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Effect of PhADT3 RNAi downregulation in wild-type and <t>PhCM2</t> RNAi petunia flowers. a , b PhADT3 mRNA ( a ) and PhADT1 mRNA ( b ) levels in wild-type and PhCM2 RNAi line 15 petunia flowers. For each genetic background, black and white bars represent flowers infiltrated with empty vector or the PhADT3 RNAi construct, respectively. Data are presented as a percentage relative to the corresponding empty vector reference. c , d Levels of phenylalanine ( c ) and phenylalanine-derived volatiles ( d ) in petunia flowers of wild-type and PhCM2 RNAi line 15 infiltrated with agrobacterium carrying the empty vector (black bars) or the PhADT3 RNAi construct (white bars). For ( c , d ) data are presented as a percentage relative to the WT empty vector reference. Data are means ± SE ( n = 6 biological replicates). * P
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Images

1) Product Images from "Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants"

Article Title: Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants

Journal: Nature Communications

doi: 10.1038/s41467-018-07969-2

Effect of PhADT3 RNAi downregulation in wild-type and PhCM2 RNAi petunia flowers. a , b PhADT3 mRNA ( a ) and PhADT1 mRNA ( b ) levels in wild-type and PhCM2 RNAi line 15 petunia flowers. For each genetic background, black and white bars represent flowers infiltrated with empty vector or the PhADT3 RNAi construct, respectively. Data are presented as a percentage relative to the corresponding empty vector reference. c , d Levels of phenylalanine ( c ) and phenylalanine-derived volatiles ( d ) in petunia flowers of wild-type and PhCM2 RNAi line 15 infiltrated with agrobacterium carrying the empty vector (black bars) or the PhADT3 RNAi construct (white bars). For ( c , d ) data are presented as a percentage relative to the WT empty vector reference. Data are means ± SE ( n = 6 biological replicates). * P
Figure Legend Snippet: Effect of PhADT3 RNAi downregulation in wild-type and PhCM2 RNAi petunia flowers. a , b PhADT3 mRNA ( a ) and PhADT1 mRNA ( b ) levels in wild-type and PhCM2 RNAi line 15 petunia flowers. For each genetic background, black and white bars represent flowers infiltrated with empty vector or the PhADT3 RNAi construct, respectively. Data are presented as a percentage relative to the corresponding empty vector reference. c , d Levels of phenylalanine ( c ) and phenylalanine-derived volatiles ( d ) in petunia flowers of wild-type and PhCM2 RNAi line 15 infiltrated with agrobacterium carrying the empty vector (black bars) or the PhADT3 RNAi construct (white bars). For ( c , d ) data are presented as a percentage relative to the WT empty vector reference. Data are means ± SE ( n = 6 biological replicates). * P

Techniques Used: Plasmid Preparation, Construct, Derivative Assay

Metabolic effects of PhCM2 RNAi downregulation in petunia flowers. a PhCM2 mRNA levels in 2-day-old petunia petals at 20:00 h. Expression levels in transgenic lines (white bars) are shown as a percentage of PhCM2 expression in control (black bar) petals set as 100% ( n ≥ 3 biological replicates). b CM activities with 500 μM chorismate in crude extracts (striped bars), and cytosolic (gray bars) and plastidial (back bars) fractions prepared from corollas of 1- to 3-day-old wild type and PhCM2 RNAi petunia flowers harvested at 10:00 h ( n = 3 biological replicates). c Internal pools of shikimate, prephenate, arogenate, and aromatic amino acids as well as total volatile emission ( n ≥ 6 biological replicates). Metabolite levels were measured in 2-day-old petunia petals at 20:00 h and are shown in nmol/gFW. Pink background indicates metabolites with potential dual subcellular localization, in the cytosol and plastids. Emitted volatiles were collected from 2-day-old wild-type and transgenic PhCM2 RNAi petunia flowers from 18:00 h to 22:00 h. Black and white bars represent wild-type and transgenic lines, respectively. Data are means ± SE. *P
Figure Legend Snippet: Metabolic effects of PhCM2 RNAi downregulation in petunia flowers. a PhCM2 mRNA levels in 2-day-old petunia petals at 20:00 h. Expression levels in transgenic lines (white bars) are shown as a percentage of PhCM2 expression in control (black bar) petals set as 100% ( n ≥ 3 biological replicates). b CM activities with 500 μM chorismate in crude extracts (striped bars), and cytosolic (gray bars) and plastidial (back bars) fractions prepared from corollas of 1- to 3-day-old wild type and PhCM2 RNAi petunia flowers harvested at 10:00 h ( n = 3 biological replicates). c Internal pools of shikimate, prephenate, arogenate, and aromatic amino acids as well as total volatile emission ( n ≥ 6 biological replicates). Metabolite levels were measured in 2-day-old petunia petals at 20:00 h and are shown in nmol/gFW. Pink background indicates metabolites with potential dual subcellular localization, in the cytosol and plastids. Emitted volatiles were collected from 2-day-old wild-type and transgenic PhCM2 RNAi petunia flowers from 18:00 h to 22:00 h. Black and white bars represent wild-type and transgenic lines, respectively. Data are means ± SE. *P

Techniques Used: Expressing, Transgenic Assay

Metabolic modeling of phenylalanine biosynthetic pathways in control and PhCM2 RNAi petunia flowers. Flux models representing the phenylalanine biosynthetic network in 2-day-old control and PhCM2 RNAi petunia flowers. Metabolic modeling was performed using pool sizes and isotopic abundances of phenylalanine and tyrosine, and measurements of phenylalanine-derived volatile emission (consumption flux, v c , blue lines) from petunia petals supplied with 10 mM 15 N-tyrosine for up to 6 h starting at 18:00 h ( n = 3 biological replicates). v 1 flux through the plastidial arogenate pathway (green lines), v 2 flux through the cytosolic phenylpyruvate (pink lines, also is enlarged in inserts). The v 2 / v 1 ratio is shown by black lines. Solid lines are estimated values and dotted lines are standard deviation for each flux value
Figure Legend Snippet: Metabolic modeling of phenylalanine biosynthetic pathways in control and PhCM2 RNAi petunia flowers. Flux models representing the phenylalanine biosynthetic network in 2-day-old control and PhCM2 RNAi petunia flowers. Metabolic modeling was performed using pool sizes and isotopic abundances of phenylalanine and tyrosine, and measurements of phenylalanine-derived volatile emission (consumption flux, v c , blue lines) from petunia petals supplied with 10 mM 15 N-tyrosine for up to 6 h starting at 18:00 h ( n = 3 biological replicates). v 1 flux through the plastidial arogenate pathway (green lines), v 2 flux through the cytosolic phenylpyruvate (pink lines, also is enlarged in inserts). The v 2 / v 1 ratio is shown by black lines. Solid lines are estimated values and dotted lines are standard deviation for each flux value

Techniques Used: Derivative Assay, Standard Deviation

2) Product Images from "A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System"

Article Title: A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System

Journal: Eukaryotic Cell

doi: 10.1128/EC.1.6.915-925.2002

In vitro protein binding assay of Pf MSP-1 with PfMSRP-1 and PfMSRP-2. (A) (Top) Coomassie gel (lane 1, GST-PfMSP-1; lane 2, His-PfMSRP-2) and Western blot analysis of fractions from a binding reaction using a mouse anti-His antibody (lane 1′, GST-PfMSP-1; lane 2′, His-PfMSRP-2; lane S, supernatant; lanes W1 to W3, washes 1 to 3; lane P, eluted fraction). (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSRP-2) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-His antibody. (B) (Top) Coomassie gel (lane 1, GST-PfMSRP-1; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody. (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody.
Figure Legend Snippet: In vitro protein binding assay of Pf MSP-1 with PfMSRP-1 and PfMSRP-2. (A) (Top) Coomassie gel (lane 1, GST-PfMSP-1; lane 2, His-PfMSRP-2) and Western blot analysis of fractions from a binding reaction using a mouse anti-His antibody (lane 1′, GST-PfMSP-1; lane 2′, His-PfMSRP-2; lane S, supernatant; lanes W1 to W3, washes 1 to 3; lane P, eluted fraction). (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSRP-2) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-His antibody. (B) (Top) Coomassie gel (lane 1, GST-PfMSRP-1; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody. (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody.

Techniques Used: In Vitro, Protein Binding, Western Blot, Binding Assay

Colocalization of PfMSRP-1 and PfMSRP-2 with P. falciparum MSP-1 as demonstrated by immunofluorescence of thin blood smears of asynchronous cultures of P. falciparum 3D7 prepared as described in Materials and Methods. (a) Slides were incubated with a mouse anti-PfMSRP-1 serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (b and f) Slides were incubated with a guinea pig anti-Pf83a serum and a rhodamine-labeled donkey anti-guinea pig IgG secondary antibody. (c) Overlay of panels a and b. (d) Bright-field image of panels a, b, and c. (e) Slides were incubated with a mouse anti-PfMSRP-2 serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (g) Overlay of panels e and f. (h) Bright-field image of panels e, f, and g. (i) Slides were incubated with a preimmune guinea pig serum and a rhodamine-labeled donkey anti-guinea pig IgG secondary antibody. (j) Bright-field image of panel i. (k) Slides were incubated with normal mouse serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (l) Bright field image of panel k.
Figure Legend Snippet: Colocalization of PfMSRP-1 and PfMSRP-2 with P. falciparum MSP-1 as demonstrated by immunofluorescence of thin blood smears of asynchronous cultures of P. falciparum 3D7 prepared as described in Materials and Methods. (a) Slides were incubated with a mouse anti-PfMSRP-1 serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (b and f) Slides were incubated with a guinea pig anti-Pf83a serum and a rhodamine-labeled donkey anti-guinea pig IgG secondary antibody. (c) Overlay of panels a and b. (d) Bright-field image of panels a, b, and c. (e) Slides were incubated with a mouse anti-PfMSRP-2 serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (g) Overlay of panels e and f. (h) Bright-field image of panels e, f, and g. (i) Slides were incubated with a preimmune guinea pig serum and a rhodamine-labeled donkey anti-guinea pig IgG secondary antibody. (j) Bright-field image of panel i. (k) Slides were incubated with normal mouse serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (l) Bright field image of panel k.

Techniques Used: Immunofluorescence, Incubation, Labeling

(A) Northern blot analysis of total RNA isolated from asynchronous cultures of P. falciparum strain 3D7. Blots were probed using PfMSRP-1 (lane 1), PfMSRP-2 (lane 2), and PfMSRP-3 (lane 3). (B) RNA slot blots containing total RNA isolated from synchronized cultures of P. falciparum strain 3D7 as described in Materials and Methods. The filters were probed with PfMSRP-1, PfMSRP-2, PfMSRP-3, and a probe corresponding to the 5′ region of MSP-1. The blot was also probed with a primer specific for the P. falciparum small ribosomal subunit to normalize loading. Lanes 1, 15.0 μg of RNA; lanes 2, 10.0 μg of RNA; lanes 3, 5.0 μg of RNA; lanes 4, 1.0 μg of RNA.
Figure Legend Snippet: (A) Northern blot analysis of total RNA isolated from asynchronous cultures of P. falciparum strain 3D7. Blots were probed using PfMSRP-1 (lane 1), PfMSRP-2 (lane 2), and PfMSRP-3 (lane 3). (B) RNA slot blots containing total RNA isolated from synchronized cultures of P. falciparum strain 3D7 as described in Materials and Methods. The filters were probed with PfMSRP-1, PfMSRP-2, PfMSRP-3, and a probe corresponding to the 5′ region of MSP-1. The blot was also probed with a primer specific for the P. falciparum small ribosomal subunit to normalize loading. Lanes 1, 15.0 μg of RNA; lanes 2, 10.0 μg of RNA; lanes 3, 5.0 μg of RNA; lanes 4, 1.0 μg of RNA.

Techniques Used: Northern Blot, Isolation

Lack of cross-reactivity of antisera directed against recombinant PfMSRP-1 and PfMSRP-2. A 50.0-ng portion of PfMSRP-1 is run on each blot in lanes 1 to 4, and 50.0 ng of PfMSRP-2 is run on each of the blots in lanes 5 to 8. Lane 1, polyclonal mouse anti-GST IgG antibody as a positive control; lanes 2 and 7, mouse anti-PfMSRP-1; lanes 3 and 6, mouse anti-PfMSRP-2; lane 4, Coomassie stain of recombinant PfMSRP-1 used in the studies; lane 5, monoclonal mouse anti-His antibody; lane 8, Coomassie stain of recombinant PfMSRP-2 used in the studies. PfMSRP-1 and PfMSRP-2 did not show any reactivity when incubated with preimmune mouse serum or with the goat anti-mouse horseradish peroxidase-conjugated secondary antibody alone. The same molecular weight marker is run in lanes 1 and 5.
Figure Legend Snippet: Lack of cross-reactivity of antisera directed against recombinant PfMSRP-1 and PfMSRP-2. A 50.0-ng portion of PfMSRP-1 is run on each blot in lanes 1 to 4, and 50.0 ng of PfMSRP-2 is run on each of the blots in lanes 5 to 8. Lane 1, polyclonal mouse anti-GST IgG antibody as a positive control; lanes 2 and 7, mouse anti-PfMSRP-1; lanes 3 and 6, mouse anti-PfMSRP-2; lane 4, Coomassie stain of recombinant PfMSRP-1 used in the studies; lane 5, monoclonal mouse anti-His antibody; lane 8, Coomassie stain of recombinant PfMSRP-2 used in the studies. PfMSRP-1 and PfMSRP-2 did not show any reactivity when incubated with preimmune mouse serum or with the goat anti-mouse horseradish peroxidase-conjugated secondary antibody alone. The same molecular weight marker is run in lanes 1 and 5.

Techniques Used: Recombinant, Positive Control, Staining, Incubation, Molecular Weight, Marker

3) Product Images from "A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair *"

Article Title: A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M900571200

The location of the interaction domains in the primary sequence of G. stearothermophilus UvrA and UvrB.
Figure Legend Snippet: The location of the interaction domains in the primary sequence of G. stearothermophilus UvrA and UvrB.

Techniques Used: Sequencing

4) Product Images from "Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment"

Article Title: Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

Journal: Cancer Medicine

doi: 10.1002/cam4.320

Characterization of KM10 scFv generated from MS-1 mAb. (A) Schematic representation of the generated scFv expression vector. (B) The purity of the used KM10 scFv was analyzed in native PAGE electrophoresis. Lane 1, molecular weight marker; Lane 2, purified KM10 scFv. (C) Bound KM10 scFv in Aggrus-derived P4262 peptide-coated plates were detected using peroxidase-conjugated anti-FLAG antibody. Data are presented as the means ± SDs of triplicate measurements. (D) CHO cells transfected with empty vectors (CHO/Mock) or Aggrus expression plasmid (CHO/Aggrus), as well as H226 and PC-10 cells were treated with 5 μ g/mL of KM10 scFv (upper panels) or MS-1 mAb (lower panels). The cells were then treated with Alexa Fluor 488-conjugated secondary antibody. The broken lines represent the cells treated with secondary antibody alone and the bold lines represent KM10 scFv- or MS-1 mAb-treated cells. (E) The indicated five concentrations of KM10 scFv were passed over the chips containing immobilized recombinant human Aggrus-Fc protein using the Biacore X100 system for protein interaction analysis.
Figure Legend Snippet: Characterization of KM10 scFv generated from MS-1 mAb. (A) Schematic representation of the generated scFv expression vector. (B) The purity of the used KM10 scFv was analyzed in native PAGE electrophoresis. Lane 1, molecular weight marker; Lane 2, purified KM10 scFv. (C) Bound KM10 scFv in Aggrus-derived P4262 peptide-coated plates were detected using peroxidase-conjugated anti-FLAG antibody. Data are presented as the means ± SDs of triplicate measurements. (D) CHO cells transfected with empty vectors (CHO/Mock) or Aggrus expression plasmid (CHO/Aggrus), as well as H226 and PC-10 cells were treated with 5 μ g/mL of KM10 scFv (upper panels) or MS-1 mAb (lower panels). The cells were then treated with Alexa Fluor 488-conjugated secondary antibody. The broken lines represent the cells treated with secondary antibody alone and the bold lines represent KM10 scFv- or MS-1 mAb-treated cells. (E) The indicated five concentrations of KM10 scFv were passed over the chips containing immobilized recombinant human Aggrus-Fc protein using the Biacore X100 system for protein interaction analysis.

Techniques Used: Generated, Mass Spectrometry, Expressing, Plasmid Preparation, Clear Native PAGE, Electrophoresis, Molecular Weight, Marker, Purification, Derivative Assay, Transfection, Recombinant

Effect of KM10 and K-11 scFvs on Aggrus-induced platelet aggregation. (A) The purity of the used K-11 scFv was analyzed in native PAGE electrophoresis. Lane 1, molecular weight marker; Lane 2, purified K-11 scFv. (B) Bound KM10 and K-11 scFvs in P4262 peptide-coated plates were detected using peroxidase-conjugated anti-FLAG antibody. Data are presented as means ± SDs of triplicate measurements. * P
Figure Legend Snippet: Effect of KM10 and K-11 scFvs on Aggrus-induced platelet aggregation. (A) The purity of the used K-11 scFv was analyzed in native PAGE electrophoresis. Lane 1, molecular weight marker; Lane 2, purified K-11 scFv. (B) Bound KM10 and K-11 scFvs in P4262 peptide-coated plates were detected using peroxidase-conjugated anti-FLAG antibody. Data are presented as means ± SDs of triplicate measurements. * P

Techniques Used: Clear Native PAGE, Electrophoresis, Molecular Weight, Marker, Purification

Affinity maturation by phage display technology. (A) Amino acid sequences of the obtained phages are described in single-letter code. Amino acids that matched with KM10 scFv sequence are represented by dash. The number of obtained phage colonies was described in parentheses. CDR, complementarity-determining region; FR, framework region. (B) Phages displaying mutated scFv were added to plate coated with human Aggrus-derived P4262 peptide and then incubated with peroxidase-conjugated anti-M13 phage mAb. Data are presented as means ± SDs of triplicate measurements. NS, not significant. * P
Figure Legend Snippet: Affinity maturation by phage display technology. (A) Amino acid sequences of the obtained phages are described in single-letter code. Amino acids that matched with KM10 scFv sequence are represented by dash. The number of obtained phage colonies was described in parentheses. CDR, complementarity-determining region; FR, framework region. (B) Phages displaying mutated scFv were added to plate coated with human Aggrus-derived P4262 peptide and then incubated with peroxidase-conjugated anti-M13 phage mAb. Data are presented as means ± SDs of triplicate measurements. NS, not significant. * P

Techniques Used: Sequencing, Derivative Assay, Incubation

5) Product Images from "The Self-Interaction of a Nodavirus Replicase Is Enhanced by Mitochondrial Membrane Lipids"

Article Title: The Self-Interaction of a Nodavirus Replicase Is Enhanced by Mitochondrial Membrane Lipids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0089628

Characterization of the stimulating effect of MMLs on protein A self-interaction activity. (A) The in vitro translation His-protA fragments aa 1–254/M1 (K91A, W92A, and R93A) and aa 1–254/M2 (S163A, R165A and Y169A) were incubated without (left) or with the MMLs (right) and subjected to Nycodenz flotation. (B) MBP-protA fragment aa 1–254 was used to pull-down His-protA fragments aa 1–254/M1 (lanes 2–5) and aa 1–254/M2 (lanes 3–8) in the increasing concentrations of MMLs (lanes 3–5 and 7–8). The concentrations of MMLs are indicated above each lane. The wt protein A fragment aa 1–254 was used as the control (lane 1). (C) MBP-protA was used to pull-down 1 µM His-protA FHV at the increasing concentrations of MML (lanes 3–5), or increasing concentrations of His-protA FHV at the 2 µg/µl MMLs (lanes 6–8). The concentrations of His-protA FHV and MMLs are indicated above. The WhNV protein A self-interaction was used as the control (lane 1).
Figure Legend Snippet: Characterization of the stimulating effect of MMLs on protein A self-interaction activity. (A) The in vitro translation His-protA fragments aa 1–254/M1 (K91A, W92A, and R93A) and aa 1–254/M2 (S163A, R165A and Y169A) were incubated without (left) or with the MMLs (right) and subjected to Nycodenz flotation. (B) MBP-protA fragment aa 1–254 was used to pull-down His-protA fragments aa 1–254/M1 (lanes 2–5) and aa 1–254/M2 (lanes 3–8) in the increasing concentrations of MMLs (lanes 3–5 and 7–8). The concentrations of MMLs are indicated above each lane. The wt protein A fragment aa 1–254 was used as the control (lane 1). (C) MBP-protA was used to pull-down 1 µM His-protA FHV at the increasing concentrations of MML (lanes 3–5), or increasing concentrations of His-protA FHV at the 2 µg/µl MMLs (lanes 6–8). The concentrations of His-protA FHV and MMLs are indicated above. The WhNV protein A self-interaction was used as the control (lane 1).

Techniques Used: Activity Assay, In Vitro, Incubation

Expression of recombinant WhNV protein A. (A) Schematic representation of the expression strategy of WhNV protein A. Protein A ORF was cloned into pMAL-c2X and expressed as C-terminal fusion proteins with MBP (MBP-protA). To manipulate the vector, we mutated a BamH I restriction endonuclease site on protein A ORF sequences. (B) SDS-PAGE analysis of purified recombinant protein A from E. coli . Lane 1, Marker; lane 2, MBP protein alone; lane 3, MBP-protA.
Figure Legend Snippet: Expression of recombinant WhNV protein A. (A) Schematic representation of the expression strategy of WhNV protein A. Protein A ORF was cloned into pMAL-c2X and expressed as C-terminal fusion proteins with MBP (MBP-protA). To manipulate the vector, we mutated a BamH I restriction endonuclease site on protein A ORF sequences. (B) SDS-PAGE analysis of purified recombinant protein A from E. coli . Lane 1, Marker; lane 2, MBP protein alone; lane 3, MBP-protA.

Techniques Used: Expressing, Recombinant, Clone Assay, Plasmid Preparation, SDS Page, Purification, Marker

6) Product Images from "Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)"

Article Title: Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)

Journal: Oncogene

doi: 10.1038/onc.2014.194

Resveratrol interacts with G3BP1 at the NTF2-like domain ( a ) Resveratrol interacts with G3BP1. Recombinant full-length G3BP1 (FL-rG3BP1; 200 ng) or whole cell lysates from SK-MEL-5 cells (500 µg) were incubated with control or resveratrol-conjugated Sepharose 4B beads, and then the proteins bound to the beads were analyzed by Western blotting. ( b ) Resveratrol interacts with G3BP1 at the NTF2-like domain. The purified NTF2-like domain (residues 1–139) or RRM domain (residues 339–421) of G3BP1 (400 µg) was incubated with control or resveratrol-conjugated Sepharose 4B beads. The binding proteins were subjected to SDS-PAGE and then stained with Coomassie Blue. Most of the RRM domain protein appeared in the flow-through fraction and washed out during the washing steps. PD: pull-down, FT: flow-through. ( c ) Computational docking model of resveratrol with the NTF2-like domain of G3BP1. The structure is shown in ribbon diagram with overlapped surface representation in yellow color (insert). Resveratrol molecule is shown as green sticks, and amino acid residues surrounding resveratrol are shown as cyan sticks. Oxygens are colored red and nitrogens are colored blue. ( d ) Resveratrol interacts with G3BP1 at Val11, Phe33 and Phe124. HEK 293 cells were transfected with the indicated plasmids for 48 h. Whole cell lysates were incubated with control or resveratrol-conjugated Sepharose 4B beads, and then the proteins bound to the beads were analyzed by Western blotting.
Figure Legend Snippet: Resveratrol interacts with G3BP1 at the NTF2-like domain ( a ) Resveratrol interacts with G3BP1. Recombinant full-length G3BP1 (FL-rG3BP1; 200 ng) or whole cell lysates from SK-MEL-5 cells (500 µg) were incubated with control or resveratrol-conjugated Sepharose 4B beads, and then the proteins bound to the beads were analyzed by Western blotting. ( b ) Resveratrol interacts with G3BP1 at the NTF2-like domain. The purified NTF2-like domain (residues 1–139) or RRM domain (residues 339–421) of G3BP1 (400 µg) was incubated with control or resveratrol-conjugated Sepharose 4B beads. The binding proteins were subjected to SDS-PAGE and then stained with Coomassie Blue. Most of the RRM domain protein appeared in the flow-through fraction and washed out during the washing steps. PD: pull-down, FT: flow-through. ( c ) Computational docking model of resveratrol with the NTF2-like domain of G3BP1. The structure is shown in ribbon diagram with overlapped surface representation in yellow color (insert). Resveratrol molecule is shown as green sticks, and amino acid residues surrounding resveratrol are shown as cyan sticks. Oxygens are colored red and nitrogens are colored blue. ( d ) Resveratrol interacts with G3BP1 at Val11, Phe33 and Phe124. HEK 293 cells were transfected with the indicated plasmids for 48 h. Whole cell lysates were incubated with control or resveratrol-conjugated Sepharose 4B beads, and then the proteins bound to the beads were analyzed by Western blotting.

Techniques Used: Recombinant, Incubation, Western Blot, Purification, Binding Assay, SDS Page, Staining, Flow Cytometry, Transfection

7) Product Images from "Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons"

Article Title: Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons

Journal: PLoS ONE

doi: 10.1371/journal.pone.0110846

Direct interaction of hnRNP R and SMN. (A) Purification scheme of recombinant hnRNP R and SMN expressed as His-tagged proteins in E. coli strain BL21. (B) Affinity purification profile on a fast protein liquid chromatography (FPLC) of hnRNP R and SDS-PAGE of recombinant hnRNP R purification steps visualized by silver staining. (C) Affinity purification profile on a FPLC of SMN and SDS-PAGE of recombinant SMN purification steps visualized by colloidal staining. (D) Coimmunoprecipitation of recombinant SMN and hnRNP R.
Figure Legend Snippet: Direct interaction of hnRNP R and SMN. (A) Purification scheme of recombinant hnRNP R and SMN expressed as His-tagged proteins in E. coli strain BL21. (B) Affinity purification profile on a fast protein liquid chromatography (FPLC) of hnRNP R and SDS-PAGE of recombinant hnRNP R purification steps visualized by silver staining. (C) Affinity purification profile on a FPLC of SMN and SDS-PAGE of recombinant SMN purification steps visualized by colloidal staining. (D) Coimmunoprecipitation of recombinant SMN and hnRNP R.

Techniques Used: Purification, Recombinant, Affinity Purification, Fast Protein Liquid Chromatography, SDS Page, Silver Staining, Staining

Colocalization of Smn and hnRNP R in vivo in E18 motoneurons and axon terminals. (A) Representative cross section from E18 spinal cord stained against Smn, hnRNP R and ChAT (scale bar: 10 µm). Superimposed colocalizing points are highlighted in white. Smn signals were mainly found in the cytosol, with very few positive spots in the nuclei. HnRNP R immunoreactivity was observed in the nucleus and in the cytosol. Colocalization of Smn and hnRNP R was detected in the cytosol, especially in axonal initiation segments (PCC 0.27±0.03, MOC 0.81±0.01, N = 8). (B) Whole mount preparations from Diaphragm muscles from E18 mouse embryos stained against Smn, hnRNP R, ω-BTX and DAPI (scale bar: 2 µm). Both Smn and hnRNP R immunoreactivity were detected at these defined sites showing partial overlap (PCC 0.24±0.04, MOC 0.54±0.02, N = 6).
Figure Legend Snippet: Colocalization of Smn and hnRNP R in vivo in E18 motoneurons and axon terminals. (A) Representative cross section from E18 spinal cord stained against Smn, hnRNP R and ChAT (scale bar: 10 µm). Superimposed colocalizing points are highlighted in white. Smn signals were mainly found in the cytosol, with very few positive spots in the nuclei. HnRNP R immunoreactivity was observed in the nucleus and in the cytosol. Colocalization of Smn and hnRNP R was detected in the cytosol, especially in axonal initiation segments (PCC 0.27±0.03, MOC 0.81±0.01, N = 8). (B) Whole mount preparations from Diaphragm muscles from E18 mouse embryos stained against Smn, hnRNP R, ω-BTX and DAPI (scale bar: 2 µm). Both Smn and hnRNP R immunoreactivity were detected at these defined sites showing partial overlap (PCC 0.24±0.04, MOC 0.54±0.02, N = 6).

Techniques Used: In Vivo, Staining, Periodic Counter-current Chromatography

Coimmunoprecipitation of Smn and hnRNP R in primary motoneurons and native spinal cord. (A) 1 000 000 primary motoneurons were cultured for 7DIV on laminin-111. Cytosolic and soluble nuclear fractions were subjected to a pull-down with either Smn or hnRNP R antibodies, respectively. Coprecipitation of hnRNP R or Smn, respectively, was determined revealing an interaction of Smn and hnRNP R, particularly in the cytosolic fraction of embryonic mouse motoneurons (eluate lane). Smn was not detectable in the soluble nuclear fraction of motoneurons. HnRNP R was found both in nuclear and cytosolic extracts. For immunoprecipitation experiments a C-terminal antibody directed against hnRNP R (Abcam) was used [29] . Supernatants still contained some Smn or hnRNP R protein, respectively, suggesting that the interaction appears not to be exclusive as demonstrated by immunofluorescence colocalization analysis. No signal was obtained in the washing solution. Successful fractionation was controlled by α tubulin (cytosol) and histone H3 (nucleus) (right panel). (B) Fractionation of spinal cord tissue from E18 mouse embryos revealed a similar result as shown in (A). In the cytosolic fraction hnRNP R IP pulled-down Smn protein and vice versa . Nuclear Smn was not detected in the soluble, but in the corresponding insoluble nuclear fraction (right panel, lower blot). In contrast, nuclear hnRNP R was not found in the insoluble nuclear fraction. Cytosolic and nuclear extracts were validated by α tubulin and histone H3. (C) HEK293T cells were cultured and cytosolic and soluble nuclear fractions were prepared. Smn and hnRNP R were detected in cytosolic extracts as well as in soluble nuclear fractions. The pull down of Smn and hnRNP R, respectively, was successful (eluate lane, IP), but hnRNP R or Smn, respectively, could not be coprecipitated, neither from cytosolic nor from nuclear extracts. Successful fractionation was verified by GAPDH (cytosolic) and histone H3 (nucleus) (right panel).
Figure Legend Snippet: Coimmunoprecipitation of Smn and hnRNP R in primary motoneurons and native spinal cord. (A) 1 000 000 primary motoneurons were cultured for 7DIV on laminin-111. Cytosolic and soluble nuclear fractions were subjected to a pull-down with either Smn or hnRNP R antibodies, respectively. Coprecipitation of hnRNP R or Smn, respectively, was determined revealing an interaction of Smn and hnRNP R, particularly in the cytosolic fraction of embryonic mouse motoneurons (eluate lane). Smn was not detectable in the soluble nuclear fraction of motoneurons. HnRNP R was found both in nuclear and cytosolic extracts. For immunoprecipitation experiments a C-terminal antibody directed against hnRNP R (Abcam) was used [29] . Supernatants still contained some Smn or hnRNP R protein, respectively, suggesting that the interaction appears not to be exclusive as demonstrated by immunofluorescence colocalization analysis. No signal was obtained in the washing solution. Successful fractionation was controlled by α tubulin (cytosol) and histone H3 (nucleus) (right panel). (B) Fractionation of spinal cord tissue from E18 mouse embryos revealed a similar result as shown in (A). In the cytosolic fraction hnRNP R IP pulled-down Smn protein and vice versa . Nuclear Smn was not detected in the soluble, but in the corresponding insoluble nuclear fraction (right panel, lower blot). In contrast, nuclear hnRNP R was not found in the insoluble nuclear fraction. Cytosolic and nuclear extracts were validated by α tubulin and histone H3. (C) HEK293T cells were cultured and cytosolic and soluble nuclear fractions were prepared. Smn and hnRNP R were detected in cytosolic extracts as well as in soluble nuclear fractions. The pull down of Smn and hnRNP R, respectively, was successful (eluate lane, IP), but hnRNP R or Smn, respectively, could not be coprecipitated, neither from cytosolic nor from nuclear extracts. Successful fractionation was verified by GAPDH (cytosolic) and histone H3 (nucleus) (right panel).

Techniques Used: Cell Culture, Immunoprecipitation, Immunofluorescence, Fractionation

Colocalization of Smn and hnRNP R proteins in embryonic motoneurons. Representative images of cell bodies, axons and growth cones of primary embryonic motoneurons cultured on laminin-111 (A) and laminin-221/211 (B) for 5DIV and stained against Smn and hnRNP R (scale bar: 5 µm). Superimposed colocalizing points are highlighted in white. (C) No differences were observed with respect to colocalization and subcellular distribution of hnRNP R between these two investigated laminin isoforms. Representative images of cell bodies, axons and growth cones of motoneurons cultured on laminin-111 for either 3DIV (D) or 7DIV (E) and labeled against Smn and hnRNP R (scale bar: 5 µm). Both the degree of overlap between Smn and hnRNP R and the subcellular distribution of hnRNP R were regulated over time. The relative ratio of cytosolic versus nuclear hnRNP R immunoreactivity was significantly enhanced by 63% (P = 0.0173, t = 3.914, DF = 4) in motoneuron cell bodies cultured for 7DIV (1.63±0.16, n = 5, N = 46) in comparison to 3DIV (set as ‘1’; n = 5, N = 37). (F) After 7DIV (PCC 0.65±0.02, MOC 0.75±0.01, n = 5, N = 45) colocalization of Smn and hnRNP R in motoneuron cell bodies was higher (PCC P = 0.0112, t = 4.453, DF = 4; MOC P = 0.0086, t = 4.807, DF = 4) than after 3DIV (PCC 0.56±0.03, MOC 0.68±0.02, n = 5, N = 36). In axons the degree of overlap and correlation did not change (PCC P = 0.1504, t = 1.776, DF = 4; MOC P = 0.1449, t = 1.808, DF = 4) over time (3DIV PCC 0.43±0.04, MOC 0.55±0.03, n = 5, N = 36; 7DIV PCC 0.46±0.04, MOC 0.58±0.03, n = 5, N = 46), whereas in axonal growth cones a significant modification of the correlation (PCC P = 0.0467, t = 2.844, DF = 4; MOC P = 0.1565, t = 1.742, DF = 4) of both proteins was detected (3DIV PCC 0.38±0.03, MOC 0.52±0.02, n = 5, N = 37; 7DIV PCC 0.45±0.02, MOC 0.56±0.02, n = 5, N = 34).
Figure Legend Snippet: Colocalization of Smn and hnRNP R proteins in embryonic motoneurons. Representative images of cell bodies, axons and growth cones of primary embryonic motoneurons cultured on laminin-111 (A) and laminin-221/211 (B) for 5DIV and stained against Smn and hnRNP R (scale bar: 5 µm). Superimposed colocalizing points are highlighted in white. (C) No differences were observed with respect to colocalization and subcellular distribution of hnRNP R between these two investigated laminin isoforms. Representative images of cell bodies, axons and growth cones of motoneurons cultured on laminin-111 for either 3DIV (D) or 7DIV (E) and labeled against Smn and hnRNP R (scale bar: 5 µm). Both the degree of overlap between Smn and hnRNP R and the subcellular distribution of hnRNP R were regulated over time. The relative ratio of cytosolic versus nuclear hnRNP R immunoreactivity was significantly enhanced by 63% (P = 0.0173, t = 3.914, DF = 4) in motoneuron cell bodies cultured for 7DIV (1.63±0.16, n = 5, N = 46) in comparison to 3DIV (set as ‘1’; n = 5, N = 37). (F) After 7DIV (PCC 0.65±0.02, MOC 0.75±0.01, n = 5, N = 45) colocalization of Smn and hnRNP R in motoneuron cell bodies was higher (PCC P = 0.0112, t = 4.453, DF = 4; MOC P = 0.0086, t = 4.807, DF = 4) than after 3DIV (PCC 0.56±0.03, MOC 0.68±0.02, n = 5, N = 36). In axons the degree of overlap and correlation did not change (PCC P = 0.1504, t = 1.776, DF = 4; MOC P = 0.1449, t = 1.808, DF = 4) over time (3DIV PCC 0.43±0.04, MOC 0.55±0.03, n = 5, N = 36; 7DIV PCC 0.46±0.04, MOC 0.58±0.03, n = 5, N = 46), whereas in axonal growth cones a significant modification of the correlation (PCC P = 0.0467, t = 2.844, DF = 4; MOC P = 0.1565, t = 1.742, DF = 4) of both proteins was detected (3DIV PCC 0.38±0.03, MOC 0.52±0.02, n = 5, N = 37; 7DIV PCC 0.45±0.02, MOC 0.56±0.02, n = 5, N = 34).

Techniques Used: Cell Culture, Staining, Labeling, Periodic Counter-current Chromatography, Modification

Localization of Smn and hnRNP R at neuromuscular junctions from E18, P4 and adult Diaphragm . Whole mount preparations from Diaphragm muscles from developmental (E18) (A, C, left panels), postnatal (P4) (A, C, middle panels) and adult (3 months) (A, C, right panels) stages were performed (scale bar: 2 µm (C, left panel), 5 µm). (A) Muscles were stained against ω-BTX, SynPhys, DAPI and Smn protein. (A, left panel) At E18 Smn was highly enriched in presynaptic structures identified by SynPhys immunoreactivity. Few spots appeared in postsynaptic nuclei. (A, middle panel) Smn-positive signals were also detected in P4 motor endplates coresiding with SynPhys staining. Postsynaptic nuclei showed faint Smn immunoreactivity. (A, right panel) In 3 month old mice (adult stage) less Smn-positive signals were noticed as described before [53] , [56] . The few immunoreactive particles were predominantly located in presynaptic structures visualized by SynPhys staining. (B) Single optical slices of the P4 neuromuscular synapse highlighted the co-occurring SynPhys and Smn signals (scale bar: 5 µm). (C) Muscles were stained against ω-BTX, SynPhys, DAPI and hnRNP R. HnRNP R was codistributed with SynPhys in presynaptic compartments at E18 (left panel), P4 (middle panel) and adult stage (right panel). HnRNP R was also detected in postsynaptic structures revealing stronger immunoreactivity at these sites in comparison to Smn. (D) Single optical slices of the P4 motor endplate emphasized the presynaptic localization of hnRNP R (scale bar: 5 µm).
Figure Legend Snippet: Localization of Smn and hnRNP R at neuromuscular junctions from E18, P4 and adult Diaphragm . Whole mount preparations from Diaphragm muscles from developmental (E18) (A, C, left panels), postnatal (P4) (A, C, middle panels) and adult (3 months) (A, C, right panels) stages were performed (scale bar: 2 µm (C, left panel), 5 µm). (A) Muscles were stained against ω-BTX, SynPhys, DAPI and Smn protein. (A, left panel) At E18 Smn was highly enriched in presynaptic structures identified by SynPhys immunoreactivity. Few spots appeared in postsynaptic nuclei. (A, middle panel) Smn-positive signals were also detected in P4 motor endplates coresiding with SynPhys staining. Postsynaptic nuclei showed faint Smn immunoreactivity. (A, right panel) In 3 month old mice (adult stage) less Smn-positive signals were noticed as described before [53] , [56] . The few immunoreactive particles were predominantly located in presynaptic structures visualized by SynPhys staining. (B) Single optical slices of the P4 neuromuscular synapse highlighted the co-occurring SynPhys and Smn signals (scale bar: 5 µm). (C) Muscles were stained against ω-BTX, SynPhys, DAPI and hnRNP R. HnRNP R was codistributed with SynPhys in presynaptic compartments at E18 (left panel), P4 (middle panel) and adult stage (right panel). HnRNP R was also detected in postsynaptic structures revealing stronger immunoreactivity at these sites in comparison to Smn. (D) Single optical slices of the P4 motor endplate emphasized the presynaptic localization of hnRNP R (scale bar: 5 µm).

Techniques Used: Staining, Mouse Assay

Subcellular distribution of Smn and hnRNP R in isolated embryonic motoneurons. (A) Motoneurons showed reduced Smn protein levels upon lentiviral knockdown of Smn. Uninfected or GFP-infected mouse embryonic motoneurons were used as controls. Levels of calnexin and hnRNP R were not affected. For this experiment a C-terminal antibody directed against hnRNP R was used as reported recently [29] . This antibody recognizes distinct hnRNP R isoforms. (B) Representative images of motoneurons cultured for 7DIV and labeled against Smn (scale bar: 10 µm). GFP-transfected controls revealed immunoreactive signals for Smn in the cytosol, in neuronal processes and in Gem-like nuclear structures. Upon lentiviral Smn knockdown both cytosolic Smn immunoreactivity (Uninfected set as ‘1’, n = 4, N = 51; GFP 1.02±0.04, n = 4, N = 60; sh-Smn 0.34±0.02, n = 4, N = 74; P
Figure Legend Snippet: Subcellular distribution of Smn and hnRNP R in isolated embryonic motoneurons. (A) Motoneurons showed reduced Smn protein levels upon lentiviral knockdown of Smn. Uninfected or GFP-infected mouse embryonic motoneurons were used as controls. Levels of calnexin and hnRNP R were not affected. For this experiment a C-terminal antibody directed against hnRNP R was used as reported recently [29] . This antibody recognizes distinct hnRNP R isoforms. (B) Representative images of motoneurons cultured for 7DIV and labeled against Smn (scale bar: 10 µm). GFP-transfected controls revealed immunoreactive signals for Smn in the cytosol, in neuronal processes and in Gem-like nuclear structures. Upon lentiviral Smn knockdown both cytosolic Smn immunoreactivity (Uninfected set as ‘1’, n = 4, N = 51; GFP 1.02±0.04, n = 4, N = 60; sh-Smn 0.34±0.02, n = 4, N = 74; P

Techniques Used: Isolation, Infection, Cell Culture, Labeling, Transfection

8) Product Images from "Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus"

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002361

LL-37 signaling depends on a functional CsrS to induce GAS resistance to opsonophagocytic killing. Wild type strain 854 (WT), isogenic csrS mutants 854 csrS TM (TM), 854 csrS Ω ( csrS Ω), 854 H280A (H280A), and 854 H280A,TM (H280A TM), and isogenic csrR deletion mutant 854Δ csrR (Δ csrR ) were grown in the absence (open symbols) or presence (filled symbols) of 100 nM LL-37. Bacteria were then mixed with human peripheral blood leukocytes for 1 h in the presence of 10% human serum as complement source. Values represent the log of mean fold-change in cfu. Each symbol represents a single experiment performed in duplicate. When exposed to LL-37, wild type 854 showed a significant increase in resistance to phagocytic killing compared to untreated bacteria (P
Figure Legend Snippet: LL-37 signaling depends on a functional CsrS to induce GAS resistance to opsonophagocytic killing. Wild type strain 854 (WT), isogenic csrS mutants 854 csrS TM (TM), 854 csrS Ω ( csrS Ω), 854 H280A (H280A), and 854 H280A,TM (H280A TM), and isogenic csrR deletion mutant 854Δ csrR (Δ csrR ) were grown in the absence (open symbols) or presence (filled symbols) of 100 nM LL-37. Bacteria were then mixed with human peripheral blood leukocytes for 1 h in the presence of 10% human serum as complement source. Values represent the log of mean fold-change in cfu. Each symbol represents a single experiment performed in duplicate. When exposed to LL-37, wild type 854 showed a significant increase in resistance to phagocytic killing compared to untreated bacteria (P

Techniques Used: Functional Assay, Mutagenesis

CsrS is associated with the cell membrane and contains a surface-exposed domain. A) Western blot analysis of membrane and cytoplasmic fractions isolated from whole cell lysates of GAS wild type strain 854 (WT), isogenic csrS deficient mutant strain 854 csrS Ω (SΩ), and 854 csrS TM (TM) that expresses CsrS with 3 point mutations in the predicted extracellular domain. Specific antisera against CsrS, an unrelated membrane protein OpuABC, and CsrR were used to detect the respective proteins in both fractions. B) Biotin labeling via a disulfide linker of surface-exposed proteins in whole cells of wild type strain 854 (WT), 854 csrS Ω (SΩ) and 854 csrS TM (TM). After lysis of labeled cells, biotinylated proteins were captured on a NeutrAvidin column and then eluted by reducing the disulfide linker. Specific antisera detected CsrS in the eluted fraction, as expected for a surface-exposed protein, and CsrR in the flow-through, as expected for a cytoplasmic protein. As a control, wild type 854 cells were treated similarly, but without biotin labeling (NB). Results shown in both panels are representative of at least two independent experiments.
Figure Legend Snippet: CsrS is associated with the cell membrane and contains a surface-exposed domain. A) Western blot analysis of membrane and cytoplasmic fractions isolated from whole cell lysates of GAS wild type strain 854 (WT), isogenic csrS deficient mutant strain 854 csrS Ω (SΩ), and 854 csrS TM (TM) that expresses CsrS with 3 point mutations in the predicted extracellular domain. Specific antisera against CsrS, an unrelated membrane protein OpuABC, and CsrR were used to detect the respective proteins in both fractions. B) Biotin labeling via a disulfide linker of surface-exposed proteins in whole cells of wild type strain 854 (WT), 854 csrS Ω (SΩ) and 854 csrS TM (TM). After lysis of labeled cells, biotinylated proteins were captured on a NeutrAvidin column and then eluted by reducing the disulfide linker. Specific antisera detected CsrS in the eluted fraction, as expected for a surface-exposed protein, and CsrR in the flow-through, as expected for a cytoplasmic protein. As a control, wild type 854 cells were treated similarly, but without biotin labeling (NB). Results shown in both panels are representative of at least two independent experiments.

Techniques Used: Western Blot, Isolation, Mutagenesis, Labeling, Lysis, Flow Cytometry

9) Product Images from "Ultrastructural Characterization of Olfactory Sensilla and Immunolocalization of Odorant Binding and Chemosensory Proteins from an Ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae)"

Article Title: Ultrastructural Characterization of Olfactory Sensilla and Immunolocalization of Odorant Binding and Chemosensory Proteins from an Ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae)

Journal: International Journal of Biological Sciences

doi:

Immunolocalization of chemosensitive sensilla on S. guani antennae . (A) Transverse cross-section of LSB. (B) LSB shows strong labeling with anti-OBP1. (C) Transverse cross-section of ST-I. (D) ST-I are moderately labeled with anti-OBP-1, but gold granules are restricted to the sub-cuticular space around the sensillum lymph (sl). (E) The cross-section of the basal portion of the ST-II. (F) anti-OBP1 is widely distributed throughout the sensillum lymph (sf) of ST-II. (G) SP are moderately labeled by anti-OBP1, and gold granules are restricted at wall pores (p) and molting channels (mc). (H) Transverse cross-section of DWPS-II. (I) DWPS-II are specifically labeled with anti-OBP2. The gold granules are concentrated at the sensillum lymph (ls) as well as the wall-pores. (J) Transverse cross-section of DWPS-I. (K) DWPS-I are heavily labeled with anti-CSP1. The gold granules are exclusively located at the outer sensillum lymph (osl) cavity without dendrites, but not in the inner sensillum lymph (isl) cavity. (L) Longitudinal cross-section of SP. (M) SP are labeled with anti-CSP-1in sensillum lymph (sl) and wall pores (p). Scale bars: A=1µm, B=1µm, C=1 µm, D=500nm, E=1 µm, F=1 µm, G=1.4 µm, H=200nm, I=833nm, J=500nm, K=500nm, L=1µm, M=0.5µm.
Figure Legend Snippet: Immunolocalization of chemosensitive sensilla on S. guani antennae . (A) Transverse cross-section of LSB. (B) LSB shows strong labeling with anti-OBP1. (C) Transverse cross-section of ST-I. (D) ST-I are moderately labeled with anti-OBP-1, but gold granules are restricted to the sub-cuticular space around the sensillum lymph (sl). (E) The cross-section of the basal portion of the ST-II. (F) anti-OBP1 is widely distributed throughout the sensillum lymph (sf) of ST-II. (G) SP are moderately labeled by anti-OBP1, and gold granules are restricted at wall pores (p) and molting channels (mc). (H) Transverse cross-section of DWPS-II. (I) DWPS-II are specifically labeled with anti-OBP2. The gold granules are concentrated at the sensillum lymph (ls) as well as the wall-pores. (J) Transverse cross-section of DWPS-I. (K) DWPS-I are heavily labeled with anti-CSP1. The gold granules are exclusively located at the outer sensillum lymph (osl) cavity without dendrites, but not in the inner sensillum lymph (isl) cavity. (L) Longitudinal cross-section of SP. (M) SP are labeled with anti-CSP-1in sensillum lymph (sl) and wall pores (p). Scale bars: A=1µm, B=1µm, C=1 µm, D=500nm, E=1 µm, F=1 µm, G=1.4 µm, H=200nm, I=833nm, J=500nm, K=500nm, L=1µm, M=0.5µm.

Techniques Used: Labeling

10) Product Images from "Immuno-targeting the multifunctional CD38 using nanobody"

Article Title: Immuno-targeting the multifunctional CD38 using nanobody

Journal: Scientific Reports

doi: 10.1038/srep27055

Designing and characterization of immunotoxin. ( a ) The structure of 1053-PE38. ( b ) SDS-PAGE analysis of pure 1053-PE38 and GFPNb-PE38. The cytotoxicity of 1053-PE38 ( c ) and GFPNb-PE38 ( d ), as a control, on MM cell lines, with or without RA pre-treatment measured by WST-1 assay and analyzed by Graphpad. The experiments were performed four times.
Figure Legend Snippet: Designing and characterization of immunotoxin. ( a ) The structure of 1053-PE38. ( b ) SDS-PAGE analysis of pure 1053-PE38 and GFPNb-PE38. The cytotoxicity of 1053-PE38 ( c ) and GFPNb-PE38 ( d ), as a control, on MM cell lines, with or without RA pre-treatment measured by WST-1 assay and analyzed by Graphpad. The experiments were performed four times.

Techniques Used: SDS Page, WST-1 Assay

CD38 expression level of primary MM and the corresponding effects of the 1053-PE38. ( a ) CD38 expression in five different MM patient samples and PWBCs were analyzed by 1053-EGFP staining method, together with LP-1 and CD38-KO as relative expression controls. ( b ) The cytotoxicity of 1053-PE38 after 3-day treatment was analyzed by calcein staining followed by FACS. Three repeats were performed on each batch of samples.
Figure Legend Snippet: CD38 expression level of primary MM and the corresponding effects of the 1053-PE38. ( a ) CD38 expression in five different MM patient samples and PWBCs were analyzed by 1053-EGFP staining method, together with LP-1 and CD38-KO as relative expression controls. ( b ) The cytotoxicity of 1053-PE38 after 3-day treatment was analyzed by calcein staining followed by FACS. Three repeats were performed on each batch of samples.

Techniques Used: Expressing, Staining, FACS

11) Product Images from "Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD) *"

Article Title: Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.417576

Constructs of FAF1 and VCP mutants studied. A , FAF1 WT; FAF1(TFPR → AG), VCP-interacting loop ( 618 TFPR 621 ) of FAF1 UBX domain was shortened to AG; FAF1(82–650), FAF1 UBA domain (1–47) was deleted; FAF1 ΔUBX, FAF1 UBX domain
Figure Legend Snippet: Constructs of FAF1 and VCP mutants studied. A , FAF1 WT; FAF1(TFPR → AG), VCP-interacting loop ( 618 TFPR 621 ) of FAF1 UBX domain was shortened to AG; FAF1(82–650), FAF1 UBA domain (1–47) was deleted; FAF1 ΔUBX, FAF1 UBX domain

Techniques Used: Construct

12) Product Images from "Structural basis for Ragulator functioning as a scaffold in membrane-anchoring of Rag GTPases and mTORC1"

Article Title: Structural basis for Ragulator functioning as a scaffold in membrane-anchoring of Rag GTPases and mTORC1

Journal: Nature Communications

doi: 10.1038/s41467-017-01567-4

Mapping of the Rag GTPase-interacting sites on the Ragulator complex. a Co-IP assays of WT and p18 variants with the other Ragulator components and RagA GDP –RagC GTP . b A ribbon diagram of the MP1-p14 heterodimer showing the locations of mutations used in the mapping of the Rag GTPase-interacting site on the MP1-p14 heterodimer. c Co-IP assays of WT and mutant p18, MP1 or p14 with the other Ragulator components and RagA GDP –RagC GTP . d Localization of RagA GDP –RagC GTP in cells over-expressing the Ragulator complex with WT and mutant p18, MP1 or p14. Scale bar represents 10 μm. e Co-IP assays of WT and mutant p18, MP1, or p14 with the other Ragulator components and C17orf59
Figure Legend Snippet: Mapping of the Rag GTPase-interacting sites on the Ragulator complex. a Co-IP assays of WT and p18 variants with the other Ragulator components and RagA GDP –RagC GTP . b A ribbon diagram of the MP1-p14 heterodimer showing the locations of mutations used in the mapping of the Rag GTPase-interacting site on the MP1-p14 heterodimer. c Co-IP assays of WT and mutant p18, MP1 or p14 with the other Ragulator components and RagA GDP –RagC GTP . d Localization of RagA GDP –RagC GTP in cells over-expressing the Ragulator complex with WT and mutant p18, MP1 or p14. Scale bar represents 10 μm. e Co-IP assays of WT and mutant p18, MP1, or p14 with the other Ragulator components and C17orf59

Techniques Used: Co-Immunoprecipitation Assay, Mutagenesis, Expressing

Validation of the interactions among different Ragulator components. a Co-IP assays to validate the interactions of p18 with the other four components. FLAG-tagged CASTOR1, the cytoplasmic arginine sensor that has no direct interaction with the Ragulator or the Rag GTPases, was used as a negative control. The p18 14–161 variant that lacks the N-terminal lipid-modification region was denoted as the wild-type (WT) protein. WT p18 was fused with a FLAG tag and the p18 variants were fused with a FLAG-GST tag to improve expression. b Co-IP assays to validate the interactions of p18 with C7orf59 and HBXIP. Mutation L119R or V132D on p18 significantly impairs the interactions with the other four components; as negative controls, mutations V118R and R134D exhibit no notable effect on the interactions because the two residues have no direct interactions with C7orf59-HBXIP. c Cellular co-localization of WT and mutant p18 with the other four components. The interacting regions of p18 for MP1, HBXIP, and C7orf59 are essential for the assembly of the Ragulator complex, but the region for p14 is not. Scale bar represents 10 μm
Figure Legend Snippet: Validation of the interactions among different Ragulator components. a Co-IP assays to validate the interactions of p18 with the other four components. FLAG-tagged CASTOR1, the cytoplasmic arginine sensor that has no direct interaction with the Ragulator or the Rag GTPases, was used as a negative control. The p18 14–161 variant that lacks the N-terminal lipid-modification region was denoted as the wild-type (WT) protein. WT p18 was fused with a FLAG tag and the p18 variants were fused with a FLAG-GST tag to improve expression. b Co-IP assays to validate the interactions of p18 with C7orf59 and HBXIP. Mutation L119R or V132D on p18 significantly impairs the interactions with the other four components; as negative controls, mutations V118R and R134D exhibit no notable effect on the interactions because the two residues have no direct interactions with C7orf59-HBXIP. c Cellular co-localization of WT and mutant p18 with the other four components. The interacting regions of p18 for MP1, HBXIP, and C7orf59 are essential for the assembly of the Ragulator complex, but the region for p14 is not. Scale bar represents 10 μm

Techniques Used: Co-Immunoprecipitation Assay, Negative Control, Variant Assay, Modification, FLAG-tag, Expressing, Mutagenesis

The Ragulator functions as a scaffold to recruit the Rag GTPases to the lysosomal membrane in mTORC1 signaling. a Working model for the functional role of the Ragulator complex in targeting the Rag GTPases to the lysosome. The model is constructed based on the structures of the Ragulator complex, the modeled Rag GTPases from yeast Gtr1–Gtr2 complex (PDB code 4ARZ), and the cryo-EM structure of mTORC1 (PDB code 5H64). b Superposition of the structures of the Ragulator and EGO-TC (PDB code 4XPM) complexes. The Ragulator components are colored as in Fig. 1a , and Ego1, Ego2, and Ego3 of the EGO-TC are colored in red, gray, and eggshell, respectively. c The Ragulator and EGO-TC complexes share similar structural and functional features in mTORC1/TORC1 signaling. Ego1, Ego2, and Ego3 of the EGO-TC appear to be the counterparts of p18, HBXIP, and p14 of the Ragulator
Figure Legend Snippet: The Ragulator functions as a scaffold to recruit the Rag GTPases to the lysosomal membrane in mTORC1 signaling. a Working model for the functional role of the Ragulator complex in targeting the Rag GTPases to the lysosome. The model is constructed based on the structures of the Ragulator complex, the modeled Rag GTPases from yeast Gtr1–Gtr2 complex (PDB code 4ARZ), and the cryo-EM structure of mTORC1 (PDB code 5H64). b Superposition of the structures of the Ragulator and EGO-TC (PDB code 4XPM) complexes. The Ragulator components are colored as in Fig. 1a , and Ego1, Ego2, and Ego3 of the EGO-TC are colored in red, gray, and eggshell, respectively. c The Ragulator and EGO-TC complexes share similar structural and functional features in mTORC1/TORC1 signaling. Ego1, Ego2, and Ego3 of the EGO-TC appear to be the counterparts of p18, HBXIP, and p14 of the Ragulator

Techniques Used: Functional Assay, Construct

13) Product Images from "The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons"

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

Journal: Scientific Reports

doi: 10.1038/s41598-018-31785-9

Acetylation of CDK5 at K33 causes a loss of kinase activity due to impaired ATP binding. ( a , b ) HEK293 cells were transfected with either FLAG-tagged wild type mouse CDK5 (WT), an acetyl-null mutant (K33R; KR) or a mimetic mutant (K33Q; KQ) of CDK5 in the presence of ( a ) p35-HA or ( b ) p25-HA. Lysates were immunoprecipitated (IPed) with an anti-FLAG antibody and then subjected to an in vitro phosphorylation assay using histone H1 as a substrate. The resulting phosphorylated H1 (P-H1) was visualized via immunoblot analysis (IB) with an anti-phospho-H1 antibody. Coomassie brilliant blue (CBB) staining for H1 was used as a loading control. Immunoprecipitates or whole cell lysates (WCLs) were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for WCL. ( c ) Bacterially purified, recombinant His-tagged CDK5 WT or K33-acetylated CDK5 (Ac-CDK5; Ac) was subjected to an in vitro phosphorylation assay in the presence of H1, [γ- 32 P]ATP and the indicated doses of recombinant p25. The resulting phosphorylated H1 was visualized by autoradiography. Inputs were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. ( d ) Recombinant His-CDK5 WT or His-Ac-CDK5 was incubated with or without resin conjugated to ATP. After washing, the resulting ATP-bound CDK5 was resolved by SDS-PAGE and visualized by IB with an anti-His antibody. Input signals were measured by IB with the indicated antibodies. ( e . X-axis represents the varying concentration of mant-ATP as indicated. Y-axis represents the relative fluorescence intensity of specific binding, where Bmax is maximum specific binding and Kd is equilibrium binding constant. Wilcoxon matched-pairs rank test was employed to test the binding difference between CDK5 WT and Ac-CDK5 ( ** P = 0.004; Spearman correlation coefficient, rs = 0.976; n = 3). ( f ) Lysates from HEK293 cells expressing p35-FLAG or p25-FLAG were incubated with recombinant His-CDK5 WT or His-Ac-CDK5 bound to Ni-NTA beads. Reaction mixtures were subjected to pull-down and subsequent IB with the indicated antibodies. An anti-FLAG antibody was employed to visualize the extent of CDK5-bound p35 or p25. WCLs were subjected to IB with the indicated antibodies. ( g ) Recombinant His-CDK5 WT plus increasing amounts of recombinant His-Ac-CDK5 was subjected to an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Phosphorylated H1 levels were visualized by autoradiography.
Figure Legend Snippet: Acetylation of CDK5 at K33 causes a loss of kinase activity due to impaired ATP binding. ( a , b ) HEK293 cells were transfected with either FLAG-tagged wild type mouse CDK5 (WT), an acetyl-null mutant (K33R; KR) or a mimetic mutant (K33Q; KQ) of CDK5 in the presence of ( a ) p35-HA or ( b ) p25-HA. Lysates were immunoprecipitated (IPed) with an anti-FLAG antibody and then subjected to an in vitro phosphorylation assay using histone H1 as a substrate. The resulting phosphorylated H1 (P-H1) was visualized via immunoblot analysis (IB) with an anti-phospho-H1 antibody. Coomassie brilliant blue (CBB) staining for H1 was used as a loading control. Immunoprecipitates or whole cell lysates (WCLs) were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for WCL. ( c ) Bacterially purified, recombinant His-tagged CDK5 WT or K33-acetylated CDK5 (Ac-CDK5; Ac) was subjected to an in vitro phosphorylation assay in the presence of H1, [γ- 32 P]ATP and the indicated doses of recombinant p25. The resulting phosphorylated H1 was visualized by autoradiography. Inputs were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. ( d ) Recombinant His-CDK5 WT or His-Ac-CDK5 was incubated with or without resin conjugated to ATP. After washing, the resulting ATP-bound CDK5 was resolved by SDS-PAGE and visualized by IB with an anti-His antibody. Input signals were measured by IB with the indicated antibodies. ( e . X-axis represents the varying concentration of mant-ATP as indicated. Y-axis represents the relative fluorescence intensity of specific binding, where Bmax is maximum specific binding and Kd is equilibrium binding constant. Wilcoxon matched-pairs rank test was employed to test the binding difference between CDK5 WT and Ac-CDK5 ( ** P = 0.004; Spearman correlation coefficient, rs = 0.976; n = 3). ( f ) Lysates from HEK293 cells expressing p35-FLAG or p25-FLAG were incubated with recombinant His-CDK5 WT or His-Ac-CDK5 bound to Ni-NTA beads. Reaction mixtures were subjected to pull-down and subsequent IB with the indicated antibodies. An anti-FLAG antibody was employed to visualize the extent of CDK5-bound p35 or p25. WCLs were subjected to IB with the indicated antibodies. ( g ) Recombinant His-CDK5 WT plus increasing amounts of recombinant His-Ac-CDK5 was subjected to an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Phosphorylated H1 levels were visualized by autoradiography.

Techniques Used: Activity Assay, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, In Vitro, Phosphorylation Assay, Staining, SDS Page, Purification, Recombinant, Autoradiography, Incubation, Concentration Assay, Fluorescence, Expressing

14) Product Images from "The Small GTPase Rap1 is a Modulator of Hedgehog Signaling"

Article Title: The Small GTPase Rap1 is a Modulator of Hedgehog Signaling

Journal: Developmental biology

doi: 10.1016/j.ydbio.2015.10.020

Rap1 over-expression modulates Hh signaling in vivo
Figure Legend Snippet: Rap1 over-expression modulates Hh signaling in vivo

Techniques Used: Over Expression, In Vivo

Rap1 genetically interacts with the Hh pathway
Figure Legend Snippet: Rap1 genetically interacts with the Hh pathway

Techniques Used:

Rap1 suppresses hh Mrt
Figure Legend Snippet: Rap1 suppresses hh Mrt

Techniques Used:

Manipulation of Rap1 expression alters Hh signaling in vitro
Figure Legend Snippet: Manipulation of Rap1 expression alters Hh signaling in vitro

Techniques Used: Expressing, In Vitro

Hh signaling induces Rap1-GTP exchange
Figure Legend Snippet: Hh signaling induces Rap1-GTP exchange

Techniques Used:

Rap1 enhances SmoA
Figure Legend Snippet: Rap1 enhances SmoA

Techniques Used:

15) Product Images from "Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy"

Article Title: Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1038/mtm.2014.47

Effect of incubation volume and medium on the protein nanovector complex size and transfection efficiency. HNRK and HKRN modular protein nanovectors were allowed to self-assembly during a 20-minute incubation of HNRK and HKRN proteins with plasmid DNA in either 200 μl OPTIPRO medium or 30 μl PBS. Assembled nanovectors were then used to transfect HEK293T cells and transfection efficiencies were quantified 1 day later by flow cytometry ( a, b ). HNRK nanovector stability was analyzed after self-assembly in 200 μl OPTIPRO medium or 30 μl phosphate-buffered saline, and for 20 minutes to 5 hours incubation periods. The products formed were analyzed by dynamic light scattering ( c ) or transmission electron microscopy ( d ) as indicated. Transfection efficiency of HNRK nanovector after self-assembling in 200 μl OPTIPRO medium for 20 minutes to 5 hours was analyzed by flow cytometry 1 day after transfection (100% represents transfection efficiency for nanovectors formed in the 20 minutes incubation time). * P
Figure Legend Snippet: Effect of incubation volume and medium on the protein nanovector complex size and transfection efficiency. HNRK and HKRN modular protein nanovectors were allowed to self-assembly during a 20-minute incubation of HNRK and HKRN proteins with plasmid DNA in either 200 μl OPTIPRO medium or 30 μl PBS. Assembled nanovectors were then used to transfect HEK293T cells and transfection efficiencies were quantified 1 day later by flow cytometry ( a, b ). HNRK nanovector stability was analyzed after self-assembly in 200 μl OPTIPRO medium or 30 μl phosphate-buffered saline, and for 20 minutes to 5 hours incubation periods. The products formed were analyzed by dynamic light scattering ( c ) or transmission electron microscopy ( d ) as indicated. Transfection efficiency of HNRK nanovector after self-assembling in 200 μl OPTIPRO medium for 20 minutes to 5 hours was analyzed by flow cytometry 1 day after transfection (100% represents transfection efficiency for nanovectors formed in the 20 minutes incubation time). * P

Techniques Used: Incubation, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Transmission Assay, Electron Microscopy

In vitro transfection and transduction efficiency of vectors. HEK293T cells were incubated for 4 hours with DNA, lipofectamine (LIPO), or modular protein nanovectors at different protein/DNA mass ratios (ratios from 2.5 to 7.5 are shown), and transfection efficiencies ( a ) and relative fluorescence intensity per cell ( b ) were quantified 1 day later by flow cytometry. DNA concentrations used (2 μg/well, 2.4 × 10 11 copies of gfp gene) were the same in all cases so as to compare transfection efficiencies. HNRK and HKRN vectors were purified from insoluble bacterial fractions and compared with HNRK obtained from soluble bacterial fractions (HNRKs). In parallel experiments, HEK293T cells were incubated with lentiviral vectors at different multiplicities of infections (MOIs) (at MOI 1, there are 1.4 × 10 6 to 1.4 × 10 7 copies of the gfp gene) and transduction efficiencies ( c ) and relative fluorescence intensity per cell ( d ) were quantified at 1 and 3 days post-transduction (dpt). a, b : * P
Figure Legend Snippet: In vitro transfection and transduction efficiency of vectors. HEK293T cells were incubated for 4 hours with DNA, lipofectamine (LIPO), or modular protein nanovectors at different protein/DNA mass ratios (ratios from 2.5 to 7.5 are shown), and transfection efficiencies ( a ) and relative fluorescence intensity per cell ( b ) were quantified 1 day later by flow cytometry. DNA concentrations used (2 μg/well, 2.4 × 10 11 copies of gfp gene) were the same in all cases so as to compare transfection efficiencies. HNRK and HKRN vectors were purified from insoluble bacterial fractions and compared with HNRK obtained from soluble bacterial fractions (HNRKs). In parallel experiments, HEK293T cells were incubated with lentiviral vectors at different multiplicities of infections (MOIs) (at MOI 1, there are 1.4 × 10 6 to 1.4 × 10 7 copies of the gfp gene) and transduction efficiencies ( c ) and relative fluorescence intensity per cell ( d ) were quantified at 1 and 3 days post-transduction (dpt). a, b : * P

Techniques Used: In Vitro, Transfection, Transduction, Incubation, Fluorescence, Flow Cytometry, Cytometry, Purification

Effects of chloroquine and fetal bovine serum (FBS) on transfection efficiency of protein nanovectors. HEK293T cells were incubated for 4 hours with the modular protein nanovectors HNRK ( a, c ) or HKRN ( b, d ) in the absence or presence of the endosome disrupting agent chloroquine ( a, b ), or in absence or presence of 10% FBS ( c, d ). One day later, transfection efficiencies were quantified by flow cytometry. * P
Figure Legend Snippet: Effects of chloroquine and fetal bovine serum (FBS) on transfection efficiency of protein nanovectors. HEK293T cells were incubated for 4 hours with the modular protein nanovectors HNRK ( a, c ) or HKRN ( b, d ) in the absence or presence of the endosome disrupting agent chloroquine ( a, b ), or in absence or presence of 10% FBS ( c, d ). One day later, transfection efficiencies were quantified by flow cytometry. * P

Techniques Used: Transfection, Incubation, Flow Cytometry, Cytometry

16) Product Images from "Identification of Trans-4-Hydroxy-L-Proline as a Compatible Solute and Its Biosynthesis and Molecular Characterization in Halobacillus halophilus"

Article Title: Identification of Trans-4-Hydroxy-L-Proline as a Compatible Solute and Its Biosynthesis and Molecular Characterization in Halobacillus halophilus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02054

Physical maps of the proline and Hyp biosynthetic gene clusters showing the open reading frames, promoters, and terminators (A) and the relative transcriptional expression of H. halophilus proH and PH-4 gene in the presence of different NaCl concentrations (B,C) and salts (D) . The putative functions of HBHAL_RS03960, HBHAL_RS03965, HBHAL_RS03970, and HBHAL_RS11735 were predicted to be pyrroline-5-carboxylate reductase (ProH), glutamate-5-kinase (ProJ), glutamate-5-semialdehyde dehydrogenase (ProA), and proline 4-hydroxylase (PH-4, this study), respectively. Cells grown in marine broth with different NaCl concentrations or salts were used in the transcriptional analysis, and their relative expression in the presence of different NaCl concentrations and salts were calculated based on expressional levels at 0.4 M and 1.0 M NaCl, respectively. The levels of malate dehydrogenase transcript (HBHAL_RS13485, mdh ) were used for normalization of total RNA templates.
Figure Legend Snippet: Physical maps of the proline and Hyp biosynthetic gene clusters showing the open reading frames, promoters, and terminators (A) and the relative transcriptional expression of H. halophilus proH and PH-4 gene in the presence of different NaCl concentrations (B,C) and salts (D) . The putative functions of HBHAL_RS03960, HBHAL_RS03965, HBHAL_RS03970, and HBHAL_RS11735 were predicted to be pyrroline-5-carboxylate reductase (ProH), glutamate-5-kinase (ProJ), glutamate-5-semialdehyde dehydrogenase (ProA), and proline 4-hydroxylase (PH-4, this study), respectively. Cells grown in marine broth with different NaCl concentrations or salts were used in the transcriptional analysis, and their relative expression in the presence of different NaCl concentrations and salts were calculated based on expressional levels at 0.4 M and 1.0 M NaCl, respectively. The levels of malate dehydrogenase transcript (HBHAL_RS13485, mdh ) were used for normalization of total RNA templates.

Techniques Used: Expressing

Circos plot showing the relationships between the products of three candidate genes ( HBHAL_RS02420 , HBHAL_RS11735 , HBHAL_RS15600 ) and proteins annotated as PH-4 in GenBank. The plot was generated with Circoletto. The ribbons between the three candidate proteins and GenBank PH-4 proteins represent their local alignments and the ribbon colors indicate their amino acid sequence identities (no ribbons,
Figure Legend Snippet: Circos plot showing the relationships between the products of three candidate genes ( HBHAL_RS02420 , HBHAL_RS11735 , HBHAL_RS15600 ) and proteins annotated as PH-4 in GenBank. The plot was generated with Circoletto. The ribbons between the three candidate proteins and GenBank PH-4 proteins represent their local alignments and the ribbon colors indicate their amino acid sequence identities (no ribbons,

Techniques Used: Generated, Sequencing

Expressional analysis of HBHAL_RS11735 using a pET28a construct in E. coli BL21 (DE3) cells (A) and PH-4 activity assay of the overexpressed gene product (B) . (A) A pET28a-HBHAL_RS11735 construct was overexpressed in E. coli BL21 (DE3) cells, and crude protein extracts of these cells were separated by SDS-PAGE. lane M, protein size marker; lane 1, total protein from uninduced cells carrying the HBHAL_RS11735 gene; lane 2, total protein from induced cells carrying the HBHAL_RS11735 gene; lanes 3, soluble protein from induced cells carrying the HBHAL_RS11735 gene. (B) PH-4 activity assay of soluble protein from E. coli cells overexpressing the HBHAL_RS11735 gene. PH-4 activity is expressed as enzyme units per mg·wet cell weight, and cells of E. coli BL21 (DE3) without the HBHAL_RS11735 gene and H. halophilus DSM 2266 cells were used as negative and positive controls, respectively.
Figure Legend Snippet: Expressional analysis of HBHAL_RS11735 using a pET28a construct in E. coli BL21 (DE3) cells (A) and PH-4 activity assay of the overexpressed gene product (B) . (A) A pET28a-HBHAL_RS11735 construct was overexpressed in E. coli BL21 (DE3) cells, and crude protein extracts of these cells were separated by SDS-PAGE. lane M, protein size marker; lane 1, total protein from uninduced cells carrying the HBHAL_RS11735 gene; lane 2, total protein from induced cells carrying the HBHAL_RS11735 gene; lanes 3, soluble protein from induced cells carrying the HBHAL_RS11735 gene. (B) PH-4 activity assay of soluble protein from E. coli cells overexpressing the HBHAL_RS11735 gene. PH-4 activity is expressed as enzyme units per mg·wet cell weight, and cells of E. coli BL21 (DE3) without the HBHAL_RS11735 gene and H. halophilus DSM 2266 cells were used as negative and positive controls, respectively.

Techniques Used: Construct, Activity Assay, SDS Page, Marker

17) Product Images from "Purification, crystallization and X-ray diffraction analysis of a novel ring-cleaving enzyme (BoxCC) from Burkholderia xenovorans LB400"

Article Title: Purification, crystallization and X-ray diffraction analysis of a novel ring-cleaving enzyme (BoxCC) from Burkholderia xenovorans LB400

Journal:

doi: 10.1107/S1744309108010919

Superdex 200 16/60 gel-filtration analysis showing that BoxC C (red peak) forms a stable dimer of ∼120 kDa in solution. The blue peaks represent protein standards: peak I, conalbumin (75 kDa); peak II, ovalbumin (43 kDa).
Figure Legend Snippet: Superdex 200 16/60 gel-filtration analysis showing that BoxC C (red peak) forms a stable dimer of ∼120 kDa in solution. The blue peaks represent protein standards: peak I, conalbumin (75 kDa); peak II, ovalbumin (43 kDa).

Techniques Used: Filtration

( a ) Native BoxC C crystal grown in 23% PEG 3350 buffered with 100 m M Tris pH 8.5. ( b ) X-ray diffraction image of the native BoxC C crystal collected to 1.5 Å resolution.
Figure Legend Snippet: ( a ) Native BoxC C crystal grown in 23% PEG 3350 buffered with 100 m M Tris pH 8.5. ( b ) X-ray diffraction image of the native BoxC C crystal collected to 1.5 Å resolution.

Techniques Used:

18) Product Images from "An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex"

Article Title: An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

Journal: Journal of Cell Science

doi: 10.1242/jcs.222034

Rabin8 cooperates with VARP in ciliary trafficking of VAMP7. (A–E) Retinas labeled with anti-VARP (r) and anti-VAMP7 (m) (A); anti-VAMP7 (m) (red), anti-Rabin8 (r) (blue), followed by anti-VARP conjugated to Alexa Fluor 488 (green) (B); anti-Rabin8 (r) (red) and anti-GM130(m) (blue) and anti-VARP-488 (green) (C); anti-rhodopsin 11D5 (m) (green) and anti-VARP (r) (red) (D); anti-rhodopsin 11D5 (m) (red), anti-Rabin8(r) (blue) and anti-VARP-488 (green) (E). The boxed area is repeated in insets to show individual staining patterns separately. Arrows in B indicate carriers containing VAMP7, whereas a yellow arrowhead indicates carries that appear in the process of fusion; arrow in C indicates the Golgi; arrows in D indicate nascent RTCs. Scale bar: 5 µm for A–E and 2.5 µm for insets in A and D. (F) GST–VAMP7 CD, or GST alone (Ctrl), was incubated with 6His–Rabin8 followed by increasing concentrations of purified recombinant VARP or vice versa. Bound proteins were immunoblotted (IB), as indicated. (G) GST–Rabin8 or GST alone (Ctrl) was incubated with or without recombinant VARP and/or VAMP7. Bound proteins and GST fusion proteins were detected as above. M, myoid region; E, ellipsoid region; G, Golgi, N, nucleus; m, mouse antibody; r rabbit antibody.
Figure Legend Snippet: Rabin8 cooperates with VARP in ciliary trafficking of VAMP7. (A–E) Retinas labeled with anti-VARP (r) and anti-VAMP7 (m) (A); anti-VAMP7 (m) (red), anti-Rabin8 (r) (blue), followed by anti-VARP conjugated to Alexa Fluor 488 (green) (B); anti-Rabin8 (r) (red) and anti-GM130(m) (blue) and anti-VARP-488 (green) (C); anti-rhodopsin 11D5 (m) (green) and anti-VARP (r) (red) (D); anti-rhodopsin 11D5 (m) (red), anti-Rabin8(r) (blue) and anti-VARP-488 (green) (E). The boxed area is repeated in insets to show individual staining patterns separately. Arrows in B indicate carriers containing VAMP7, whereas a yellow arrowhead indicates carries that appear in the process of fusion; arrow in C indicates the Golgi; arrows in D indicate nascent RTCs. Scale bar: 5 µm for A–E and 2.5 µm for insets in A and D. (F) GST–VAMP7 CD, or GST alone (Ctrl), was incubated with 6His–Rabin8 followed by increasing concentrations of purified recombinant VARP or vice versa. Bound proteins were immunoblotted (IB), as indicated. (G) GST–Rabin8 or GST alone (Ctrl) was incubated with or without recombinant VARP and/or VAMP7. Bound proteins and GST fusion proteins were detected as above. M, myoid region; E, ellipsoid region; G, Golgi, N, nucleus; m, mouse antibody; r rabbit antibody.

Techniques Used: Labeling, Staining, Incubation, Purification, Recombinant

19) Product Images from "Structural basis and mechanism of the unfolding-induced activation of HdeA, a bacterial acid response chaperone"

Article Title: Structural basis and mechanism of the unfolding-induced activation of HdeA, a bacterial acid response chaperone

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.006398

Representative 19 F NMR spectra of HdeA. Representative 19 F NMR spectra of HdeA mutants with 19 F probes incorporated at sites 7, 16, 35, 49, 55, and 63 in the free state ( left ) or in complex with the client protein MalE ( right ) at pH 2.0. The spectra acquired in a buffer containing 90% H 2 O and 10% D 2 O are shown in blue or green for the free or complexed state, and those acquired in 100% D 2 O are shown in red or yellow for the free or complexed state. Enlarged view of the peaks are shown as insets .
Figure Legend Snippet: Representative 19 F NMR spectra of HdeA. Representative 19 F NMR spectra of HdeA mutants with 19 F probes incorporated at sites 7, 16, 35, 49, 55, and 63 in the free state ( left ) or in complex with the client protein MalE ( right ) at pH 2.0. The spectra acquired in a buffer containing 90% H 2 O and 10% D 2 O are shown in blue or green for the free or complexed state, and those acquired in 100% D 2 O are shown in red or yellow for the free or complexed state. Enlarged view of the peaks are shown as insets .

Techniques Used: Nuclear Magnetic Resonance

20) Product Images from "N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro"

Article Title: N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro

Journal: Journal of molecular and cellular cardiology

doi: 10.1016/j.yjmcc.2016.09.003

Validation of pro- and anti-inflammatory responses upon treatment with N-terminal cMyBP-C fragments a–e) Murine macrophages were treated for 6 h with 500 ng/ml C0C2, C0C1f, C0C1, or C0-L fragments, or 1 µg/ml LPS. mRNA levels of TNFα, IL-6, IL-1β, VCAM1, and ICAM1 were measured by qRT-PCR. f) Murine macrophages were treated with C0C1f peptide for 3, 6, 9, 24, or 72 h. g–i) Murine macrophages were treated for 6 h with C0C2, C0C1f, C0C1, or C0-L fragment and LPS; mRNA levels of Arg1, IL-10, and TGFβ were measured by qRT-PCR. Values shown are mean ± SEM; statistical analysis was performed using Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc test, * p
Figure Legend Snippet: Validation of pro- and anti-inflammatory responses upon treatment with N-terminal cMyBP-C fragments a–e) Murine macrophages were treated for 6 h with 500 ng/ml C0C2, C0C1f, C0C1, or C0-L fragments, or 1 µg/ml LPS. mRNA levels of TNFα, IL-6, IL-1β, VCAM1, and ICAM1 were measured by qRT-PCR. f) Murine macrophages were treated with C0C1f peptide for 3, 6, 9, 24, or 72 h. g–i) Murine macrophages were treated for 6 h with C0C2, C0C1f, C0C1, or C0-L fragment and LPS; mRNA levels of Arg1, IL-10, and TGFβ were measured by qRT-PCR. Values shown are mean ± SEM; statistical analysis was performed using Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc test, * p

Techniques Used: Quantitative RT-PCR

21) Product Images from "Raver1 interactions with Vinculin and RNA Suggest a Feed-Forward Pathway in Directing mRNA to Focal Adhesions"

Article Title: Raver1 interactions with Vinculin and RNA Suggest a Feed-Forward Pathway in Directing mRNA to Focal Adhesions

Journal: Structure (London, England : 1993)

doi: 10.1016/j.str.2009.04.010

Structure of the Vinculin:Raver1 Complex.
Figure Legend Snippet: Structure of the Vinculin:Raver1 Complex.

Techniques Used:

Crystal Structures of Human Vinculin in Complex with Raver1
Figure Legend Snippet: Crystal Structures of Human Vinculin in Complex with Raver1

Techniques Used:

Vinculin Functions as a Scaffold for the Production of Adhesion Components by the Translational Machinery at Nascent Focal Adhesions.
Figure Legend Snippet: Vinculin Functions as a Scaffold for the Production of Adhesion Components by the Translational Machinery at Nascent Focal Adhesions.

Techniques Used:

Crystal Structures of Human Vinculin in Complex with Raver1
Figure Legend Snippet: Crystal Structures of Human Vinculin in Complex with Raver1

Techniques Used:

The RRM1 Domain of Raver1 Directs its Interactions with Vinculin.
Figure Legend Snippet: The RRM1 Domain of Raver1 Directs its Interactions with Vinculin.

Techniques Used:

Structural Constraints and Alterations Provoked by the Vinculin-Raver1 Interaction.
Figure Legend Snippet: Structural Constraints and Alterations Provoked by the Vinculin-Raver1 Interaction.

Techniques Used:

Raver1 Residues that Direct Interactions with Vinculin are Required for Vinculin and Raver1 Co-Localization in Cells.
Figure Legend Snippet: Raver1 Residues that Direct Interactions with Vinculin are Required for Vinculin and Raver1 Co-Localization in Cells.

Techniques Used:

The Vinculin:Raver1 Complex is Permissive for Binding to F-actin.
Figure Legend Snippet: The Vinculin:Raver1 Complex is Permissive for Binding to F-actin.

Techniques Used: Binding Assay

Domain Organization of human Vinculin ( top ) and human Raver1 ( bottom ) . Vinculin domains Vh1, Vh2, Vh3, and Vt2 together comprise the vinculin head domain VH. NES, nuclear export sequence; NLS, nuclear localization signal.
Figure Legend Snippet: Domain Organization of human Vinculin ( top ) and human Raver1 ( bottom ) . Vinculin domains Vh1, Vh2, Vh3, and Vt2 together comprise the vinculin head domain VH. NES, nuclear export sequence; NLS, nuclear localization signal.

Techniques Used: Sequencing

22) Product Images from "The subcellular localization of yeast glycogen synthase is dependent upon glycogen content"

Article Title: The subcellular localization of yeast glycogen synthase is dependent upon glycogen content

Journal: Canadian journal of microbiology

doi: 10.1139/w10-027

Expression of non-phosphorylatable glycogen synthase mutants restores glycogen storage to yeast strain lacking glycogenin
Figure Legend Snippet: Expression of non-phosphorylatable glycogen synthase mutants restores glycogen storage to yeast strain lacking glycogenin

Techniques Used: Expressing

23) Product Images from "Disease Resistance and Abiotic Stress Tolerance in Rice Are Inversely Modulated by an Abscisic Acid-Inducible Mitogen-Activated Protein Kinase W⃞"

Article Title: Disease Resistance and Abiotic Stress Tolerance in Rice Are Inversely Modulated by an Abscisic Acid-Inducible Mitogen-Activated Protein Kinase W⃞

Journal: The Plant Cell

doi: 10.1105/tpc.008714

OsMAPK5-RI Lines Exhibited Enhanced Resistance to the Bacterial Pathogen B. glumae . Leaf sheaths of 1-month-old control and T1 transgenic seedlings were inoculated with B. glumae (1 × 10 6 colony-forming units). At least 10 hygromycin-positive transgenic seedlings per line were used in each experiment. Experiments were repeated twice with similar results. (A) Disease resistance evaluation based on lesion size at 7 days after inoculation. See supplemental data online for photographs of disease symptoms. (B) Disease resistance evaluation based on bacterial growth in planta at 7 days after inoculation. (C) MBP in gel kinase assay of immunoprecipitated OsMAPK5 from leaf tissues at 7 days after inoculation.
Figure Legend Snippet: OsMAPK5-RI Lines Exhibited Enhanced Resistance to the Bacterial Pathogen B. glumae . Leaf sheaths of 1-month-old control and T1 transgenic seedlings were inoculated with B. glumae (1 × 10 6 colony-forming units). At least 10 hygromycin-positive transgenic seedlings per line were used in each experiment. Experiments were repeated twice with similar results. (A) Disease resistance evaluation based on lesion size at 7 days after inoculation. See supplemental data online for photographs of disease symptoms. (B) Disease resistance evaluation based on bacterial growth in planta at 7 days after inoculation. (C) MBP in gel kinase assay of immunoprecipitated OsMAPK5 from leaf tissues at 7 days after inoculation.

Techniques Used: Transgenic Assay, Kinase Assay, Immunoprecipitation

Constitutive Expression of PR1 and PR10 in OsMAPK5-RI Transgenic Lines. Total RNA was isolated from 2-week-old control and T1 transgenic seedlings grown under normal conditions. The RNA gel blot (10 μg of RNA per lane) was probed sequentially with the PR1b , PR10 , and rice 25S rRNA.
Figure Legend Snippet: Constitutive Expression of PR1 and PR10 in OsMAPK5-RI Transgenic Lines. Total RNA was isolated from 2-week-old control and T1 transgenic seedlings grown under normal conditions. The RNA gel blot (10 μg of RNA per lane) was probed sequentially with the PR1b , PR10 , and rice 25S rRNA.

Techniques Used: Expressing, Transgenic Assay, Isolation, Western Blot

Genomic Organization, Alternative Splicing, Recombinant Proteins, and Autophosphorylation Activity of OsMAPK5 . (A) DNA gel blot analysis of the OsMAPK5 gene. Total DNA from cv Drew (4 μg for each lane) was digested individually with EcoRI, HindIII, PstI, and XbaI and hybridized with a gene-specific probe covering the region from nucleotide 999 to the 3′ end of the OsMAPK5a cDNA. (B) RT-PCR analysis using a primer pair covering the differentiated regions of the OsMAPK5a and OsMAPK5b cDNAs. The blast fungus–induced (2 days after infection) mRNAs from cv Drew were used for RT-PCR analysis (lane 1). The cDNAs of OsMAPK5a and OsMAPK5b also were used for PCR with the same primer pair (lanes 2 and 3). M, DNA size markers. (C) In vitro expression of OsMAPK5a and OsMAPK5b, and specificity of the OsMAPK5 antibody. One hundred nanograms of total protein from E. coli (left lanes) or 10 ng of affinity-purified fusion protein of His-OsMAPK5a and His-OsMAPK5b (right lanes) was separated by 10% SDS-PAGE and detected with the anti-OsMAPK5 antibody. (D) In vitro autophosphorylation assay of the affinity-purified fusion proteins His-OsMAPK5a and His-OsMAPK5b.
Figure Legend Snippet: Genomic Organization, Alternative Splicing, Recombinant Proteins, and Autophosphorylation Activity of OsMAPK5 . (A) DNA gel blot analysis of the OsMAPK5 gene. Total DNA from cv Drew (4 μg for each lane) was digested individually with EcoRI, HindIII, PstI, and XbaI and hybridized with a gene-specific probe covering the region from nucleotide 999 to the 3′ end of the OsMAPK5a cDNA. (B) RT-PCR analysis using a primer pair covering the differentiated regions of the OsMAPK5a and OsMAPK5b cDNAs. The blast fungus–induced (2 days after infection) mRNAs from cv Drew were used for RT-PCR analysis (lane 1). The cDNAs of OsMAPK5a and OsMAPK5b also were used for PCR with the same primer pair (lanes 2 and 3). M, DNA size markers. (C) In vitro expression of OsMAPK5a and OsMAPK5b, and specificity of the OsMAPK5 antibody. One hundred nanograms of total protein from E. coli (left lanes) or 10 ng of affinity-purified fusion protein of His-OsMAPK5a and His-OsMAPK5b (right lanes) was separated by 10% SDS-PAGE and detected with the anti-OsMAPK5 antibody. (D) In vitro autophosphorylation assay of the affinity-purified fusion proteins His-OsMAPK5a and His-OsMAPK5b.

Techniques Used: Recombinant, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Infection, Polymerase Chain Reaction, In Vitro, Expressing, Affinity Purification, SDS Page

OsMAPK5-RI Lines Exhibited Enhanced Resistance to the Blast Fungus. Experiments were repeated two times with similar results. (A) Blast resistance evaluation of T0 transgenic plants by the spot inoculation method. Shown are typical disease symptoms on leaves of control plants (CK) as well as overexpression (OX) and dsRNAi (RI) transgenic plants at 6 days after inoculation with fungal isolate IC17-18/1. (B) at 5 days after inoculation. (C) Blast resistance evaluation of T1 transgenic plants based on lesion numbers per infected leaf at 5 days after inoculation. (D) ). (E) MBP in gel kinase assay of immunoprecipitated OsMAPK5 from leaf tissues of control and transgenic lines at 5 days after inoculation.
Figure Legend Snippet: OsMAPK5-RI Lines Exhibited Enhanced Resistance to the Blast Fungus. Experiments were repeated two times with similar results. (A) Blast resistance evaluation of T0 transgenic plants by the spot inoculation method. Shown are typical disease symptoms on leaves of control plants (CK) as well as overexpression (OX) and dsRNAi (RI) transgenic plants at 6 days after inoculation with fungal isolate IC17-18/1. (B) at 5 days after inoculation. (C) Blast resistance evaluation of T1 transgenic plants based on lesion numbers per infected leaf at 5 days after inoculation. (D) ). (E) MBP in gel kinase assay of immunoprecipitated OsMAPK5 from leaf tissues of control and transgenic lines at 5 days after inoculation.

Techniques Used: Transgenic Assay, Over Expression, Infection, Kinase Assay, Immunoprecipitation

Induction of OsMAPK5 , Its Protein, and Kinase Activity by Drought, Salt, and Low Temperature. Rice tissues from the same time course were used for RNA gel blot, immunoblot, and kinase activity analyses. Experiments were repeated three times using samples from independent treatments. (A) RNA gel blot analyses of OsMAPK5 expression in 2-week-old seedlings subjected to drought (water withheld for up to 5 days), salt (200 mM NaCl), or cold (4°C) stress. For drought and salt stresses, RNA from both root and leaf tissues was extracted at the times specified. Only leaf tissues were collected for the cold treatment. (B) Immunoblot analyses of OsMAPK5 under drought (root tissues), salt (root tissues), and cold (leaf tissues) stresses. (C) MBP in gel kinase activity assay of immunoprecipitated OsMAPK5 under drought (root tissues), salt (root tissues), and cold (leaf tissues) stresses.
Figure Legend Snippet: Induction of OsMAPK5 , Its Protein, and Kinase Activity by Drought, Salt, and Low Temperature. Rice tissues from the same time course were used for RNA gel blot, immunoblot, and kinase activity analyses. Experiments were repeated three times using samples from independent treatments. (A) RNA gel blot analyses of OsMAPK5 expression in 2-week-old seedlings subjected to drought (water withheld for up to 5 days), salt (200 mM NaCl), or cold (4°C) stress. For drought and salt stresses, RNA from both root and leaf tissues was extracted at the times specified. Only leaf tissues were collected for the cold treatment. (B) Immunoblot analyses of OsMAPK5 under drought (root tissues), salt (root tissues), and cold (leaf tissues) stresses. (C) MBP in gel kinase activity assay of immunoprecipitated OsMAPK5 under drought (root tissues), salt (root tissues), and cold (leaf tissues) stresses.

Techniques Used: Activity Assay, Western Blot, Expressing, Kinase Assay, Immunoprecipitation

Induction of OsMAPK5 , Its Protein, and Kinase Activity by Abscisic Acid and Wounding. Experiments were repeated twice using samples from independent treatments. (A) RNA gel blot analysis of OsMAPK5 expression in 2-week-old seedlings treated with 0.1 mM abscisic acid (ABA), 1 mM SA, 0.1 mM JA, or wounding. Total RNAs were extracted at the times specified. The same blots were probed with the cDNA of PBZ1 ), to assess the effectiveness of the chemical treatments because PBZ1 ). (B) Immunoblot analysis of OsMAPK5 in 2-week-old seedlings treated with 0.1 mM abscisic acid, 1 mM SA, 0.1 mM JA, or wounding. (C) MBP in gel kinase activity of immunoprecipitated OsMAPK5 from 2-week-old seedlings treated with 0.1 mM abscisic acid, 1 mM SA, 0.1 mM JA, or wounding.
Figure Legend Snippet: Induction of OsMAPK5 , Its Protein, and Kinase Activity by Abscisic Acid and Wounding. Experiments were repeated twice using samples from independent treatments. (A) RNA gel blot analysis of OsMAPK5 expression in 2-week-old seedlings treated with 0.1 mM abscisic acid (ABA), 1 mM SA, 0.1 mM JA, or wounding. Total RNAs were extracted at the times specified. The same blots were probed with the cDNA of PBZ1 ), to assess the effectiveness of the chemical treatments because PBZ1 ). (B) Immunoblot analysis of OsMAPK5 in 2-week-old seedlings treated with 0.1 mM abscisic acid, 1 mM SA, 0.1 mM JA, or wounding. (C) MBP in gel kinase activity of immunoprecipitated OsMAPK5 from 2-week-old seedlings treated with 0.1 mM abscisic acid, 1 mM SA, 0.1 mM JA, or wounding.

Techniques Used: Activity Assay, Western Blot, Expressing, Immunoprecipitation

Altered Tolerance of OsMAPK5-OX and OsMAPK5-RI Transgenic Plants to Cold, Salt, and Drought. (A) Percentage of surviving seedlings after treatment with cold (4°C for 3 days followed by normal growth conditions for recovery), salt (200 mM NaCl for up to 4 days), or drought (withholding water for up to 6 days). At least 40 hygromycin-positive T1 transgenic seedlings were used in each experiment. All experiments were repeated twice. Statistical analysis ( t test) was performed to evaluate the levels of cold, salt, and drought tolerance based on the percentage of surviving seedlings in the overexpression or suppression lines versus the control line after the abiotic treatments. See supplemental data online for photographs of rice seedlings after cold, salt, and drought treatments. (B) MBP in gel kinase assay of immunoprecipitated OsMAPK5 from mixed leaf tissue sampling at different times under cold (6, 12, and 24 h), salinity (6, 12, and 24 h), or drought (2, 3, and 4 day) stresses. The relative MBP kinase activities of control and transgenic lines and the control line were calculated based on phosphorimager quantification of band intensity.
Figure Legend Snippet: Altered Tolerance of OsMAPK5-OX and OsMAPK5-RI Transgenic Plants to Cold, Salt, and Drought. (A) Percentage of surviving seedlings after treatment with cold (4°C for 3 days followed by normal growth conditions for recovery), salt (200 mM NaCl for up to 4 days), or drought (withholding water for up to 6 days). At least 40 hygromycin-positive T1 transgenic seedlings were used in each experiment. All experiments were repeated twice. Statistical analysis ( t test) was performed to evaluate the levels of cold, salt, and drought tolerance based on the percentage of surviving seedlings in the overexpression or suppression lines versus the control line after the abiotic treatments. See supplemental data online for photographs of rice seedlings after cold, salt, and drought treatments. (B) MBP in gel kinase assay of immunoprecipitated OsMAPK5 from mixed leaf tissue sampling at different times under cold (6, 12, and 24 h), salinity (6, 12, and 24 h), or drought (2, 3, and 4 day) stresses. The relative MBP kinase activities of control and transgenic lines and the control line were calculated based on phosphorimager quantification of band intensity.

Techniques Used: Transgenic Assay, Over Expression, Kinase Assay, Immunoprecipitation, Sampling

24) Product Images from "Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro"

Article Title: Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

Journal: Journal of the American Chemical Society

doi: 10.1021/ja901306q

Max-E47 hybrids inhibit native MaxbHLHZ activation from the E-box (MY1H). ( A ) The HIS3 assay of the inhibition of native MaxbHLHZ by the Max-E47 hybrids. Plates a–d are transformations plated on SD/-H/-L + 10 mM 3-AT plates, which were incubated
Figure Legend Snippet: Max-E47 hybrids inhibit native MaxbHLHZ activation from the E-box (MY1H). ( A ) The HIS3 assay of the inhibition of native MaxbHLHZ by the Max-E47 hybrids. Plates a–d are transformations plated on SD/-H/-L + 10 mM 3-AT plates, which were incubated

Techniques Used: Activation Assay, Inhibition, Incubation

Max-E47 hybrids activate transcription from the E-box (Y1H). ( A ) The HIS3 assay of Max-E47 hybrids expressed in pGAD424. SD/-H/-L + 10 mM 3-AT plates were incubated at 30 °C for 6 days. a . pGAD424 (negative control); clean. b . pGAD424/native MaxbHLHZ
Figure Legend Snippet: Max-E47 hybrids activate transcription from the E-box (Y1H). ( A ) The HIS3 assay of Max-E47 hybrids expressed in pGAD424. SD/-H/-L + 10 mM 3-AT plates were incubated at 30 °C for 6 days. a . pGAD424 (negative control); clean. b . pGAD424/native MaxbHLHZ

Techniques Used: Incubation, Negative Control

25) Product Images from "Expression, Localization, and Biochemical Characterization of Nicotinamide Mononucleotide Adenylyltransferase 2 *"

Article Title: Expression, Localization, and Biochemical Characterization of Nicotinamide Mononucleotide Adenylyltransferase 2 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.178913

Nmnat2 is a neuronal protein and expression is developmentally regulated. A , to assess the specificity of an in-house antibody targeted against full-length Nmnat2, Neuro-2a, a neuroblastoma cell line reported to express Nmnat2 transcript (BioGPS), were infected with lentiviruses (MOI of 1) expressing either shRNA against Nmnat2 ( lanes A and B ) or EGFP ( lane C ). Total lysate from each was compared by Western blot. B , to evaluate the tissue expression of Nmnat2, lysate from various tissues were analyzed by Western blot. Note that a slightly slower migrating band is observed in several tissues including brain, marked with an asterisk ; this band is nonspecific as shown in C. C , comparison of brain lysate from mice in which both Nmnat2 alleles have been trapped ( Tr/Tr ) versus wild-type controls (+/+). Each lane represents a single animal. As expected (see text), Nmnat2 expression in gene trap mouse brain is ∼50% of wild-type controls. Note the same nonspecific band observed in B , marked with an asterisk. D , to evaluate the cellular expression of Nmnat2, lysates from various cell types were analyzed by Western blot. Nmnat2 is detected in a human neuroblastoma cell line SH-SY5Y, but not in primary glia, U87 or U343, which are glioblastoma cell lines, or either of the negative controls NIH3T3 or HEK293T. In SH-SY5Y, Nmnat2 is dramatically up-regulated upon differentiation. E , Nmnat2 expression increases during maturation of primary cortical neurons similarly to synaptobrevin 2, a critical synaptic vesicle protein. F , expression of Nmnat2 during mouse development. Mouse brain lysate was obtained from a pair of male and female mice at birth and every 4 days thereafter as indicated. For each panel, an equal amount of protein was loaded in each lane; actin, GAPDH, or VCP are used as loading controls. An asterisk indicates a nonspecific band. NF , neurofilament; Syb 2 , synaptobrevin 2; DIV , days in vitro ; Diff. , differentiated; Undiff. , undifferentiated. HC , heavy chain.
Figure Legend Snippet: Nmnat2 is a neuronal protein and expression is developmentally regulated. A , to assess the specificity of an in-house antibody targeted against full-length Nmnat2, Neuro-2a, a neuroblastoma cell line reported to express Nmnat2 transcript (BioGPS), were infected with lentiviruses (MOI of 1) expressing either shRNA against Nmnat2 ( lanes A and B ) or EGFP ( lane C ). Total lysate from each was compared by Western blot. B , to evaluate the tissue expression of Nmnat2, lysate from various tissues were analyzed by Western blot. Note that a slightly slower migrating band is observed in several tissues including brain, marked with an asterisk ; this band is nonspecific as shown in C. C , comparison of brain lysate from mice in which both Nmnat2 alleles have been trapped ( Tr/Tr ) versus wild-type controls (+/+). Each lane represents a single animal. As expected (see text), Nmnat2 expression in gene trap mouse brain is ∼50% of wild-type controls. Note the same nonspecific band observed in B , marked with an asterisk. D , to evaluate the cellular expression of Nmnat2, lysates from various cell types were analyzed by Western blot. Nmnat2 is detected in a human neuroblastoma cell line SH-SY5Y, but not in primary glia, U87 or U343, which are glioblastoma cell lines, or either of the negative controls NIH3T3 or HEK293T. In SH-SY5Y, Nmnat2 is dramatically up-regulated upon differentiation. E , Nmnat2 expression increases during maturation of primary cortical neurons similarly to synaptobrevin 2, a critical synaptic vesicle protein. F , expression of Nmnat2 during mouse development. Mouse brain lysate was obtained from a pair of male and female mice at birth and every 4 days thereafter as indicated. For each panel, an equal amount of protein was loaded in each lane; actin, GAPDH, or VCP are used as loading controls. An asterisk indicates a nonspecific band. NF , neurofilament; Syb 2 , synaptobrevin 2; DIV , days in vitro ; Diff. , differentiated; Undiff. , undifferentiated. HC , heavy chain.

Techniques Used: Expressing, Infection, shRNA, Western Blot, Mouse Assay, In Vitro

Endogenous Nmnat2 localizes to synaptic terminals. A , schematic of synaptosome purification protocol. Synaptosomes are detached nerve terminals formed during homogenization and contain membranes derived from both pre- and postsynaptic compartments. For P1, S1, P11, S11, P200, and S200, P refers to pellet, and S refers to supernatant, whereas numbers refer to the force of centrifugation for each step ( e.g. S200 , supernatant resulting from 200,000 × g spin). B , Western blot analysis of fractionated mouse brains; an equal amount of protein was loaded for each sample. An asterisk indicates a nonspecific band. NRX , neurexin; Syb 2 , synaptobrevin 2; synap. , synaptosome; SV , synaptic vesicles; SPM , synaptic plasma membrane; mito , mitochondria.
Figure Legend Snippet: Endogenous Nmnat2 localizes to synaptic terminals. A , schematic of synaptosome purification protocol. Synaptosomes are detached nerve terminals formed during homogenization and contain membranes derived from both pre- and postsynaptic compartments. For P1, S1, P11, S11, P200, and S200, P refers to pellet, and S refers to supernatant, whereas numbers refer to the force of centrifugation for each step ( e.g. S200 , supernatant resulting from 200,000 × g spin). B , Western blot analysis of fractionated mouse brains; an equal amount of protein was loaded for each sample. An asterisk indicates a nonspecific band. NRX , neurexin; Syb 2 , synaptobrevin 2; synap. , synaptosome; SV , synaptic vesicles; SPM , synaptic plasma membrane; mito , mitochondria.

Techniques Used: Purification, Homogenization, Derivative Assay, Centrifugation, Western Blot

Nmnat2 localizes to Rab7 late endosomes and trans- Golgi network in HeLa cells. ) ( top panel ); TGN46, a trans- ) ( middle panel ) ( bottom panel ). Dotted lines indicate regions in the cytoplasm where Rab7 and Nmnat2 are closely associated. Arrows indicate where Nmnat2 does not co-localize with Rab7. Arrowhead indicates specific vesicle where Nmnat2 colocalizes with Rab7. An asterisk marks the nucleus in each cell. Scale bar , 10 μm.
Figure Legend Snippet: Nmnat2 localizes to Rab7 late endosomes and trans- Golgi network in HeLa cells. ) ( top panel ); TGN46, a trans- ) ( middle panel ) ( bottom panel ). Dotted lines indicate regions in the cytoplasm where Rab7 and Nmnat2 are closely associated. Arrows indicate where Nmnat2 does not co-localize with Rab7. Arrowhead indicates specific vesicle where Nmnat2 colocalizes with Rab7. An asterisk marks the nucleus in each cell. Scale bar , 10 μm.

Techniques Used:

Exogenous Nmnat2 is toxic to primary neurons. A , primary cortical neurons were infected with lentivirus expressing either Nmnat2-FLAG or Nmnat2 C164S/C165S-FLAG. Approximately 4 days after infection, cultures were fixed and probed with an anti-FLAG antibody. Nmnat2, but not Nmnat2 C164S/C165S, is toxic to neurons, which results in massive neuronal death; arrows indicate neurons shown in the inset . For comparison, a glial cell in each image is marked with an asterisk. Scale bar , 50 μm. B , primary cortical neurons were infected with lentivirus (MOI ∼ 3) expressing Nmnat2-EGFP. Nmnat2 localizes to Golgi (as in HeLa cells) but also dendrites and axons ( scale bar , 10 μm). Shown is a magnified view of a dendrite ( lower panel ; scale bar , 2 μm).
Figure Legend Snippet: Exogenous Nmnat2 is toxic to primary neurons. A , primary cortical neurons were infected with lentivirus expressing either Nmnat2-FLAG or Nmnat2 C164S/C165S-FLAG. Approximately 4 days after infection, cultures were fixed and probed with an anti-FLAG antibody. Nmnat2, but not Nmnat2 C164S/C165S, is toxic to neurons, which results in massive neuronal death; arrows indicate neurons shown in the inset . For comparison, a glial cell in each image is marked with an asterisk. Scale bar , 50 μm. B , primary cortical neurons were infected with lentivirus (MOI ∼ 3) expressing Nmnat2-EGFP. Nmnat2 localizes to Golgi (as in HeLa cells) but also dendrites and axons ( scale bar , 10 μm). Shown is a magnified view of a dendrite ( lower panel ; scale bar , 2 μm).

Techniques Used: Infection, Expressing

Nmnat2 is a palmitoylated peripheral membrane protein. A , sequence alignment reveals Cys 164 and Cys 165 as potential targets of palmitoylation. Palmitoylation lacks a well defined motif but occurs on cysteines frequently with nearby basic residues. Cys 164 and Cys 165 are highlighted in red , whereas nearby conserved basic residues are in boldface italic. B , HEK293T were transfected and metabolically labeled with [ 3 H]palmitate. Following immunoprecipitation, incorporation of radiolabel was assessed by in-gel fluorography. To assess the efficiency of immunoprecipitation, a 1:10 dilution of eluted protein was analyzed by Western blot ( WB ). CC , Cys 164 and Cys 165 ; SS , Ser 164 and Ser 165 . C , HeLa were infected with lentiviruses (MOI of 3) expressing either Nmnat2-EGFP, Nmnat2 C164S/C165S-EGFP, or EGFP and then fixed and probed for GM130, a well characterized cis- ). D , infected HeLa cells were permeabilized with digitonin prior to fixation to assess the solubility of wild-type Nmnat2 and C164S/C165S mutant. Note that under these conditions, the nuclear membrane remains intact. Scale bar , 10 μm.
Figure Legend Snippet: Nmnat2 is a palmitoylated peripheral membrane protein. A , sequence alignment reveals Cys 164 and Cys 165 as potential targets of palmitoylation. Palmitoylation lacks a well defined motif but occurs on cysteines frequently with nearby basic residues. Cys 164 and Cys 165 are highlighted in red , whereas nearby conserved basic residues are in boldface italic. B , HEK293T were transfected and metabolically labeled with [ 3 H]palmitate. Following immunoprecipitation, incorporation of radiolabel was assessed by in-gel fluorography. To assess the efficiency of immunoprecipitation, a 1:10 dilution of eluted protein was analyzed by Western blot ( WB ). CC , Cys 164 and Cys 165 ; SS , Ser 164 and Ser 165 . C , HeLa were infected with lentiviruses (MOI of 3) expressing either Nmnat2-EGFP, Nmnat2 C164S/C165S-EGFP, or EGFP and then fixed and probed for GM130, a well characterized cis- ). D , infected HeLa cells were permeabilized with digitonin prior to fixation to assess the solubility of wild-type Nmnat2 and C164S/C165S mutant. Note that under these conditions, the nuclear membrane remains intact. Scale bar , 10 μm.

Techniques Used: Sequencing, Transfection, Metabolic Labelling, Labeling, Immunoprecipitation, Western Blot, Infection, Expressing, Solubility, Mutagenesis

26) Product Images from "Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling"

Article Title: Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling

Journal: Scientific Reports

doi: 10.1038/srep11088

Ha83 deletion analysis in HzAM1 cells. ( A ) Microscopy analysis of viral replication in HzAM1 cells. Fluorescence microscopy shows the progression of viral infection in HzAM1 cells transfected with HaWT ( polyhedrin repaired HaBacHZ8 bacmid), Ha83KO ( ha83 deleted HearNPV mutant), and Ha83Rep (Ha83KO reinserted a ha83 in the polyhedrin locus) from 24 to 72 h p.t. An additional infection fluorescence microscopy shows the infectivity of viral infection in HzAM1 cells infected with HaWT, Ha83KO, and Ha83Rep at 72 h p.i. Light microscopy shows the formation of occlusion bodies in HaWT, Ha83KO, and Ha83Rep infected cells at 96 h p.i. ( B ) Virus growth curves as determined by TCID 50 endpoint dilution assays. For the transfection growth curves, HzAM1 cells were transfected with HaWT, Ha83KO, and Ha83Rep bacmid DNA. The supernatants were then harvested at the indicated time points p. t., and titers were determined using TCID 50 assays. Each data point was determined from the average of three independent transfections, and error bars represent the standard deviations. ( C ) Real-time PCR analysis of viral DNA replication. HzAM1 cells were transfected with each bacmid DNA (HaWT, Ha83KO, or Ha83Rep). At the designated time points, total intracellular DNA was extracted and analyzed by real-time PCR. The y-axis value indicates the number of viral DNA genome copies within each sample. The graph shows the results of three independent replication assays and each replication was tested for another three times, with error bars indicating the standard deviations. Statistically significant differences are indicated by asterisks (* P ⊠ 0.05).
Figure Legend Snippet: Ha83 deletion analysis in HzAM1 cells. ( A ) Microscopy analysis of viral replication in HzAM1 cells. Fluorescence microscopy shows the progression of viral infection in HzAM1 cells transfected with HaWT ( polyhedrin repaired HaBacHZ8 bacmid), Ha83KO ( ha83 deleted HearNPV mutant), and Ha83Rep (Ha83KO reinserted a ha83 in the polyhedrin locus) from 24 to 72 h p.t. An additional infection fluorescence microscopy shows the infectivity of viral infection in HzAM1 cells infected with HaWT, Ha83KO, and Ha83Rep at 72 h p.i. Light microscopy shows the formation of occlusion bodies in HaWT, Ha83KO, and Ha83Rep infected cells at 96 h p.i. ( B ) Virus growth curves as determined by TCID 50 endpoint dilution assays. For the transfection growth curves, HzAM1 cells were transfected with HaWT, Ha83KO, and Ha83Rep bacmid DNA. The supernatants were then harvested at the indicated time points p. t., and titers were determined using TCID 50 assays. Each data point was determined from the average of three independent transfections, and error bars represent the standard deviations. ( C ) Real-time PCR analysis of viral DNA replication. HzAM1 cells were transfected with each bacmid DNA (HaWT, Ha83KO, or Ha83Rep). At the designated time points, total intracellular DNA was extracted and analyzed by real-time PCR. The y-axis value indicates the number of viral DNA genome copies within each sample. The graph shows the results of three independent replication assays and each replication was tested for another three times, with error bars indicating the standard deviations. Statistically significant differences are indicated by asterisks (* P ⊠ 0.05).

Techniques Used: Microscopy, Fluorescence, Infection, Transfection, Mutagenesis, Light Microscopy, Real-time Polymerase Chain Reaction

27) Product Images from "Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion *"

Article Title: Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.411934

Identification of residues in the JD that mediate intramolecular interactions with CamLD and calcium-dependent activation of PfCDPK1. A , multiple sequence alignment of JDs from related CDPKs. The JD of PfCDPK1 was aligned with JDs of PfCDPK4, TgCDPK1,
Figure Legend Snippet: Identification of residues in the JD that mediate intramolecular interactions with CamLD and calcium-dependent activation of PfCDPK1. A , multiple sequence alignment of JDs from related CDPKs. The JD of PfCDPK1 was aligned with JDs of PfCDPK4, TgCDPK1,

Techniques Used: Activation Assay, Sequencing

A model for PfCDPK1 activation and inhibition of its activity by P3 peptide. In presence of Ca 2+ , PfCDPK1 undergoes a conformational change so that the CamLD interacts with the P3 region of the JD and exposes the substrate binding catalytic site in the
Figure Legend Snippet: A model for PfCDPK1 activation and inhibition of its activity by P3 peptide. In presence of Ca 2+ , PfCDPK1 undergoes a conformational change so that the CamLD interacts with the P3 region of the JD and exposes the substrate binding catalytic site in the

Techniques Used: Activation Assay, Inhibition, Activity Assay, Binding Assay

Intramolecular interactions between the CamLD and JD of PfCDPK1. The CONTACT program from the CCP4 package was used to investigate the possible interactions of Phe 350 , Leu 356 , and Phe 363 –Ile 364 with residues from CamLD in PfCDPK1 with 4 Å
Figure Legend Snippet: Intramolecular interactions between the CamLD and JD of PfCDPK1. The CONTACT program from the CCP4 package was used to investigate the possible interactions of Phe 350 , Leu 356 , and Phe 363 –Ile 364 with residues from CamLD in PfCDPK1 with 4 Å

Techniques Used:

P3 peptide serves as a binding site for CamLD and inhibits kinase activity of PfCDPK1. A , schematic of PfCDPK1 and peptides from the JD. The JD of PfCDPK1 was dissected into three nonoverlapping peptides, P1, P2, and P3. B , CamLD of PfCDPK1 binding the
Figure Legend Snippet: P3 peptide serves as a binding site for CamLD and inhibits kinase activity of PfCDPK1. A , schematic of PfCDPK1 and peptides from the JD. The JD of PfCDPK1 was dissected into three nonoverlapping peptides, P1, P2, and P3. B , CamLD of PfCDPK1 binding the

Techniques Used: Binding Assay, Activity Assay

28) Product Images from "A Neuron-Specific Antiviral Mechanism Prevents Lethal Flaviviral Infection of Mosquitoes"

Article Title: A Neuron-Specific Antiviral Mechanism Prevents Lethal Flaviviral Infection of Mosquitoes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004848

AaHig interrupts flaviviral endocytosis into mosquito cells. (A-B) Viral attachment assay. The serial concentration of purified AaHig protein was premixed with 5 M.O.I. DENV-2 on ice, and then the Aag2 (A) and C6/36 (B) cells were consequently preadsorbed with the mixture for a time course at 4oC. The cells were washed 5 times by cold PBS and collected at certain time points for total RNA isolation. (C-D) Viral entry assay. The serial concentration of purified AaHig protein was premixed with 5 M.O.I. DENV-2 on ice, and then the mixture was transferred into the cultured Aag2 cells (C) at 28°C and C6/36 (D) cells at 30°C respectively. The cells were stringently washed 5 times by PBS at room temperature, and then collected at serial time points for detection. (A-D) For the assay at 48 hours, the cells were washed 5 times after 1 hr incubation at 4°C (A and B) or 28°C /30°C (C and D), and consequently cultured at 28°C (Aag2) or 30°C (C6/36) for an additional 48 hrs. The viral genome was determined by Taqman qPCR and normalized by A . aegypti actin . The data were presented as the mean ± standard error. The result was combined from 3 independent experiments. *, p
Figure Legend Snippet: AaHig interrupts flaviviral endocytosis into mosquito cells. (A-B) Viral attachment assay. The serial concentration of purified AaHig protein was premixed with 5 M.O.I. DENV-2 on ice, and then the Aag2 (A) and C6/36 (B) cells were consequently preadsorbed with the mixture for a time course at 4oC. The cells were washed 5 times by cold PBS and collected at certain time points for total RNA isolation. (C-D) Viral entry assay. The serial concentration of purified AaHig protein was premixed with 5 M.O.I. DENV-2 on ice, and then the mixture was transferred into the cultured Aag2 cells (C) at 28°C and C6/36 (D) cells at 30°C respectively. The cells were stringently washed 5 times by PBS at room temperature, and then collected at serial time points for detection. (A-D) For the assay at 48 hours, the cells were washed 5 times after 1 hr incubation at 4°C (A and B) or 28°C /30°C (C and D), and consequently cultured at 28°C (Aag2) or 30°C (C6/36) for an additional 48 hrs. The viral genome was determined by Taqman qPCR and normalized by A . aegypti actin . The data were presented as the mean ± standard error. The result was combined from 3 independent experiments. *, p

Techniques Used: Concentration Assay, Purification, Isolation, Cell Culture, Incubation, Real-time Polymerase Chain Reaction

Both the viral interaction and membrane bound of AaHig are essential for the AaHig antiviral activity. (A-B) Construction and expression of recombinant AaHig truncations. The schematic representation of AaHig truncations with sequential depletion of modules is shown in the panel A. The fragments of AaHig truncated genes were constructed into pAc5.1/V5-His A vector to express in Drosophila S2 cells (pAc-AaHig-A ~ pAc-AaHig-F). The S2 cells transfected by the empty vector were used as a mock control. The expression in the supernatant was detected by western blotting with anti-V5 mAb (B). (C) The interaction between AaHig truncated peptides and the DENV-2 E protein in a co-IP assay. The cell supernatant was individually incubated with purified DENV-2 E protein. The supernatant from the pAc-GFP transfected cells was used as a mock control. The protein complex was pulled down with an anti-V5 antibody and probed using an anti-FLAG-HRP antibody. The experiment was reproduced 3 times. (D) The detection of binding capacity between AaHig fragments and DENV-2 E protein by ELISA. The supernatant of pAc-AaHigs and pAc-GFP transfected cells was used to assess the binding activity with DENV-2 E proteins. The binding was probed using the anti-V5 mAb. The data were presented as the mean ± standard error. The experiment was repeated 3 times with the similar result. (E) AaHig resists DENV-2 infection in A . aegypti Aag2 cells. The pAc-AaHig-Full recombinant plasmid was transfected into Aag2 cells. The pAc-GFP was used as negative control. After 48 hrs, the 0.01 M.O.I. DENV-2 was added into the cells and the viral load was determined by Taqman qPCR and normalized by A . aegypti actin . The data were presented as the mean ± standard error. The result was combined from 3 independent experiments. (F) The role of AaHig truncated peptides in DENV-2 infection of Aag2 cells. A variety of AaHig truncations were expressed in Aag2 cells. After 0.01 M.O.I. DENV-2 infection, the viruses were determined by Taqman qPCR and normalized against A . aegypti actin . The data were presented as the mean ± standard error. The result was combined from 2 independent experiments. (G) The schematic representation of AaHig-G truncation. (H) The interaction between AaHig-G peptide and the DENV-2 E protein in a co-IP assay. The gene of AaHig-G was cloned into pMT/BiP/V5-His A and expressed in S2 cells. The cell supernatant with AaHig-G was individually incubated with purified DENV-2 E protein. The supernatant from the pMT/BiP/V5-His A vector transfected cells was used as a mock control. The transfection of pMT-AaHig-Full served as a positive control. The protein complex was pulled down with an anti-V5 antibody and probed using an anti-FLAG-HRP antibody. The experiment was reproduced 3 times. (I) The role of AaHig-G in DENV-2 infection of Aag2 cells. Both AaHig-G and AaHig-Full cloned in pMT/BiP/V5-His A vector were ectopically expressed in S2 cells. The conditional medium premixed with 0.01 M.O.I. DENV-2 was added into mosquito Aag2 cells for infection. The infectivity were determined by Taqman qPCR and normalized against A . aegypti actin . The data were presented as the mean ± standard error. The result was combined from 3 independent experiments.
Figure Legend Snippet: Both the viral interaction and membrane bound of AaHig are essential for the AaHig antiviral activity. (A-B) Construction and expression of recombinant AaHig truncations. The schematic representation of AaHig truncations with sequential depletion of modules is shown in the panel A. The fragments of AaHig truncated genes were constructed into pAc5.1/V5-His A vector to express in Drosophila S2 cells (pAc-AaHig-A ~ pAc-AaHig-F). The S2 cells transfected by the empty vector were used as a mock control. The expression in the supernatant was detected by western blotting with anti-V5 mAb (B). (C) The interaction between AaHig truncated peptides and the DENV-2 E protein in a co-IP assay. The cell supernatant was individually incubated with purified DENV-2 E protein. The supernatant from the pAc-GFP transfected cells was used as a mock control. The protein complex was pulled down with an anti-V5 antibody and probed using an anti-FLAG-HRP antibody. The experiment was reproduced 3 times. (D) The detection of binding capacity between AaHig fragments and DENV-2 E protein by ELISA. The supernatant of pAc-AaHigs and pAc-GFP transfected cells was used to assess the binding activity with DENV-2 E proteins. The binding was probed using the anti-V5 mAb. The data were presented as the mean ± standard error. The experiment was repeated 3 times with the similar result. (E) AaHig resists DENV-2 infection in A . aegypti Aag2 cells. The pAc-AaHig-Full recombinant plasmid was transfected into Aag2 cells. The pAc-GFP was used as negative control. After 48 hrs, the 0.01 M.O.I. DENV-2 was added into the cells and the viral load was determined by Taqman qPCR and normalized by A . aegypti actin . The data were presented as the mean ± standard error. The result was combined from 3 independent experiments. (F) The role of AaHig truncated peptides in DENV-2 infection of Aag2 cells. A variety of AaHig truncations were expressed in Aag2 cells. After 0.01 M.O.I. DENV-2 infection, the viruses were determined by Taqman qPCR and normalized against A . aegypti actin . The data were presented as the mean ± standard error. The result was combined from 2 independent experiments. (G) The schematic representation of AaHig-G truncation. (H) The interaction between AaHig-G peptide and the DENV-2 E protein in a co-IP assay. The gene of AaHig-G was cloned into pMT/BiP/V5-His A and expressed in S2 cells. The cell supernatant with AaHig-G was individually incubated with purified DENV-2 E protein. The supernatant from the pMT/BiP/V5-His A vector transfected cells was used as a mock control. The transfection of pMT-AaHig-Full served as a positive control. The protein complex was pulled down with an anti-V5 antibody and probed using an anti-FLAG-HRP antibody. The experiment was reproduced 3 times. (I) The role of AaHig-G in DENV-2 infection of Aag2 cells. Both AaHig-G and AaHig-Full cloned in pMT/BiP/V5-His A vector were ectopically expressed in S2 cells. The conditional medium premixed with 0.01 M.O.I. DENV-2 was added into mosquito Aag2 cells for infection. The infectivity were determined by Taqman qPCR and normalized against A . aegypti actin . The data were presented as the mean ± standard error. The result was combined from 3 independent experiments.

Techniques Used: Activity Assay, Expressing, Recombinant, Construct, Plasmid Preparation, Transfection, Western Blot, Co-Immunoprecipitation Assay, Incubation, Purification, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection, Negative Control, Real-time Polymerase Chain Reaction, Clone Assay, Positive Control

AaHig directly recognizes dengue virus. (A) Expression and purification of AaHig from Drosophila S2 cells. The full length AaHig was cloned into the pMT/BiP/V5-His A expression vector. The recombinant plasmid was transfected into Drosophila S2 cells, and the expression was probed using an anti-V5 mAb. The supernatant from mock-transfected S2 cells was used as the mock control (Right Panel). Recombinant AaHig protein, produced in Drosophila cells, was purified using a Ni-His column (Left Panel). (B) AaHig interacted with DENV-2 E protein in a co-immunoprecipitation (co-IP) assay. The purified AaHig (V5) and DENV-2 E (FLAG) proteins were used to investigate the interaction of the proteins. We reproduced the experiment 3 times. (C) AaHig captured DENV-2 virions in an ELISA assay. The binding was probed using the flavivirus E mAb 4G2. The data were presented as the mean ± standard error. The experiment was reproduced 3 times. (D) AaHig interfaced with DENV virions in the infected mosquito cells. pAc-AaHig was transfected in Aag2 cells, and subsequently the cells were infected by 5 M.O.I. DENV-2 at 12 hrs post transfection. The uninfected cells transfected by pAc-AaHig were used as a mock control. After 48 hrs infection, the cells were lysated and an anti-flaviviral E 4G2 mAb was added into the lysate for the pull-down assay. We reproduced the experiment 3 times. (E) The co-staining between AaHig and DENV-2 in the A . aegypti brain. The mosquito brains were dissected from uninfected mocks and infected mosquitoes at 6 days post infection to undergo immunofluorescence staining. AaHig was stained with anti-mouse IgG Alexa-546 (Red), and the DENV-2 E protein was stained using anti-human IgG Alexa-488 (Green). Nuclei were stained with To-Pro-3 iodide (Blue). Images were examined using the 63×objective lens of a Zeiss LSM 780 meta confocal. (F) The expression of ectodomains in the DENV-2 E protein. The genes of ED1+ED2 (1-296AA) and ED3 (297-400AA) of DENV-2 E protein were cloned into pMT/BiP/V5-His A vector and the encoded peptides were expressed in Drosophila S2 cells. The supernatant from empty vector-transfected S2 cells was used as a mock. The recombinant peptides were detected with an anti-FLAG antibody via western blotting. (G) The ectodomains of DENV-2 E protein interacted with AaHig in a co-IP assay. The protein complex was pulled down with an anti-FLAG antibody and detected using an anti-V5-HRP antibody. We reproduced the experiment 3 times. (H) The ectodomains of DENV-2 E protein interfaced with AaHig by an ELISA assay. The binding was probed using an anti-V5-HRP antibody. The data were presented as the mean ± standard error. The experiment was reproduced 3 times. (I) The interaction between AaHig and the linear motifs of ED1/ED2. The linear motifs of ED1 and ED2 were sequentially deleted from the ectodomains. The five truncations were cloned into the pMT/BiP/V5-His A vector, and subsequently expressed in the S2 cell supernatant. The protein complex was pulled down with an anti-FLAG antibody and detected using an anti-V5-HRP antibody. We reproduced the experiment 3 times. (J) Inoculation of AaHig impaired DENV-2 infectivity in A . aegypti . Two laddered concentrations of purified AaHig protein were premixed with 10 M.I.D. 50 of DENV-2 for mosquito thoracic microinjection. The infected mosquitoes were sacrificed at 3 (i) and 6 (ii) days post infection. The viral load was determined by Taqman qPCR and normalized by A . aegypti actin . The results were combined from 3 independent experiments. One dot represents 1 mosquito and the horizontal line represents the median value in the figures. The statistical analysis was done with the Mann-Whitney test.
Figure Legend Snippet: AaHig directly recognizes dengue virus. (A) Expression and purification of AaHig from Drosophila S2 cells. The full length AaHig was cloned into the pMT/BiP/V5-His A expression vector. The recombinant plasmid was transfected into Drosophila S2 cells, and the expression was probed using an anti-V5 mAb. The supernatant from mock-transfected S2 cells was used as the mock control (Right Panel). Recombinant AaHig protein, produced in Drosophila cells, was purified using a Ni-His column (Left Panel). (B) AaHig interacted with DENV-2 E protein in a co-immunoprecipitation (co-IP) assay. The purified AaHig (V5) and DENV-2 E (FLAG) proteins were used to investigate the interaction of the proteins. We reproduced the experiment 3 times. (C) AaHig captured DENV-2 virions in an ELISA assay. The binding was probed using the flavivirus E mAb 4G2. The data were presented as the mean ± standard error. The experiment was reproduced 3 times. (D) AaHig interfaced with DENV virions in the infected mosquito cells. pAc-AaHig was transfected in Aag2 cells, and subsequently the cells were infected by 5 M.O.I. DENV-2 at 12 hrs post transfection. The uninfected cells transfected by pAc-AaHig were used as a mock control. After 48 hrs infection, the cells were lysated and an anti-flaviviral E 4G2 mAb was added into the lysate for the pull-down assay. We reproduced the experiment 3 times. (E) The co-staining between AaHig and DENV-2 in the A . aegypti brain. The mosquito brains were dissected from uninfected mocks and infected mosquitoes at 6 days post infection to undergo immunofluorescence staining. AaHig was stained with anti-mouse IgG Alexa-546 (Red), and the DENV-2 E protein was stained using anti-human IgG Alexa-488 (Green). Nuclei were stained with To-Pro-3 iodide (Blue). Images were examined using the 63×objective lens of a Zeiss LSM 780 meta confocal. (F) The expression of ectodomains in the DENV-2 E protein. The genes of ED1+ED2 (1-296AA) and ED3 (297-400AA) of DENV-2 E protein were cloned into pMT/BiP/V5-His A vector and the encoded peptides were expressed in Drosophila S2 cells. The supernatant from empty vector-transfected S2 cells was used as a mock. The recombinant peptides were detected with an anti-FLAG antibody via western blotting. (G) The ectodomains of DENV-2 E protein interacted with AaHig in a co-IP assay. The protein complex was pulled down with an anti-FLAG antibody and detected using an anti-V5-HRP antibody. We reproduced the experiment 3 times. (H) The ectodomains of DENV-2 E protein interfaced with AaHig by an ELISA assay. The binding was probed using an anti-V5-HRP antibody. The data were presented as the mean ± standard error. The experiment was reproduced 3 times. (I) The interaction between AaHig and the linear motifs of ED1/ED2. The linear motifs of ED1 and ED2 were sequentially deleted from the ectodomains. The five truncations were cloned into the pMT/BiP/V5-His A vector, and subsequently expressed in the S2 cell supernatant. The protein complex was pulled down with an anti-FLAG antibody and detected using an anti-V5-HRP antibody. We reproduced the experiment 3 times. (J) Inoculation of AaHig impaired DENV-2 infectivity in A . aegypti . Two laddered concentrations of purified AaHig protein were premixed with 10 M.I.D. 50 of DENV-2 for mosquito thoracic microinjection. The infected mosquitoes were sacrificed at 3 (i) and 6 (ii) days post infection. The viral load was determined by Taqman qPCR and normalized by A . aegypti actin . The results were combined from 3 independent experiments. One dot represents 1 mosquito and the horizontal line represents the median value in the figures. The statistical analysis was done with the Mann-Whitney test.

Techniques Used: Expressing, Purification, Clone Assay, Plasmid Preparation, Recombinant, Transfection, Produced, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Infection, Pull Down Assay, Staining, Immunofluorescence, Western Blot, Real-time Polymerase Chain Reaction, MANN-WHITNEY

The high expression of AaHig in the brain of A . aegypti . (A-B) The expression of AaHig in various tissues of A . aegypti . The abundance of AaHig was assessed via SYBR Green qPCR (A) and immuno-blotting with an AaHig antibody (B). (A) The total RNA was isolated from mosquito tissues to determine AaHig expression by SYBR Green qPCR and normalized by A . aegypti actin ( AAEL011197 ). The qPCR primers were described in the S1 Table . Data were represented as the mean ± standard error. (B) A variety of tissues were dissected from female A . aegypti . 50 μg of total protein from tissue lysates was loaded into each lane. The detection of A . aegypti actin was used as the internal control. (C) Secretory property of AaHig. The full length of AaHig gene (1bp-2436bp) was inserted into the expression vector pAc5.1/V5-His A with a V5 tag. The recombinant DNA plasmid (pAc-AaHig) was transfected into S2 cells. AaHig expression was detected with anti-V5 antibody in the cell lysate and supernatant. (D) AaHig staining in A . aegypti Aag2 cells. Aag2 is an A . aegypti cell lineage of embryonic origin. The pAc-AaHig recombinant plasmid was transfected into Aag2 cells. AaHig was stained by anti-V5 antibody and anti-mouse IgG Alexa-546 (Red). The plasma membrane was stained by the Wheat Germ Agglutinin (WGA) conjugated with Alexa-488 (Green). Nuclei were stained with To-Pro-3 iodide (Blue). Images were examined using the 63×objective lens of a Zeiss LSM 780 meta confocal. (E) AaHig is localized on the cell surface of neural cells of the A . aegypti brain. The brains were dissected from female A . aegypti for the in situ staining. An anti-HRP rabbit polyclonal antibody with anti-rabbit IgG Alexa-488 was used for surface staining of mosquito neural cells (Green). The AaHig protein was detected by an anti-AaHig mouse polyclonal antibody and anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide. Images were examined using the 10× and 63×objective lens of a Zeiss LSM 780 meta confocal. AL, antennal lobes; OL, optic lobes.
Figure Legend Snippet: The high expression of AaHig in the brain of A . aegypti . (A-B) The expression of AaHig in various tissues of A . aegypti . The abundance of AaHig was assessed via SYBR Green qPCR (A) and immuno-blotting with an AaHig antibody (B). (A) The total RNA was isolated from mosquito tissues to determine AaHig expression by SYBR Green qPCR and normalized by A . aegypti actin ( AAEL011197 ). The qPCR primers were described in the S1 Table . Data were represented as the mean ± standard error. (B) A variety of tissues were dissected from female A . aegypti . 50 μg of total protein from tissue lysates was loaded into each lane. The detection of A . aegypti actin was used as the internal control. (C) Secretory property of AaHig. The full length of AaHig gene (1bp-2436bp) was inserted into the expression vector pAc5.1/V5-His A with a V5 tag. The recombinant DNA plasmid (pAc-AaHig) was transfected into S2 cells. AaHig expression was detected with anti-V5 antibody in the cell lysate and supernatant. (D) AaHig staining in A . aegypti Aag2 cells. Aag2 is an A . aegypti cell lineage of embryonic origin. The pAc-AaHig recombinant plasmid was transfected into Aag2 cells. AaHig was stained by anti-V5 antibody and anti-mouse IgG Alexa-546 (Red). The plasma membrane was stained by the Wheat Germ Agglutinin (WGA) conjugated with Alexa-488 (Green). Nuclei were stained with To-Pro-3 iodide (Blue). Images were examined using the 63×objective lens of a Zeiss LSM 780 meta confocal. (E) AaHig is localized on the cell surface of neural cells of the A . aegypti brain. The brains were dissected from female A . aegypti for the in situ staining. An anti-HRP rabbit polyclonal antibody with anti-rabbit IgG Alexa-488 was used for surface staining of mosquito neural cells (Green). The AaHig protein was detected by an anti-AaHig mouse polyclonal antibody and anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide. Images were examined using the 10× and 63×objective lens of a Zeiss LSM 780 meta confocal. AL, antennal lobes; OL, optic lobes.

Techniques Used: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Isolation, Plasmid Preparation, Recombinant, Transfection, Staining, Whole Genome Amplification, In Situ

The role of AaHig in flaviviruses infection of A . aegypti . Immuno-blockade of AaHig enhanced the DENV-2 (A-B) and JEV (C-D) infections in the whole bodies (i) and heads (ii) of mosquitoes. The murine AaHig antibody, in the 10-fold serial dilutions, was premixed with 10 M.I.D. 50 viruses to co-microinject into the thorax of mosquitoes. The treated mosquitoes were sacrificed to examine the viral load in the whole mosquito bodies (i) and heads (ii) at 3 (A, C) and 6 (B, D) days post-infection by TaqMan qPCR and normalized against A . aegypti actin . The results were reproduced by 3 times. One dot represents 1 mosquito/head and the horizontal line represents the median of the results. The data were analyzed statistically using the non-parametric Mann-Whitney test.
Figure Legend Snippet: The role of AaHig in flaviviruses infection of A . aegypti . Immuno-blockade of AaHig enhanced the DENV-2 (A-B) and JEV (C-D) infections in the whole bodies (i) and heads (ii) of mosquitoes. The murine AaHig antibody, in the 10-fold serial dilutions, was premixed with 10 M.I.D. 50 viruses to co-microinject into the thorax of mosquitoes. The treated mosquitoes were sacrificed to examine the viral load in the whole mosquito bodies (i) and heads (ii) at 3 (A, C) and 6 (B, D) days post-infection by TaqMan qPCR and normalized against A . aegypti actin . The results were reproduced by 3 times. One dot represents 1 mosquito/head and the horizontal line represents the median of the results. The data were analyzed statistically using the non-parametric Mann-Whitney test.

Techniques Used: Infection, Real-time Polymerase Chain Reaction, MANN-WHITNEY

29) Product Images from "Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus"

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002361

LL-37 signaling depends on a functional CsrS to induce GAS resistance to opsonophagocytic killing. Wild type strain 854 (WT), isogenic csrS mutants 854 csrS TM (TM), 854 csrS Ω ( csrS Ω), 854 H280A (H280A), and 854 H280A,TM (H280A TM), and isogenic csrR deletion mutant 854Δ csrR (Δ csrR ) were grown in the absence (open symbols) or presence (filled symbols) of 100 nM LL-37. Bacteria were then mixed with human peripheral blood leukocytes for 1 h in the presence of 10% human serum as complement source. Values represent the log of mean fold-change in cfu. Each symbol represents a single experiment performed in duplicate. When exposed to LL-37, wild type 854 showed a significant increase in resistance to phagocytic killing compared to untreated bacteria (P
Figure Legend Snippet: LL-37 signaling depends on a functional CsrS to induce GAS resistance to opsonophagocytic killing. Wild type strain 854 (WT), isogenic csrS mutants 854 csrS TM (TM), 854 csrS Ω ( csrS Ω), 854 H280A (H280A), and 854 H280A,TM (H280A TM), and isogenic csrR deletion mutant 854Δ csrR (Δ csrR ) were grown in the absence (open symbols) or presence (filled symbols) of 100 nM LL-37. Bacteria were then mixed with human peripheral blood leukocytes for 1 h in the presence of 10% human serum as complement source. Values represent the log of mean fold-change in cfu. Each symbol represents a single experiment performed in duplicate. When exposed to LL-37, wild type 854 showed a significant increase in resistance to phagocytic killing compared to untreated bacteria (P

Techniques Used: Functional Assay, Mutagenesis

CsrS is associated with the cell membrane and contains a surface-exposed domain. A) Western blot analysis of membrane and cytoplasmic fractions isolated from whole cell lysates of GAS wild type strain 854 (WT), isogenic csrS deficient mutant strain 854 csrS Ω (SΩ), and 854 csrS TM (TM) that expresses CsrS with 3 point mutations in the predicted extracellular domain. Specific antisera against CsrS, an unrelated membrane protein OpuABC, and CsrR were used to detect the respective proteins in both fractions. B) Biotin labeling via a disulfide linker of surface-exposed proteins in whole cells of wild type strain 854 (WT), 854 csrS Ω (SΩ) and 854 csrS TM (TM). After lysis of labeled cells, biotinylated proteins were captured on a NeutrAvidin column and then eluted by reducing the disulfide linker. Specific antisera detected CsrS in the eluted fraction, as expected for a surface-exposed protein, and CsrR in the flow-through, as expected for a cytoplasmic protein. As a control, wild type 854 cells were treated similarly, but without biotin labeling (NB). Results shown in both panels are representative of at least two independent experiments.
Figure Legend Snippet: CsrS is associated with the cell membrane and contains a surface-exposed domain. A) Western blot analysis of membrane and cytoplasmic fractions isolated from whole cell lysates of GAS wild type strain 854 (WT), isogenic csrS deficient mutant strain 854 csrS Ω (SΩ), and 854 csrS TM (TM) that expresses CsrS with 3 point mutations in the predicted extracellular domain. Specific antisera against CsrS, an unrelated membrane protein OpuABC, and CsrR were used to detect the respective proteins in both fractions. B) Biotin labeling via a disulfide linker of surface-exposed proteins in whole cells of wild type strain 854 (WT), 854 csrS Ω (SΩ) and 854 csrS TM (TM). After lysis of labeled cells, biotinylated proteins were captured on a NeutrAvidin column and then eluted by reducing the disulfide linker. Specific antisera detected CsrS in the eluted fraction, as expected for a surface-exposed protein, and CsrR in the flow-through, as expected for a cytoplasmic protein. As a control, wild type 854 cells were treated similarly, but without biotin labeling (NB). Results shown in both panels are representative of at least two independent experiments.

Techniques Used: Western Blot, Isolation, Mutagenesis, Labeling, Lysis, Flow Cytometry

30) Product Images from "Identification of Highly Specific scFvs against Total Adiponectin for Diagnostic Purposes"

Article Title: Identification of Highly Specific scFvs against Total Adiponectin for Diagnostic Purposes

Journal: Biology

doi: 10.3390/biology6020026

Binding activity for adiponectin of the expressed scFvs. The expressed scFvs were extracted from periplasma and an equal amount of total proteins (10 µg/µL) was used directly for measurement of the adiponectin-binding capacity in an ELISA. ScFvH5 showed the highest binding activity for recombinant adiponectin in ELISA. Bovine serum albumin (BSA) was used as a negative control.
Figure Legend Snippet: Binding activity for adiponectin of the expressed scFvs. The expressed scFvs were extracted from periplasma and an equal amount of total proteins (10 µg/µL) was used directly for measurement of the adiponectin-binding capacity in an ELISA. ScFvH5 showed the highest binding activity for recombinant adiponectin in ELISA. Bovine serum albumin (BSA) was used as a negative control.

Techniques Used: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control

Binding activity of purified scFvH5. Recombinant adiponectin (1 µg) was coated in a microtiter plate. For antigen detection, the purified scFvH5 was serially diluted from 10 µg to 0.1 µg/1 mL. The binding activity of the scFvH5 was detected with protein L-POD. The ELISA results are given as mean ± SD values from triple determinations.
Figure Legend Snippet: Binding activity of purified scFvH5. Recombinant adiponectin (1 µg) was coated in a microtiter plate. For antigen detection, the purified scFvH5 was serially diluted from 10 µg to 0.1 µg/1 mL. The binding activity of the scFvH5 was detected with protein L-POD. The ELISA results are given as mean ± SD values from triple determinations.

Techniques Used: Binding Assay, Activity Assay, Purification, Recombinant, Enzyme-linked Immunosorbent Assay

SDS-PAGE analysis of the purified recombinant adiponectin. The full-length adiponectin without LD was expressed as a 6×His fused protein in E. coli and was purified using Ni-NTA based purification. After separating in an SDS-PAGE, the gel was stained with Coomassie Brilliant Blue. E1 and E2: eluate 1 and 2, respectively; M: protein marker.
Figure Legend Snippet: SDS-PAGE analysis of the purified recombinant adiponectin. The full-length adiponectin without LD was expressed as a 6×His fused protein in E. coli and was purified using Ni-NTA based purification. After separating in an SDS-PAGE, the gel was stained with Coomassie Brilliant Blue. E1 and E2: eluate 1 and 2, respectively; M: protein marker.

Techniques Used: SDS Page, Purification, Recombinant, Staining, Marker

Sensitivity and Specificity of scFvH5. Sensitivity and specificity of scFvH5 were verified by ELISA using 0.1 µg/mL of scFvH5. In addition to the coated recombinant adiponectin, protein Cq1 and human insulin were used for determining the specificity, while the different amounts of the coated proteins were used for determining the sensitivity of scFvH5. The ELISA results are given as mean ± SD values from triple determinations. Insulin was used as a general negative control.
Figure Legend Snippet: Sensitivity and Specificity of scFvH5. Sensitivity and specificity of scFvH5 were verified by ELISA using 0.1 µg/mL of scFvH5. In addition to the coated recombinant adiponectin, protein Cq1 and human insulin were used for determining the specificity, while the different amounts of the coated proteins were used for determining the sensitivity of scFvH5. The ELISA results are given as mean ± SD values from triple determinations. Insulin was used as a general negative control.

Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control

The purified scFvH5 can bind to globular and native adiponectin. In an ELISA, 100 µL of recombinant globular or native adiponectin (Biovendor) or C1q as negative control (1 µg/mL each) were coated on a microtiter plate and different concentrations of scFvH5 were used for antigen. The ELISA results are given as mean ± SD values from triple determinations.
Figure Legend Snippet: The purified scFvH5 can bind to globular and native adiponectin. In an ELISA, 100 µL of recombinant globular or native adiponectin (Biovendor) or C1q as negative control (1 µg/mL each) were coated on a microtiter plate and different concentrations of scFvH5 were used for antigen. The ELISA results are given as mean ± SD values from triple determinations.

Techniques Used: Purification, Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control

31) Product Images from "Identification of Trans-4-Hydroxy-L-Proline as a Compatible Solute and Its Biosynthesis and Molecular Characterization in Halobacillus halophilus"

Article Title: Identification of Trans-4-Hydroxy-L-Proline as a Compatible Solute and Its Biosynthesis and Molecular Characterization in Halobacillus halophilus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02054

Concentrations of major intracellular organic compounds in H. halophilus grown in marine broth with different NaCl concentrations as quantified by HPLC.
Figure Legend Snippet: Concentrations of major intracellular organic compounds in H. halophilus grown in marine broth with different NaCl concentrations as quantified by HPLC.

Techniques Used: High Performance Liquid Chromatography

Physical maps of the proline and Hyp biosynthetic gene clusters showing the open reading frames, promoters, and terminators (A) and the relative transcriptional expression of H. halophilus proH and PH-4 gene in the presence of different NaCl concentrations (B,C) and salts (D) . The putative functions of HBHAL_RS03960, HBHAL_RS03965, HBHAL_RS03970, and HBHAL_RS11735 were predicted to be pyrroline-5-carboxylate reductase (ProH), glutamate-5-kinase (ProJ), glutamate-5-semialdehyde dehydrogenase (ProA), and proline 4-hydroxylase (PH-4, this study), respectively. Cells grown in marine broth with different NaCl concentrations or salts were used in the transcriptional analysis, and their relative expression in the presence of different NaCl concentrations and salts were calculated based on expressional levels at 0.4 M and 1.0 M NaCl, respectively. The levels of malate dehydrogenase transcript (HBHAL_RS13485, mdh ) were used for normalization of total RNA templates.
Figure Legend Snippet: Physical maps of the proline and Hyp biosynthetic gene clusters showing the open reading frames, promoters, and terminators (A) and the relative transcriptional expression of H. halophilus proH and PH-4 gene in the presence of different NaCl concentrations (B,C) and salts (D) . The putative functions of HBHAL_RS03960, HBHAL_RS03965, HBHAL_RS03970, and HBHAL_RS11735 were predicted to be pyrroline-5-carboxylate reductase (ProH), glutamate-5-kinase (ProJ), glutamate-5-semialdehyde dehydrogenase (ProA), and proline 4-hydroxylase (PH-4, this study), respectively. Cells grown in marine broth with different NaCl concentrations or salts were used in the transcriptional analysis, and their relative expression in the presence of different NaCl concentrations and salts were calculated based on expressional levels at 0.4 M and 1.0 M NaCl, respectively. The levels of malate dehydrogenase transcript (HBHAL_RS13485, mdh ) were used for normalization of total RNA templates.

Techniques Used: Expressing

1 H-NMR spectra of extracts from H. halophilus cells grown in marine broth containing 0.4 (A) , 1.0 (B) , and 2.5 M (C) NaCl. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS; 5 mM) was used as the internal standard. Intracellular organic compounds from the 1 H-NMR spectra were identified with the Chenomx NMR suite. 1 H-NMR peaks corresponding to glutamate (Glu), glutamine (Gln), glycine betaine (GB), proline (Pro), glycine (Gly), and trans-4-hydroxy- L-proline (Hyp) are indicated. Unidentified intracellular organic compounds (Un) that responded to changes in NaCl concentrations are also shown.
Figure Legend Snippet: 1 H-NMR spectra of extracts from H. halophilus cells grown in marine broth containing 0.4 (A) , 1.0 (B) , and 2.5 M (C) NaCl. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS; 5 mM) was used as the internal standard. Intracellular organic compounds from the 1 H-NMR spectra were identified with the Chenomx NMR suite. 1 H-NMR peaks corresponding to glutamate (Glu), glutamine (Gln), glycine betaine (GB), proline (Pro), glycine (Gly), and trans-4-hydroxy- L-proline (Hyp) are indicated. Unidentified intracellular organic compounds (Un) that responded to changes in NaCl concentrations are also shown.

Techniques Used: Nuclear Magnetic Resonance

Expressional analysis of HBHAL_RS11735 using a pET28a construct in E. coli BL21 (DE3) cells (A) and PH-4 activity assay of the overexpressed gene product (B) . (A) A pET28a-HBHAL_RS11735 construct was overexpressed in E. coli BL21 (DE3) cells, and crude protein extracts of these cells were separated by SDS-PAGE. lane M, protein size marker; lane 1, total protein from uninduced cells carrying the HBHAL_RS11735 gene; lane 2, total protein from induced cells carrying the HBHAL_RS11735 gene; lanes 3, soluble protein from induced cells carrying the HBHAL_RS11735 gene. (B) PH-4 activity assay of soluble protein from E. coli cells overexpressing the HBHAL_RS11735 gene. PH-4 activity is expressed as enzyme units per mg·wet cell weight, and cells of E. coli BL21 (DE3) without the HBHAL_RS11735 gene and H. halophilus DSM 2266 cells were used as negative and positive controls, respectively.
Figure Legend Snippet: Expressional analysis of HBHAL_RS11735 using a pET28a construct in E. coli BL21 (DE3) cells (A) and PH-4 activity assay of the overexpressed gene product (B) . (A) A pET28a-HBHAL_RS11735 construct was overexpressed in E. coli BL21 (DE3) cells, and crude protein extracts of these cells were separated by SDS-PAGE. lane M, protein size marker; lane 1, total protein from uninduced cells carrying the HBHAL_RS11735 gene; lane 2, total protein from induced cells carrying the HBHAL_RS11735 gene; lanes 3, soluble protein from induced cells carrying the HBHAL_RS11735 gene. (B) PH-4 activity assay of soluble protein from E. coli cells overexpressing the HBHAL_RS11735 gene. PH-4 activity is expressed as enzyme units per mg·wet cell weight, and cells of E. coli BL21 (DE3) without the HBHAL_RS11735 gene and H. halophilus DSM 2266 cells were used as negative and positive controls, respectively.

Techniques Used: Construct, Activity Assay, SDS Page, Marker

32) Product Images from "Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency"

Article Title: Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

Journal: Nature Communications

doi: 10.1038/ncomms8134

Fabrication and characterization of linearly opened GFP oligomers. ( a ) Schematic representation of the construction of linearly opened GFP oligomers. CapGFP is designed to contain the GFP 11 strand connected to the N terminus of full-length GFP (1–11) with a His tag. Both CapGFP and GFP monomer (without His tag) were co-expressed in cells and opened oligomers were purified by His-affinity purifications. ( b ) Native PAGE analysis of open and circular forms of GFP oligomers. ( c ) Analysis of discrete opened oligomers from dimer to pentadecamer by native PAGE. ( d ) In-vitro assemblies of opened GFP oligomers with the GFP 1–10 fragment. Linearly opened GFP trimer, tetramer and pentamer were reacted with excess GFP 1–10 and resulting protein assemblies were analysed in a native PAGE gel. ( e ) TEM images of opened GFP trimer and tetramer. ( f ) TEM images and schematic drawing of MBP-displayed opened GFP trimer. A possible protein arrangement in a representative TEM image (a copy of the first image) was suggested with dotted yellow circles. Scale bars, 10 nm.
Figure Legend Snippet: Fabrication and characterization of linearly opened GFP oligomers. ( a ) Schematic representation of the construction of linearly opened GFP oligomers. CapGFP is designed to contain the GFP 11 strand connected to the N terminus of full-length GFP (1–11) with a His tag. Both CapGFP and GFP monomer (without His tag) were co-expressed in cells and opened oligomers were purified by His-affinity purifications. ( b ) Native PAGE analysis of open and circular forms of GFP oligomers. ( c ) Analysis of discrete opened oligomers from dimer to pentadecamer by native PAGE. ( d ) In-vitro assemblies of opened GFP oligomers with the GFP 1–10 fragment. Linearly opened GFP trimer, tetramer and pentamer were reacted with excess GFP 1–10 and resulting protein assemblies were analysed in a native PAGE gel. ( e ) TEM images of opened GFP trimer and tetramer. ( f ) TEM images and schematic drawing of MBP-displayed opened GFP trimer. A possible protein arrangement in a representative TEM image (a copy of the first image) was suggested with dotted yellow circles. Scale bars, 10 nm.

Techniques Used: Purification, Clear Native PAGE, In Vitro, Transmission Electron Microscopy

33) Product Images from "Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens"

Article Title: Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

Journal: Nature

doi: 10.1038/s41586-018-0653-6

Unrooted maximum likelihood phylogeny of representative NrdF (RNR subclass Ib radical-generating subunit) sequences. All RefSeq NrdF sequences were clustered at 75% identity to reduce redundancy and a maximum likelihood phylogeny was estimated. Sequences with non-canonical amino acids in the positions involved in coordinating the metal center of the enzyme formed a well-supported clan in the NrdF2 group of sequences. We identified two variants, one in which three of the glutamates were replaced by Gln, Ser, and Lys (NrdF2.QSK) and the other in which they were replaced by Val, Pro and Lys (NrdF2.VPK). Both variants thus have a substitution of a Lys for the normally metal-bridging Glu (residue 213 in M. florum NrdF2.VPK). Together, the two variants form a well-supported (96% bootstrap support) clan in the phylogeny inside the NrdF2 diversity. The NrdF2.VPK clan appears to be derived from the NrdF2.QSK clan. Behind the sequences in the tree are a set of sequences more than 75% identical to each represented sequence. The VPK and QSK sequences in the phylogeny represent 138 and 182 sequences in RefSeq respectively.
Figure Legend Snippet: Unrooted maximum likelihood phylogeny of representative NrdF (RNR subclass Ib radical-generating subunit) sequences. All RefSeq NrdF sequences were clustered at 75% identity to reduce redundancy and a maximum likelihood phylogeny was estimated. Sequences with non-canonical amino acids in the positions involved in coordinating the metal center of the enzyme formed a well-supported clan in the NrdF2 group of sequences. We identified two variants, one in which three of the glutamates were replaced by Gln, Ser, and Lys (NrdF2.QSK) and the other in which they were replaced by Val, Pro and Lys (NrdF2.VPK). Both variants thus have a substitution of a Lys for the normally metal-bridging Glu (residue 213 in M. florum NrdF2.VPK). Together, the two variants form a well-supported (96% bootstrap support) clan in the phylogeny inside the NrdF2 diversity. The NrdF2.VPK clan appears to be derived from the NrdF2.QSK clan. Behind the sequences in the tree are a set of sequences more than 75% identical to each represented sequence. The VPK and QSK sequences in the phylogeny represent 138 and 182 sequences in RefSeq respectively.

Techniques Used: Derivative Assay, Sequencing

34) Product Images from "Inhibitors of Trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease"

Article Title: Inhibitors of Trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0006180

TcSir2rp1 structural model and docking with compound 9. A ) The 3.5 Å structural model shows a large Rossmann-fold domain (composed of 6 parallel β-strands, sandwiched between 2 layers of α-helices), and a small zinc binding domain. The substrate acetylated peptide p53 is bound to the cleft between the small and the large domains. B-C ) The substrate p53 peptide was removed from the structure and docking studies conducted with compound 9 . Several conformations (only 2 shown here) of compound 9 were possible in a putative ligand binding site close to the NAD binding site.
Figure Legend Snippet: TcSir2rp1 structural model and docking with compound 9. A ) The 3.5 Å structural model shows a large Rossmann-fold domain (composed of 6 parallel β-strands, sandwiched between 2 layers of α-helices), and a small zinc binding domain. The substrate acetylated peptide p53 is bound to the cleft between the small and the large domains. B-C ) The substrate p53 peptide was removed from the structure and docking studies conducted with compound 9 . Several conformations (only 2 shown here) of compound 9 were possible in a putative ligand binding site close to the NAD binding site.

Techniques Used: Binding Assay, Ligand Binding Assay

35) Product Images from "Nonnucleoside Reverse Transcriptase Inhibitor-Resistant HIV Is Stimulated by Efavirenz during Early Stages of Infection ▿"

Article Title: Nonnucleoside Reverse Transcriptase Inhibitor-Resistant HIV Is Stimulated by Efavirenz during Early Stages of Infection ▿

Journal: Journal of Virology

doi: 10.1128/JVI.05116-11

Effects of EFV on RT content of wild-type and mutant virions. WT or mutant (L74V+K101E+G190S [LES], K101E+G190S [ES], D10, and D10MMTT) DNA was used to transfect 293 cells in the presence or absence of 400 or 800 nM EFV. The pCMS-EGFP plasmid was cotransfected
Figure Legend Snippet: Effects of EFV on RT content of wild-type and mutant virions. WT or mutant (L74V+K101E+G190S [LES], K101E+G190S [ES], D10, and D10MMTT) DNA was used to transfect 293 cells in the presence or absence of 400 or 800 nM EFV. The pCMS-EGFP plasmid was cotransfected

Techniques Used: Mutagenesis, Plasmid Preparation

Effects of NVP and ETR on the stimulation of K101E+G190S genotypes. (A and B) NL4-3XXES, NL4-3XXD10, and NL4-3XXD10MMTT were made by transient transfection in 293 or 293T cells in the absence of NVP or ETR. PM1 T cells were infected for 6 days in the
Figure Legend Snippet: Effects of NVP and ETR on the stimulation of K101E+G190S genotypes. (A and B) NL4-3XXES, NL4-3XXD10, and NL4-3XXD10MMTT were made by transient transfection in 293 or 293T cells in the absence of NVP or ETR. PM1 T cells were infected for 6 days in the

Techniques Used: Transfection, Infection

Effects of EFV on the heterodimerization of WT and mutant RT subunits in vitro . The 6×His-tagged p51 (His-p51) and untagged p66 were expressed separately in E. coli for wild-type or mutant RT (G190S, G190S+K101E [ES], and L74V+K101E+G190S [LES]).
Figure Legend Snippet: Effects of EFV on the heterodimerization of WT and mutant RT subunits in vitro . The 6×His-tagged p51 (His-p51) and untagged p66 were expressed separately in E. coli for wild-type or mutant RT (G190S, G190S+K101E [ES], and L74V+K101E+G190S [LES]).

Techniques Used: Mutagenesis, In Vitro

36) Product Images from "Insight into the PrPC - > PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants"

Article Title: Insight into the PrPC - > PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants

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

doi: 10.1073/pnas.0400014101

Structural consequences of scrapie-sensitivity-related mutations. The blue grid corresponds to the 1.0-σ level contour of the 2Fo – Fc omit map. Maps are calculated by using data in the 15.0- to 2.5-Å-resolution range for the ARQ and VRQ variants and the 15.0- to 2.8-Å-resolution range for the ARR variant. Hydrogen bonds are displayed as dashed lines, and distances of the atoms involved are reported. ( Upper ) A136V. The Fo VRQ – Fo ARQ difference map, represented as red and green grids (contour levels: –5.0 σ and 5.0 σ, respectively) is superimposed on the ARQ 2Fo – Fc omit map. ( Lower ) Q171R.
Figure Legend Snippet: Structural consequences of scrapie-sensitivity-related mutations. The blue grid corresponds to the 1.0-σ level contour of the 2Fo – Fc omit map. Maps are calculated by using data in the 15.0- to 2.5-Å-resolution range for the ARQ and VRQ variants and the 15.0- to 2.8-Å-resolution range for the ARR variant. Hydrogen bonds are displayed as dashed lines, and distances of the atoms involved are reported. ( Upper ) A136V. The Fo VRQ – Fo ARQ difference map, represented as red and green grids (contour levels: –5.0 σ and 5.0 σ, respectively) is superimposed on the ARQ 2Fo – Fc omit map. ( Lower ) Q171R.

Techniques Used: Variant Assay

37) Product Images from "ZMYND11 links histone H3.3 K36 trimethylation to transcription elongation and tumor suppression"

Article Title: ZMYND11 links histone H3.3 K36 trimethylation to transcription elongation and tumor suppression

Journal: Nature

doi: 10.1038/nature13045

Comparison of ZMYND11 PWWP with other Kme3-binding PWWP domains a-b , Stereo view of ZMYND11 PWWP domain in superimposition with H3K36me3-bound BRPF1 PWWP (PDB ID: 2X4W) ( a ) and H3K79me3-bound HDGF PWWP (PDB ID: 3QJ6) ( b ). c , Sequence alignment of Kme3-binding PWWP domains. Conserved residues are in blue box; identical residues are shaded in red. Underscored dots: residues forming the aromatic cage. Sequence alignment was produced using the ESPript. d , Western analysis of histone peptide pulldowns with indicated point mutants in the context of Flag-tagged full-length ZMYND11 and biotinylated peptides. e , Western blot analysis of the protein-ChIP assays in cells expressing Flag-tagged full-length ZMYND11 or the indicated mutants. f , Point mutations in ZMYND11 bromo-ZnF-PWWP domains do not affect protein folding. Circular dichroism (CD) spectroscopy analysis of the WT ZMYND11 BP domains and indicated mutants used in this study. g , Steady-state analysis of the biolayer interferometry (BLI) sensorgrams of ZMYND11 BP binding to unmethylated (black square) and fully methylated (blue triangle) 22-mer duplex DNA derived from the Widom 601 sequence. Unmethylated duplex DNA: 5’-CAGCTGAACATCGCTTTTGATG-3’; fully methylated duplex DNA: 5’-CAGCTGAACAT[5medC]GCTTTTGATG-3’.
Figure Legend Snippet: Comparison of ZMYND11 PWWP with other Kme3-binding PWWP domains a-b , Stereo view of ZMYND11 PWWP domain in superimposition with H3K36me3-bound BRPF1 PWWP (PDB ID: 2X4W) ( a ) and H3K79me3-bound HDGF PWWP (PDB ID: 3QJ6) ( b ). c , Sequence alignment of Kme3-binding PWWP domains. Conserved residues are in blue box; identical residues are shaded in red. Underscored dots: residues forming the aromatic cage. Sequence alignment was produced using the ESPript. d , Western analysis of histone peptide pulldowns with indicated point mutants in the context of Flag-tagged full-length ZMYND11 and biotinylated peptides. e , Western blot analysis of the protein-ChIP assays in cells expressing Flag-tagged full-length ZMYND11 or the indicated mutants. f , Point mutations in ZMYND11 bromo-ZnF-PWWP domains do not affect protein folding. Circular dichroism (CD) spectroscopy analysis of the WT ZMYND11 BP domains and indicated mutants used in this study. g , Steady-state analysis of the biolayer interferometry (BLI) sensorgrams of ZMYND11 BP binding to unmethylated (black square) and fully methylated (blue triangle) 22-mer duplex DNA derived from the Widom 601 sequence. Unmethylated duplex DNA: 5’-CAGCTGAACATCGCTTTTGATG-3’; fully methylated duplex DNA: 5’-CAGCTGAACAT[5medC]GCTTTTGATG-3’.

Techniques Used: Binding Assay, Sequencing, Produced, Western Blot, Chromatin Immunoprecipitation, Expressing, Spectroscopy, Methylation, Derivative Assay

Crystal structures of ZMYND11 BP domains a , Sequence and structure-based secondary structural assignment of the ZMYND11 tandem Bromo-PWWP (BP) domains. Dashed box (magenta) and dashed line (black): unmodeled sequence in the free and complex structures, respectively; Cyan shading: Basic residues within ZA loop (LZA); Magenta shading: residues corresponding to canonical acetyl-lysine recognition motif; Black box: residues mutated to facilitate co-crystal formation. Black dot: aromatic caging residues; Aster: H3 hydrogen bonding residues with magenta unique to H3.3 variant. b , Burial of the K36me3-binding aromatic cage by an adjacent bromodomain during crystal packing. Two acidic residues, D234 and E236, contribute to such packing contacts through electrostatic interaction with the positive surface patch (blue) of PWWP. c , An overall view of superimposed free and H3K36me3 peptide-bound ZMYND11 bromo-PWWP double mutant. d , Western blot analysis of histone peptide pulldowns with WT ZMYND11 PBP and the PBP-D234A/E236A mutant. e . ITC curves of the histone H3.3K36me3 peptide titrated into ZMYND11 BP-D234A/E236A mutant. f . Solvent accessible surface representations of ZYMND11 bromo-ZnF-PWWP in its free state. Note the tight integration of the paired modules. g , Ribbon view of bromo-ZnF-PWWP with basic lysine/arginine clusters highlighted as yellow sticks. h , Superimposition of ZMYND11 bromodomain with H4K16ac-bound BPTF bromodomain (PDB ID: 3QZT). i , Surface representation of BPTF bound to H4K16ac and its comparison with ZMYND11 bromodomain. The Kac pocket is missing in ZMYND11 bromo due to the occurrence of Y231. Note the positive residue clusters around the peptide binding surface of ZMYND11 bromo (right). j , Zinc coordination sphere of the newly identified ZnF motif of ZMYND11. Note the burial of non-zinc-coordinating C274 at the hydrophobic interface between ZnF (salmon) and PWWP (blue) of ZMYND11. k , Encapsulation of ZnF (salmon) by bromo (green) and PWWP (blue) in stereo view. Dashed line denotes hydrogen bonding or zinc coordinating interactions. Note the burial of hydrophobic residues including F262, L264, F273, C274 and Y275 from ZnF at the bromo-ZnF and ZnF-PWWP interfaces. l , Structural alignment of ZMYND11 ZnF-PWWP (salmon and blue) with Pdp1 PWWP (PDB ID: 2L89) (magenta) and BRPF1 PWWP (PDB ID: 2X4W) (cyan) showing the structural overlaps of ZMYND11 ZnF with Pdp1 α3 and BRPF1 β2-β3 insertion.
Figure Legend Snippet: Crystal structures of ZMYND11 BP domains a , Sequence and structure-based secondary structural assignment of the ZMYND11 tandem Bromo-PWWP (BP) domains. Dashed box (magenta) and dashed line (black): unmodeled sequence in the free and complex structures, respectively; Cyan shading: Basic residues within ZA loop (LZA); Magenta shading: residues corresponding to canonical acetyl-lysine recognition motif; Black box: residues mutated to facilitate co-crystal formation. Black dot: aromatic caging residues; Aster: H3 hydrogen bonding residues with magenta unique to H3.3 variant. b , Burial of the K36me3-binding aromatic cage by an adjacent bromodomain during crystal packing. Two acidic residues, D234 and E236, contribute to such packing contacts through electrostatic interaction with the positive surface patch (blue) of PWWP. c , An overall view of superimposed free and H3K36me3 peptide-bound ZMYND11 bromo-PWWP double mutant. d , Western blot analysis of histone peptide pulldowns with WT ZMYND11 PBP and the PBP-D234A/E236A mutant. e . ITC curves of the histone H3.3K36me3 peptide titrated into ZMYND11 BP-D234A/E236A mutant. f . Solvent accessible surface representations of ZYMND11 bromo-ZnF-PWWP in its free state. Note the tight integration of the paired modules. g , Ribbon view of bromo-ZnF-PWWP with basic lysine/arginine clusters highlighted as yellow sticks. h , Superimposition of ZMYND11 bromodomain with H4K16ac-bound BPTF bromodomain (PDB ID: 3QZT). i , Surface representation of BPTF bound to H4K16ac and its comparison with ZMYND11 bromodomain. The Kac pocket is missing in ZMYND11 bromo due to the occurrence of Y231. Note the positive residue clusters around the peptide binding surface of ZMYND11 bromo (right). j , Zinc coordination sphere of the newly identified ZnF motif of ZMYND11. Note the burial of non-zinc-coordinating C274 at the hydrophobic interface between ZnF (salmon) and PWWP (blue) of ZMYND11. k , Encapsulation of ZnF (salmon) by bromo (green) and PWWP (blue) in stereo view. Dashed line denotes hydrogen bonding or zinc coordinating interactions. Note the burial of hydrophobic residues including F262, L264, F273, C274 and Y275 from ZnF at the bromo-ZnF and ZnF-PWWP interfaces. l , Structural alignment of ZMYND11 ZnF-PWWP (salmon and blue) with Pdp1 PWWP (PDB ID: 2L89) (magenta) and BRPF1 PWWP (PDB ID: 2X4W) (cyan) showing the structural overlaps of ZMYND11 ZnF with Pdp1 α3 and BRPF1 β2-β3 insertion.

Techniques Used: Sequencing, Variant Assay, Binding Assay, Mutagenesis, Western Blot

Crystal structure of ZMYND11 BP in complex with H3.1K36me3 and its comparison with the H3.3K36me3-bound complex a . ITC curves of H3.3K36me3 or H3.1K36me3 peptides titrated into ZMYND11 BP and PBP domains. Titration c -values are 1.41 for BP-H3.3, 0.15 for BP-H3.1, 1.26 for PBP-H3.3 and 0.16 for PBP-H3.1, respectively. The “n” value was fixed at 1 for curve fitting. b , Western blot analysis of peptide pulldowns under stringent binding condition. c . Western blot analysis of Flag IP in cells co-expressing Flag-H3.3 or H3.1 and Myc-ZMYND11. d , Structure of ZMYND11-BP in complex with H3.1K36me3 peptide. BP is in surface representation with bromo, ZnF and PWWP colored green, salmon, and blue respectively. 2Fo-Fc omit map around H3 peptide, Polyethylene glycol (PEG) and phosphate (PO4) are shown as cyan mesh contoured at 1σ level. e and f , Simulated annealing Fo-Fc omit map countered at 2.5σ level around the histone segments containing H3.1Ala31 ( e ) or H3.3Ser31( f ) in complex with ZMYND11 BP. Residues R168, H250, E251, E254, N266, R268, R309, R317 of BP, a bridging water (Wat), and segment “Ala29-Val35” of histone H3.1 or H3.3 peptides were omitted for simulated annealing (starting temperature 2500 K and 500 cooling steps) map calculation by the program Phenix. Magenta dashes, hydrogen bonds. Note that the Nε atom of R268 side chain ( e ) and side chains of R168 ( e, f ) displayed poor densities, suggesting their conformational flexibility. g , Western analysis of histone peptide pulldowns with indicated point mutants and biotinylated peptides. h , Structural alignment of ZMYND11 BP-H3.3K36me3 (blue), ZMYND11 BP-H3.1K36me3 (salmon), and BRPF1 PWWP-H3.1K36me3 (PDB ID: 2X4W)(yellow). i , Structural alignment of H3K36me3-bound ZMYND11 PWWP (blue), PHF19 Tudor (PDB ID: 4BD3) (red) and PHF1 Tudor (PDB ID: 4HCZ) (cyan). Both BP and H3 peptides are presented as backbone coils, with key residues depicted as stick.
Figure Legend Snippet: Crystal structure of ZMYND11 BP in complex with H3.1K36me3 and its comparison with the H3.3K36me3-bound complex a . ITC curves of H3.3K36me3 or H3.1K36me3 peptides titrated into ZMYND11 BP and PBP domains. Titration c -values are 1.41 for BP-H3.3, 0.15 for BP-H3.1, 1.26 for PBP-H3.3 and 0.16 for PBP-H3.1, respectively. The “n” value was fixed at 1 for curve fitting. b , Western blot analysis of peptide pulldowns under stringent binding condition. c . Western blot analysis of Flag IP in cells co-expressing Flag-H3.3 or H3.1 and Myc-ZMYND11. d , Structure of ZMYND11-BP in complex with H3.1K36me3 peptide. BP is in surface representation with bromo, ZnF and PWWP colored green, salmon, and blue respectively. 2Fo-Fc omit map around H3 peptide, Polyethylene glycol (PEG) and phosphate (PO4) are shown as cyan mesh contoured at 1σ level. e and f , Simulated annealing Fo-Fc omit map countered at 2.5σ level around the histone segments containing H3.1Ala31 ( e ) or H3.3Ser31( f ) in complex with ZMYND11 BP. Residues R168, H250, E251, E254, N266, R268, R309, R317 of BP, a bridging water (Wat), and segment “Ala29-Val35” of histone H3.1 or H3.3 peptides were omitted for simulated annealing (starting temperature 2500 K and 500 cooling steps) map calculation by the program Phenix. Magenta dashes, hydrogen bonds. Note that the Nε atom of R268 side chain ( e ) and side chains of R168 ( e, f ) displayed poor densities, suggesting their conformational flexibility. g , Western analysis of histone peptide pulldowns with indicated point mutants and biotinylated peptides. h , Structural alignment of ZMYND11 BP-H3.3K36me3 (blue), ZMYND11 BP-H3.1K36me3 (salmon), and BRPF1 PWWP-H3.1K36me3 (PDB ID: 2X4W)(yellow). i , Structural alignment of H3K36me3-bound ZMYND11 PWWP (blue), PHF19 Tudor (PDB ID: 4BD3) (red) and PHF1 Tudor (PDB ID: 4HCZ) (cyan). Both BP and H3 peptides are presented as backbone coils, with key residues depicted as stick.

Techniques Used: Titration, Western Blot, Binding Assay, Expressing

Molecular basis for H3.3K36me3 recognition by ZMYND11 bromo-PWWP domains
Figure Legend Snippet: Molecular basis for H3.3K36me3 recognition by ZMYND11 bromo-PWWP domains

Techniques Used:

38) Product Images from "Structure of GUN4 from Chlamydomonas reinhardtii"

Article Title: Structure of GUN4 from Chlamydomonas reinhardtii

Journal: Acta Crystallographica. Section F, Structural Biology Communications

doi: 10.1107/S2053230X15012248

Crystal structure of GUN4 from C. reinhardtii . ( a ) Three different perspectives are shown. The α2/α3 and α6/α7 loops are coloured pink and orange, respectively. The side chains of Pro216 and Thr218 within the α6/α7
Figure Legend Snippet: Crystal structure of GUN4 from C. reinhardtii . ( a ) Three different perspectives are shown. The α2/α3 and α6/α7 loops are coloured pink and orange, respectively. The side chains of Pro216 and Thr218 within the α6/α7

Techniques Used:

Porphyrin-binding cleft of GUN4 from C. reinhardtii . GUN4 has been trimmed of the α2/α3 and α6/α7 loops to reveal the porphyrin-binding cleft. Both a front-on (left) and a side view (right) are presented. The side chains
Figure Legend Snippet: Porphyrin-binding cleft of GUN4 from C. reinhardtii . GUN4 has been trimmed of the α2/α3 and α6/α7 loops to reveal the porphyrin-binding cleft. Both a front-on (left) and a side view (right) are presented. The side chains

Techniques Used: Binding Assay

Sequence alignment of GUN4 performed with ClustalW 2. Highlighted in yellow is the portion of C. reinhardtii GUN4 that is resolvable in the crystal structure. The underlined region shows peptide coverage from MSMS proteomic analysis of the crystals. Residues
Figure Legend Snippet: Sequence alignment of GUN4 performed with ClustalW 2. Highlighted in yellow is the portion of C. reinhardtii GUN4 that is resolvable in the crystal structure. The underlined region shows peptide coverage from MSMS proteomic analysis of the crystals. Residues

Techniques Used: Sequencing

39) Product Images from "Interaction of the Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall Polymer of Geobacillus stearothermophilus PV72 Assessed by Real-Time Surface Plasmon Resonance Biosensor Technology"

Article Title: Interaction of the Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall Polymer of Geobacillus stearothermophilus PV72 Assessed by Real-Time Surface Plasmon Resonance Biosensor Technology

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.6.1758-1768.2004

Dependence of the free-to-total SCWP ratio on the amount of 3SLH (squares) and rSbsB (triangles) added in equilibrated mixtures containing the glycan at a constant 1 mg/liter. The fraction of free SCWP in the solutions was calculated from the initial rates measured for its binding to a sensor surface with immobilized rSbsB. Data represent the mean ± SD of duplicate measurements.
Figure Legend Snippet: Dependence of the free-to-total SCWP ratio on the amount of 3SLH (squares) and rSbsB (triangles) added in equilibrated mixtures containing the glycan at a constant 1 mg/liter. The fraction of free SCWP in the solutions was calculated from the initial rates measured for its binding to a sensor surface with immobilized rSbsB. Data represent the mean ± SD of duplicate measurements.

Techniques Used: Binding Assay

SDS-PAGE analysis of SDS extracts from whole cells of E. coli BL21(DE3) before (lane a) and 5 h after (lanes b and d) induction of expression of the genes encoding rSbsB (lane b) and Δ3SLH-SbsB (lane d). In lanes f and g, SDS extracts from whole cells of E. coli HMS174(DE3) before (lane f) and 5 h after (lane g) induction of expression of the gene encoding 3SLH are shown. Lanes c, e, and h show purified rSbsB (c), Δ3SLH-SbsB (e), and 3SLH (h).
Figure Legend Snippet: SDS-PAGE analysis of SDS extracts from whole cells of E. coli BL21(DE3) before (lane a) and 5 h after (lanes b and d) induction of expression of the genes encoding rSbsB (lane b) and Δ3SLH-SbsB (lane d). In lanes f and g, SDS extracts from whole cells of E. coli HMS174(DE3) before (lane f) and 5 h after (lane g) induction of expression of the gene encoding 3SLH are shown. Lanes c, e, and h show purified rSbsB (c), Δ3SLH-SbsB (e), and 3SLH (h).

Techniques Used: SDS Page, Expressing, Purification

Dependence of the free-to-total 3SLH (squares) and rSbsB (triangles) ratios on the amount of SCWP added in equilibrated mixtures containing the corresponding sample at a constant 3 nM. The fraction of free protein in the solutions was calculated from the initial rates measured for its binding to a sensor surface with immobilized SCWP. Data represent the mean ± SD of duplicate measurements.
Figure Legend Snippet: Dependence of the free-to-total 3SLH (squares) and rSbsB (triangles) ratios on the amount of SCWP added in equilibrated mixtures containing the corresponding sample at a constant 3 nM. The fraction of free protein in the solutions was calculated from the initial rates measured for its binding to a sensor surface with immobilized SCWP. Data represent the mean ± SD of duplicate measurements.

Techniques Used: Binding Assay

40) Product Images from "Interaction of the Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall Polymer of Geobacillus stearothermophilus PV72 Assessed by Real-Time Surface Plasmon Resonance Biosensor Technology"

Article Title: Interaction of the Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall Polymer of Geobacillus stearothermophilus PV72 Assessed by Real-Time Surface Plasmon Resonance Biosensor Technology

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.6.1758-1768.2004

Dependence of the free-to-total SCWP ratio on the amount of 3SLH (squares) and rSbsB (triangles) added in equilibrated mixtures containing the glycan at a constant 1 mg/liter. The fraction of free SCWP in the solutions was calculated from the initial rates measured for its binding to a sensor surface with immobilized rSbsB. Data represent the mean ± SD of duplicate measurements.
Figure Legend Snippet: Dependence of the free-to-total SCWP ratio on the amount of 3SLH (squares) and rSbsB (triangles) added in equilibrated mixtures containing the glycan at a constant 1 mg/liter. The fraction of free SCWP in the solutions was calculated from the initial rates measured for its binding to a sensor surface with immobilized rSbsB. Data represent the mean ± SD of duplicate measurements.

Techniques Used: Binding Assay

SDS-PAGE analysis of SDS extracts from whole cells of E. coli BL21(DE3) before (lane a) and 5 h after (lanes b and d) induction of expression of the genes encoding rSbsB (lane b) and Δ3SLH-SbsB (lane d). In lanes f and g, SDS extracts from whole cells of E. coli HMS174(DE3) before (lane f) and 5 h after (lane g) induction of expression of the gene encoding 3SLH are shown. Lanes c, e, and h show purified rSbsB (c), Δ3SLH-SbsB (e), and 3SLH (h).
Figure Legend Snippet: SDS-PAGE analysis of SDS extracts from whole cells of E. coli BL21(DE3) before (lane a) and 5 h after (lanes b and d) induction of expression of the genes encoding rSbsB (lane b) and Δ3SLH-SbsB (lane d). In lanes f and g, SDS extracts from whole cells of E. coli HMS174(DE3) before (lane f) and 5 h after (lane g) induction of expression of the gene encoding 3SLH are shown. Lanes c, e, and h show purified rSbsB (c), Δ3SLH-SbsB (e), and 3SLH (h).

Techniques Used: SDS Page, Expressing, Purification

Dependence of the free-to-total 3SLH (squares) and rSbsB (triangles) ratios on the amount of SCWP added in equilibrated mixtures containing the corresponding sample at a constant 3 nM. The fraction of free protein in the solutions was calculated from the initial rates measured for its binding to a sensor surface with immobilized SCWP. Data represent the mean ± SD of duplicate measurements.
Figure Legend Snippet: Dependence of the free-to-total 3SLH (squares) and rSbsB (triangles) ratios on the amount of SCWP added in equilibrated mixtures containing the corresponding sample at a constant 3 nM. The fraction of free protein in the solutions was calculated from the initial rates measured for its binding to a sensor surface with immobilized SCWP. Data represent the mean ± SD of duplicate measurements.

Techniques Used: Binding Assay

Related Articles

Clone Assay:

Article Title: Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons
Article Snippet: .. Purification of murine recombinant hnRNP R and SMN protein His-tagged hnRNP R and SMN full length proteins were expressed in E. coli after cloning the corresponding cDNA constructs into the pET-28a and pET-32a vector system (Novagen, Madison, WI), respectively. ..

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus
Article Snippet: .. Expression of recombinant Csr proteins components and development of antisera Full-length CsrR and N-terminal truncated CsrS (CsrSΔ1-231) and were fused separately to a N-terminal His6 tag by cloning into overexpression vector pET-28a (Novagen) PCR-amplified DNA fragments obtained with primer pairs JL-48/JL-49 and HTW 37/46, respectively. .. Following overexpression by IPTG induction, recombinant proteins were affinity purified using Ni2+ -NTA resin (Qiagen) under native conditions (His6 -CsrR) or under denaturing conditions (His6 -CsrSΔ1-231) according to the manufacturer's protocol.

Article Title: Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)
Article Snippet: .. Purification of recombinant G3BP1 and USP10 The NTF2-like (residues 1–139) and RRM (residues 339–421) domains of hG3BP (NP-005745) were cloned into the pET-28a vector (Novagen, Madison, WI, USA). .. The NTF2-like domain of G3BP1 was expressed in Codon Plus (DE3) RIPL E.coli (Stratagene) and then harvested after 2–2.5 h growth at 37°C following induction with 1 mM isopropyl-1-thio-β-galactopyranoside (IPTG).

Article Title: Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants
Article Snippet: .. Enzyme assays For biochemical characterization of mature PhCM1 (minus predicted transit peptide) and PhCM2 as well as mature PhADT3L and PhADT3S, the coding region of the corresponding gene was amplified using forward and reverse primers (Supplementary Table ) containing Nde I and Bam HI restriction sites, respectively, for directional cloning into pET-28a expression vector (Novagen, Madison, WI) in-frame with an N-terminal 6XHis-tag. .. After sequence verification, recombinant proteins were produced in E. coli and purified on Ni-NTA resin (Qiagen, Hilden, Germany) .

Article Title: A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair *
Article Snippet: .. Expression and Purification of G. stearothermophilus UvrA and UvrB Interaction Domain Complex —The DNA sequences encoding the interaction domains ( ) were amplified from the plasmids containing the genes for full-length UvrA and UvrB , cloned into pET-28a (+) (Novagen; see ), and confirmed by sequencing. .. The UvrA and UvrB domain expression constructs contained residues 131–245 of UvrA and residues 149–250 of UvrB, respectively, with an N-terminal His6 tag and a thrombin cleavage site.

Positron Emission Tomography:

Article Title: The Self-Interaction of a Nodavirus Replicase Is Enhanced by Mitochondrial Membrane Lipids
Article Snippet: .. For in vitro translation, WhNV and FHV protein A ORF was inserted into pET-28a (Novagen, Germany ), respectively. .. Mutations were introduced into protein A ORF via PCR-mediated mutagenesis as described previously , .

Article Title: A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System
Article Snippet: .. PfMSRP-2 was expressed in E. coli BL-21 DE3 Codon Plus cells by using the pET-28a plasmid vector (Novagen). .. The recombinant protein was expressed as an amino-terminal fusion with a six-histidine tag.

Article Title: Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons
Article Snippet: .. Purification of murine recombinant hnRNP R and SMN protein His-tagged hnRNP R and SMN full length proteins were expressed in E. coli after cloning the corresponding cDNA constructs into the pET-28a and pET-32a vector system (Novagen, Madison, WI), respectively. ..

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus
Article Snippet: .. Expression of recombinant Csr proteins components and development of antisera Full-length CsrR and N-terminal truncated CsrS (CsrSΔ1-231) and were fused separately to a N-terminal His6 tag by cloning into overexpression vector pET-28a (Novagen) PCR-amplified DNA fragments obtained with primer pairs JL-48/JL-49 and HTW 37/46, respectively. .. Following overexpression by IPTG induction, recombinant proteins were affinity purified using Ni2+ -NTA resin (Qiagen) under native conditions (His6 -CsrR) or under denaturing conditions (His6 -CsrSΔ1-231) according to the manufacturer's protocol.

Article Title: Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)
Article Snippet: .. Purification of recombinant G3BP1 and USP10 The NTF2-like (residues 1–139) and RRM (residues 339–421) domains of hG3BP (NP-005745) were cloned into the pET-28a vector (Novagen, Madison, WI, USA). .. The NTF2-like domain of G3BP1 was expressed in Codon Plus (DE3) RIPL E.coli (Stratagene) and then harvested after 2–2.5 h growth at 37°C following induction with 1 mM isopropyl-1-thio-β-galactopyranoside (IPTG).

Article Title: Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment
Article Snippet: .. The generated KM10 scFv gene was then subcloned into a pET-28a vector (Novagen, Madison, WI) in frame with a (His)6 tag sequence to ensure purification. .. The affinity maturated K-11 scFv gene was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using KM10 scFv in pET-28a vector as a template.

Article Title: Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants
Article Snippet: .. Enzyme assays For biochemical characterization of mature PhCM1 (minus predicted transit peptide) and PhCM2 as well as mature PhADT3L and PhADT3S, the coding region of the corresponding gene was amplified using forward and reverse primers (Supplementary Table ) containing Nde I and Bam HI restriction sites, respectively, for directional cloning into pET-28a expression vector (Novagen, Madison, WI) in-frame with an N-terminal 6XHis-tag. .. After sequence verification, recombinant proteins were produced in E. coli and purified on Ni-NTA resin (Qiagen, Hilden, Germany) .

Article Title: A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair *
Article Snippet: .. Expression and Purification of G. stearothermophilus UvrA and UvrB Interaction Domain Complex —The DNA sequences encoding the interaction domains ( ) were amplified from the plasmids containing the genes for full-length UvrA and UvrB , cloned into pET-28a (+) (Novagen; see ), and confirmed by sequencing. .. The UvrA and UvrB domain expression constructs contained residues 131–245 of UvrA and residues 149–250 of UvrB, respectively, with an N-terminal His6 tag and a thrombin cleavage site.

In Vitro:

Article Title: The Self-Interaction of a Nodavirus Replicase Is Enhanced by Mitochondrial Membrane Lipids
Article Snippet: .. For in vitro translation, WhNV and FHV protein A ORF was inserted into pET-28a (Novagen, Germany ), respectively. .. Mutations were introduced into protein A ORF via PCR-mediated mutagenesis as described previously , .

Construct:

Article Title: Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons
Article Snippet: .. Purification of murine recombinant hnRNP R and SMN protein His-tagged hnRNP R and SMN full length proteins were expressed in E. coli after cloning the corresponding cDNA constructs into the pET-28a and pET-32a vector system (Novagen, Madison, WI), respectively. ..

Purification:

Article Title: Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons
Article Snippet: .. Purification of murine recombinant hnRNP R and SMN protein His-tagged hnRNP R and SMN full length proteins were expressed in E. coli after cloning the corresponding cDNA constructs into the pET-28a and pET-32a vector system (Novagen, Madison, WI), respectively. ..

Article Title: Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)
Article Snippet: .. Purification of recombinant G3BP1 and USP10 The NTF2-like (residues 1–139) and RRM (residues 339–421) domains of hG3BP (NP-005745) were cloned into the pET-28a vector (Novagen, Madison, WI, USA). .. The NTF2-like domain of G3BP1 was expressed in Codon Plus (DE3) RIPL E.coli (Stratagene) and then harvested after 2–2.5 h growth at 37°C following induction with 1 mM isopropyl-1-thio-β-galactopyranoside (IPTG).

Article Title: Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment
Article Snippet: .. The generated KM10 scFv gene was then subcloned into a pET-28a vector (Novagen, Madison, WI) in frame with a (His)6 tag sequence to ensure purification. .. The affinity maturated K-11 scFv gene was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using KM10 scFv in pET-28a vector as a template.

Article Title: A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair *
Article Snippet: .. Expression and Purification of G. stearothermophilus UvrA and UvrB Interaction Domain Complex —The DNA sequences encoding the interaction domains ( ) were amplified from the plasmids containing the genes for full-length UvrA and UvrB , cloned into pET-28a (+) (Novagen; see ), and confirmed by sequencing. .. The UvrA and UvrB domain expression constructs contained residues 131–245 of UvrA and residues 149–250 of UvrB, respectively, with an N-terminal His6 tag and a thrombin cleavage site.

Polymerase Chain Reaction:

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus
Article Snippet: .. Expression of recombinant Csr proteins components and development of antisera Full-length CsrR and N-terminal truncated CsrS (CsrSΔ1-231) and were fused separately to a N-terminal His6 tag by cloning into overexpression vector pET-28a (Novagen) PCR-amplified DNA fragments obtained with primer pairs JL-48/JL-49 and HTW 37/46, respectively. .. Following overexpression by IPTG induction, recombinant proteins were affinity purified using Ni2+ -NTA resin (Qiagen) under native conditions (His6 -CsrR) or under denaturing conditions (His6 -CsrSΔ1-231) according to the manufacturer's protocol.

Generated:

Article Title: Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment
Article Snippet: .. The generated KM10 scFv gene was then subcloned into a pET-28a vector (Novagen, Madison, WI) in frame with a (His)6 tag sequence to ensure purification. .. The affinity maturated K-11 scFv gene was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using KM10 scFv in pET-28a vector as a template.

Amplification:

Article Title: Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants
Article Snippet: .. Enzyme assays For biochemical characterization of mature PhCM1 (minus predicted transit peptide) and PhCM2 as well as mature PhADT3L and PhADT3S, the coding region of the corresponding gene was amplified using forward and reverse primers (Supplementary Table ) containing Nde I and Bam HI restriction sites, respectively, for directional cloning into pET-28a expression vector (Novagen, Madison, WI) in-frame with an N-terminal 6XHis-tag. .. After sequence verification, recombinant proteins were produced in E. coli and purified on Ni-NTA resin (Qiagen, Hilden, Germany) .

Article Title: A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair *
Article Snippet: .. Expression and Purification of G. stearothermophilus UvrA and UvrB Interaction Domain Complex —The DNA sequences encoding the interaction domains ( ) were amplified from the plasmids containing the genes for full-length UvrA and UvrB , cloned into pET-28a (+) (Novagen; see ), and confirmed by sequencing. .. The UvrA and UvrB domain expression constructs contained residues 131–245 of UvrA and residues 149–250 of UvrB, respectively, with an N-terminal His6 tag and a thrombin cleavage site.

Expressing:

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus
Article Snippet: .. Expression of recombinant Csr proteins components and development of antisera Full-length CsrR and N-terminal truncated CsrS (CsrSΔ1-231) and were fused separately to a N-terminal His6 tag by cloning into overexpression vector pET-28a (Novagen) PCR-amplified DNA fragments obtained with primer pairs JL-48/JL-49 and HTW 37/46, respectively. .. Following overexpression by IPTG induction, recombinant proteins were affinity purified using Ni2+ -NTA resin (Qiagen) under native conditions (His6 -CsrR) or under denaturing conditions (His6 -CsrSΔ1-231) according to the manufacturer's protocol.

Article Title: Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants
Article Snippet: .. Enzyme assays For biochemical characterization of mature PhCM1 (minus predicted transit peptide) and PhCM2 as well as mature PhADT3L and PhADT3S, the coding region of the corresponding gene was amplified using forward and reverse primers (Supplementary Table ) containing Nde I and Bam HI restriction sites, respectively, for directional cloning into pET-28a expression vector (Novagen, Madison, WI) in-frame with an N-terminal 6XHis-tag. .. After sequence verification, recombinant proteins were produced in E. coli and purified on Ni-NTA resin (Qiagen, Hilden, Germany) .

Article Title: A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair *
Article Snippet: .. Expression and Purification of G. stearothermophilus UvrA and UvrB Interaction Domain Complex —The DNA sequences encoding the interaction domains ( ) were amplified from the plasmids containing the genes for full-length UvrA and UvrB , cloned into pET-28a (+) (Novagen; see ), and confirmed by sequencing. .. The UvrA and UvrB domain expression constructs contained residues 131–245 of UvrA and residues 149–250 of UvrB, respectively, with an N-terminal His6 tag and a thrombin cleavage site.

Sequencing:

Article Title: Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment
Article Snippet: .. The generated KM10 scFv gene was then subcloned into a pET-28a vector (Novagen, Madison, WI) in frame with a (His)6 tag sequence to ensure purification. .. The affinity maturated K-11 scFv gene was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using KM10 scFv in pET-28a vector as a template.

Article Title: A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair *
Article Snippet: .. Expression and Purification of G. stearothermophilus UvrA and UvrB Interaction Domain Complex —The DNA sequences encoding the interaction domains ( ) were amplified from the plasmids containing the genes for full-length UvrA and UvrB , cloned into pET-28a (+) (Novagen; see ), and confirmed by sequencing. .. The UvrA and UvrB domain expression constructs contained residues 131–245 of UvrA and residues 149–250 of UvrB, respectively, with an N-terminal His6 tag and a thrombin cleavage site.

Recombinant:

Article Title: Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons
Article Snippet: .. Purification of murine recombinant hnRNP R and SMN protein His-tagged hnRNP R and SMN full length proteins were expressed in E. coli after cloning the corresponding cDNA constructs into the pET-28a and pET-32a vector system (Novagen, Madison, WI), respectively. ..

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus
Article Snippet: .. Expression of recombinant Csr proteins components and development of antisera Full-length CsrR and N-terminal truncated CsrS (CsrSΔ1-231) and were fused separately to a N-terminal His6 tag by cloning into overexpression vector pET-28a (Novagen) PCR-amplified DNA fragments obtained with primer pairs JL-48/JL-49 and HTW 37/46, respectively. .. Following overexpression by IPTG induction, recombinant proteins were affinity purified using Ni2+ -NTA resin (Qiagen) under native conditions (His6 -CsrR) or under denaturing conditions (His6 -CsrSΔ1-231) according to the manufacturer's protocol.

Article Title: Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)
Article Snippet: .. Purification of recombinant G3BP1 and USP10 The NTF2-like (residues 1–139) and RRM (residues 339–421) domains of hG3BP (NP-005745) were cloned into the pET-28a vector (Novagen, Madison, WI, USA). .. The NTF2-like domain of G3BP1 was expressed in Codon Plus (DE3) RIPL E.coli (Stratagene) and then harvested after 2–2.5 h growth at 37°C following induction with 1 mM isopropyl-1-thio-β-galactopyranoside (IPTG).

Over Expression:

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus
Article Snippet: .. Expression of recombinant Csr proteins components and development of antisera Full-length CsrR and N-terminal truncated CsrS (CsrSΔ1-231) and were fused separately to a N-terminal His6 tag by cloning into overexpression vector pET-28a (Novagen) PCR-amplified DNA fragments obtained with primer pairs JL-48/JL-49 and HTW 37/46, respectively. .. Following overexpression by IPTG induction, recombinant proteins were affinity purified using Ni2+ -NTA resin (Qiagen) under native conditions (His6 -CsrR) or under denaturing conditions (His6 -CsrSΔ1-231) according to the manufacturer's protocol.

Plasmid Preparation:

Article Title: A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System
Article Snippet: .. PfMSRP-2 was expressed in E. coli BL-21 DE3 Codon Plus cells by using the pET-28a plasmid vector (Novagen). .. The recombinant protein was expressed as an amino-terminal fusion with a six-histidine tag.

Article Title: Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons
Article Snippet: .. Purification of murine recombinant hnRNP R and SMN protein His-tagged hnRNP R and SMN full length proteins were expressed in E. coli after cloning the corresponding cDNA constructs into the pET-28a and pET-32a vector system (Novagen, Madison, WI), respectively. ..

Article Title: Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus
Article Snippet: .. Expression of recombinant Csr proteins components and development of antisera Full-length CsrR and N-terminal truncated CsrS (CsrSΔ1-231) and were fused separately to a N-terminal His6 tag by cloning into overexpression vector pET-28a (Novagen) PCR-amplified DNA fragments obtained with primer pairs JL-48/JL-49 and HTW 37/46, respectively. .. Following overexpression by IPTG induction, recombinant proteins were affinity purified using Ni2+ -NTA resin (Qiagen) under native conditions (His6 -CsrR) or under denaturing conditions (His6 -CsrSΔ1-231) according to the manufacturer's protocol.

Article Title: Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)
Article Snippet: .. Purification of recombinant G3BP1 and USP10 The NTF2-like (residues 1–139) and RRM (residues 339–421) domains of hG3BP (NP-005745) were cloned into the pET-28a vector (Novagen, Madison, WI, USA). .. The NTF2-like domain of G3BP1 was expressed in Codon Plus (DE3) RIPL E.coli (Stratagene) and then harvested after 2–2.5 h growth at 37°C following induction with 1 mM isopropyl-1-thio-β-galactopyranoside (IPTG).

Article Title: Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment
Article Snippet: .. The generated KM10 scFv gene was then subcloned into a pET-28a vector (Novagen, Madison, WI) in frame with a (His)6 tag sequence to ensure purification. .. The affinity maturated K-11 scFv gene was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using KM10 scFv in pET-28a vector as a template.

Article Title: Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants
Article Snippet: .. Enzyme assays For biochemical characterization of mature PhCM1 (minus predicted transit peptide) and PhCM2 as well as mature PhADT3L and PhADT3S, the coding region of the corresponding gene was amplified using forward and reverse primers (Supplementary Table ) containing Nde I and Bam HI restriction sites, respectively, for directional cloning into pET-28a expression vector (Novagen, Madison, WI) in-frame with an N-terminal 6XHis-tag. .. After sequence verification, recombinant proteins were produced in E. coli and purified on Ni-NTA resin (Qiagen, Hilden, Germany) .

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    Millipore plasmid pet 28a
    Validation of nucleic acid digestion by pepsin. a , Digestion of various DNA and RNA by commercial porcine pepsin. Lane 1, 2: salmon sperm DNA; Lane 3, 4: λ DNA; Lane 5, 6: <t>pET-28a;</t> Lane 7, 8: M13mp18; Lane 9, 10: RNA ladder. Other conditions: 4.0 mg ml −1 of pepsin, NaH 2 PO 4 buffer (25 mM, pH 3.8, including 200 mM NaCl), 37 °C, 5 h. For RNA, the digestion time was 1 h. b , Effect of alkaline conditions on pepsin NA digestion. Lane 1, original λ DNA; Lane 2, digested by active pepsin; Lane 3, digested by NaOH-pretreated pepsin. c, Digestion of λ DNA by commercial porcine pepsin (Lane P), recombinant pepsin (Lane rP) and mutant pepsin (Lane mP). Conditions: 0.15 mg ml −1 enzymes, pH 3.8, 37 °C, 12 h. A 0.8% agarose gel was used for electrophoresis.
    Plasmid Pet 28a, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid pet 28a/product/Millipore
    Average 90 stars, based on 361 article reviews
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    plasmid pet 28a - by Bioz Stars, 2020-08
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    93
    Millipore pet 28a uap56
    Validation of nucleic acid digestion by pepsin. a , Digestion of various DNA and RNA by commercial porcine pepsin. Lane 1, 2: salmon sperm DNA; Lane 3, 4: λ DNA; Lane 5, 6: <t>pET-28a;</t> Lane 7, 8: M13mp18; Lane 9, 10: RNA ladder. Other conditions: 4.0 mg ml −1 of pepsin, NaH 2 PO 4 buffer (25 mM, pH 3.8, including 200 mM NaCl), 37 °C, 5 h. For RNA, the digestion time was 1 h. b , Effect of alkaline conditions on pepsin NA digestion. Lane 1, original λ DNA; Lane 2, digested by active pepsin; Lane 3, digested by NaOH-pretreated pepsin. c, Digestion of λ DNA by commercial porcine pepsin (Lane P), recombinant pepsin (Lane rP) and mutant pepsin (Lane mP). Conditions: 0.15 mg ml −1 enzymes, pH 3.8, 37 °C, 12 h. A 0.8% agarose gel was used for electrophoresis.
    Pet 28a Uap56, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pet 28a uap56/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pet 28a uap56 - by Bioz Stars, 2020-08
    93/100 stars
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    Validation of nucleic acid digestion by pepsin. a , Digestion of various DNA and RNA by commercial porcine pepsin. Lane 1, 2: salmon sperm DNA; Lane 3, 4: λ DNA; Lane 5, 6: pET-28a; Lane 7, 8: M13mp18; Lane 9, 10: RNA ladder. Other conditions: 4.0 mg ml −1 of pepsin, NaH 2 PO 4 buffer (25 mM, pH 3.8, including 200 mM NaCl), 37 °C, 5 h. For RNA, the digestion time was 1 h. b , Effect of alkaline conditions on pepsin NA digestion. Lane 1, original λ DNA; Lane 2, digested by active pepsin; Lane 3, digested by NaOH-pretreated pepsin. c, Digestion of λ DNA by commercial porcine pepsin (Lane P), recombinant pepsin (Lane rP) and mutant pepsin (Lane mP). Conditions: 0.15 mg ml −1 enzymes, pH 3.8, 37 °C, 12 h. A 0.8% agarose gel was used for electrophoresis.

    Journal: Scientific Reports

    Article Title: Digestion of Nucleic Acids Starts in the Stomach

    doi: 10.1038/srep11936

    Figure Lengend Snippet: Validation of nucleic acid digestion by pepsin. a , Digestion of various DNA and RNA by commercial porcine pepsin. Lane 1, 2: salmon sperm DNA; Lane 3, 4: λ DNA; Lane 5, 6: pET-28a; Lane 7, 8: M13mp18; Lane 9, 10: RNA ladder. Other conditions: 4.0 mg ml −1 of pepsin, NaH 2 PO 4 buffer (25 mM, pH 3.8, including 200 mM NaCl), 37 °C, 5 h. For RNA, the digestion time was 1 h. b , Effect of alkaline conditions on pepsin NA digestion. Lane 1, original λ DNA; Lane 2, digested by active pepsin; Lane 3, digested by NaOH-pretreated pepsin. c, Digestion of λ DNA by commercial porcine pepsin (Lane P), recombinant pepsin (Lane rP) and mutant pepsin (Lane mP). Conditions: 0.15 mg ml −1 enzymes, pH 3.8, 37 °C, 12 h. A 0.8% agarose gel was used for electrophoresis.

    Article Snippet: Plasmid pET-28a (5.4 kb) was purchased from EMD Chemicals Inc. (Novagen, D00131614) with concentration of 500 μg ml−1 , and was diluted into 210 μg ml−1 before use.

    Techniques: Positron Emission Tomography, Recombinant, Mutagenesis, Agarose Gel Electrophoresis, Electrophoresis