three experiments. In stationary mode ( G–I ), the cell line was incubated for 20 min with bacterial suspension without any shaking. In dynamic mode ( D–F ), bacterial suspension was passed over the cell line in enforced flow conditions for 20 min to generate shear stress of 0.1 pN μm −2 . With chloramphenicol ( J–L ), adherence performed identically as in stationary mode , but the bacterial cells before addition to T24 cells were preincubated in medium containing 300 μ m of chloramphenicol to block receptor binding sites on the Dr fimbriae. Adhered bacteria were visualized by GFP-dependent green fluorescence. The bars denote 20 μm. Images are representative of three independent experiments.
Figure Legend Snippet: Detection of surface located Dr fimbriae in the E. coli AAEC191 strains encoded in the pCC90, pCC90DraE-ΔSS, and pCC90D54stop (DraE negative mutant) plasmids, denoted as DraE , DraE -Δ SS , and DraE-stop , respectively. A–C , for Dr fimbriae, immunofluorescence examination with rabbit anti-DraE and goat TRITC-labeled anti-rabbit antibodies of Dr fimbriae at the surface of AAEC191A/pCC90 ( A ), AAEC191A/pCC90DraE-ΔSS ( B ), and control AAEC191A/pCC90D54stop ( C ) strains. The left and right halves of B and C represent fluorescence and white light views of the same bacterial sample. D–L , microscopic fluorescence detection of Dr fimbriae-mediated adherence of AAEC191A/pCC90/pGFP ( D , G , and J ), AAEC191A/pCC90DraE-ΔSS/pGFP ( E , H , and K ) and control AAEC191A/pCC90D54stop/pGFP ( F , I , and L ) strains to cancer T24 urinary bladder cells. Bacterial adherence were performed in three experiments. In stationary mode ( G–I ), the cell line was incubated for 20 min with bacterial suspension without any shaking. In dynamic mode ( D–F ), bacterial suspension was passed over the cell line in enforced flow conditions for 20 min to generate shear stress of 0.1 pN μm −2 . With chloramphenicol ( J–L ), adherence performed identically as in stationary mode , but the bacterial cells before addition to T24 cells were preincubated in medium containing 300 μ m of chloramphenicol to block receptor binding sites on the Dr fimbriae. Adhered bacteria were visualized by GFP-dependent green fluorescence. The bars denote 20 μm. Images are representative of three independent experiments.

Techniques Used: Mutagenesis, Immunofluorescence, Labeling, Fluorescence, Incubation, Flow Cytometry, Blocking Assay, Binding Assay

Western blot detection of potential DraE and DraE-ΔSS oligomers in the periplasmic fractions isolated from E. coli BL21(DE3)/pET30b-sygDraBE and BL21(DE3)/pET30b-sygDraBE-ΔSS strains, respectively. Samples in lanes 100 and 25 were incubated with Laemmli buffer at 100 and 25 °C, respectively, followed by electrophoresis (SDS–15% polyacrylamide gel). The open and filled arrowheads denote monomeric fully unfolded and oligomeric SDS-resistant forms of DraE, respectively. Immunodetection was performed using primary rabbit polyclonal anti-DraE antibodies, secondary goat labeled with horseradish peroxidase anti-rabbit antibodies and diaminobenzidine as a reaction substrate. Lane M contained a PageRuler prestained protein ladder (Fermentas), which included 10-, 15-, 25-, 35-, 40-, 55-, 70-, 100-, 130-, and 170-kDa compounds.
Figure Legend Snippet: Western blot detection of potential DraE and DraE-ΔSS oligomers in the periplasmic fractions isolated from E. coli BL21(DE3)/pET30b-sygDraBE and BL21(DE3)/pET30b-sygDraBE-ΔSS strains, respectively. Samples in lanes 100 and 25 were incubated with Laemmli buffer at 100 and 25 °C, respectively, followed by electrophoresis (SDS–15% polyacrylamide gel). The open and filled arrowheads denote monomeric fully unfolded and oligomeric SDS-resistant forms of DraE, respectively. Immunodetection was performed using primary rabbit polyclonal anti-DraE antibodies, secondary goat labeled with horseradish peroxidase anti-rabbit antibodies and diaminobenzidine as a reaction substrate. Lane M contained a PageRuler prestained protein ladder (Fermentas), which included 10-, 15-, 25-, 35-, 40-, 55-, 70-, 100-, 130-, and 170-kDa compounds.

Techniques Used: Western Blot, Isolation, Incubation, Electrophoresis, Immunodetection, Labeling

Force-extension curves from mechanical unfolding MD simulations ( dashed lines ), shown along with the unfolding work profiles ( solid lines ; equal to the integral of the pulling force) of DraE-sc and DraE-sc-ΔSS. Sample structures corresponding to the respective end-to-end distances are shown, with green spheres indicating the centers of mass used to define this distance coordinate. Note that the chosen pulling direction, marked with arrows , should correspond to forces exerted on an extended fimbrial polymer when bacteria are subject to shear forces. All values are averaged over 10 independent simulation runs. Triangles indicate strand dissociation events in which strand A ( top ) or strand G ( bottom ) detach from the core B–F region in individual simulations and are color-coded according to the presence or absence of the disulfide bond; e.g. the position of an orange top triangle corresponds to an end-to-end distance at which strand A dissociated in the reduced protein form (without the disulfide bond).
Figure Legend Snippet: Force-extension curves from mechanical unfolding MD simulations ( dashed lines ), shown along with the unfolding work profiles ( solid lines ; equal to the integral of the pulling force) of DraE-sc and DraE-sc-ΔSS. Sample structures corresponding to the respective end-to-end distances are shown, with green spheres indicating the centers of mass used to define this distance coordinate. Note that the chosen pulling direction, marked with arrows , should correspond to forces exerted on an extended fimbrial polymer when bacteria are subject to shear forces. All values are averaged over 10 independent simulation runs. Triangles indicate strand dissociation events in which strand A ( top ) or strand G ( bottom ) detach from the core B–F region in individual simulations and are color-coded according to the presence or absence of the disulfide bond; e.g. the position of an orange top triangle corresponds to an end-to-end distance at which strand A dissociated in the reduced protein form (without the disulfide bond).

Techniques Used:

30) Product Images from "Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins"

Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0189546

Quantitative SPR measurements of cdh23 molecules binding to pcdh15. (A) Left, association and dissociation of the cdh23(WT)-pcdh15(WT) complex. Experimental data (sensorgrams) are represented in a gradient of green to blue colors for different concentrations of cdh23(WT) as labeled. Red lines indicate fitted model parameters (RMSD = 2.89). Injection peaks were removed and not fitted. Top three traces correspond to 5, 10, and 15 μM, respectively, but the 10 μM trace is not labeled for clarity. Black arrow indicates the position of equilibrium SPR signal (R eq ). Middle panel shows the fitting of R eq to a Langmuir binding isotherm at different concentrations of analyte. Measurements for selected concentrations were done in duplicates. Right panel shows a heat map of the k off and K D distribution from the global fit of all traces in corresponding leftmost panel. The signal density of the peaks in the k off and K D distribution plot can directly be discerned from their color, which is scaled according to the color bar on the right side of the distribution plot [ 48 ]. (B) Association and dissociation curves for the T15E-G16D complex shown as in (A) (RMSD = 11.08). Top three traces correspond to 15, 20, and 25 μM, respectively, but the 15 and 20 μM traces are not labeled for clarity. The data were analyzed with the EVILFIT algorithm and the Biacore evaluation software.
Figure Legend Snippet: Quantitative SPR measurements of cdh23 molecules binding to pcdh15. (A) Left, association and dissociation of the cdh23(WT)-pcdh15(WT) complex. Experimental data (sensorgrams) are represented in a gradient of green to blue colors for different concentrations of cdh23(WT) as labeled. Red lines indicate fitted model parameters (RMSD = 2.89). Injection peaks were removed and not fitted. Top three traces correspond to 5, 10, and 15 μM, respectively, but the 10 μM trace is not labeled for clarity. Black arrow indicates the position of equilibrium SPR signal (R eq ). Middle panel shows the fitting of R eq to a Langmuir binding isotherm at different concentrations of analyte. Measurements for selected concentrations were done in duplicates. Right panel shows a heat map of the k off and K D distribution from the global fit of all traces in corresponding leftmost panel. The signal density of the peaks in the k off and K D distribution plot can directly be discerned from their color, which is scaled according to the color bar on the right side of the distribution plot [ 48 ]. (B) Association and dissociation curves for the T15E-G16D complex shown as in (A) (RMSD = 11.08). Top three traces correspond to 15, 20, and 25 μM, respectively, but the 15 and 20 μM traces are not labeled for clarity. The data were analyzed with the EVILFIT algorithm and the Biacore evaluation software.

Techniques Used: SPR Assay, Binding Assay, Labeling, Injection, Software

Heterotetrameric tip link made of CDH23 bound to PCDH15. (A) The tip link is formed by a CDH23 parallel dimer interacting tip-to-tip with a PCDH15 parallel dimer [ 39 ]. These proteins feature 27 and 11 extracellular cadherin (EC) repeats, respectively. (B) Ribbon diagram of mouse cdh23 (blue) bound to pcdh15 (magenta) with Ca 2+ ions as green spheres (PDB ID: 4APX). Sites of deafness-causing mutations R113 and I108 in PCDH15 are shown in stick representation and circled. (C D) Detail of I108 (C) and R113 (D) with surrounding residues in the cdh23 and pcdh15 interface.
Figure Legend Snippet: Heterotetrameric tip link made of CDH23 bound to PCDH15. (A) The tip link is formed by a CDH23 parallel dimer interacting tip-to-tip with a PCDH15 parallel dimer [ 39 ]. These proteins feature 27 and 11 extracellular cadherin (EC) repeats, respectively. (B) Ribbon diagram of mouse cdh23 (blue) bound to pcdh15 (magenta) with Ca 2+ ions as green spheres (PDB ID: 4APX). Sites of deafness-causing mutations R113 and I108 in PCDH15 are shown in stick representation and circled. (C D) Detail of I108 (C) and R113 (D) with surrounding residues in the cdh23 and pcdh15 interface.

Techniques Used:

Variation of ΔT m at increasing ratios of cdh23 and pcdh15. (A) Variation of ΔT m for WT and two deafness-related complexes at increasing concentration ratios. The WT ΔT m increases from ~2 to ~5.5°C from 1:1 to 5:1 ratios. The deafness mutants have ΔT m
Figure Legend Snippet: Variation of ΔT m at increasing ratios of cdh23 and pcdh15. (A) Variation of ΔT m for WT and two deafness-related complexes at increasing concentration ratios. The WT ΔT m increases from ~2 to ~5.5°C from 1:1 to 5:1 ratios. The deafness mutants have ΔT m

Techniques Used: Concentration Assay

Rate and equilibrium constants for different mutants vs ΔΔT m . (A-C) K D vs ΔΔT m (A), k off vs ΔΔT m (B), and k on vs ΔΔT m (C) for various cdh23-pcdh15 complexes (WT-WT: light blue; T15E-WT: red; WT-G16D: yellow; T15E-G16D: light green, F7E-WT: cyan; S76V-WT: navy blue; WT-Q165L: pink). Squares and circles represent data points for ΔΔT m at 1:1 and 5:1 ratios, respectively. The WT-WT complex lies at the origin. Data points along the dashed diagonal line in A support ΔΔT m as a good predictor of K D , while data points off the diagonal indicate exceptions (most notably F7E-WT). Vertical error bars represent standard deviation for measurements of the rate or equilibrium constant of that mutant. Horizontal error bars represent the standard deviation for the ΔT m measurement.
Figure Legend Snippet: Rate and equilibrium constants for different mutants vs ΔΔT m . (A-C) K D vs ΔΔT m (A), k off vs ΔΔT m (B), and k on vs ΔΔT m (C) for various cdh23-pcdh15 complexes (WT-WT: light blue; T15E-WT: red; WT-G16D: yellow; T15E-G16D: light green, F7E-WT: cyan; S76V-WT: navy blue; WT-Q165L: pink). Squares and circles represent data points for ΔΔT m at 1:1 and 5:1 ratios, respectively. The WT-WT complex lies at the origin. Data points along the dashed diagonal line in A support ΔΔT m as a good predictor of K D , while data points off the diagonal indicate exceptions (most notably F7E-WT). Vertical error bars represent standard deviation for measurements of the rate or equilibrium constant of that mutant. Horizontal error bars represent the standard deviation for the ΔT m measurement.

Techniques Used: Standard Deviation, Mutagenesis

Thermal melting curves of cdh23 and pcdh15 variants monitored with SYPRO orange. (A) Upper panels show normalized fluorescence for melting curves with cdh23(WT) in cyan, pcdh15(WT) in magenta, and the mixture in black. Lower panels show the derivative of the normalized melting curve. Concentration ratios from left to right are: 1:1, 2:1, 3:1, 4:1, 5:1 (cdh23:pcdh15). (B-C) Overlay of the melting curves for pcdh15(WT) (B) and for pcdh15(R113G) (C) at indicated cdh23:pcdh15 ratios. The curves were normalized to pcdh15 only (first peak).
Figure Legend Snippet: Thermal melting curves of cdh23 and pcdh15 variants monitored with SYPRO orange. (A) Upper panels show normalized fluorescence for melting curves with cdh23(WT) in cyan, pcdh15(WT) in magenta, and the mixture in black. Lower panels show the derivative of the normalized melting curve. Concentration ratios from left to right are: 1:1, 2:1, 3:1, 4:1, 5:1 (cdh23:pcdh15). (B-C) Overlay of the melting curves for pcdh15(WT) (B) and for pcdh15(R113G) (C) at indicated cdh23:pcdh15 ratios. The curves were normalized to pcdh15 only (first peak).

Techniques Used: Fluorescence, Concentration Assay

Mapping of rationally designed mutation sites on the structure of the cdh23 and pcdh15 complex. (A) Surface representation of cdh23 (blue and cyan) bound to pcdh15 (purple and pink; PDB ID: 4APX). (B) Cdh23 and pcdh15 interaction surfaces exposed with mutation sites labeled. Residues labeled in red are mutated in inherited deafness. Underlined labels indicate sites that belong to paired mutant complexes cdh23(H11K)-pcdh15(Q218E) and cdh23(T79E)-pcdh15(H91R).
Figure Legend Snippet: Mapping of rationally designed mutation sites on the structure of the cdh23 and pcdh15 complex. (A) Surface representation of cdh23 (blue and cyan) bound to pcdh15 (purple and pink; PDB ID: 4APX). (B) Cdh23 and pcdh15 interaction surfaces exposed with mutation sites labeled. Residues labeled in red are mutated in inherited deafness. Underlined labels indicate sites that belong to paired mutant complexes cdh23(H11K)-pcdh15(Q218E) and cdh23(T79E)-pcdh15(H91R).

Techniques Used: Mutagenesis, Labeling

31) Product Images from "Salvage of the 5-deoxyribose byproduct of radical SAM enzymes"

Article Title: Salvage of the 5-deoxyribose byproduct of radical SAM enzymes

Journal: Nature Communications

doi: 10.1038/s41467-018-05589-4

Activities of the B. thuringiensis kinase (DrdK), isomerase (DrdI), and aldolase (DrdA). a Coupled assay of DHAP formation from 5-deoxyribose when DrdK (K), DrdI (I), DrdA (A), and ATP are present in the reaction. DHAP formation was tracked via glycerol 3-phosphate dehydrogenase-catalyzed oxidation of NADH. Standard deviation for each point was
Figure Legend Snippet: Activities of the B. thuringiensis kinase (DrdK), isomerase (DrdI), and aldolase (DrdA). a Coupled assay of DHAP formation from 5-deoxyribose when DrdK (K), DrdI (I), DrdA (A), and ATP are present in the reaction. DHAP formation was tracked via glycerol 3-phosphate dehydrogenase-catalyzed oxidation of NADH. Standard deviation for each point was

Techniques Used: Standard Deviation

Growth and metabolic phenotypes of drdA and drdI deletant strains. a Deleting drdA or drdI exacerbates 5-deoxyribose toxicity. A disc containing 10 µL of 1 M 5-deoxyribose was applied to lawns of B. thuringiensis wild type (WT) or deletant cells. Note the larger zone of growth inhibition for the deletant strains. b Similarly, adding 1 mM 5-deoxyribose (5-dR) to liquid medium reduced the growth rate of the deletant strains significantly more than the wild type. Data are means of four replicates or three replicates for the Δ drdI strain; error bars are the s.d. Significance was determined by t -test, * P
Figure Legend Snippet: Growth and metabolic phenotypes of drdA and drdI deletant strains. a Deleting drdA or drdI exacerbates 5-deoxyribose toxicity. A disc containing 10 µL of 1 M 5-deoxyribose was applied to lawns of B. thuringiensis wild type (WT) or deletant cells. Note the larger zone of growth inhibition for the deletant strains. b Similarly, adding 1 mM 5-deoxyribose (5-dR) to liquid medium reduced the growth rate of the deletant strains significantly more than the wild type. Data are means of four replicates or three replicates for the Δ drdI strain; error bars are the s.d. Significance was determined by t -test, * P

Techniques Used: Inhibition

32) Product Images from "Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus"

Article Title: Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200841

DNA binding activity of Rep68 and TrwC/Rep. (A) Rep68 and TrwC/Rep DNA substrates used for binding assays. Rep68 substrates were an AAV ori heteroduplex, and an equivalent AAV ori substrate mutated in the RBS sequence (RBSmut). trs is shown in boldface; nicking site is indicated by a slash. RBS is highlighted in bold italic. The AAVS1 minimal sequence (30) is presented below. TrwC/Rep chimera substrates were oligonucleotides oriTw (25+8) and oriTw (25+8) mutated in the inverted repeat IR (IRmut). Horizontal lines and nucleotides highlighted in boldface show sequence requirements for binding and nicking activities [ 77 ]. The nic site is represented by a slash. The IR recognized during binding is indicated with arrows; distal and proximal arms are shown. * represents the radioactively labelled strand. (B) and (C) EMSA assays with His-Rep68 and His-TrwC/Rep chimera, respectively. 30 fmol of the indicated radiolabelled substrates were incubated either with 100 ng of His-Rep68 or 200 ng of His-TrwC/Rep. All reactions contain 400 ng of poly(dI-dC) as nonspecific DNA. Competition assays were done using cold competitor DNA at 10- to 90-fold molar excess. Products were analysed on a native 6% polyacrylamide gel. Percentage of bound substrate (“% shift”) was calculated using Image Quant TL software. Bound and unbound products of the reaction are indicated with arrows. The DNA binding percentage is a representative example obtained from 3 independent experiments (n = 3). (D) Fluorescence polarization assay with oriTw (25+8) DNA labelled with carboxyfluorescein at 5 mM concentration. Binding was performed in 25 mM HEPES (pH 7.0), 200 mM NaCl at room temperature. Data analysis was performed as described by Yoon-Robarts et al . [ 57 ].
Figure Legend Snippet: DNA binding activity of Rep68 and TrwC/Rep. (A) Rep68 and TrwC/Rep DNA substrates used for binding assays. Rep68 substrates were an AAV ori heteroduplex, and an equivalent AAV ori substrate mutated in the RBS sequence (RBSmut). trs is shown in boldface; nicking site is indicated by a slash. RBS is highlighted in bold italic. The AAVS1 minimal sequence (30) is presented below. TrwC/Rep chimera substrates were oligonucleotides oriTw (25+8) and oriTw (25+8) mutated in the inverted repeat IR (IRmut). Horizontal lines and nucleotides highlighted in boldface show sequence requirements for binding and nicking activities [ 77 ]. The nic site is represented by a slash. The IR recognized during binding is indicated with arrows; distal and proximal arms are shown. * represents the radioactively labelled strand. (B) and (C) EMSA assays with His-Rep68 and His-TrwC/Rep chimera, respectively. 30 fmol of the indicated radiolabelled substrates were incubated either with 100 ng of His-Rep68 or 200 ng of His-TrwC/Rep. All reactions contain 400 ng of poly(dI-dC) as nonspecific DNA. Competition assays were done using cold competitor DNA at 10- to 90-fold molar excess. Products were analysed on a native 6% polyacrylamide gel. Percentage of bound substrate (“% shift”) was calculated using Image Quant TL software. Bound and unbound products of the reaction are indicated with arrows. The DNA binding percentage is a representative example obtained from 3 independent experiments (n = 3). (D) Fluorescence polarization assay with oriTw (25+8) DNA labelled with carboxyfluorescein at 5 mM concentration. Binding was performed in 25 mM HEPES (pH 7.0), 200 mM NaCl at room temperature. Data analysis was performed as described by Yoon-Robarts et al . [ 57 ].

Techniques Used: Binding Assay, Activity Assay, Sequencing, Incubation, Software, Fluorescence, Concentration Assay

DNA helicase activity of Rep68 and TrwC/Rep. Increasing amounts of Rep68 and TrwC/Rep chimeric protein (0, 1, 10, 100 ng of protein) were assayed in the presence or absence of ATP. “boil” indicates heat-denatured substrate, used as a positive control. Products of the reaction were resolved on 12% native polyacrylamide gels. The diagram to the left of the gel shows the position of the substrate (partial duplex M13 DNA) and unwound labelled ssDNA. The percent of unwound substrate was quantified using the Image Quant TL software. The helicase efficiency percentage is a representative example obtained from 3 independent experiments (n = 3).
Figure Legend Snippet: DNA helicase activity of Rep68 and TrwC/Rep. Increasing amounts of Rep68 and TrwC/Rep chimeric protein (0, 1, 10, 100 ng of protein) were assayed in the presence or absence of ATP. “boil” indicates heat-denatured substrate, used as a positive control. Products of the reaction were resolved on 12% native polyacrylamide gels. The diagram to the left of the gel shows the position of the substrate (partial duplex M13 DNA) and unwound labelled ssDNA. The percent of unwound substrate was quantified using the Image Quant TL software. The helicase efficiency percentage is a representative example obtained from 3 independent experiments (n = 3).

Techniques Used: Activity Assay, Positive Control, Software

Design and validation of the TrwC/Rep chimeric protein. (A) Schematic representation of Rep68, TrwC and the TrwC/Rep chimera. For each protein, overall configuration and functional domains are shown. The position of the HUH and Y2 motif, and the NLS (black bar) are indicated. Amino acid (aa) positions are indicated above each protein. (B) Western blot analysis of Rep68 and TrwC/Rep chimera expressed in 293T cells. Molecular weight in kDa is shown on the right. Antibodies used to detect proteins are shown on the left. (C) Localization of TrwC, Rep68 and TrwC/Rep chimera in 293T cells visualized by immunofluorescence microscopy. Images are shown at 60X magnification. (D) Assessment of the purity of His-Rep68 and His-TrwC/Rep chimera purified by nickel affinity and gel filtration chromatography. 200 ng of protein was loaded onto a 12% SDS-PAGE gel and subsequently stained with Coomassie Brilliant blue to visualize the quality of the purified protein. Marker (M) is shown on the left; molecular weight from top to bottom is: 250, 150, 100, 75, 50 kDa.
Figure Legend Snippet: Design and validation of the TrwC/Rep chimeric protein. (A) Schematic representation of Rep68, TrwC and the TrwC/Rep chimera. For each protein, overall configuration and functional domains are shown. The position of the HUH and Y2 motif, and the NLS (black bar) are indicated. Amino acid (aa) positions are indicated above each protein. (B) Western blot analysis of Rep68 and TrwC/Rep chimera expressed in 293T cells. Molecular weight in kDa is shown on the right. Antibodies used to detect proteins are shown on the left. (C) Localization of TrwC, Rep68 and TrwC/Rep chimera in 293T cells visualized by immunofluorescence microscopy. Images are shown at 60X magnification. (D) Assessment of the purity of His-Rep68 and His-TrwC/Rep chimera purified by nickel affinity and gel filtration chromatography. 200 ng of protein was loaded onto a 12% SDS-PAGE gel and subsequently stained with Coomassie Brilliant blue to visualize the quality of the purified protein. Marker (M) is shown on the left; molecular weight from top to bottom is: 250, 150, 100, 75, 50 kDa.

Techniques Used: Functional Assay, Western Blot, Molecular Weight, Immunofluorescence, Microscopy, Purification, Filtration, Chromatography, SDS Page, Staining, Marker

Rep68 and TrwC/Rep nick sc plasmid DNA containing binding and nicking sites. (A) Schematic representation of the scDNA nicking assay. When incubating a sc plasmid with an endonuclease, the protein nicks specifically inducing relaxation of the plasmid (open circular, oc), which will result in slower migrating DNA species when visualised on an agarose gel. (B) His-Rep68 and His-TrwC/Rep-mediated scDNA nicking. 100 ng of the plasmid containing the target sequence indicated on top of the gels was incubated as described in Materials and Methods with either purified His-Rep68 (left panels) or His-TrwC/Rep chimera (right panels). Products were resolved on 1% agarose gels, subsequently stained with ethidium bromide. “-“: sc plasmid incubated in the reaction buffer and processed as described above, but in the absence of protein. sc and oc products are indicated with arrows. The oc/sc ratios of quantified DNA bands are indicated at the bottom of the panels. The presented gels are representative of 3 independent experiments (n = 3).
Figure Legend Snippet: Rep68 and TrwC/Rep nick sc plasmid DNA containing binding and nicking sites. (A) Schematic representation of the scDNA nicking assay. When incubating a sc plasmid with an endonuclease, the protein nicks specifically inducing relaxation of the plasmid (open circular, oc), which will result in slower migrating DNA species when visualised on an agarose gel. (B) His-Rep68 and His-TrwC/Rep-mediated scDNA nicking. 100 ng of the plasmid containing the target sequence indicated on top of the gels was incubated as described in Materials and Methods with either purified His-Rep68 (left panels) or His-TrwC/Rep chimera (right panels). Products were resolved on 1% agarose gels, subsequently stained with ethidium bromide. “-“: sc plasmid incubated in the reaction buffer and processed as described above, but in the absence of protein. sc and oc products are indicated with arrows. The oc/sc ratios of quantified DNA bands are indicated at the bottom of the panels. The presented gels are representative of 3 independent experiments (n = 3).

Techniques Used: Plasmid Preparation, Binding Assay, Agarose Gel Electrophoresis, Sequencing, Incubation, Purification, Staining

33) Product Images from "Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment"

Article Title: Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment

Journal: PLoS ONE

doi: 10.1371/journal.pone.0196254

Antibody detection of newly isolated CPG2 relative to Ps CPG2. A. Dot blot using anti His tag antibody and using anti Xen CPG2 antibody where 1, 2, and 3 are pure protein (Xen CPG2 and Ps CPG2) at concentrations (0.05, 0.1, and 0.2 mg/mL). B. Dot blot at different concentration of anti Xen CPG2 antibody where 1, 2, and 3 are blotting at dilutions 1:20 000, 1:10 000 and 1:3000 in blocking buffer. C. SDS-PAGE and Western blot analysis of the pure protein (Xen CPG2 and Ps CPG2) where M is PageRuler™ Unstained Protein Ladder (10–200 kDa), lanes 1, 2, 3 are 0.25, 0.1, and 0.05 mg/mL of Xen CPG2 and lanes 4, 5, 6 are the same series of protein concentrations of Ps CPG2.
Figure Legend Snippet: Antibody detection of newly isolated CPG2 relative to Ps CPG2. A. Dot blot using anti His tag antibody and using anti Xen CPG2 antibody where 1, 2, and 3 are pure protein (Xen CPG2 and Ps CPG2) at concentrations (0.05, 0.1, and 0.2 mg/mL). B. Dot blot at different concentration of anti Xen CPG2 antibody where 1, 2, and 3 are blotting at dilutions 1:20 000, 1:10 000 and 1:3000 in blocking buffer. C. SDS-PAGE and Western blot analysis of the pure protein (Xen CPG2 and Ps CPG2) where M is PageRuler™ Unstained Protein Ladder (10–200 kDa), lanes 1, 2, 3 are 0.25, 0.1, and 0.05 mg/mL of Xen CPG2 and lanes 4, 5, 6 are the same series of protein concentrations of Ps CPG2.

Techniques Used: Isolation, Dot Blot, Concentration Assay, Blocking Assay, SDS Page, Western Blot

Homology modeling of Xen CPG2. A. Electrostatic surface presentation of the Xen CPG2 tetramer. Positive and negative charges are shown in blue and red, respectively. B. Color rendering of a Xen CPG2 monomer. Helix in cyan, b-sheet in pink, loops in brown and amino acids which differ from the model carboxypeptidase G2 are shown as yellow sticks. Rendering was performed using PyMol. C. Stereoview of the alignment of CPG2 Pseudomonas sp. Strain RS-16 (blue cartoon, PDB ID 1CG2) with the model of Xen CPG2 (green cartoon, RMS = 0.084 (374 to 374 atoms)).
Figure Legend Snippet: Homology modeling of Xen CPG2. A. Electrostatic surface presentation of the Xen CPG2 tetramer. Positive and negative charges are shown in blue and red, respectively. B. Color rendering of a Xen CPG2 monomer. Helix in cyan, b-sheet in pink, loops in brown and amino acids which differ from the model carboxypeptidase G2 are shown as yellow sticks. Rendering was performed using PyMol. C. Stereoview of the alignment of CPG2 Pseudomonas sp. Strain RS-16 (blue cartoon, PDB ID 1CG2) with the model of Xen CPG2 (green cartoon, RMS = 0.084 (374 to 374 atoms)).

Techniques Used:

Purification of recombinant new glucarpidase relative to Ps CPG2. Coomassie blue staining of a 10% SDS-PAGE gel. M; Size markers in kiloDaltons; M1 is PageRuler Prestained Protein Ladder (10 to 180 kDa) and M2 is SeeBlue Plus prestained standard (↱3 to 198 kDa). a) Xen CPG2 purification; lane 1 is total soluble fraction of glucarpidase after centrifugation; 2, flow through; 3–4, wash 1 and wash 5; 5–9, eluted fractions from the Ni-NTA column with 400 mM imidazole. b) Ps CPG2 purification; lanes 1, 2, 3 are total, flow through, wash, lanes 4–6 are elution fractions.
Figure Legend Snippet: Purification of recombinant new glucarpidase relative to Ps CPG2. Coomassie blue staining of a 10% SDS-PAGE gel. M; Size markers in kiloDaltons; M1 is PageRuler Prestained Protein Ladder (10 to 180 kDa) and M2 is SeeBlue Plus prestained standard (↱3 to 198 kDa). a) Xen CPG2 purification; lane 1 is total soluble fraction of glucarpidase after centrifugation; 2, flow through; 3–4, wash 1 and wash 5; 5–9, eluted fractions from the Ni-NTA column with 400 mM imidazole. b) Ps CPG2 purification; lanes 1, 2, 3 are total, flow through, wash, lanes 4–6 are elution fractions.

Techniques Used: Purification, Recombinant, Staining, SDS Page, Centrifugation, Flow Cytometry

CD spectra and high voltage of Xen CPG2 and Ps CPG2. A) Combined CD spectra data of Xen CPG2 and Ps CPG2 in molar ellipticity relative the wavelength in far UV region, the spectra obtained by dragging their spectral data over each other, where smooth 0 is molar ellipticity of Xen CPG2 and smooth 1 is for CD spectra of Ps CPG2. All Spectral data are corrected for the baseline buffer, B) represents the combined High voltage (HV) for both enzymes where average 0 is Xen CPG2 and average 1 is Ps CPG2. Also the table shows the calculated protein secondary structure of Xen CPG2 and Ps CPG2 by CDNN deconvolution analysis using their CD spectral data.
Figure Legend Snippet: CD spectra and high voltage of Xen CPG2 and Ps CPG2. A) Combined CD spectra data of Xen CPG2 and Ps CPG2 in molar ellipticity relative the wavelength in far UV region, the spectra obtained by dragging their spectral data over each other, where smooth 0 is molar ellipticity of Xen CPG2 and smooth 1 is for CD spectra of Ps CPG2. All Spectral data are corrected for the baseline buffer, B) represents the combined High voltage (HV) for both enzymes where average 0 is Xen CPG2 and average 1 is Ps CPG2. Also the table shows the calculated protein secondary structure of Xen CPG2 and Ps CPG2 by CDNN deconvolution analysis using their CD spectral data.

Techniques Used:

34) Product Images from "Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse muscle"

Article Title: Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse muscle

Journal:

doi: 10.1113/jphysiol.2005.100032

hIGF-1–TTC increases NMJ size and morphological complexity
Figure Legend Snippet: hIGF-1–TTC increases NMJ size and morphological complexity

Techniques Used:

hIGF-1–TTC does not change muscle MHC composition
Figure Legend Snippet: hIGF-1–TTC does not change muscle MHC composition

Techniques Used:

Purification steps for hIGF-1–TTC
Figure Legend Snippet: Purification steps for hIGF-1–TTC

Techniques Used: Purification

hIGF-1–TTC increases specific force in EDL single intact muscle fibres from aged mice
Figure Legend Snippet: hIGF-1–TTC increases specific force in EDL single intact muscle fibres from aged mice

Techniques Used: Mouse Assay

In vivo uptake and retrograde transport of hIGF-1–TTC
Figure Legend Snippet: In vivo uptake and retrograde transport of hIGF-1–TTC

Techniques Used: In Vivo

Binding and internalization of hIGF-1–TTC in vitro
Figure Legend Snippet: Binding and internalization of hIGF-1–TTC in vitro

Techniques Used: Binding Assay, In Vitro

Mitogenic activity of hIGF-1–TTC
Figure Legend Snippet: Mitogenic activity of hIGF-1–TTC

Techniques Used: Activity Assay

35) Product Images from "NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition"

Article Title: NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition

Journal:

doi: 10.1110/ps.051844006

Sequence and structural alignment of flaviviruses. ( A ) Sequence alignment of domain III of the five flaviviruses (TBE, LI, JE, WN, and DEN-2) against LGT-E-D3. Mutations that have led to escape from antibody neutralization for other tick-borne (TBE and
Figure Legend Snippet: Sequence and structural alignment of flaviviruses. ( A ) Sequence alignment of domain III of the five flaviviruses (TBE, LI, JE, WN, and DEN-2) against LGT-E-D3. Mutations that have led to escape from antibody neutralization for other tick-borne (TBE and

Techniques Used: Sequencing, Neutralization

36) Product Images from "Ataxin-2 Modulates the Levels of Grb2 and Src but Not Ras Signaling"

Article Title: Ataxin-2 Modulates the Levels of Grb2 and Src but Not Ras Signaling

Journal: Journal of Molecular Neuroscience

doi: 10.1007/s12031-012-9949-4

Co-IP analysis of the ATXN2–Grb2 complex. a Endogenous ATXN2 is associated with endogenous Grb2 in mouse brain. Brain homogenates were fractionated by differential velocity centrifugation. The cytosolic fraction was utilized to carry out coimmunoprecipitation assays with anti-ATXN2 antibody and the corresponding Ig isotype as control. Immunoblotting was performed using antibodies against ATXN2 and Grb2. ( b – d ) Grb2 interacts with ATXN2 through its C-terminal SH3 motif. b Diagram of human Grb2 and its different domains. c Myc-ATXN2 overexpressed in HEK-293 cells was incubated with GSH affinity beads charged with GST-Grb2 or fusions with its respective subfragments. Loading density of GST or GST-fused proteins on the affinity beads was controlled with anti-GST antibody. d A repetition of this experiment with overexpressed mutant ATXN2 (Q74) yielded identical results
Figure Legend Snippet: Co-IP analysis of the ATXN2–Grb2 complex. a Endogenous ATXN2 is associated with endogenous Grb2 in mouse brain. Brain homogenates were fractionated by differential velocity centrifugation. The cytosolic fraction was utilized to carry out coimmunoprecipitation assays with anti-ATXN2 antibody and the corresponding Ig isotype as control. Immunoblotting was performed using antibodies against ATXN2 and Grb2. ( b – d ) Grb2 interacts with ATXN2 through its C-terminal SH3 motif. b Diagram of human Grb2 and its different domains. c Myc-ATXN2 overexpressed in HEK-293 cells was incubated with GSH affinity beads charged with GST-Grb2 or fusions with its respective subfragments. Loading density of GST or GST-fused proteins on the affinity beads was controlled with anti-GST antibody. d A repetition of this experiment with overexpressed mutant ATXN2 (Q74) yielded identical results

Techniques Used: Co-Immunoprecipitation Assay, Centrifugation, Incubation, Mutagenesis

37) Product Images from "The water lily genome and the early evolution of flowering plants"

Article Title: The water lily genome and the early evolution of flowering plants

Journal: Nature

doi: 10.1038/s41586-019-1852-5

Floral scent and biosynthesis in N. colorata . a , Gas chromatogram of floral volatiles from the flower of N. colorata . The internal standard (IS) is nonyl acetate. Methyl esters are in blue; terpenes are in red. Floral scent was measured three times independently with similar results. b , Phylogenetic tree of terpene synthases from N. colorata and representative plants showing the subfamilies from a–h and x. c , Expression analysis of SABATH genes of N. colorata showed that NC11G0120830 had the highest expression level in petal. d , Relative activity of Escherichia coli -expressed NC11G0120830 with six fatty acids as substrates, with the activity on decanoic acid set at 1.0. Data are mean ± s.d. of three independent measurements. e , The presence (+) and absence (−) of sesquiterpenes and methyl decanoate as floral scent compounds and their respective biosynthetic genes in four major lineages of angiosperms when known. DAMT , decanoic acid methyltranferase.
Figure Legend Snippet: Floral scent and biosynthesis in N. colorata . a , Gas chromatogram of floral volatiles from the flower of N. colorata . The internal standard (IS) is nonyl acetate. Methyl esters are in blue; terpenes are in red. Floral scent was measured three times independently with similar results. b , Phylogenetic tree of terpene synthases from N. colorata and representative plants showing the subfamilies from a–h and x. c , Expression analysis of SABATH genes of N. colorata showed that NC11G0120830 had the highest expression level in petal. d , Relative activity of Escherichia coli -expressed NC11G0120830 with six fatty acids as substrates, with the activity on decanoic acid set at 1.0. Data are mean ± s.d. of three independent measurements. e , The presence (+) and absence (−) of sesquiterpenes and methyl decanoate as floral scent compounds and their respective biosynthetic genes in four major lineages of angiosperms when known. DAMT , decanoic acid methyltranferase.

Techniques Used: Expressing, Activity Assay

38) Product Images from "The Function of UreB in Klebsiella aerogenes Urease"

Article Title: The Function of UreB in Klebsiella aerogenes Urease

Journal: Biochemistry

doi: 10.1021/bi2011064

In vivo interactions of MBP-UreD with (UreABC) 3 , UreB, (UreAC) 3 , and UreBΔ1-19. (A) SDS-PAGE depicting the interactions of MBP-UreD with (UreABC) 3 , UreB, and (UreAC) 3 . E. coli BL21-Gold(DE3) cells were co-transformed with pCDF-MBP-UreD (encoding
Figure Legend Snippet: In vivo interactions of MBP-UreD with (UreABC) 3 , UreB, (UreAC) 3 , and UreBΔ1-19. (A) SDS-PAGE depicting the interactions of MBP-UreD with (UreABC) 3 , UreB, and (UreAC) 3 . E. coli BL21-Gold(DE3) cells were co-transformed with pCDF-MBP-UreD (encoding

Techniques Used: In Vivo, SDS Page, Transformation Assay

39) Product Images from "DiGeorge Critical Region 8 (DGCR8) Is a Double-cysteine-ligated Heme Protein"

Article Title: DiGeorge Critical Region 8 (DGCR8) Is a Double-cysteine-ligated Heme Protein

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.180844

Electronic absorption and MCD spectra of the ferric heme-bound frog DGCR8 HBD-His 6 . The electronic absorption spectrum ( top panel ) was taken at room temperature using the dimeric frog HBD-His 6 at 12.2 μ m concentration in a buffer containing 50
Figure Legend Snippet: Electronic absorption and MCD spectra of the ferric heme-bound frog DGCR8 HBD-His 6 . The electronic absorption spectrum ( top panel ) was taken at room temperature using the dimeric frog HBD-His 6 at 12.2 μ m concentration in a buffer containing 50

Techniques Used: Concentration Assay

Cys-352 from both subunits in a DGCR8 dimer serve as the axial ligands to ferric heme. Electronic absorption spectra of hHBD C430S (10 μ m ) titrated with MeHgAc. The MeHgAc was added at steps of 0.5 molar equivalent of ferric heme-bound HBD dimer.
Figure Legend Snippet: Cys-352 from both subunits in a DGCR8 dimer serve as the axial ligands to ferric heme. Electronic absorption spectra of hHBD C430S (10 μ m ) titrated with MeHgAc. The MeHgAc was added at steps of 0.5 molar equivalent of ferric heme-bound HBD dimer.

Techniques Used:

EPR spectrum of the ferric heme-bound frog DGCR8 HBD-His 6 protein. The frog HBD-His 6 protein (153 μ m ) was in 50 m m EPPS (pH 8.0) and 400 m m NaCl. The spectrum represents an average of 10 scans taken at 10 K, with 9.383 GHz microwave frequency,
Figure Legend Snippet: EPR spectrum of the ferric heme-bound frog DGCR8 HBD-His 6 protein. The frog HBD-His 6 protein (153 μ m ) was in 50 m m EPPS (pH 8.0) and 400 m m NaCl. The spectrum represents an average of 10 scans taken at 10 K, with 9.383 GHz microwave frequency,

Techniques Used: Electron Paramagnetic Resonance

The sixth ligand to ferric heme in DGCR8 is not a methionine. A , mass spectra of native and SeMet-labeled frog DGCR8 HBD-His 6 indicates nearly complete substitution of Met by SeMet. The difference in their m / z peaks, 239, is consistent with all five methionine
Figure Legend Snippet: The sixth ligand to ferric heme in DGCR8 is not a methionine. A , mass spectra of native and SeMet-labeled frog DGCR8 HBD-His 6 indicates nearly complete substitution of Met by SeMet. The difference in their m / z peaks, 239, is consistent with all five methionine

Techniques Used: Labeling

Schematic of how the DGCR8 HBD binds ferric heme.
Figure Legend Snippet: Schematic of how the DGCR8 HBD binds ferric heme.

Techniques Used:

40) Product Images from "Arabidopsis CULLIN4-Damaged DNA Binding Protein 1 Interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexes to Regulate Photomorphogenesis and Flowering Time [C] CULLIN4-Damaged DNA Binding Protein 1 Interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexes to Regulate Photomorphogenesis and Flowering Time [C] [W]"

Article Title: Arabidopsis CULLIN4-Damaged DNA Binding Protein 1 Interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexes to Regulate Photomorphogenesis and Flowering Time [C] CULLIN4-Damaged DNA Binding Protein 1 Interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexes to Regulate Photomorphogenesis and Flowering Time [C] [W]

Journal: The Plant Cell

doi: 10.1105/tpc.109.065490

DDB1A and DDB1B Interact with COP1 and SPA Proteins in Vitro.
Figure Legend Snippet: DDB1A and DDB1B Interact with COP1 and SPA Proteins in Vitro.

Techniques Used: In Vitro

Related Articles

Clone Assay:

Article Title: A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein
Article Snippet: .. Protein Purification and Crystallization Mature IL18 and YLDV-IL18BP (residues 20–136) were individually cloned into a modified pET vector as SUMO fusion proteins with N-terminal 6×His tags and expressed in E. coli BL21 (DE3) gold (Stratagene ) or Rosetta-Gami 2 (Invitrogen ) strains, respectively. .. An IL18 mutant (C38S, C68S, C76S, C127S, K67A, E69A, K70A, I71A) with substitutions of four nonessential cysteines and four additional surface residues opposite to the IL18BP binding interface, IL18 (8S), and the triple-cysteine mutant of YLDV-IL18BP (residues 22–136, C87S, C132S) were cloned and expressed in the same way as WT proteins.

Centrifugation:

Article Title: The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi
Article Snippet: .. The lysates were clarified by centrifugation at 12,000 g for 10 min. For coexpression experiments, Arf1p (T31N) and Arf1p (Q71L) were coexpressed with the COOH-terminal 126 amino acids of Rud3p in the polycistronic vector pOPCG and also expressed in the E. coli BL21-GOLD (DE3) strain (Stratagene). .. Affinity chromatography with immobilized GTPases GST-Arf1p, GST-Arf3p, or GST-Arl1p were purified, and then loaded with either GDP or GTP-γ-S as described previously ( ).

Positron Emission Tomography:

Article Title: A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein
Article Snippet: .. Protein Purification and Crystallization Mature IL18 and YLDV-IL18BP (residues 20–136) were individually cloned into a modified pET vector as SUMO fusion proteins with N-terminal 6×His tags and expressed in E. coli BL21 (DE3) gold (Stratagene ) or Rosetta-Gami 2 (Invitrogen ) strains, respectively. .. An IL18 mutant (C38S, C68S, C76S, C127S, K67A, E69A, K70A, I71A) with substitutions of four nonessential cysteines and four additional surface residues opposite to the IL18BP binding interface, IL18 (8S), and the triple-cysteine mutant of YLDV-IL18BP (residues 22–136, C87S, C132S) were cloned and expressed in the same way as WT proteins.

Article Title: Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass
Article Snippet: .. Gene Expression and Protein Purification The recombinant pET-46 Ek/LIC plasmids harboring the genes coding for CbCelA-TM1, CbCdx1A, CbXyn10A, and CbHsp18 and the pET-28 plasmid harboring the gene encoding MkHistone1 were transformed individually into E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Stratagene, La Jolla, CA) and selected on LB agar plates supplemented with 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. .. This culture was transferred into a 1-liter LB medium containing the same concentration of both antibiotics and shaken at a speed of 250 rpm at 37°C until the optical density at 600 nm (OD600 ) reached 0.3.

Mutagenesis:

Article Title: Probing the Determinants of Diacylglycerol Binding Affinity in C1B domain of Protein Kinase C?
Article Snippet: .. Mutagenic DNA for the Y123W C1Bα mutant was constructed from the wt C1Bα gene using a Stratagene QuickChange™ site-directed mutagenesis kit and suitable PCR primers. .. Purified wt and mutagenic plasmids were transformed into BL21(DE3) E. coli cells.

Construct:

Article Title: Probing the Determinants of Diacylglycerol Binding Affinity in C1B domain of Protein Kinase C?
Article Snippet: .. Mutagenic DNA for the Y123W C1Bα mutant was constructed from the wt C1Bα gene using a Stratagene QuickChange™ site-directed mutagenesis kit and suitable PCR primers. .. Purified wt and mutagenic plasmids were transformed into BL21(DE3) E. coli cells.

Article Title: Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms
Article Snippet: .. The expression and purification of the GC-1 bound TRα-LBD was accomplished as described in Nunes et al. [ ]. hTRβ The human TRβ1-LBD construct, which includes amino-acid residues Glu202-Asp461 (NCBI protein accession No. NP000452), was expressed in E. coli strain BL21(DE3) (Stratagene). ..

Purification:

Article Title: New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats
Article Snippet: .. Recombinant protein p68 ΔCt2 and eIF4A3 were expressed in E. coli BL21 (DE3) and successively purified on calmodulin resin (Stratagene) and on nickel sepharose (GE-healthcare) as previously described ( ). ..

Article Title: Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases
Article Snippet: .. Expression and purification of proteins The P. abyssi L7Ae, Nop5p and aFib proteins were expressed from recombinant pET15b vectors ( ) in Escherichia coli BL21 (DE3) RIL (Stratagene) cells induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG). .. After sonication, bulk of E. coli proteins was removed by thermodenaturation at 65°C for 15 min and spinning down.

Article Title: RECQL5 plays co-operative and complementary roles with WRN syndrome helicase
Article Snippet: .. Recombinant proteins Recombinant RECQL5 protein and the ATPase dead K58R RECQL5 were purified from E scherichia coli by overexpression as fusion proteins with a self-cleaving intein–chitin-binding domain in BL21(DE3) CodonPlus RIPL strain (Stratagene, Agilent Technologies, Santa Clara, CA, USA), as previously described ( ). .. Recombinant histidine-tagged WRN protein was purified using a baculovirus/insect cell expression system, as previously described ( ).

Article Title: Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms
Article Snippet: .. The expression and purification of the GC-1 bound TRα-LBD was accomplished as described in Nunes et al. [ ]. hTRβ The human TRβ1-LBD construct, which includes amino-acid residues Glu202-Asp461 (NCBI protein accession No. NP000452), was expressed in E. coli strain BL21(DE3) (Stratagene). ..

Protein Purification:

Article Title: A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein
Article Snippet: .. Protein Purification and Crystallization Mature IL18 and YLDV-IL18BP (residues 20–136) were individually cloned into a modified pET vector as SUMO fusion proteins with N-terminal 6×His tags and expressed in E. coli BL21 (DE3) gold (Stratagene ) or Rosetta-Gami 2 (Invitrogen ) strains, respectively. .. An IL18 mutant (C38S, C68S, C76S, C127S, K67A, E69A, K70A, I71A) with substitutions of four nonessential cysteines and four additional surface residues opposite to the IL18BP binding interface, IL18 (8S), and the triple-cysteine mutant of YLDV-IL18BP (residues 22–136, C87S, C132S) were cloned and expressed in the same way as WT proteins.

Article Title: Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass
Article Snippet: .. Gene Expression and Protein Purification The recombinant pET-46 Ek/LIC plasmids harboring the genes coding for CbCelA-TM1, CbCdx1A, CbXyn10A, and CbHsp18 and the pET-28 plasmid harboring the gene encoding MkHistone1 were transformed individually into E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Stratagene, La Jolla, CA) and selected on LB agar plates supplemented with 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. .. This culture was transferred into a 1-liter LB medium containing the same concentration of both antibiotics and shaken at a speed of 250 rpm at 37°C until the optical density at 600 nm (OD600 ) reached 0.3.

Modification:

Article Title: A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein
Article Snippet: .. Protein Purification and Crystallization Mature IL18 and YLDV-IL18BP (residues 20–136) were individually cloned into a modified pET vector as SUMO fusion proteins with N-terminal 6×His tags and expressed in E. coli BL21 (DE3) gold (Stratagene ) or Rosetta-Gami 2 (Invitrogen ) strains, respectively. .. An IL18 mutant (C38S, C68S, C76S, C127S, K67A, E69A, K70A, I71A) with substitutions of four nonessential cysteines and four additional surface residues opposite to the IL18BP binding interface, IL18 (8S), and the triple-cysteine mutant of YLDV-IL18BP (residues 22–136, C87S, C132S) were cloned and expressed in the same way as WT proteins.

Expressing:

Article Title: Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases
Article Snippet: .. Expression and purification of proteins The P. abyssi L7Ae, Nop5p and aFib proteins were expressed from recombinant pET15b vectors ( ) in Escherichia coli BL21 (DE3) RIL (Stratagene) cells induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG). .. After sonication, bulk of E. coli proteins was removed by thermodenaturation at 65°C for 15 min and spinning down.

Article Title: Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass
Article Snippet: .. Gene Expression and Protein Purification The recombinant pET-46 Ek/LIC plasmids harboring the genes coding for CbCelA-TM1, CbCdx1A, CbXyn10A, and CbHsp18 and the pET-28 plasmid harboring the gene encoding MkHistone1 were transformed individually into E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Stratagene, La Jolla, CA) and selected on LB agar plates supplemented with 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. .. This culture was transferred into a 1-liter LB medium containing the same concentration of both antibiotics and shaken at a speed of 250 rpm at 37°C until the optical density at 600 nm (OD600 ) reached 0.3.

Article Title: Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms
Article Snippet: .. The expression and purification of the GC-1 bound TRα-LBD was accomplished as described in Nunes et al. [ ]. hTRβ The human TRβ1-LBD construct, which includes amino-acid residues Glu202-Asp461 (NCBI protein accession No. NP000452), was expressed in E. coli strain BL21(DE3) (Stratagene). ..

Polymerase Chain Reaction:

Article Title: Probing the Determinants of Diacylglycerol Binding Affinity in C1B domain of Protein Kinase C?
Article Snippet: .. Mutagenic DNA for the Y123W C1Bα mutant was constructed from the wt C1Bα gene using a Stratagene QuickChange™ site-directed mutagenesis kit and suitable PCR primers. .. Purified wt and mutagenic plasmids were transformed into BL21(DE3) E. coli cells.

Crystallization Assay:

Article Title: A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein
Article Snippet: .. Protein Purification and Crystallization Mature IL18 and YLDV-IL18BP (residues 20–136) were individually cloned into a modified pET vector as SUMO fusion proteins with N-terminal 6×His tags and expressed in E. coli BL21 (DE3) gold (Stratagene ) or Rosetta-Gami 2 (Invitrogen ) strains, respectively. .. An IL18 mutant (C38S, C68S, C76S, C127S, K67A, E69A, K70A, I71A) with substitutions of four nonessential cysteines and four additional surface residues opposite to the IL18BP binding interface, IL18 (8S), and the triple-cysteine mutant of YLDV-IL18BP (residues 22–136, C87S, C132S) were cloned and expressed in the same way as WT proteins.

Recombinant:

Article Title: New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats
Article Snippet: .. Recombinant protein p68 ΔCt2 and eIF4A3 were expressed in E. coli BL21 (DE3) and successively purified on calmodulin resin (Stratagene) and on nickel sepharose (GE-healthcare) as previously described ( ). ..

Article Title: Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases
Article Snippet: .. Expression and purification of proteins The P. abyssi L7Ae, Nop5p and aFib proteins were expressed from recombinant pET15b vectors ( ) in Escherichia coli BL21 (DE3) RIL (Stratagene) cells induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG). .. After sonication, bulk of E. coli proteins was removed by thermodenaturation at 65°C for 15 min and spinning down.

Article Title: Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass
Article Snippet: .. Gene Expression and Protein Purification The recombinant pET-46 Ek/LIC plasmids harboring the genes coding for CbCelA-TM1, CbCdx1A, CbXyn10A, and CbHsp18 and the pET-28 plasmid harboring the gene encoding MkHistone1 were transformed individually into E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Stratagene, La Jolla, CA) and selected on LB agar plates supplemented with 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. .. This culture was transferred into a 1-liter LB medium containing the same concentration of both antibiotics and shaken at a speed of 250 rpm at 37°C until the optical density at 600 nm (OD600 ) reached 0.3.

Article Title: RECQL5 plays co-operative and complementary roles with WRN syndrome helicase
Article Snippet: .. Recombinant proteins Recombinant RECQL5 protein and the ATPase dead K58R RECQL5 were purified from E scherichia coli by overexpression as fusion proteins with a self-cleaving intein–chitin-binding domain in BL21(DE3) CodonPlus RIPL strain (Stratagene, Agilent Technologies, Santa Clara, CA, USA), as previously described ( ). .. Recombinant histidine-tagged WRN protein was purified using a baculovirus/insect cell expression system, as previously described ( ).

Over Expression:

Article Title: RECQL5 plays co-operative and complementary roles with WRN syndrome helicase
Article Snippet: .. Recombinant proteins Recombinant RECQL5 protein and the ATPase dead K58R RECQL5 were purified from E scherichia coli by overexpression as fusion proteins with a self-cleaving intein–chitin-binding domain in BL21(DE3) CodonPlus RIPL strain (Stratagene, Agilent Technologies, Santa Clara, CA, USA), as previously described ( ). .. Recombinant histidine-tagged WRN protein was purified using a baculovirus/insect cell expression system, as previously described ( ).

Transformation Assay:

Article Title: Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass
Article Snippet: .. Gene Expression and Protein Purification The recombinant pET-46 Ek/LIC plasmids harboring the genes coding for CbCelA-TM1, CbCdx1A, CbXyn10A, and CbHsp18 and the pET-28 plasmid harboring the gene encoding MkHistone1 were transformed individually into E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Stratagene, La Jolla, CA) and selected on LB agar plates supplemented with 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. .. This culture was transferred into a 1-liter LB medium containing the same concentration of both antibiotics and shaken at a speed of 250 rpm at 37°C until the optical density at 600 nm (OD600 ) reached 0.3.

Plasmid Preparation:

Article Title: A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein
Article Snippet: .. Protein Purification and Crystallization Mature IL18 and YLDV-IL18BP (residues 20–136) were individually cloned into a modified pET vector as SUMO fusion proteins with N-terminal 6×His tags and expressed in E. coli BL21 (DE3) gold (Stratagene ) or Rosetta-Gami 2 (Invitrogen ) strains, respectively. .. An IL18 mutant (C38S, C68S, C76S, C127S, K67A, E69A, K70A, I71A) with substitutions of four nonessential cysteines and four additional surface residues opposite to the IL18BP binding interface, IL18 (8S), and the triple-cysteine mutant of YLDV-IL18BP (residues 22–136, C87S, C132S) were cloned and expressed in the same way as WT proteins.

Article Title: Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass
Article Snippet: .. Gene Expression and Protein Purification The recombinant pET-46 Ek/LIC plasmids harboring the genes coding for CbCelA-TM1, CbCdx1A, CbXyn10A, and CbHsp18 and the pET-28 plasmid harboring the gene encoding MkHistone1 were transformed individually into E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Stratagene, La Jolla, CA) and selected on LB agar plates supplemented with 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. .. This culture was transferred into a 1-liter LB medium containing the same concentration of both antibiotics and shaken at a speed of 250 rpm at 37°C until the optical density at 600 nm (OD600 ) reached 0.3.

Article Title: The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi
Article Snippet: .. The lysates were clarified by centrifugation at 12,000 g for 10 min. For coexpression experiments, Arf1p (T31N) and Arf1p (Q71L) were coexpressed with the COOH-terminal 126 amino acids of Rud3p in the polycistronic vector pOPCG and also expressed in the E. coli BL21-GOLD (DE3) strain (Stratagene). .. Affinity chromatography with immobilized GTPases GST-Arf1p, GST-Arf3p, or GST-Arl1p were purified, and then loaded with either GDP or GTP-γ-S as described previously ( ).

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    Stratagene e coli bl21
    SDS-PAGE analysis of purified protein recombinant SllB in E. coli <t>BL21</t> cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.
    E Coli Bl21, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene e coli bl21 gold
    In vivo interactions of MBP-UreD with (UreABC) 3 , UreB, (UreAC) 3 , and UreBΔ1-19. (A) SDS-PAGE depicting the interactions of MBP-UreD with (UreABC) 3 , UreB, and (UreAC) 3 . E. coli <t>BL21-Gold(DE3)</t> cells were co-transformed with pCDF-MBP-UreD (encoding
    E Coli Bl21 Gold, supplied by Stratagene, used in various techniques. Bioz Stars score: 90/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene e coli bl21 de3 ripl cells
    A biochemical approach to dissect portal function. (a) The plasmids containing g20 variants (WT or mutants) were transformed into E. coli <t>BL21</t> (DE3) <t>RIPL</t> strain for IPTG induced expression of the respective gp20 protein (green). (b) The E. coli cells
    E Coli Bl21 De3 Ripl Cells, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SDS-PAGE analysis of purified protein recombinant SllB in E. coli BL21 cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: SDS-PAGE analysis of purified protein recombinant SllB in E. coli BL21 cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

    Techniques: SDS Page, Purification, Recombinant, Marker

    A transmission electron microscopic observation on the E. coli BL21 with recombinant protein. (A) Normal E. coli BL21 was treated as control. (B) The E. coli BL21 cells recombinant S-layer protein. (C) Crystal lattice structures on surface of the E. coli BL21 cells recombinant S-layer protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: A transmission electron microscopic observation on the E. coli BL21 with recombinant protein. (A) Normal E. coli BL21 was treated as control. (B) The E. coli BL21 cells recombinant S-layer protein. (C) Crystal lattice structures on surface of the E. coli BL21 cells recombinant S-layer protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

    Techniques: Transmission Assay, Recombinant

    Expression of sllB in E. coli BL21 cells. (A) SDS-PAGE analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; lane M represent Takara Protein Marker (Broad). (B) Western blot analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; M, Takara Protein Marker (Broad). Lane M1, Precision plus protein standards; lane M2, perfect protein marker. Note the band pointed with arrows is the recombinant S-layer protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: Expression of sllB in E. coli BL21 cells. (A) SDS-PAGE analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; lane M represent Takara Protein Marker (Broad). (B) Western blot analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; M, Takara Protein Marker (Broad). Lane M1, Precision plus protein standards; lane M2, perfect protein marker. Note the band pointed with arrows is the recombinant S-layer protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

    Techniques: Expressing, SDS Page, Marker, Western Blot, Recombinant

    Abl kinase domain and Abl SH3-SH2-Kinase single chain multi-domain expression in bacteria and their purification analysed by SDS-PAGE and Western blot. (A) SDS-PAGE of Abl kinase: whole-cell lysate (lane 1); soluble proteins after centrifuge (lane 2); proteins bound on Co 2+ resin (lane3); retaining proteins on Co 2+ resin after thrombin cleavage (lane 4). (B) Tyrosine phosphorylation status: whole-cell lysate of BL21 with GroEL expression (lane 1); whole-cell lysate of BL21 with GroEL and Abl kinase domain co-expression (lane 2); proteins bound on Co 2+ resin (lane 3); proteins on Co 2+ resin after thrombin cleavage (lane 4); proteins eluted from the affinity resin (lane 5); protein from Lane 5 after CIP treatment (lane 6); dephosphorylated Abl kinase domain after purification (lane 7). The upper panel is Coomassie blue stained SDS-PAGE, the lower panel is an immunoblot with anti-phosphotyrosine antibody. (C) SDS-PAGE of Abl SH3-SH2-kinase: cell lysate before and after centrifuge (land 1 and 2); proteins remained on and eluted from the TALON resin after thrombin cleavage (land 3 and 4); after ionic exchange purification (lane 5); after both ionic exchange and size exclusion purification (lane 6); NMR sample before and after measurement (lane 7 and 8).

    Journal: Molecular bioSystems

    Article Title: Abl Kinase Constructs Expressed in Bacteria: facilitation of structural and functional studies including segmental labeling by expressed protein ligation

    doi: 10.1039/c2mb25051a

    Figure Lengend Snippet: Abl kinase domain and Abl SH3-SH2-Kinase single chain multi-domain expression in bacteria and their purification analysed by SDS-PAGE and Western blot. (A) SDS-PAGE of Abl kinase: whole-cell lysate (lane 1); soluble proteins after centrifuge (lane 2); proteins bound on Co 2+ resin (lane3); retaining proteins on Co 2+ resin after thrombin cleavage (lane 4). (B) Tyrosine phosphorylation status: whole-cell lysate of BL21 with GroEL expression (lane 1); whole-cell lysate of BL21 with GroEL and Abl kinase domain co-expression (lane 2); proteins bound on Co 2+ resin (lane 3); proteins on Co 2+ resin after thrombin cleavage (lane 4); proteins eluted from the affinity resin (lane 5); protein from Lane 5 after CIP treatment (lane 6); dephosphorylated Abl kinase domain after purification (lane 7). The upper panel is Coomassie blue stained SDS-PAGE, the lower panel is an immunoblot with anti-phosphotyrosine antibody. (C) SDS-PAGE of Abl SH3-SH2-kinase: cell lysate before and after centrifuge (land 1 and 2); proteins remained on and eluted from the TALON resin after thrombin cleavage (land 3 and 4); after ionic exchange purification (lane 5); after both ionic exchange and size exclusion purification (lane 6); NMR sample before and after measurement (lane 7 and 8).

    Article Snippet: Plasmid containing SH3-SH2-kinase, pMAL/SH32(C2A)kin and plasmid containing kinase domain for ligation, pMAL/SspDnaAblCkinase were simply transformed into E. coli BL21 for recombinant protein expression.

    Techniques: Expressing, Purification, SDS Page, Western Blot, Staining, Nuclear Magnetic Resonance

    In vivo interactions of MBP-UreD with (UreABC) 3 , UreB, (UreAC) 3 , and UreBΔ1-19. (A) SDS-PAGE depicting the interactions of MBP-UreD with (UreABC) 3 , UreB, and (UreAC) 3 . E. coli BL21-Gold(DE3) cells were co-transformed with pCDF-MBP-UreD (encoding

    Journal: Biochemistry

    Article Title: The Function of UreB in Klebsiella aerogenes Urease

    doi: 10.1021/bi2011064

    Figure Lengend Snippet: In vivo interactions of MBP-UreD with (UreABC) 3 , UreB, (UreAC) 3 , and UreBΔ1-19. (A) SDS-PAGE depicting the interactions of MBP-UreD with (UreABC) 3 , UreB, and (UreAC) 3 . E. coli BL21-Gold(DE3) cells were co-transformed with pCDF-MBP-UreD (encoding

    Article Snippet: UreBΔ1-19 was purified from E. coli BL21-Gold(DE3) harboring pUreBΔ1-19 by a process similar to that for UreB.

    Techniques: In Vivo, SDS Page, Transformation Assay

    A biochemical approach to dissect portal function. (a) The plasmids containing g20 variants (WT or mutants) were transformed into E. coli BL21 (DE3) RIPL strain for IPTG induced expression of the respective gp20 protein (green). (b) The E. coli cells

    Journal: Journal of molecular biology

    Article Title: Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

    doi: 10.1016/j.jmb.2013.10.011

    Figure Lengend Snippet: A biochemical approach to dissect portal function. (a) The plasmids containing g20 variants (WT or mutants) were transformed into E. coli BL21 (DE3) RIPL strain for IPTG induced expression of the respective gp20 protein (green). (b) The E. coli cells

    Article Snippet: E. coli BL21 (DE3) RIPL cells containing the gp20 recombinant plasmids was induced with isopropyl-β-D-thio-galactoside (IPTG; 1 mM) for 20 min at 37°C.

    Techniques: Transformation Assay, Expressing