his tagged eglarr01  (Qiagen)

 
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    Name:
    Ni NTA Agarose
    Description:
    For purification of His tagged proteins by gravity flow chromatography Kit contents Qiagen Ni NTA Agarose 25mL 45 to 165m Bead Up to 50mg mL Binding Capacity Cell Lysate Start Material Nickel charged Resin 2 8psi max Pressure Manual Automated Processing Large Scale Sepharose CL 6B Matrix 100g to 100mg Yield 6xHis tag Affinity Chromatography Purification High Binding Affinity and High Capacity Precharged Ready to use Matrices For Purification of His tagged Proteins by Gravity flow Chromatography Benefits One step purification from crude lysate to 95 pure protein High binding affinity and high capacity Choice of purification under native or denaturing conditions Precharged ready to use matrices for any scale of purification Automated purification and assay protocol
    Catalog Number:
    30210
    Price:
    305
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    Ni NTA Agarose
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    Structured Review

    Qiagen his tagged eglarr01
    Ni NTA Agarose
    For purification of His tagged proteins by gravity flow chromatography Kit contents Qiagen Ni NTA Agarose 25mL 45 to 165m Bead Up to 50mg mL Binding Capacity Cell Lysate Start Material Nickel charged Resin 2 8psi max Pressure Manual Automated Processing Large Scale Sepharose CL 6B Matrix 100g to 100mg Yield 6xHis tag Affinity Chromatography Purification High Binding Affinity and High Capacity Precharged Ready to use Matrices For Purification of His tagged Proteins by Gravity flow Chromatography Benefits One step purification from crude lysate to 95 pure protein High binding affinity and high capacity Choice of purification under native or denaturing conditions Precharged ready to use matrices for any scale of purification Automated purification and assay protocol
    https://www.bioz.com/result/his tagged eglarr01/product/Qiagen
    Average 90 stars, based on 842 article reviews
    Price from $9.99 to $1999.99
    his tagged eglarr01 - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues"

    Article Title: A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues

    Journal: Molecules

    doi: 10.3390/molecules21070831

    The recombinant EglaRR01 enzyme (0.1 μg) showed an active band at 40.1 kDa in the CMC zymogram. The three additional bands that appeared below 40.1 kDa in the CMC zymogram were probably caused by the action of partially degraded EglaRR01. 1 : Molecular weight marker (kDa); 2 : spin column portion of partly purified endoglucanase; 3 : ammonium sulfate (40%–60%) fraction; 4 : purified endoglucanase; and 5 : purified endoglucanase showing yellow opaque region in native gel.
    Figure Legend Snippet: The recombinant EglaRR01 enzyme (0.1 μg) showed an active band at 40.1 kDa in the CMC zymogram. The three additional bands that appeared below 40.1 kDa in the CMC zymogram were probably caused by the action of partially degraded EglaRR01. 1 : Molecular weight marker (kDa); 2 : spin column portion of partly purified endoglucanase; 3 : ammonium sulfate (40%–60%) fraction; 4 : purified endoglucanase; and 5 : purified endoglucanase showing yellow opaque region in native gel.

    Techniques Used: Recombinant, Molecular Weight, Marker, Purification

    Effect of pH on the activity and stability of EglaRR01. ( a ) The optimal pH for EglaRR01 was determined by measuring the enzyme activity on 1% ( w/v ) CMC in 50 mM buffers at 65 °C with various pH values. The buffers used to establish the optimum pH and to assess pH stability were as follows: sodium acetate buffer (pH 4–6, ♦), and sodium phosphate buffer (pH 6–8, ●); and ( b ) to determine the pH stability of EglaRR01, the enzyme was incubated for 16 h at 4 °C in buffers of different pH values. The residual enzyme activity was measured under standard assay procedures. All measurements were carried out in triplicate.
    Figure Legend Snippet: Effect of pH on the activity and stability of EglaRR01. ( a ) The optimal pH for EglaRR01 was determined by measuring the enzyme activity on 1% ( w/v ) CMC in 50 mM buffers at 65 °C with various pH values. The buffers used to establish the optimum pH and to assess pH stability were as follows: sodium acetate buffer (pH 4–6, ♦), and sodium phosphate buffer (pH 6–8, ●); and ( b ) to determine the pH stability of EglaRR01, the enzyme was incubated for 16 h at 4 °C in buffers of different pH values. The residual enzyme activity was measured under standard assay procedures. All measurements were carried out in triplicate.

    Techniques Used: Activity Assay, Incubation

    Classification of EglaRR01 by nucleotide and amino acid sequence analyses. Amino acid sequences of endoglucanases, including EglaRR01, were compared and analyzed phylogenetically using a neighbor-joining method. GenBank accession numbers are in parentheses. Phylogenetic analysis showed that EglaRR01 is closely related to cellulases from an uncultured species of Enterobacter .
    Figure Legend Snippet: Classification of EglaRR01 by nucleotide and amino acid sequence analyses. Amino acid sequences of endoglucanases, including EglaRR01, were compared and analyzed phylogenetically using a neighbor-joining method. GenBank accession numbers are in parentheses. Phylogenetic analysis showed that EglaRR01 is closely related to cellulases from an uncultured species of Enterobacter .

    Techniques Used: Sequencing

    Effect of temperature on the activity and stability of EglaRR01. ( a ) Optimal temperature for EglaRR01 is 60 °C, as determined by measuring its enzymatic activity with 1% ( w/v ) CMC in 50 mM sodium acetate buffer, pH 5, at 25 to 70 °C in five degree increments; and ( b ) thermostability was determined by measuring the enzymatic activity of EglaRR01 after incubation at 30 to 70 °C in 10 degree increments for 60 min.
    Figure Legend Snippet: Effect of temperature on the activity and stability of EglaRR01. ( a ) Optimal temperature for EglaRR01 is 60 °C, as determined by measuring its enzymatic activity with 1% ( w/v ) CMC in 50 mM sodium acetate buffer, pH 5, at 25 to 70 °C in five degree increments; and ( b ) thermostability was determined by measuring the enzymatic activity of EglaRR01 after incubation at 30 to 70 °C in 10 degree increments for 60 min.

    Techniques Used: Activity Assay, Incubation

    2) Product Images from "Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor"

    Article Title: Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

    Journal: Insect biochemistry and molecular biology

    doi: 10.1016/j.ibmb.2007.05.013

    SDS-polyacrylamide gel electrophoretic analysis of M. sexta proHP21 from baculovirus-infected insect cells The purified recombinant HP21 precursor (0.2 μg) was subjected to electrophoresis on a 10% SDS polyacrylamide gel under reducing condition. The protein is visualized by silver staining ( A ). As described in Materials and methods , proHP21 was treated with buffer ( left lanes), PNGase F ( B ) or O -glycosidase ( C ), separated by 10% SDS-PAGE, and detected using HP21 antibodies. Sizes and positions of the molecular mass markers are indicated on the right .
    Figure Legend Snippet: SDS-polyacrylamide gel electrophoretic analysis of M. sexta proHP21 from baculovirus-infected insect cells The purified recombinant HP21 precursor (0.2 μg) was subjected to electrophoresis on a 10% SDS polyacrylamide gel under reducing condition. The protein is visualized by silver staining ( A ). As described in Materials and methods , proHP21 was treated with buffer ( left lanes), PNGase F ( B ) or O -glycosidase ( C ), separated by 10% SDS-PAGE, and detected using HP21 antibodies. Sizes and positions of the molecular mass markers are indicated on the right .

    Techniques Used: Infection, Purification, Recombinant, Electrophoresis, Silver Staining, SDS Page

    Role of M. sexta HP21 in the proPO activation system Purified proHP14 (40 ng, 2 μl), proHP21 (80 ng, 2 μl), βGRP2 (40 ng, 2 μl), curdlan (100 μg, 10 μl), and buffer C (14 μl) were incubated at 37°C for 1 h. After centrifugation, 5 μl of the supernatant was reacted with 5 μl of 1:10 diluted plasma from induced ( left panel, IH) or naïve ( right panel, CH) larvae, 1 μl of M. luteus ) and plotted as mean ± S.D. (n=3) in the bar graph . The pre-incubation mixture without proHP14 and/or proHP21 was used as controls.
    Figure Legend Snippet: Role of M. sexta HP21 in the proPO activation system Purified proHP14 (40 ng, 2 μl), proHP21 (80 ng, 2 μl), βGRP2 (40 ng, 2 μl), curdlan (100 μg, 10 μl), and buffer C (14 μl) were incubated at 37°C for 1 h. After centrifugation, 5 μl of the supernatant was reacted with 5 μl of 1:10 diluted plasma from induced ( left panel, IH) or naïve ( right panel, CH) larvae, 1 μl of M. luteus ) and plotted as mean ± S.D. (n=3) in the bar graph . The pre-incubation mixture without proHP14 and/or proHP21 was used as controls.

    Techniques Used: Activation Assay, Purification, Incubation, Centrifugation

    Formation of an SDS-stable complex of M. sexta HP21 and serpin-4 ProHP14 (40 ng, 2 μl), proHP21 (40 ng, 2 μl), curdlan (20 ng, 1 μl), βGRP2 (40 ng, 2 μl), serpin-4 (0.2 μg, 2 μl), and buffer C (15 μl) were incubated at 37°C for 1 h. In the controls, proHP14, proHP21, or serpin-4 in the reaction mixture was replaced by the same volume of buffer C. The reaction and control samples were separated by 7.5% SDS-PAGE under reducing condition. Immunoblot analysis was performed using 1:1,000 diluted serpin-4 ( left panel) or HP21 ( right panel) antiserum at the first antibody. a, 50 kDa serpin-4; b, 60 kDa proHP21; c, ~30 kDa HP21 heavy and light chains; *, 75 kDa complex of serpin-4 and HP21 catalytic chain. Sizes and positions of the molecular mass markers are indicated.
    Figure Legend Snippet: Formation of an SDS-stable complex of M. sexta HP21 and serpin-4 ProHP14 (40 ng, 2 μl), proHP21 (40 ng, 2 μl), curdlan (20 ng, 1 μl), βGRP2 (40 ng, 2 μl), serpin-4 (0.2 μg, 2 μl), and buffer C (15 μl) were incubated at 37°C for 1 h. In the controls, proHP14, proHP21, or serpin-4 in the reaction mixture was replaced by the same volume of buffer C. The reaction and control samples were separated by 7.5% SDS-PAGE under reducing condition. Immunoblot analysis was performed using 1:1,000 diluted serpin-4 ( left panel) or HP21 ( right panel) antiserum at the first antibody. a, 50 kDa serpin-4; b, 60 kDa proHP21; c, ~30 kDa HP21 heavy and light chains; *, 75 kDa complex of serpin-4 and HP21 catalytic chain. Sizes and positions of the molecular mass markers are indicated.

    Techniques Used: Incubation, SDS Page

    3) Product Images from "Prointerleukin-18 Is Activated by Meprin ? in Vitro andin Vivo in Intestinal Inflammation *"

    Article Title: Prointerleukin-18 Is Activated by Meprin ? in Vitro andin Vivo in Intestinal Inflammation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M802814200

    Identification of the proIL-18 site cleaved by meprin B. The meprin B cleavage site is indicated, and the nine-amino acid sequence identified by MS/MS is shown in boldface . Caspase-1 and putative PR-3 sites are also indicated. The region of caspase-3 cleavage is italicized ).
    Figure Legend Snippet: Identification of the proIL-18 site cleaved by meprin B. The meprin B cleavage site is indicated, and the nine-amino acid sequence identified by MS/MS is shown in boldface . Caspase-1 and putative PR-3 sites are also indicated. The region of caspase-3 cleavage is italicized ).

    Techniques Used: Sequencing, Mass Spectrometry

    Effect of meprin B on IL-18 activity in EL-4 cells. NF-κB activation was used to assess IL-18 bioactivity. EL-4 cells were untreated or treated for 30 min with proIL-18 (200 ng/ml) alone, meprin B ( mepB ) alone, or proIL-18 preincubated with meprin B. Cells lysates were assayed for NF-κB activation using a p65 TransAM kit. Jurkat nuclear extract served as a positive control for NF-κB activation. NF-κB activation is shown as -fold increase over unstimulated EL-4 cells ( n = 3; *, p
    Figure Legend Snippet: Effect of meprin B on IL-18 activity in EL-4 cells. NF-κB activation was used to assess IL-18 bioactivity. EL-4 cells were untreated or treated for 30 min with proIL-18 (200 ng/ml) alone, meprin B ( mepB ) alone, or proIL-18 preincubated with meprin B. Cells lysates were assayed for NF-κB activation using a p65 TransAM kit. Jurkat nuclear extract served as a positive control for NF-κB activation. NF-κB activation is shown as -fold increase over unstimulated EL-4 cells ( n = 3; *, p

    Techniques Used: Activity Assay, Activation Assay, Positive Control

    Interaction of proIL-18 with meprin B expressed in MDCK cells. Full-length meprin β was transfected into MDCK cells, and latent meprin B was activated with trypsin. After removal and inhibition of trypsin, proIL-18 was added to the culture medium and incubated for 22 h; the medium and the cell lysate were then subjected to SDS-PAGE. The upper panel is a Western blot of the culture medium using a polyclonal antibody to IL-18; the lower panel is a Western blot of the cell lysate fraction using a polyclonal antibody to meprin β. The results are typical of three independent experiments.
    Figure Legend Snippet: Interaction of proIL-18 with meprin B expressed in MDCK cells. Full-length meprin β was transfected into MDCK cells, and latent meprin B was activated with trypsin. After removal and inhibition of trypsin, proIL-18 was added to the culture medium and incubated for 22 h; the medium and the cell lysate were then subjected to SDS-PAGE. The upper panel is a Western blot of the culture medium using a polyclonal antibody to IL-18; the lower panel is a Western blot of the cell lysate fraction using a polyclonal antibody to meprin β. The results are typical of three independent experiments.

    Techniques Used: Transfection, Inhibition, Incubation, SDS Page, Western Blot

    4) Product Images from "Cytokinin Growth Responses in Arabidopsis Involve the 26S Proteasome Subunit RPN12"

    Article Title: Cytokinin Growth Responses in Arabidopsis Involve the 26S Proteasome Subunit RPN12

    Journal: The Plant Cell

    doi: 10.1105/tpc.010381

    Association of the RPN12a-1–NPTII Fusion Protein with the 26S Proteasome. The 26S proteasome was partially purified from Arabidopsis seedlings homozygous for the rpn12a-1 mutation by sequential polyethylene glycol precipitations and subjected to size exclusion FPLC. (A) Column fractions were assayed for CP peptidase activity. Only the portion of the FPLC elution profile around the peak of peptidase activity is shown. (B) Column fractions subjected to SDS-PAGE and immunoblot analysis with antibodies against RPN12a, RPN10, RPN5a, RPT2a subunits of the RP, and PBA1 and PAC1 subunits of the CP. The positions of the wild-type RPN12a and the RPN12a-NPTII fusion protein are indicated by arrowheads.
    Figure Legend Snippet: Association of the RPN12a-1–NPTII Fusion Protein with the 26S Proteasome. The 26S proteasome was partially purified from Arabidopsis seedlings homozygous for the rpn12a-1 mutation by sequential polyethylene glycol precipitations and subjected to size exclusion FPLC. (A) Column fractions were assayed for CP peptidase activity. Only the portion of the FPLC elution profile around the peak of peptidase activity is shown. (B) Column fractions subjected to SDS-PAGE and immunoblot analysis with antibodies against RPN12a, RPN10, RPN5a, RPT2a subunits of the RP, and PBA1 and PAC1 subunits of the CP. The positions of the wild-type RPN12a and the RPN12a-NPTII fusion protein are indicated by arrowheads.

    Techniques Used: Purification, Mutagenesis, Fast Protein Liquid Chromatography, Activity Assay, SDS Page

    5) Product Images from "Production of Low-Expressing Recombinant Cationic Biopolymers with High Purity"

    Article Title: Production of Low-Expressing Recombinant Cationic Biopolymers with High Purity

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2017.03.012

    A) The amounts of purified biopolymers from each 500 mL of BL21(DE3) pLysS culture (Yield). B) The SDS-PAGE picture of the Ni-NTA purified TH2G, TH4G, TH6G and TH8G. C) The quantification of biopolymer purity using Image J software. TH4G purity is not determined since the molecular weights of SlyD and TH4G are very close.
    Figure Legend Snippet: A) The amounts of purified biopolymers from each 500 mL of BL21(DE3) pLysS culture (Yield). B) The SDS-PAGE picture of the Ni-NTA purified TH2G, TH4G, TH6G and TH8G. C) The quantification of biopolymer purity using Image J software. TH4G purity is not determined since the molecular weights of SlyD and TH4G are very close.

    Techniques Used: Purification, SDS Page, Software

    A) The growth curves of BL21(DE3) bacteria transformed with TH2G and TH8G constructs with and without IPTG induction. B) The amounts of purified TH2G and TH8G from 500mL of culture. C) The SDS-PAGE picture of the purified TH2G and TH8G biopolymers. D) The quantitative analysis of impurities in purified TH2G and TH8G biopolymers using Image J software. The data are presented as mean±s.d, n=3.
    Figure Legend Snippet: A) The growth curves of BL21(DE3) bacteria transformed with TH2G and TH8G constructs with and without IPTG induction. B) The amounts of purified TH2G and TH8G from 500mL of culture. C) The SDS-PAGE picture of the purified TH2G and TH8G biopolymers. D) The quantitative analysis of impurities in purified TH2G and TH8G biopolymers using Image J software. The data are presented as mean±s.d, n=3.

    Techniques Used: Transformation Assay, Construct, Purification, SDS Page, Software

    Comparison of the yield and purity of TH8G biopolymer after expression in BL21(DE3) host and purification by Ni-NTA and TALON resins. A) The amount of purified TH8G obtained from 500 mL of culture. B) The SDS-PAGE picture of the purified TH8G. C) The quantification of TH8G purity using the Image J software. The data are presented as mean±s.d. (n= 3). * indicates significance, p
    Figure Legend Snippet: Comparison of the yield and purity of TH8G biopolymer after expression in BL21(DE3) host and purification by Ni-NTA and TALON resins. A) The amount of purified TH8G obtained from 500 mL of culture. B) The SDS-PAGE picture of the purified TH8G. C) The quantification of TH8G purity using the Image J software. The data are presented as mean±s.d. (n= 3). * indicates significance, p

    Techniques Used: Expressing, Purification, SDS Page, Software

    6) Product Images from "Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations"

    Article Title: Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074040

    Sequence-structure relationship for the EphA5 and EphA4 LBDs. (a) Alignment of the sequences of the EphA5 and EphA4 LBDs. Identical residues are colored in blue, homologous in green and different in black. Residues in the D and E β-strands are highlighted in yellow and residues in the J–K loop in pink. Two residues that are in close contact in the EphA4 structure (Ile in the D strand and Asp in the J–K-loop) and the corresponding residues in the EphA5 LBD are boxed. (b) Structure of the EphA5 LBD with spheres for Asp190 in the J–K-loop, and Glu80 in the D strand which corresponds to a Ile in the structure of the EphA4 LBD (c).
    Figure Legend Snippet: Sequence-structure relationship for the EphA5 and EphA4 LBDs. (a) Alignment of the sequences of the EphA5 and EphA4 LBDs. Identical residues are colored in blue, homologous in green and different in black. Residues in the D and E β-strands are highlighted in yellow and residues in the J–K loop in pink. Two residues that are in close contact in the EphA4 structure (Ile in the D strand and Asp in the J–K-loop) and the corresponding residues in the EphA5 LBD are boxed. (b) Structure of the EphA5 LBD with spheres for Asp190 in the J–K-loop, and Glu80 in the D strand which corresponds to a Ile in the structure of the EphA4 LBD (c).

    Techniques Used: Sequencing

    The structure of the unbound EphA5 LBD resembles that of other Eph receptors bound to ephrin ligands. (a) Superimposition of the LBD structures of unbound EphA5 (red), EphA2 (green, 3C8X), EphA4 (yellow, 3CKH) and EphB2 (blue, 3ETP). A short β-sheet is formed by the EphA4 and EphB2 residues corresponding to EphA5 residues Ala179-Ser182 and Gly189-M193 in the J–K loop (orange arrows). (b) Superimposition of the LBD structures of the unbound EphA5 (red), EphA2 in complex with ephrin-A2 (green, 3CZU), EphA4 with ephrin-A2 (cyan, 3WO3), EphA4 with ephrin-B2 (blue, 3GXU), EphB2 with ephrin-B2 (pink, 1KGY) and EphB4 with ephrin-B2 (yellow, 2HLE).
    Figure Legend Snippet: The structure of the unbound EphA5 LBD resembles that of other Eph receptors bound to ephrin ligands. (a) Superimposition of the LBD structures of unbound EphA5 (red), EphA2 (green, 3C8X), EphA4 (yellow, 3CKH) and EphB2 (blue, 3ETP). A short β-sheet is formed by the EphA4 and EphB2 residues corresponding to EphA5 residues Ala179-Ser182 and Gly189-M193 in the J–K loop (orange arrows). (b) Superimposition of the LBD structures of the unbound EphA5 (red), EphA2 in complex with ephrin-A2 (green, 3CZU), EphA4 with ephrin-A2 (cyan, 3WO3), EphA4 with ephrin-B2 (blue, 3GXU), EphB2 with ephrin-B2 (pink, 1KGY) and EphB4 with ephrin-B2 (yellow, 2HLE).

    Techniques Used:

    Structural properties and solvent accessibility of the EphA5 LBD in solution. (a) Residue-specific Cα chemical shift deviations (ΔCα = δobs – δcoil) for the EphA5 LBD. The bars for the J–K loop residues with helical conformations in the crystal structure are colored in red. (b) Superimposition of 1 H- 15 N NMR HSQC spectra for the 15 N-labeled EphA5 LBD at 25°C in 10 mM phosphate buffer, pH 6.5 (blue) and 15 min after dissolving the lyophilized sample in D 2 O (red). The disappearance of the blue HSQC peaks indicates the high exposure of the amide protons to the solvent. (c) Superimposition of HSQC spectra of the EphA5 LBD at 25°C, 15 min (blue) and 24 hours (red) after dissolving the lyophilized sample in D 2 O. (d) EphA5 LBD structure with residues whose HSQC peaks are missing even in H 2 O buffer colored in green, residues whose backbone amide protons completely exchanged within 15 min in blue, residues whose backbone amide protons persisted after 15 min but completely exchanged in 2 hours in yellow, and residues whose backbone amide protons persisted even after 2 hours in red. The very rapid exchange of their amide protons indicates that the D and E strands are highly exposed to the solvent.
    Figure Legend Snippet: Structural properties and solvent accessibility of the EphA5 LBD in solution. (a) Residue-specific Cα chemical shift deviations (ΔCα = δobs – δcoil) for the EphA5 LBD. The bars for the J–K loop residues with helical conformations in the crystal structure are colored in red. (b) Superimposition of 1 H- 15 N NMR HSQC spectra for the 15 N-labeled EphA5 LBD at 25°C in 10 mM phosphate buffer, pH 6.5 (blue) and 15 min after dissolving the lyophilized sample in D 2 O (red). The disappearance of the blue HSQC peaks indicates the high exposure of the amide protons to the solvent. (c) Superimposition of HSQC spectra of the EphA5 LBD at 25°C, 15 min (blue) and 24 hours (red) after dissolving the lyophilized sample in D 2 O. (d) EphA5 LBD structure with residues whose HSQC peaks are missing even in H 2 O buffer colored in green, residues whose backbone amide protons completely exchanged within 15 min in blue, residues whose backbone amide protons persisted after 15 min but completely exchanged in 2 hours in yellow, and residues whose backbone amide protons persisted even after 2 hours in red. The very rapid exchange of their amide protons indicates that the D and E strands are highly exposed to the solvent.

    Techniques Used: Nuclear Magnetic Resonance, Labeling

    Unique ligand-binding specificity of the EphA5 LBD. (a) Isothermal titration calorimetry profiles for the interaction of the EphA5 LBD with the WDC peptide (upper panel) and plots of the integrated values for the reaction heats (after blank subtraction and normalization to the amount of the peptide injected) versus EphA5 to WDC molar ratio (lower panel). The thermodynamic binding parameters are shown in the lower panel. (b) Inhibition of ephrin-A5 alkaline phosphatase (AP) binding to immobilized EphA5 Fc by increasing concentrations of WDC in ELISAs. Bound ephrin-A5 AP represents the ratio of the OD at 405 nm for ephrin-A5 AP bound to EphA5 Fc in the presence of the indicated concentrations of the WDC peptide and in the absence of peptide. (c) Inhibition of ephrin-A5 AP binding to EphA receptors and ephrin-B2 AP binding to EphB receptors by 100 μM WDC. Bound ephrin AP represents the ratio of the OD at 405 nm for ephrin-A5 AP or ephrin-B2 AP bound to different Eph receptor Fc proteins in the presence of WDC peptide and in the absence of peptide. The peptide substantially inhibits ephrin binding only to EphA5. Averages and standard errors from triplicate measurements are shown. (d) Superimposition of the NMR HSQC spectra of the EphA5 LBD in the absence (blue) and in the presence (red) of WDC at a molar ratio of 1:3 (EphA5:WDC). (e) Superimposition of the NMR HSQC spectra of the EphA5 LBD in the absence (blue) and in the presence (red) of C1 at a molar ratio of 1:20 (EphA5:C1).
    Figure Legend Snippet: Unique ligand-binding specificity of the EphA5 LBD. (a) Isothermal titration calorimetry profiles for the interaction of the EphA5 LBD with the WDC peptide (upper panel) and plots of the integrated values for the reaction heats (after blank subtraction and normalization to the amount of the peptide injected) versus EphA5 to WDC molar ratio (lower panel). The thermodynamic binding parameters are shown in the lower panel. (b) Inhibition of ephrin-A5 alkaline phosphatase (AP) binding to immobilized EphA5 Fc by increasing concentrations of WDC in ELISAs. Bound ephrin-A5 AP represents the ratio of the OD at 405 nm for ephrin-A5 AP bound to EphA5 Fc in the presence of the indicated concentrations of the WDC peptide and in the absence of peptide. (c) Inhibition of ephrin-A5 AP binding to EphA receptors and ephrin-B2 AP binding to EphB receptors by 100 μM WDC. Bound ephrin AP represents the ratio of the OD at 405 nm for ephrin-A5 AP or ephrin-B2 AP bound to different Eph receptor Fc proteins in the presence of WDC peptide and in the absence of peptide. The peptide substantially inhibits ephrin binding only to EphA5. Averages and standard errors from triplicate measurements are shown. (d) Superimposition of the NMR HSQC spectra of the EphA5 LBD in the absence (blue) and in the presence (red) of WDC at a molar ratio of 1:3 (EphA5:WDC). (e) Superimposition of the NMR HSQC spectra of the EphA5 LBD in the absence (blue) and in the presence (red) of C1 at a molar ratio of 1:20 (EphA5:C1).

    Techniques Used: Ligand Binding Assay, Isothermal Titration Calorimetry, Injection, Binding Assay, Inhibition, Nuclear Magnetic Resonance

    The crystal structure of the EphA5 LBD shows well defined D–E and J–K loops. (a) Crystal structure of the EphA5 LBD. Residues Ala179-Ser182 and Gly189-M193 in the J–K loop, which adopt unusual helical-like conformations, are displayed in red. Two disulfide bridges Cys137-Cys147 and Cys102-Cys220, are displayed in orange. (b, c) Electron density maps for the D–E and J–K loops, showing that all residues are well defined.
    Figure Legend Snippet: The crystal structure of the EphA5 LBD shows well defined D–E and J–K loops. (a) Crystal structure of the EphA5 LBD. Residues Ala179-Ser182 and Gly189-M193 in the J–K loop, which adopt unusual helical-like conformations, are displayed in red. Two disulfide bridges Cys137-Cys147 and Cys102-Cys220, are displayed in orange. (b, c) Electron density maps for the D–E and J–K loops, showing that all residues are well defined.

    Techniques Used:

    15 N backbone dynamics for the EphA5 LBD on the ps-ns time scale. (a) Generalized squared order parameter (S 2 ) derived from the Model-free analysis of the relaxation data for EphA5. Red indicates residues with S 2
    Figure Legend Snippet: 15 N backbone dynamics for the EphA5 LBD on the ps-ns time scale. (a) Generalized squared order parameter (S 2 ) derived from the Model-free analysis of the relaxation data for EphA5. Red indicates residues with S 2

    Techniques Used: Derivative Assay

    Distinctive dynamic behaviors of the EphA5 LBD as revealed by molecular dynamics simulations. (a) Trajectories of root-mean-square deviations (RMSD) of heavy atoms in three independent molecular dynamics simulations. (b) Trajectories of root-mean-square fluctuations (RMSF) of the Cα atoms computed for three independent simulations, with average values and standard deviations calculated over 30 ns for each simulation. (c) EphA5 LBD structure with the residues having RMSF > average in green and those > 2-fold the average in red.
    Figure Legend Snippet: Distinctive dynamic behaviors of the EphA5 LBD as revealed by molecular dynamics simulations. (a) Trajectories of root-mean-square deviations (RMSD) of heavy atoms in three independent molecular dynamics simulations. (b) Trajectories of root-mean-square fluctuations (RMSF) of the Cα atoms computed for three independent simulations, with average values and standard deviations calculated over 30 ns for each simulation. (c) EphA5 LBD structure with the residues having RMSF > average in green and those > 2-fold the average in red.

    Techniques Used:

    7) Product Images from "Stable complex formation of CENP-B with the CENP-A nucleosome"

    Article Title: Stable complex formation of CENP-B with the CENP-A nucleosome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv405

    CENP-B binds to the CENP-A and H3.1 nucleosomes. ( A ) Schematic representation of CENP-B DBD binding to nucleosomes. ( B ) Electrophoretic mobility shift assay. The H3.1, CENP-A and H3.1 CATD nucleosomes (lanes 1, 3 and 5, respectively) and those complexed with the CENP-B DBD (lanes 2, 4 and 6, respectively) were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis with ethidium bromide staining. ( C ) Protein contents of the H3.1, CENP-A and H3.1 CATD nucleosomes (lanes 2, 4 and 6, respectively) and those complexed with the CENP-B DBD (lanes 3, 5 and 7, respectively), analyzed by SDS-15% polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.
    Figure Legend Snippet: CENP-B binds to the CENP-A and H3.1 nucleosomes. ( A ) Schematic representation of CENP-B DBD binding to nucleosomes. ( B ) Electrophoretic mobility shift assay. The H3.1, CENP-A and H3.1 CATD nucleosomes (lanes 1, 3 and 5, respectively) and those complexed with the CENP-B DBD (lanes 2, 4 and 6, respectively) were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis with ethidium bromide staining. ( C ) Protein contents of the H3.1, CENP-A and H3.1 CATD nucleosomes (lanes 2, 4 and 6, respectively) and those complexed with the CENP-B DBD (lanes 3, 5 and 7, respectively), analyzed by SDS-15% polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Polyacrylamide Gel Electrophoresis, Staining

    The CENP-B DBD specifically binds to the CENP-A-H4 complex. ( A ) Schematic representation of the pull-down assay with the His 6 -tagged CENP-B DBD and the CENP-A-H4 or H3.1-H4 complex. ( B ) The His 6 -tagged CENP-B DBD (50 nM) complexed with a 21 base-pair DNA was incubated with the CENP-A-H4 or H3.1-H4 complex (50 nM). The proteins pulled down with the Ni-NTA agarose beads were analyzed by 16% SDS-PAGE with Coomassie Brilliant Blue staining. Lane 1 indicates the molecular mass markers. Lane 2: His 6 -tagged CENP-B DBD (50% of input). Lane 3: the H3.1-H4 complex (20% of input). Lane 4: the CENP-A-H4 complex (20% of input). Lanes 5 and 6 represent the pull-down experiments with the H3.1-H4 complex, in the absence and presence of the His 6 -tagged CENP-B DBD, respectively. Lanes 7 and 8 represent the pull-down experiments with the CENP-A-H4 complex, in the absence and presence of the His 6 -tagged CENP-B DBD, respectively.
    Figure Legend Snippet: The CENP-B DBD specifically binds to the CENP-A-H4 complex. ( A ) Schematic representation of the pull-down assay with the His 6 -tagged CENP-B DBD and the CENP-A-H4 or H3.1-H4 complex. ( B ) The His 6 -tagged CENP-B DBD (50 nM) complexed with a 21 base-pair DNA was incubated with the CENP-A-H4 or H3.1-H4 complex (50 nM). The proteins pulled down with the Ni-NTA agarose beads were analyzed by 16% SDS-PAGE with Coomassie Brilliant Blue staining. Lane 1 indicates the molecular mass markers. Lane 2: His 6 -tagged CENP-B DBD (50% of input). Lane 3: the H3.1-H4 complex (20% of input). Lane 4: the CENP-A-H4 complex (20% of input). Lanes 5 and 6 represent the pull-down experiments with the H3.1-H4 complex, in the absence and presence of the His 6 -tagged CENP-B DBD, respectively. Lanes 7 and 8 represent the pull-down experiments with the CENP-A-H4 complex, in the absence and presence of the His 6 -tagged CENP-B DBD, respectively.

    Techniques Used: Pull Down Assay, Incubation, SDS Page, Staining

    The CENP-B DBD does not stably bind to the H3.1 CATD nucleosome. ( A ) Schematic representation of H3.1, CENP-A and H3.1 CATD . Cylinders indicate the regions that form an α-helix structure in nucleosomes. ( B ) The H3.1, CENP-A, or H3.1 CATD nucleosomes (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of a naked 166 base-pair α-satellite DNA containing the proximal CENP-B box sequence. Lanes 1–6, 7–12 and 13–18 indicate the experiments with the H3.1, CENP-A and H3.1 CATD nucleosomes, respectively. The amounts of naked 166 base-pair α-satellite DNA concentrations are 0 ng (lanes 2, 8 and 14), 50 ng (lanes 3, 9 and 15), 75 ng (lanes 4, 10 and 16), 100 ng (lanes 5, 11 and 17) and 125 ng (lanes 6, 12 and 18). Lanes 1, 7 and 13 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( C ) Graphic representation of the experiments shown in panel (B). Averages of four independent experiments are shown with standard deviation values. ( D ) The pull-down assay with the H3.1 CATD -H4 complex. The experiments were performed as in Figure 4B . Lane 1 indicates the molecular mass markers. Lane 2: His 6 -tagged CENP-B DBD (50% of input). Lane 3: the CENP-A-H4 complex (20% of input). Lane 4: the H3.1 CATD -H4 complex (20% of input). Lanes 5 and 6 represent the pull-down experiments with the CENP-A-H4 complex, in the absence and presence of the His 6 -tagged CENP-B DBD, respectively. Lanes 7 and 8 represent the pull-down experiments with the H3.1 CATD -H4 complex, in the absence and presence of the His 6 -tagged CENP-B DBD, respectively.
    Figure Legend Snippet: The CENP-B DBD does not stably bind to the H3.1 CATD nucleosome. ( A ) Schematic representation of H3.1, CENP-A and H3.1 CATD . Cylinders indicate the regions that form an α-helix structure in nucleosomes. ( B ) The H3.1, CENP-A, or H3.1 CATD nucleosomes (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of a naked 166 base-pair α-satellite DNA containing the proximal CENP-B box sequence. Lanes 1–6, 7–12 and 13–18 indicate the experiments with the H3.1, CENP-A and H3.1 CATD nucleosomes, respectively. The amounts of naked 166 base-pair α-satellite DNA concentrations are 0 ng (lanes 2, 8 and 14), 50 ng (lanes 3, 9 and 15), 75 ng (lanes 4, 10 and 16), 100 ng (lanes 5, 11 and 17) and 125 ng (lanes 6, 12 and 18). Lanes 1, 7 and 13 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( C ) Graphic representation of the experiments shown in panel (B). Averages of four independent experiments are shown with standard deviation values. ( D ) The pull-down assay with the H3.1 CATD -H4 complex. The experiments were performed as in Figure 4B . Lane 1 indicates the molecular mass markers. Lane 2: His 6 -tagged CENP-B DBD (50% of input). Lane 3: the CENP-A-H4 complex (20% of input). Lane 4: the H3.1 CATD -H4 complex (20% of input). Lanes 5 and 6 represent the pull-down experiments with the CENP-A-H4 complex, in the absence and presence of the His 6 -tagged CENP-B DBD, respectively. Lanes 7 and 8 represent the pull-down experiments with the H3.1 CATD -H4 complex, in the absence and presence of the His 6 -tagged CENP-B DBD, respectively.

    Techniques Used: Stable Transfection, Incubation, Sequencing, Standard Deviation, Pull Down Assay

    CENP-B binds more stably to the proximal DNA region of the CENP-A nucleosome. ( A ) Schematic representation of the proximal and distal CENP-B box locations, relative to the CENP-A nucleosome (dotted ellipses). The upper and lower panels illustrate the nucleosomes with the 166 base-pair α-satellite DNA (used in Figures 1 and 2 ) and the 166 base-pair α-satellite (-20) DNA, respectively. ( B ) Electrophoretic mobility shift assay. The H3.1 and CENP-A nucleosomes (lanes 1, 3, 5 and 7) and those complexed with the CENP-B DBD (lanes 2, 4, 6 and 8, respectively) were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis with ethidium bromide staining. Lanes 1, 2, 5 and 6 indicate the H3.1 nucleosomes, and lanes 3, 4, 7 and 8 indicate the CENP-A nucleosomes. Lanes 1–4 and lanes 5–8 are experiments with the 166 base-pair α-satellite DNA and the 166 base-pair α-satellite (-20) DNA, respectively. ( C ) The H3.1 (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of the naked 166 base-pair α-satellite DNA. The amounts of naked 166 base-pair α-satellite DNA are 0 ng (lanes 2 and 8), 50 ng (lanes 3 and 9), 75 ng (lanes 4 and 10), 100 ng (lanes 5 and 11) and 125 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( D ) Graphic representation of the experiments shown in panel (C). The amounts (%) of H3.1 nucleosomes complexed with CENP-B DBD were plotted against the amounts of competitor DNA. Averages of three independent experiments are shown with standard deviation values. ( E ) The CENP-A nucleosomes (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of the naked 166 base-pair α-satellite DNA. The amounts of naked 166 base-pair α-satellite DNA are 0 ng (lanes 2 and 8), 50 ng (lanes 3 and 9), 75 ng (lanes 4 and 10), 100 ng (lanes 5 and 11) and 125 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( F ) Graphic representation of the experiments shown in panel (E). The amounts (%) of CENP-A nucleosomes complexed with CENP-B DBD were plotted against the amounts of competitor DNA. Averages of three independent experiments are shown with standard deviation values.
    Figure Legend Snippet: CENP-B binds more stably to the proximal DNA region of the CENP-A nucleosome. ( A ) Schematic representation of the proximal and distal CENP-B box locations, relative to the CENP-A nucleosome (dotted ellipses). The upper and lower panels illustrate the nucleosomes with the 166 base-pair α-satellite DNA (used in Figures 1 and 2 ) and the 166 base-pair α-satellite (-20) DNA, respectively. ( B ) Electrophoretic mobility shift assay. The H3.1 and CENP-A nucleosomes (lanes 1, 3, 5 and 7) and those complexed with the CENP-B DBD (lanes 2, 4, 6 and 8, respectively) were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis with ethidium bromide staining. Lanes 1, 2, 5 and 6 indicate the H3.1 nucleosomes, and lanes 3, 4, 7 and 8 indicate the CENP-A nucleosomes. Lanes 1–4 and lanes 5–8 are experiments with the 166 base-pair α-satellite DNA and the 166 base-pair α-satellite (-20) DNA, respectively. ( C ) The H3.1 (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of the naked 166 base-pair α-satellite DNA. The amounts of naked 166 base-pair α-satellite DNA are 0 ng (lanes 2 and 8), 50 ng (lanes 3 and 9), 75 ng (lanes 4 and 10), 100 ng (lanes 5 and 11) and 125 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( D ) Graphic representation of the experiments shown in panel (C). The amounts (%) of H3.1 nucleosomes complexed with CENP-B DBD were plotted against the amounts of competitor DNA. Averages of three independent experiments are shown with standard deviation values. ( E ) The CENP-A nucleosomes (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of the naked 166 base-pair α-satellite DNA. The amounts of naked 166 base-pair α-satellite DNA are 0 ng (lanes 2 and 8), 50 ng (lanes 3 and 9), 75 ng (lanes 4 and 10), 100 ng (lanes 5 and 11) and 125 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( F ) Graphic representation of the experiments shown in panel (E). The amounts (%) of CENP-A nucleosomes complexed with CENP-B DBD were plotted against the amounts of competitor DNA. Averages of three independent experiments are shown with standard deviation values.

    Techniques Used: Stable Transfection, Electrophoretic Mobility Shift Assay, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Standard Deviation

    CENP-B binds to the CENP-A nucleosome more stably than the H3.1 nucleosome. ( A ) Schematic representation of the competitive CENP-B retention assay. ( B ) The H3.1 or CENP-A nucleosomes (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of a naked 166 base-pair DNA containing the CENP-B box sequence. Lanes 1–6 and lanes 7–12 indicate the experiments with the H3.1 and CENP-A nucleosomes, respectively. The amounts of naked 166 base-pair DNA are 0 ng (lanes 2 and 8), 25 ng (lanes 3 and 9), 50 ng (lanes 4 and 10), 75 ng (lanes 5 and 11), and 100 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( C ) Graphic representation of the experiments shown in panel (B). The amounts (%) of nucleosomes complexed with the CENP-B DBD were plotted against the amounts of competitor DNA. Averages of four independent experiments are shown with standard deviation values.
    Figure Legend Snippet: CENP-B binds to the CENP-A nucleosome more stably than the H3.1 nucleosome. ( A ) Schematic representation of the competitive CENP-B retention assay. ( B ) The H3.1 or CENP-A nucleosomes (containing 100 ng DNA) complexed with the CENP-B DBD were incubated in the presence of a naked 166 base-pair DNA containing the CENP-B box sequence. Lanes 1–6 and lanes 7–12 indicate the experiments with the H3.1 and CENP-A nucleosomes, respectively. The amounts of naked 166 base-pair DNA are 0 ng (lanes 2 and 8), 25 ng (lanes 3 and 9), 50 ng (lanes 4 and 10), 75 ng (lanes 5 and 11), and 100 ng (lanes 6 and 12). Lanes 1 and 7 indicate control experiments without the CENP-B DBD and the naked 166 base-pair DNA. ( C ) Graphic representation of the experiments shown in panel (B). The amounts (%) of nucleosomes complexed with the CENP-B DBD were plotted against the amounts of competitor DNA. Averages of four independent experiments are shown with standard deviation values.

    Techniques Used: Stable Transfection, Incubation, Sequencing, Standard Deviation

    CENP-B binding to alphoid DNA enhances CENP-A retention on nucleosomes. ( A ) Schematic diagram of the CENP-A preassembly followed by the ChIP assay. After co-transfection with the wild type and mutant CENP-B box alphoid tetO DNAs, and a Halo-CENP-A expressing plasmid, the cells expressing tetR-EYFP-HJURP were cultured in medium containing doxycycline for 24 hr. To deposit the maximum level of CENP-A nucleosomes by the tethering of tetR-EYFP-HJURP at tetO sites on the transfected alphoid tetO DNAs, the cells were cultured with doxycycline-free medium for 24 hr. Then, doxycycline was added to the medium to quench the CENP-A deposition. The cells were harvested after 0, 24, 48 and 96 hr of culture in medium containing doxycycline. The ChIP assay and the quantitative PCR/competitive PCR were then performed. The black line indicates the culture with medium containing doxycycline. The red line indicates the culture with doxycycline-free medium, to deposit CENP-A. ( B ) The relative copy number of the total alphoid tetO array, quantitated by real-time PCR. The ChIP assay was performed with anti-CENP-A, anti-CENP-B, anti-histone H3, and anti-GFP (also recognizing EYFP) antibodies. The bars show the relative rates of recovery of the total alphoid tetO DNA against the input DNA. Error bars represent the SEM (n = 3). ( C ) DNA samples recovered after the ChIP assay were analyzed by competitive PCR. The relative enrichment of the wild type CENP-B box alphoid tetO DNA versus the mutant CENP-B box alphoid tetO DNA is shown below the gel images. White arrowheads indicate the PCR fragments from the wild type CENP-B box alphoid tetO DNA. Gray arrowheads indicate the PCR fragments from the mutant CENP-B box alphoid tetO DNA. The values of CENP-A and H3 are normalized by the input DNA. Error bars represent the SEM (n = 3). P-values obtained with the t-test are indicated.
    Figure Legend Snippet: CENP-B binding to alphoid DNA enhances CENP-A retention on nucleosomes. ( A ) Schematic diagram of the CENP-A preassembly followed by the ChIP assay. After co-transfection with the wild type and mutant CENP-B box alphoid tetO DNAs, and a Halo-CENP-A expressing plasmid, the cells expressing tetR-EYFP-HJURP were cultured in medium containing doxycycline for 24 hr. To deposit the maximum level of CENP-A nucleosomes by the tethering of tetR-EYFP-HJURP at tetO sites on the transfected alphoid tetO DNAs, the cells were cultured with doxycycline-free medium for 24 hr. Then, doxycycline was added to the medium to quench the CENP-A deposition. The cells were harvested after 0, 24, 48 and 96 hr of culture in medium containing doxycycline. The ChIP assay and the quantitative PCR/competitive PCR were then performed. The black line indicates the culture with medium containing doxycycline. The red line indicates the culture with doxycycline-free medium, to deposit CENP-A. ( B ) The relative copy number of the total alphoid tetO array, quantitated by real-time PCR. The ChIP assay was performed with anti-CENP-A, anti-CENP-B, anti-histone H3, and anti-GFP (also recognizing EYFP) antibodies. The bars show the relative rates of recovery of the total alphoid tetO DNA against the input DNA. Error bars represent the SEM (n = 3). ( C ) DNA samples recovered after the ChIP assay were analyzed by competitive PCR. The relative enrichment of the wild type CENP-B box alphoid tetO DNA versus the mutant CENP-B box alphoid tetO DNA is shown below the gel images. White arrowheads indicate the PCR fragments from the wild type CENP-B box alphoid tetO DNA. Gray arrowheads indicate the PCR fragments from the mutant CENP-B box alphoid tetO DNA. The values of CENP-A and H3 are normalized by the input DNA. Error bars represent the SEM (n = 3). P-values obtained with the t-test are indicated.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Cotransfection, Mutagenesis, Expressing, Plasmid Preparation, Cell Culture, Transfection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    The CENP-A nucleosome preassembly level reaches a maximum after 24h under HJURP tethering conditions. ( A ) Schematic diagram of the CENP-A nucleosome preassembly and the ChIP real-time PCR/competitive PCR analysis. HT1080 cells stably expressing tetR-EYFP-HJURP were transfected with 50 kb alphoid tetO BAC DNAs and Halo-CENP-A expressing plasmid DNA. To generate CENP-A nucleosome preassembly on the transfected alphoid tetO DNAs by the tethering of tetR-EYFP-HJURP, the cells were cultured in doxycycline-free medium. The CENP-A nucleosome assembly levels were analyzed by a ChIP assay. The DNA samples recovered by ChIP were quantitated by real-time PCR. Then, the PCR products (competitively amplified wild type and mutant CENP-B box alphoid tetO DNAs) were digested with EcoRV and analyzed by agarose gel electrophoresis ( 43 , 67 ). ( B ) Example of a competitive PCR control. The wild type and mutant CENP-B box alphoid tetO BAC DNAs were mixed at different ratios, and amplified competitively with the same primer set by PCR. The white arrowhead indicates the PCR fragment from the wild type CENP-B box alphoid tetO DNA. The gray arrowhead indicates the PCR fragment from the mutant CENP-B box alphoid tetO DNA. ( C ) Schematic diagram of the CENP-A deposition and the transient ChIP assay. The cells transfected with the alphoid tetO BAC DNA mixture were cultured with medium containing doxycycline for 24 hr. To analyze CENP-A nucleosome deposition at tetO sites on the transfected alphoid tetO DNAs, the cells were cultured with doxycycline-free medium for 0–48 hr. The ChIP assay was then performed with anti-CENP-A and anti-CENP-B antibodies. The relative copy number of the total alphoid tetO array was quantitated by real-time PCR. The black line indicates the culture with medium containing doxycycline. The red line indicates the culture with doxycycline-free medium. ( D ) CENP-A preassembly levels on the total alphoid tetO DNAs, determined by the ChIP analysis. The relative copy numbers of the total alphoid tetO array recovered by the beads with the anti-CENP-A antibody or anti-CENP-B antibody, or without antibody, were quantitated by real-time PCR, and are displayed as graphs. Error bars represent the SEM (n = 3). P -values obtained with the t -test are indicated.
    Figure Legend Snippet: The CENP-A nucleosome preassembly level reaches a maximum after 24h under HJURP tethering conditions. ( A ) Schematic diagram of the CENP-A nucleosome preassembly and the ChIP real-time PCR/competitive PCR analysis. HT1080 cells stably expressing tetR-EYFP-HJURP were transfected with 50 kb alphoid tetO BAC DNAs and Halo-CENP-A expressing plasmid DNA. To generate CENP-A nucleosome preassembly on the transfected alphoid tetO DNAs by the tethering of tetR-EYFP-HJURP, the cells were cultured in doxycycline-free medium. The CENP-A nucleosome assembly levels were analyzed by a ChIP assay. The DNA samples recovered by ChIP were quantitated by real-time PCR. Then, the PCR products (competitively amplified wild type and mutant CENP-B box alphoid tetO DNAs) were digested with EcoRV and analyzed by agarose gel electrophoresis ( 43 , 67 ). ( B ) Example of a competitive PCR control. The wild type and mutant CENP-B box alphoid tetO BAC DNAs were mixed at different ratios, and amplified competitively with the same primer set by PCR. The white arrowhead indicates the PCR fragment from the wild type CENP-B box alphoid tetO DNA. The gray arrowhead indicates the PCR fragment from the mutant CENP-B box alphoid tetO DNA. ( C ) Schematic diagram of the CENP-A deposition and the transient ChIP assay. The cells transfected with the alphoid tetO BAC DNA mixture were cultured with medium containing doxycycline for 24 hr. To analyze CENP-A nucleosome deposition at tetO sites on the transfected alphoid tetO DNAs, the cells were cultured with doxycycline-free medium for 0–48 hr. The ChIP assay was then performed with anti-CENP-A and anti-CENP-B antibodies. The relative copy number of the total alphoid tetO array was quantitated by real-time PCR. The black line indicates the culture with medium containing doxycycline. The red line indicates the culture with doxycycline-free medium. ( D ) CENP-A preassembly levels on the total alphoid tetO DNAs, determined by the ChIP analysis. The relative copy numbers of the total alphoid tetO array recovered by the beads with the anti-CENP-A antibody or anti-CENP-B antibody, or without antibody, were quantitated by real-time PCR, and are displayed as graphs. Error bars represent the SEM (n = 3). P -values obtained with the t -test are indicated.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Stable Transfection, Expressing, Transfection, BAC Assay, Plasmid Preparation, Cell Culture, Amplification, Mutagenesis, Agarose Gel Electrophoresis

    8) Product Images from "Rab31 expression levels modulate tumor-relevant characteristics of breast cancer cells"

    Article Title: Rab31 expression levels modulate tumor-relevant characteristics of breast cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-11-62

    Rab31 overexpression reduces the invasive capacity of cells in vitro. Stably transfected MDA-MB-231 cells were seeded into the upper compartments of Matrigel™-coated invasion chambers. After 24 h of incubation, invaded cells were fixed, stained, and counted. At least three independent experiments were performed in triplicates each. Results are given in %, normalized to the number of invaded vector-transfected cells. Whisker box plots indicate the 25th and 75th percentile, the vertical bars indicate the 10th and 90th percentile. The median value of at least three independent experiments is indicated by a bar within the box. Statistically significant differences (p
    Figure Legend Snippet: Rab31 overexpression reduces the invasive capacity of cells in vitro. Stably transfected MDA-MB-231 cells were seeded into the upper compartments of Matrigel™-coated invasion chambers. After 24 h of incubation, invaded cells were fixed, stained, and counted. At least three independent experiments were performed in triplicates each. Results are given in %, normalized to the number of invaded vector-transfected cells. Whisker box plots indicate the 25th and 75th percentile, the vertical bars indicate the 10th and 90th percentile. The median value of at least three independent experiments is indicated by a bar within the box. Statistically significant differences (p

    Techniques Used: Over Expression, In Vitro, Stable Transfection, Transfection, Multiple Displacement Amplification, Incubation, Staining, Plasmid Preparation, Whisker Assay

    Rab31 expression levels in stably transfected breast cancer cells. (A) pAb RT3-IgG specifically detects rab31 in cell lysates from stably transfected MDA-MB-231 and CAMA-1 breast cancer cells in Western blot analyses. Vector control, MDA-MB-231, or CAMA-1 cells were stably transfected with the empty vector pRcRSV; batch, parental MDA-MB-231 (low endogenous rab31 expression) or CAMA-1 (no endogenous rab31 expression) cells were stably transfected with the pRcRSV-based rab31 expression vector; high, medium or low, selected cell clones of batch-transfected MDA-MB-231 or CAMA-1 cells with differing high, medium or low protein levels of rab31. (B) Rab31 antigen values (ng/mg of total protein) in rab31-transfected MDA-MB-231 and CAMA-1 cells, determined by rab31-ELISA. Mean values (± SEM) of at least three independent experiments are depicted.
    Figure Legend Snippet: Rab31 expression levels in stably transfected breast cancer cells. (A) pAb RT3-IgG specifically detects rab31 in cell lysates from stably transfected MDA-MB-231 and CAMA-1 breast cancer cells in Western blot analyses. Vector control, MDA-MB-231, or CAMA-1 cells were stably transfected with the empty vector pRcRSV; batch, parental MDA-MB-231 (low endogenous rab31 expression) or CAMA-1 (no endogenous rab31 expression) cells were stably transfected with the pRcRSV-based rab31 expression vector; high, medium or low, selected cell clones of batch-transfected MDA-MB-231 or CAMA-1 cells with differing high, medium or low protein levels of rab31. (B) Rab31 antigen values (ng/mg of total protein) in rab31-transfected MDA-MB-231 and CAMA-1 cells, determined by rab31-ELISA. Mean values (± SEM) of at least three independent experiments are depicted.

    Techniques Used: Expressing, Stable Transfection, Transfection, Multiple Displacement Amplification, Western Blot, Plasmid Preparation, Clone Assay, Enzyme-linked Immunosorbent Assay

    Rab31 overexpression enhances proliferation of breast cancer cells in vitro. Cells were seeded in triplicate onto 24-well plates, detached with 0.05% EDTA-solution after 24, 48, 72 and 96 h of cultivation and counted with a Neubauer-chamber under trypan blue exclusion. Cell number at 24 h was set to 100%; increase in cell number was expressed relative to the 24 h value in %. Mean values (± SEM) of at least three independent experiments are depicted. Statistically significant differences (p
    Figure Legend Snippet: Rab31 overexpression enhances proliferation of breast cancer cells in vitro. Cells were seeded in triplicate onto 24-well plates, detached with 0.05% EDTA-solution after 24, 48, 72 and 96 h of cultivation and counted with a Neubauer-chamber under trypan blue exclusion. Cell number at 24 h was set to 100%; increase in cell number was expressed relative to the 24 h value in %. Mean values (± SEM) of at least three independent experiments are depicted. Statistically significant differences (p

    Techniques Used: Over Expression, In Vitro

    Rab31 overexpression reduces the adhesive capacity of human breast cancer cells in vitro. Stably transfected MDA-MB-231 cells (A) and CAMA-1 cells (B) were seeded on collagen type IV-coated microtiter plates. After 2 h of cell cultivation, the number of adherent cells was monitored by the hexosaminidase activity assay. At least 5 independent experiments were performed in triplicates each. The results are given in % relative to the cell number of adherent vector-transfected control cells. Whisker box plots indicate the 25th and 75th percentile, the vertical bars indicate the 10th and 90th percentile. The median value of at least 5 experiments is indicated by a bar within the box. Statistically significant differences (p
    Figure Legend Snippet: Rab31 overexpression reduces the adhesive capacity of human breast cancer cells in vitro. Stably transfected MDA-MB-231 cells (A) and CAMA-1 cells (B) were seeded on collagen type IV-coated microtiter plates. After 2 h of cell cultivation, the number of adherent cells was monitored by the hexosaminidase activity assay. At least 5 independent experiments were performed in triplicates each. The results are given in % relative to the cell number of adherent vector-transfected control cells. Whisker box plots indicate the 25th and 75th percentile, the vertical bars indicate the 10th and 90th percentile. The median value of at least 5 experiments is indicated by a bar within the box. Statistically significant differences (p

    Techniques Used: Over Expression, In Vitro, Stable Transfection, Transfection, Multiple Displacement Amplification, Activity Assay, Plasmid Preparation, Whisker Assay

    Overexpression of rab31 affects lung colonization and metastatic growth of human breast cancer cells. Stably transfected, lacZ -tagged, MDA-MB-231 cells were inoculated into nude mice via tail vein injection. Mice received either vector control cells (grey column, n = 7) or batch-transfected rab31 cells (green column, n = 7). Animals were sacrificed at day 35 after injection, lungs were collected and stained with X-Gal. Metastases were counted in lungs of the mice. Whisker box plots indicate the 25th and 75th percentile, the vertical bars indicate the 10th and 90th percentile. Results are expressed as the median number of metastases. The median value is indicated by a bar within the box. Statistically significant differences (p
    Figure Legend Snippet: Overexpression of rab31 affects lung colonization and metastatic growth of human breast cancer cells. Stably transfected, lacZ -tagged, MDA-MB-231 cells were inoculated into nude mice via tail vein injection. Mice received either vector control cells (grey column, n = 7) or batch-transfected rab31 cells (green column, n = 7). Animals were sacrificed at day 35 after injection, lungs were collected and stained with X-Gal. Metastases were counted in lungs of the mice. Whisker box plots indicate the 25th and 75th percentile, the vertical bars indicate the 10th and 90th percentile. Results are expressed as the median number of metastases. The median value is indicated by a bar within the box. Statistically significant differences (p

    Techniques Used: Over Expression, Stable Transfection, Transfection, Multiple Displacement Amplification, Mouse Assay, Injection, Plasmid Preparation, Staining, Whisker Assay

    Characterization of polyclonal antibodies directed to human rab31. (A) The reaction pattern of polyclonal antibody (pAb) RT3-IgG (black) and of the pre-immune rabbit serum (gray) was analyzed by a ‘one-sided ELISA’ assay [ 19 ]. rab31-His, purified recombinant, histidine-tagged rab31, used for immunization; GST-rab31, purified recombinant GST-rab31 fusion protein; BSA, bovine serum albumin. (B) In Western blot analyses, pAb RT3-IgG strongly reacts with recombinant rab31 and not at all or only weakly with the highly homologous Rab proteins rab5 and rab22A, respectively. (C) Immunohistochemical staining of paraffin-embedded, formalin-fixed breast cancer specimens with pAb RT4-IgG. Specific immunostaining is observed in the cytoplasm as well as in the nucleus of cancer cells (upper panel, see arrow), stromal cells are stained less frequently (lower panel, see arrow).
    Figure Legend Snippet: Characterization of polyclonal antibodies directed to human rab31. (A) The reaction pattern of polyclonal antibody (pAb) RT3-IgG (black) and of the pre-immune rabbit serum (gray) was analyzed by a ‘one-sided ELISA’ assay [ 19 ]. rab31-His, purified recombinant, histidine-tagged rab31, used for immunization; GST-rab31, purified recombinant GST-rab31 fusion protein; BSA, bovine serum albumin. (B) In Western blot analyses, pAb RT3-IgG strongly reacts with recombinant rab31 and not at all or only weakly with the highly homologous Rab proteins rab5 and rab22A, respectively. (C) Immunohistochemical staining of paraffin-embedded, formalin-fixed breast cancer specimens with pAb RT4-IgG. Specific immunostaining is observed in the cytoplasm as well as in the nucleus of cancer cells (upper panel, see arrow), stromal cells are stained less frequently (lower panel, see arrow).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Western Blot, Immunohistochemistry, Staining, Immunostaining

    9) Product Images from "Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella"

    Article Title: Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200704069

    CrPKD2 is involved in mating. (A) The construct used for RNAi included 1,000 bp of the CrPKD2 cDNA (5′ UTR and first two exons) appended antisense to the corresponding genomic fragment, driven by the native promoter. AphVIII is a selectable marker gene driven by the HSP70A-RbcS2 fusion promoter ( Sizova et al., 2001 ). (B) Immunoblot analysis shows a reduced amount of CrPKD2 in RNAi transformants Ri45 and Ri46 compared with wild-type cells. An HSP70B antibody was used as a loading control ( Schroda et al., 1999 ). (C) Histogram showing the decrease in mating efficiency that occurs in RNAi transformants with reduced levels of CrPKD2. Bars represent the mean ± SEM from three independent experiments. (D) Protein tyrosine kinase activity was assayed in vitro in flagellar proteins isolated from mated gametes. Samples were incubated for 30 min and analyzed on immunoblots probed with antibodies against the phosphotyrosine residue of CrPKG (α-pTyr) or α-tubulin as loading control. Phosphorylation of CrPKG is reduced in flagellar extracts of gametes of the RNAi46 strain.
    Figure Legend Snippet: CrPKD2 is involved in mating. (A) The construct used for RNAi included 1,000 bp of the CrPKD2 cDNA (5′ UTR and first two exons) appended antisense to the corresponding genomic fragment, driven by the native promoter. AphVIII is a selectable marker gene driven by the HSP70A-RbcS2 fusion promoter ( Sizova et al., 2001 ). (B) Immunoblot analysis shows a reduced amount of CrPKD2 in RNAi transformants Ri45 and Ri46 compared with wild-type cells. An HSP70B antibody was used as a loading control ( Schroda et al., 1999 ). (C) Histogram showing the decrease in mating efficiency that occurs in RNAi transformants with reduced levels of CrPKD2. Bars represent the mean ± SEM from three independent experiments. (D) Protein tyrosine kinase activity was assayed in vitro in flagellar proteins isolated from mated gametes. Samples were incubated for 30 min and analyzed on immunoblots probed with antibodies against the phosphotyrosine residue of CrPKG (α-pTyr) or α-tubulin as loading control. Phosphorylation of CrPKG is reduced in flagellar extracts of gametes of the RNAi46 strain.

    Techniques Used: Construct, Marker, Activity Assay, In Vitro, Isolation, Incubation, Western Blot

    Flagellar CrPKD2 is cleaved. (A) Diagram of the CrPKD2 protein showing the regions used to generate the Loop1 and N- and C-terminal antibodies. The arrow marks the approximate location of the CrPKD2 cleavage site that generates fragments of 120 and 90 kD. (B) Immunoblots from whole cells (Cell) and flagella (F) of wild-type cells were probed with antibodies against Loop1 or the N or C termini of CrPKD2. The Loop1 antibody reacts with three bands (210, 120, and 90 kD; lane 1) in whole cells, but only the smaller two bands are present in flagella (lane 2). The C-terminal antibody does not react with the 120-kD N-terminal fragment (lanes 3 and 4), demonstrating that this fragment does not include the C terminus of the protein. The N-terminal antibody reacts with two bands (210 and 120 kD) in whole cells and the 120-kD form in the flagella (lanes 5 and 6). Thus, full-length CrPKD2 is cleaved into two fragments (120 and 90 kD) that are present in flagella. (C) Immunoblot of transformant expressing CrPKD2–GFP. GFP-tagging results in two bands that react with the GFP antibody (lane 1): a 240-kD band (the 210-kD full-length PKD2 + 30 kD GFP) and a 120-kD band (the 90-kD C-terminal fragment + 30 kD GFP). Only the smaller band is present in flagella (lane 2). Both bands also react with the C-PKD2 antibody (lane 3), as do the untagged CrPKD2 bands also present in these cells. (D) Immunoblots of bld1 and bld2 cells (mutants that have no flagella) and cell bodies of wild-type cells (WT CB) probed with the anti–C-PKD2 antibody. The flagellar form of CrPKD2 is present in the cell bodies, indicating that cleavage occurs in the cell body. The same blot was probed with α-tubulin as a loading control.
    Figure Legend Snippet: Flagellar CrPKD2 is cleaved. (A) Diagram of the CrPKD2 protein showing the regions used to generate the Loop1 and N- and C-terminal antibodies. The arrow marks the approximate location of the CrPKD2 cleavage site that generates fragments of 120 and 90 kD. (B) Immunoblots from whole cells (Cell) and flagella (F) of wild-type cells were probed with antibodies against Loop1 or the N or C termini of CrPKD2. The Loop1 antibody reacts with three bands (210, 120, and 90 kD; lane 1) in whole cells, but only the smaller two bands are present in flagella (lane 2). The C-terminal antibody does not react with the 120-kD N-terminal fragment (lanes 3 and 4), demonstrating that this fragment does not include the C terminus of the protein. The N-terminal antibody reacts with two bands (210 and 120 kD) in whole cells and the 120-kD form in the flagella (lanes 5 and 6). Thus, full-length CrPKD2 is cleaved into two fragments (120 and 90 kD) that are present in flagella. (C) Immunoblot of transformant expressing CrPKD2–GFP. GFP-tagging results in two bands that react with the GFP antibody (lane 1): a 240-kD band (the 210-kD full-length PKD2 + 30 kD GFP) and a 120-kD band (the 90-kD C-terminal fragment + 30 kD GFP). Only the smaller band is present in flagella (lane 2). Both bands also react with the C-PKD2 antibody (lane 3), as do the untagged CrPKD2 bands also present in these cells. (D) Immunoblots of bld1 and bld2 cells (mutants that have no flagella) and cell bodies of wild-type cells (WT CB) probed with the anti–C-PKD2 antibody. The flagellar form of CrPKD2 is present in the cell bodies, indicating that cleavage occurs in the cell body. The same blot was probed with α-tubulin as a loading control.

    Techniques Used: Western Blot, Expressing

    Flagellar CrPKD2 increases when IFT is blocked. (A) Wild-type and fla10 cells were shifted to 32° for 1 or 1.5 h, and immunoblots of isolated flagella were probed with antibodies as listed. Note the rise in CrPKD2 when IFT proteins disappear in flagella from fla10 cells. (B) Cells were treated with 20 mM NaPPi to induce flagellar resorption, and flagellar proteins were probed on immunoblots as in A. CrPKD2 did not increase in these flagella during resorption under this condition.
    Figure Legend Snippet: Flagellar CrPKD2 increases when IFT is blocked. (A) Wild-type and fla10 cells were shifted to 32° for 1 or 1.5 h, and immunoblots of isolated flagella were probed with antibodies as listed. Note the rise in CrPKD2 when IFT proteins disappear in flagella from fla10 cells. (B) Cells were treated with 20 mM NaPPi to induce flagellar resorption, and flagellar proteins were probed on immunoblots as in A. CrPKD2 did not increase in these flagella during resorption under this condition.

    Techniques Used: Western Blot, Isolation

    Some of the CrPKD2–GFP moves in the flagellar membrane. (A) Fluorescence micrograph of the flagellum used to generate the kymographs of CrPKD2–GFP. Only one flagellum was illuminated. The cell body is outlined with a dotted line. (B) Kymograph of CrPKD2–GFP generated using the video (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200704069/DC1 ). (C) The lines corresponding to those seen in the kymograph were used to measure the anterograde velocity of CrPKD2–GFP. (D) Two CrPKD2–GFP particles are shown (above the solid line) moving in the anterograde direction at ∼1.6 μm/s. The dotted lines mark particles that did not move.
    Figure Legend Snippet: Some of the CrPKD2–GFP moves in the flagellar membrane. (A) Fluorescence micrograph of the flagellum used to generate the kymographs of CrPKD2–GFP. Only one flagellum was illuminated. The cell body is outlined with a dotted line. (B) Kymograph of CrPKD2–GFP generated using the video (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200704069/DC1 ). (C) The lines corresponding to those seen in the kymograph were used to measure the anterograde velocity of CrPKD2–GFP. (D) Two CrPKD2–GFP particles are shown (above the solid line) moving in the anterograde direction at ∼1.6 μm/s. The dotted lines mark particles that did not move.

    Techniques Used: Fluorescence, Generated

    Flagellar CrPKD2 increases during gametogenesis. (A) C. reinhardtii vegetative cells (V) and gametes (G) stained with N-PKD2 or C-PKD2 (green) and acetylated α-tubulin (red) antibodies. Bars, 5 μm. (B) 10 μg of flagellar protein from CC125 (mt+) vegetative cells and cells undergoing gametic differentiation in nitrogen-deficient medium was probed on immunoblots with antibodies against the Loop1 PKD2 epitope and an intermediate chain of outer arm dynein, IC2, as a loading control. The amount of CrPKD2 in the flagella increased during the course of gametic differentiation.
    Figure Legend Snippet: Flagellar CrPKD2 increases during gametogenesis. (A) C. reinhardtii vegetative cells (V) and gametes (G) stained with N-PKD2 or C-PKD2 (green) and acetylated α-tubulin (red) antibodies. Bars, 5 μm. (B) 10 μg of flagellar protein from CC125 (mt+) vegetative cells and cells undergoing gametic differentiation in nitrogen-deficient medium was probed on immunoblots with antibodies against the Loop1 PKD2 epitope and an intermediate chain of outer arm dynein, IC2, as a loading control. The amount of CrPKD2 in the flagella increased during the course of gametic differentiation.

    Techniques Used: Staining, Western Blot

    CrPKD2 is concentrated in flagellar membranes. (A) Electron micrographs of thin sections of isolated flagellar membrane vesicles. Flagellar membranes were isolated in 0.1% NP-40 and were sedimented (top) or further purified on an Optiprep gradient (bottom). (B) Immunoblots of the proteins (2.3 μg) in flagella (F) and vesicles from A purified without (1) or with (2) an Optiprep gradient were probed for an axonemal protein radial spoke protein 1, FMG-1, Loop1 PKD2, IFT 139 (IFT complex A), and IFT172 (IFT complex B). Black lines indicate that intervening lanes have been spliced out. (C) Coomassie blue–stained gel (CB) of equal protein loadings (11 μg) of flagellar fractions and immunoblots probed with FMG-1 and Loop1 PKD2 antibodies. FMG-1 and CrPKD2 are concentrated in flagellar membrane fractions. Some CrPKD2 remains associated with the axoneme even though the FMG-1 is released from the axoneme completely. F, flagella; S, flagellar proteins solubilized by freeze–thaw; A, axoneme extracted with 0.1 or 1% NP-40; M, corresponding flagellar membrane. (D) Electron micrograph of axonemes extracted with 1% NP-40 from Fig. 4 C. Bars: (A) 50 nm; (D) 500 nm.
    Figure Legend Snippet: CrPKD2 is concentrated in flagellar membranes. (A) Electron micrographs of thin sections of isolated flagellar membrane vesicles. Flagellar membranes were isolated in 0.1% NP-40 and were sedimented (top) or further purified on an Optiprep gradient (bottom). (B) Immunoblots of the proteins (2.3 μg) in flagella (F) and vesicles from A purified without (1) or with (2) an Optiprep gradient were probed for an axonemal protein radial spoke protein 1, FMG-1, Loop1 PKD2, IFT 139 (IFT complex A), and IFT172 (IFT complex B). Black lines indicate that intervening lanes have been spliced out. (C) Coomassie blue–stained gel (CB) of equal protein loadings (11 μg) of flagellar fractions and immunoblots probed with FMG-1 and Loop1 PKD2 antibodies. FMG-1 and CrPKD2 are concentrated in flagellar membrane fractions. Some CrPKD2 remains associated with the axoneme even though the FMG-1 is released from the axoneme completely. F, flagella; S, flagellar proteins solubilized by freeze–thaw; A, axoneme extracted with 0.1 or 1% NP-40; M, corresponding flagellar membrane. (D) Electron micrograph of axonemes extracted with 1% NP-40 from Fig. 4 C. Bars: (A) 50 nm; (D) 500 nm.

    Techniques Used: Isolation, Purification, Western Blot, Staining

    CrPKD2 is a member of the TRPP2 family. (A) Secondary structure of CrPKD2. CrPKD2 contains a coiled-coil domain at the N and C termini, an EF hand domain, six transmembrane domains (numbers denote amino acid positions), and acidic amino acid cluster domains. The pore region (P) is shown, between aa 1,342–1,377, by comparison to human and D. melanogaster PKD2 ( Tsiokas et al., 1999 ; Venglarik et al., 2004 ). Not drawn to scale. (B) The EF hand domain of CrPKD2 contains the six conserved residues (coordination vertices) involved in calcium binding (positions 1, 3, 5, 7, 9, and 12).
    Figure Legend Snippet: CrPKD2 is a member of the TRPP2 family. (A) Secondary structure of CrPKD2. CrPKD2 contains a coiled-coil domain at the N and C termini, an EF hand domain, six transmembrane domains (numbers denote amino acid positions), and acidic amino acid cluster domains. The pore region (P) is shown, between aa 1,342–1,377, by comparison to human and D. melanogaster PKD2 ( Tsiokas et al., 1999 ; Venglarik et al., 2004 ). Not drawn to scale. (B) The EF hand domain of CrPKD2 contains the six conserved residues (coordination vertices) involved in calcium binding (positions 1, 3, 5, 7, 9, and 12).

    Techniques Used: Binding Assay

    CrPKD2–GFP FRAP is reduced in the flagella of fla10 cells at restrictive temperature. (A) An ∼2-μm segment of a fla10 flagellum containing CrPKD2–GFP is shown (box in DIC panel) before bleaching, after bleaching, and after 2 min of recovery. The rest of the flagellum is not visible because it is not illuminated. (B) The fluorescence in the bleached area increased in the flagellum of fla10 cells at nonrestrictive temperature after 1 and 2 min after photobleaching. In fla10 cells at restrictive temperature, recovery only reached 9.4 versus 19.3% at permissive temperature. (C) In control ( pf18 ) cells at 22 and 32°C, fluorescence recovery is similar to that of fla10 cells at room temperature. Bar graphs represent mean ± SEM (statistics determined by t test).
    Figure Legend Snippet: CrPKD2–GFP FRAP is reduced in the flagella of fla10 cells at restrictive temperature. (A) An ∼2-μm segment of a fla10 flagellum containing CrPKD2–GFP is shown (box in DIC panel) before bleaching, after bleaching, and after 2 min of recovery. The rest of the flagellum is not visible because it is not illuminated. (B) The fluorescence in the bleached area increased in the flagellum of fla10 cells at nonrestrictive temperature after 1 and 2 min after photobleaching. In fla10 cells at restrictive temperature, recovery only reached 9.4 versus 19.3% at permissive temperature. (C) In control ( pf18 ) cells at 22 and 32°C, fluorescence recovery is similar to that of fla10 cells at room temperature. Bar graphs represent mean ± SEM (statistics determined by t test).

    Techniques Used: Fluorescence

    10) Product Images from "The Regulatory Particle of the Saccharomyces cerevisiae Proteasome"

    Article Title: The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

    Journal: Molecular and Cellular Biology

    doi:

    Proteasomal ATPases associate into a heteromeric complex. His 6 -Rpt1 was expressed in a Δ rpt1 background (DY19). Extracts from His 6 -Rpt1-expressing and wild-type (WT) control strains were partially purified on DEAE–CL-6B resin in the absence of ATP. The 500 mM NaCl eluate was fractionated on Ni-NTA affinity columns. Column fractions were subjected to immunoblotting (A) and tested for peptidase activity against Suc-LLVY-AMC (B). The epitope-tagged complex eluting at 100 mM imidazole contained a number of RP subunits (Rpt1, Rpt6, and Rpn10) (A) but lacked peptidase activity (B). The wild-type complex eluted during low-imidazole rinses. (C) Extracts from strains expressing His 6 -tagged versions of each of the six ATPases were also purified by Ni-NTA chromatography. Fractions loaded onto the Ni-NTA column (Load) were compared to fractions from the 100 mM imidazole eluate (Eluate) by immunoblotting with anti-Rpt1 and anti-Rpt6 antibodies.
    Figure Legend Snippet: Proteasomal ATPases associate into a heteromeric complex. His 6 -Rpt1 was expressed in a Δ rpt1 background (DY19). Extracts from His 6 -Rpt1-expressing and wild-type (WT) control strains were partially purified on DEAE–CL-6B resin in the absence of ATP. The 500 mM NaCl eluate was fractionated on Ni-NTA affinity columns. Column fractions were subjected to immunoblotting (A) and tested for peptidase activity against Suc-LLVY-AMC (B). The epitope-tagged complex eluting at 100 mM imidazole contained a number of RP subunits (Rpt1, Rpt6, and Rpn10) (A) but lacked peptidase activity (B). The wild-type complex eluted during low-imidazole rinses. (C) Extracts from strains expressing His 6 -tagged versions of each of the six ATPases were also purified by Ni-NTA chromatography. Fractions loaded onto the Ni-NTA column (Load) were compared to fractions from the 100 mM imidazole eluate (Eluate) by immunoblotting with anti-Rpt1 and anti-Rpt6 antibodies.

    Techniques Used: Expressing, Purification, Activity Assay, Chromatography

    The proteasome is a heteromeric complex of ATPases. His 6 -Rpt2 was expressed in a Δ rpt2 background (DY17). Extracts from His 6 -Rpt2-expressing and wild-type (WT) control strains were partially purified by DEAE–Affi-Gel Blue chromatography in the presence of 1 mM Mg-ATP. The 150 mM NaCl eluate was subjected to Ni-NTA affinity chromatography. Column fractions were immunoblotted (A) and tested for peptidase activity against Suc-LLVY-AMC (B). The epitope-tagged complex eluted at 100 mM imidazole, as indicated by immunoblotting against Rpt1, Rpt6, and Rpn10 (A) and by peptidase activity (B). The wild-type proteasome eluted during low-imidazole rinses. (C) Extracts from strains expressing His 6 -tagged versions of each of the six ATPases were individually purified by Ni-NTA chromatography. Fractions loaded onto the Ni-NTA column (Load) were compared to fractions from the 100 mM imidazole eluate (Eluate) by immunoblotting with anti-Rpt1 and anti-Rpt6 antibodies.
    Figure Legend Snippet: The proteasome is a heteromeric complex of ATPases. His 6 -Rpt2 was expressed in a Δ rpt2 background (DY17). Extracts from His 6 -Rpt2-expressing and wild-type (WT) control strains were partially purified by DEAE–Affi-Gel Blue chromatography in the presence of 1 mM Mg-ATP. The 150 mM NaCl eluate was subjected to Ni-NTA affinity chromatography. Column fractions were immunoblotted (A) and tested for peptidase activity against Suc-LLVY-AMC (B). The epitope-tagged complex eluted at 100 mM imidazole, as indicated by immunoblotting against Rpt1, Rpt6, and Rpn10 (A) and by peptidase activity (B). The wild-type proteasome eluted during low-imidazole rinses. (C) Extracts from strains expressing His 6 -tagged versions of each of the six ATPases were individually purified by Ni-NTA chromatography. Fractions loaded onto the Ni-NTA column (Load) were compared to fractions from the 100 mM imidazole eluate (Eluate) by immunoblotting with anti-Rpt1 and anti-Rpt6 antibodies.

    Techniques Used: Expressing, Purification, Chromatography, Affinity Column, Activity Assay

    11) Product Images from "Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms"

    Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-9-91

    SDS-PAGE of recombinant FumF protein . Proteins were separated by 12% (w/v) SDS-PAGE and then stained with Coomassie brilliant blue G-250. Lane 1, molecular weight standards; Lane 2, total protein of E. coli BL21(DE3)pLysS harboring empty pETBlue-2 (control); Lane 3, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 without induction by IPTG; Lane 4, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 induced by addition of 0.5 mM IPTG; Lane 5, sample purified by the Ni-NTA column method. The recombinant FumF protein is indicated by the black arrow.
    Figure Legend Snippet: SDS-PAGE of recombinant FumF protein . Proteins were separated by 12% (w/v) SDS-PAGE and then stained with Coomassie brilliant blue G-250. Lane 1, molecular weight standards; Lane 2, total protein of E. coli BL21(DE3)pLysS harboring empty pETBlue-2 (control); Lane 3, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 without induction by IPTG; Lane 4, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 induced by addition of 0.5 mM IPTG; Lane 5, sample purified by the Ni-NTA column method. The recombinant FumF protein is indicated by the black arrow.

    Techniques Used: SDS Page, Recombinant, Staining, Molecular Weight, Purification

    12) Product Images from "All trans-retinoic acid analogs promote cancer cell apoptosis through non-genomic Crabp1 mediating ERK1/2 phosphorylation"

    Article Title: All trans-retinoic acid analogs promote cancer cell apoptosis through non-genomic Crabp1 mediating ERK1/2 phosphorylation

    Journal: Scientific Reports

    doi: 10.1038/srep22396

    Crabp1-ERK1/2 activation by compounds 3 and 4 stimulates PP2A in ESC. ( A ) ERK activation elicited by RA and compounds 3 and 4 for 30 min is abolished in Crabp1 KO ESC. ( B ) Semi- in vitro kinase assay. Partially immuno-purified Crabp1 complex (bottom) activates recombinant ERK1/2 in vitro under 100 nM RA, C3, and C4 treatment for 30 min. ( C ) RA and Compound 3 decrease cell viability as detected by MTT assay with 100 nM treatments for 24 hrs. ( D ) G1 phase expansion by RA and compounds 3 and 4 in WT ESC but not in Crabp1 KO ESC. Cells were treated with RA and compounds 3 and 4 at 100 nM for 12 hrs before flow cytometric analysis. Asterisk shows significance: RA (p = 0.05), C3 (p = 0.05) and C4 (p = 0.04) versus control, and mean ± S.E.M (n = 4). ( E ) WT ESCs were transfected with scrambled (control) and siCrabp1 (Crabp1 KD) followed by RA treatment (100 nM). RA-induced phosphatase activity (detected for free phosphate) is blocked by Crabp1 knockdown. Asterisk shows significance: 30min (p = 0.01), 3hr (p = 0.002), and 6hr (p = 0.004) versus WT ESC (left). RA-stimulated PP2A activation is blocked by ERK1/2 inhibitor, FR180204; 3 hr (p = 0.03) versus vehicle (center). RA, compounds 3 and 4 stimulate PP2A activity after 100 nM treatment for 3 hrs in WT ESC but not in Crabp1 KO ESC. RA (p
    Figure Legend Snippet: Crabp1-ERK1/2 activation by compounds 3 and 4 stimulates PP2A in ESC. ( A ) ERK activation elicited by RA and compounds 3 and 4 for 30 min is abolished in Crabp1 KO ESC. ( B ) Semi- in vitro kinase assay. Partially immuno-purified Crabp1 complex (bottom) activates recombinant ERK1/2 in vitro under 100 nM RA, C3, and C4 treatment for 30 min. ( C ) RA and Compound 3 decrease cell viability as detected by MTT assay with 100 nM treatments for 24 hrs. ( D ) G1 phase expansion by RA and compounds 3 and 4 in WT ESC but not in Crabp1 KO ESC. Cells were treated with RA and compounds 3 and 4 at 100 nM for 12 hrs before flow cytometric analysis. Asterisk shows significance: RA (p = 0.05), C3 (p = 0.05) and C4 (p = 0.04) versus control, and mean ± S.E.M (n = 4). ( E ) WT ESCs were transfected with scrambled (control) and siCrabp1 (Crabp1 KD) followed by RA treatment (100 nM). RA-induced phosphatase activity (detected for free phosphate) is blocked by Crabp1 knockdown. Asterisk shows significance: 30min (p = 0.01), 3hr (p = 0.002), and 6hr (p = 0.004) versus WT ESC (left). RA-stimulated PP2A activation is blocked by ERK1/2 inhibitor, FR180204; 3 hr (p = 0.03) versus vehicle (center). RA, compounds 3 and 4 stimulate PP2A activity after 100 nM treatment for 3 hrs in WT ESC but not in Crabp1 KO ESC. RA (p

    Techniques Used: Activation Assay, In Vitro, Kinase Assay, Purification, Recombinant, MTT Assay, Flow Cytometry, Transfection, Activity Assay

    RAR-independent, rapid activation of ERK1/2 by compounds 3 and 4. ( A ) Structure of compounds. ( B ) Activation of ERK1/2 in Cos-1 assessed by western blot analyses under 100 nM for 30 min. Upper band depicts ERK1 (44 kDa) and lower band depicts ERK2 (42 kDa). ( C ) Compounds 3 and 4 do not activate RAR activity as detected by RAR reporter assay performed in Cos-1 cells treated with compounds at 250 nM for 24 hrs. ( D ) Pan-RAR antagonist AGN 193109 (100 nM, 1 hr pretreatment) fails to block rapid ERK activation in Cos-1 cells. Data ( B – D ) are representative of at least 3 independent experiments. ( E ) In vitro competition assay to displace 3 H-atRA from Crabp1 by atRA, C3, and C4. Data is displayed as counts per minute (CPM). Asterisk shows significance: RA (p
    Figure Legend Snippet: RAR-independent, rapid activation of ERK1/2 by compounds 3 and 4. ( A ) Structure of compounds. ( B ) Activation of ERK1/2 in Cos-1 assessed by western blot analyses under 100 nM for 30 min. Upper band depicts ERK1 (44 kDa) and lower band depicts ERK2 (42 kDa). ( C ) Compounds 3 and 4 do not activate RAR activity as detected by RAR reporter assay performed in Cos-1 cells treated with compounds at 250 nM for 24 hrs. ( D ) Pan-RAR antagonist AGN 193109 (100 nM, 1 hr pretreatment) fails to block rapid ERK activation in Cos-1 cells. Data ( B – D ) are representative of at least 3 independent experiments. ( E ) In vitro competition assay to displace 3 H-atRA from Crabp1 by atRA, C3, and C4. Data is displayed as counts per minute (CPM). Asterisk shows significance: RA (p

    Techniques Used: Activation Assay, Western Blot, Activity Assay, Reporter Assay, Blocking Assay, In Vitro, Competitive Binding Assay

    Crabp1-dependent activation of ERK1/2 by compounds 3 and 4 requires ligand binding. ( A ) Crabp1 expression assessed on protein (left) and mRNA (right) levels. ( B ) Western blot analysis. Compounds 3 and 4 fail to activate ERK1/2 in Crabp1-negative MOVCAR at 30 min, 100 nM. ( C ) Western blot analyses of kinetics of ERK1/2 activation by compounds (100 nM). MOVCAR cells re-expressing Crabp1 (upper panels) or empty vector (EV, lower panels) were treated with vehicle, RA, compound 3 or compound 4 for the indicated hrs. Early phase of ERK1/2 activation is detected in Crabp1 expressing cells. ( D ) Rapid ERK1/2 activation in MOVCAR re-expressing wild type or mock-mutated Crabp1 Y133F but no activation in cells re-expressing Crabp1 R131A, RA binding deficient mutant at 30 min, 100 nM treatments. ( E ) 3 H-atRA ligand binding assay reveals RA binding to wild type and Crabp1 Y133F but no binding to Crabp1 R131A proteins. *p
    Figure Legend Snippet: Crabp1-dependent activation of ERK1/2 by compounds 3 and 4 requires ligand binding. ( A ) Crabp1 expression assessed on protein (left) and mRNA (right) levels. ( B ) Western blot analysis. Compounds 3 and 4 fail to activate ERK1/2 in Crabp1-negative MOVCAR at 30 min, 100 nM. ( C ) Western blot analyses of kinetics of ERK1/2 activation by compounds (100 nM). MOVCAR cells re-expressing Crabp1 (upper panels) or empty vector (EV, lower panels) were treated with vehicle, RA, compound 3 or compound 4 for the indicated hrs. Early phase of ERK1/2 activation is detected in Crabp1 expressing cells. ( D ) Rapid ERK1/2 activation in MOVCAR re-expressing wild type or mock-mutated Crabp1 Y133F but no activation in cells re-expressing Crabp1 R131A, RA binding deficient mutant at 30 min, 100 nM treatments. ( E ) 3 H-atRA ligand binding assay reveals RA binding to wild type and Crabp1 Y133F but no binding to Crabp1 R131A proteins. *p

    Techniques Used: Activation Assay, Ligand Binding Assay, Expressing, Western Blot, Plasmid Preparation, Binding Assay, Mutagenesis

    Crabp1-dependent induction of apoptosis by compounds 3 and 4. ( A ) Images showing apoptosis of Crabp1-positive MOVCAR cells (pSIVA staining, green). Cells were treated with 100 nM for 24 hrs. Quantitation of pSIVA positive cells shown on the right: *p
    Figure Legend Snippet: Crabp1-dependent induction of apoptosis by compounds 3 and 4. ( A ) Images showing apoptosis of Crabp1-positive MOVCAR cells (pSIVA staining, green). Cells were treated with 100 nM for 24 hrs. Quantitation of pSIVA positive cells shown on the right: *p

    Techniques Used: Staining, Quantitation Assay

    13) Product Images from "HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis"

    Article Title: HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2015.18

    Effect of HAX1 on BCR-induced apoptosis. Bone marrow cells and splenocytes from four Hax1 −/− and four WT mice were stimulated with anti-IgM and cultivated over a period of 4 days. B220/eFluor/annexin V staining was performed every day. ( a ) B220 + bone marrow cells and ( b ) B220 + splenic B cells were analyzed for annexin V and eFluor staining. ( c ) Comparison of the percentages of living and dead cells; (i) living and (ii) dead bone marrow cells; (iii) living and (iv) dead splenic B cells. Abbreviations: A, annexin V; eF, eFluor; A − /eF − , living cells; A + /eF − , early apoptotic cells, A + /eF + , late apoptotic, and A − /eF + , dead cells. The means ± SD are shown; the significances were calculated using the unpaired Student's t -test.
    Figure Legend Snippet: Effect of HAX1 on BCR-induced apoptosis. Bone marrow cells and splenocytes from four Hax1 −/− and four WT mice were stimulated with anti-IgM and cultivated over a period of 4 days. B220/eFluor/annexin V staining was performed every day. ( a ) B220 + bone marrow cells and ( b ) B220 + splenic B cells were analyzed for annexin V and eFluor staining. ( c ) Comparison of the percentages of living and dead cells; (i) living and (ii) dead bone marrow cells; (iii) living and (iv) dead splenic B cells. Abbreviations: A, annexin V; eF, eFluor; A − /eF − , living cells; A + /eF − , early apoptotic cells, A + /eF + , late apoptotic, and A − /eF + , dead cells. The means ± SD are shown; the significances were calculated using the unpaired Student's t -test.

    Techniques Used: Mouse Assay, Staining

    Internalization kinetics of splenic B cells. ( a ) The decrease in mean fluorescent intensities (MFI) upon IgM-FITC internalization at 37°C from three Hax1 −/− and three WT mice is shown. The MFI of time point 0 was set at 100. The numbers of the following time points were calculated accordingly. ( b ) The increase in the MFI upon IgM-pHrodo internalization at 37°C from 3 Hax1 −/− and 3 WT is shown. The MFI value of time point 0 was subtracted from the MFI of the following time points. ( c ) One representative Hax1 −/− and WT IgM-pHrodo FACS comparison is shown and indicates internalization after 90 minutes. The means ± SD are shown; the significances were calculated using the unpaired Student's t -test.
    Figure Legend Snippet: Internalization kinetics of splenic B cells. ( a ) The decrease in mean fluorescent intensities (MFI) upon IgM-FITC internalization at 37°C from three Hax1 −/− and three WT mice is shown. The MFI of time point 0 was set at 100. The numbers of the following time points were calculated accordingly. ( b ) The increase in the MFI upon IgM-pHrodo internalization at 37°C from 3 Hax1 −/− and 3 WT is shown. The MFI value of time point 0 was subtracted from the MFI of the following time points. ( c ) One representative Hax1 −/− and WT IgM-pHrodo FACS comparison is shown and indicates internalization after 90 minutes. The means ± SD are shown; the significances were calculated using the unpaired Student's t -test.

    Techniques Used: Mouse Assay, FACS

    HAX1 interaction with the cytoplasmic domains of Ig subtypes. ( a ) The transmembrane (M1) and the cytoplasmic domains (M2) of human IgE were exchanged with the mouse IgM, IgG1, IgG2a, IgE, or IgA isotype, and the resulting chimeric plasmids were stably expressed in the A20 B lymphoma cell line. ( b ) The mean fluorescent intensities (MFI) reflecting the surface expression of the chimeric proteins are shown as bar graph, and ( c ) one representative FACS histogram is shown. ( d ) HAX1 was enriched from the stably transfected A20 cell lines using protein G Sepharose, and the binding of the chimeric human IgE/mouse tail receptors was determined using the anti-human IgE antibody. ( e ) The protein lysates used for co-IP were controlled for the presence of the HAX1 protein.
    Figure Legend Snippet: HAX1 interaction with the cytoplasmic domains of Ig subtypes. ( a ) The transmembrane (M1) and the cytoplasmic domains (M2) of human IgE were exchanged with the mouse IgM, IgG1, IgG2a, IgE, or IgA isotype, and the resulting chimeric plasmids were stably expressed in the A20 B lymphoma cell line. ( b ) The mean fluorescent intensities (MFI) reflecting the surface expression of the chimeric proteins are shown as bar graph, and ( c ) one representative FACS histogram is shown. ( d ) HAX1 was enriched from the stably transfected A20 cell lines using protein G Sepharose, and the binding of the chimeric human IgE/mouse tail receptors was determined using the anti-human IgE antibody. ( e ) The protein lysates used for co-IP were controlled for the presence of the HAX1 protein.

    Techniques Used: Stable Transfection, Expressing, FACS, Transfection, Binding Assay, Co-Immunoprecipitation Assay

    Frequencies of bone marrow lymphocytes and B220 + cells. ( a ) Percentages of cells gated from live lymphocytes from four Hax1 −/− and four WT mice. ( b ) The lymphocytes were further analyzed to determine the number of B220 + cells. ( c ) One representative comparison is shown. The means ± SD are shown; the significances were calculated using the unpaired Student's t -test.
    Figure Legend Snippet: Frequencies of bone marrow lymphocytes and B220 + cells. ( a ) Percentages of cells gated from live lymphocytes from four Hax1 −/− and four WT mice. ( b ) The lymphocytes were further analyzed to determine the number of B220 + cells. ( c ) One representative comparison is shown. The means ± SD are shown; the significances were calculated using the unpaired Student's t -test.

    Techniques Used: Mouse Assay

    Rate of cell death of Hax1 −/− and WT bone marrow cells and splenocytes. ( a ) Bone marrow cells and ( b ) splenocytes from five Hax1 −/− and five WT mice were cultured over a period of 4 days without further stimuli. The percentage of dead cells was determined every day using eFluor staining. The means ± SD are shown; the significances were calculated using the unpaired Student's t -test.
    Figure Legend Snippet: Rate of cell death of Hax1 −/− and WT bone marrow cells and splenocytes. ( a ) Bone marrow cells and ( b ) splenocytes from five Hax1 −/− and five WT mice were cultured over a period of 4 days without further stimuli. The percentage of dead cells was determined every day using eFluor staining. The means ± SD are shown; the significances were calculated using the unpaired Student's t -test.

    Techniques Used: Mouse Assay, Cell Culture, Staining

    14) Product Images from "Bromodomain protein 7 interacts with PRMT5 and PRC2, and is involved in transcriptional repression of their target genes"

    Article Title: Bromodomain protein 7 interacts with PRMT5 and PRC2, and is involved in transcriptional repression of their target genes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr170

    BRD7 co-purifies with PRMT5-containing hSWI/SNF complexes. ( A ) Nuclear extracts from either control (Ctrl) HeLa S3 (lanes 1 and 2) or HeLa S3/Fl-BAF45 cells (lanes 3 and 4) were purified by affinity chromatography using anti-flag M2 agarose beads, and 5 µl of void and peak fractions was analyzed by silver staining. ( B ) Western blot analysis was performed on 15 µl of affinity-purified control HeLa S3 (lane 2) and flag-tagged hSWI/SNF complexes (lane 3) using the indicated antibodies. The input lane shows expression of hSWI–SNF subunits in 20 µg of HeLa S3/Fl-hSWI–SNF nuclear extract. ( C ) Nuclear extract (250 µg) from HeLa S3/Fl-BAF45 cells was immunoprecipitated using either preimmune (PI) (lane 2) or immune anti-CBP antibody (lane 3), and western blot analysis was conducted using the indicated antibodies. Input represents 20 µg of HeLa S3/Fl-BAF45 nuclear extract. ( D ) RIPA extract from either control HeLa S3 or HeLaS3/His-BRD7 cells were incubated with Ni–NTA agarose beads, and after extensive washing ∼15 µl of affinity-purified control HeLa S3 (lane 2) or His-BRD7 fraction (lane 3) was analyzed by western blotting as described in (B). Input shows levels of BRD7-associated proteins in 20 µg of HeLa S3 RIPA extract. ( E ) HeLa S3/His-BRD7 RIPA extract was immunoprecipitated with either PI (lane 2) or anti-CBP antibody (lane 3), and proteins were detected by western blotting using the indicated antibodies. ( F ) Approximately 250 µg of HeLa S3 RIPA extract was immunoprecipitated with PI (lane 2) and immune anti-BRD7 antibody (lane 3), and BRD7-associated proteins were detected by western blot analysis.
    Figure Legend Snippet: BRD7 co-purifies with PRMT5-containing hSWI/SNF complexes. ( A ) Nuclear extracts from either control (Ctrl) HeLa S3 (lanes 1 and 2) or HeLa S3/Fl-BAF45 cells (lanes 3 and 4) were purified by affinity chromatography using anti-flag M2 agarose beads, and 5 µl of void and peak fractions was analyzed by silver staining. ( B ) Western blot analysis was performed on 15 µl of affinity-purified control HeLa S3 (lane 2) and flag-tagged hSWI/SNF complexes (lane 3) using the indicated antibodies. The input lane shows expression of hSWI–SNF subunits in 20 µg of HeLa S3/Fl-hSWI–SNF nuclear extract. ( C ) Nuclear extract (250 µg) from HeLa S3/Fl-BAF45 cells was immunoprecipitated using either preimmune (PI) (lane 2) or immune anti-CBP antibody (lane 3), and western blot analysis was conducted using the indicated antibodies. Input represents 20 µg of HeLa S3/Fl-BAF45 nuclear extract. ( D ) RIPA extract from either control HeLa S3 or HeLaS3/His-BRD7 cells were incubated with Ni–NTA agarose beads, and after extensive washing ∼15 µl of affinity-purified control HeLa S3 (lane 2) or His-BRD7 fraction (lane 3) was analyzed by western blotting as described in (B). Input shows levels of BRD7-associated proteins in 20 µg of HeLa S3 RIPA extract. ( E ) HeLa S3/His-BRD7 RIPA extract was immunoprecipitated with either PI (lane 2) or anti-CBP antibody (lane 3), and proteins were detected by western blotting using the indicated antibodies. ( F ) Approximately 250 µg of HeLa S3 RIPA extract was immunoprecipitated with PI (lane 2) and immune anti-BRD7 antibody (lane 3), and BRD7-associated proteins were detected by western blot analysis.

    Techniques Used: Purification, Affinity Chromatography, Silver Staining, Western Blot, Affinity Purification, Expressing, Immunoprecipitation, Incubation

    BRD7 can directly interact with hSWI/SNF and PRC2 components. ( A and B ) Equal amounts (1–2 µg) of GST, GST-PAH2 or GST-BRD7 were immobilized on glutathione agarose beads, and incubated with 35 S-labeled hSWI–SNF and PRC2 subunits. After extensive washing the retained proteins were detected by autoradiography. The input lane represents 10% of the total amount of protein used in each reaction. ( C ) Similar amounts of GST, GST-PAH2 or GST-EED were bound to glutathione agarose beads before adding 35 S-labeled PRC2 subunits and BRD7. Samples were processed and detected as described in A.
    Figure Legend Snippet: BRD7 can directly interact with hSWI/SNF and PRC2 components. ( A and B ) Equal amounts (1–2 µg) of GST, GST-PAH2 or GST-BRD7 were immobilized on glutathione agarose beads, and incubated with 35 S-labeled hSWI–SNF and PRC2 subunits. After extensive washing the retained proteins were detected by autoradiography. The input lane represents 10% of the total amount of protein used in each reaction. ( C ) Similar amounts of GST, GST-PAH2 or GST-EED were bound to glutathione agarose beads before adding 35 S-labeled PRC2 subunits and BRD7. Samples were processed and detected as described in A.

    Techniques Used: Incubation, Labeling, Autoradiography

    15) Product Images from "Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP"

    Article Title: Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-016-0282-9

    Purification and characterization of the sf GFP-AhR. a Diagram of the purification procedure using Ni + -NTA column installed on FPLC AKTA prime system. Continuous line represents the absorbance of the eluate, and the peaks of the flow-through sample and purified protein ( sf GFP-AhR) are indicated. Dashed line represents conductivity of the eluate. b Detection of the purified sf GFP and sf GFP-AhR was done after SDS-PAGE separation either by blue staining or by immune blotting using anti-GFP or anti-6 × His antibodies. The protein molecular weight ladder is in the first lane (M). c Indirect ELISA for testing the purified sf GFP-AhR and sf GFP that were immobilized (10nM) on the microplate and detected by polyclonal and monoclonal anti-GFP or anti-6 × His tag antibodies (1:2000)
    Figure Legend Snippet: Purification and characterization of the sf GFP-AhR. a Diagram of the purification procedure using Ni + -NTA column installed on FPLC AKTA prime system. Continuous line represents the absorbance of the eluate, and the peaks of the flow-through sample and purified protein ( sf GFP-AhR) are indicated. Dashed line represents conductivity of the eluate. b Detection of the purified sf GFP and sf GFP-AhR was done after SDS-PAGE separation either by blue staining or by immune blotting using anti-GFP or anti-6 × His antibodies. The protein molecular weight ladder is in the first lane (M). c Indirect ELISA for testing the purified sf GFP-AhR and sf GFP that were immobilized (10nM) on the microplate and detected by polyclonal and monoclonal anti-GFP or anti-6 × His tag antibodies (1:2000)

    Techniques Used: Purification, Fast Protein Liquid Chromatography, Flow Cytometry, SDS Page, Staining, Molecular Weight, Indirect ELISA

    Testing the interaction of the sf GFP-AhR with the TCDD by ELISA Indirect ELISA was performed using TCDD-pre-coated microplate. Anti-TCDD, sfGFP-AhR (100 nM) and sfGFP (100 nM) were added to the wells and the interaction was detected either by an anti-6 × His ( a ) or anti-GFP ( b ) antibodies. The graphical inset in each panel explains the principle of the detection method
    Figure Legend Snippet: Testing the interaction of the sf GFP-AhR with the TCDD by ELISA Indirect ELISA was performed using TCDD-pre-coated microplate. Anti-TCDD, sfGFP-AhR (100 nM) and sfGFP (100 nM) were added to the wells and the interaction was detected either by an anti-6 × His ( a ) or anti-GFP ( b ) antibodies. The graphical inset in each panel explains the principle of the detection method

    Techniques Used: Enzyme-linked Immunosorbent Assay, Indirect ELISA

    TCDD detection by the sf GFP-AhR-based competitive ELISA. a Optimal concentration of the sf GFP-AhR for the assay was determined by indirect ELISA using serial logarithmic concentrations (nM) for incubation in TCDD-pre-coated wells. b Competitive ELISA was performed on serial logarithmic concentrations (ppt) of free TCDD, which were incubated with the rabbit-a-TCDD or the sf GFP-AhR (30 nM) before being transferred into the TCDD-pre-coated wells. The detection of bound sf GFP-AhR in both types of ELISA was performed using a rabbit anti-GFP, and both rabbit sera were detected with a goat anti-rabbit-HRP. The logarithmic fit equation and the accuracy (R 2 ) are shown next to each curve
    Figure Legend Snippet: TCDD detection by the sf GFP-AhR-based competitive ELISA. a Optimal concentration of the sf GFP-AhR for the assay was determined by indirect ELISA using serial logarithmic concentrations (nM) for incubation in TCDD-pre-coated wells. b Competitive ELISA was performed on serial logarithmic concentrations (ppt) of free TCDD, which were incubated with the rabbit-a-TCDD or the sf GFP-AhR (30 nM) before being transferred into the TCDD-pre-coated wells. The detection of bound sf GFP-AhR in both types of ELISA was performed using a rabbit anti-GFP, and both rabbit sera were detected with a goat anti-rabbit-HRP. The logarithmic fit equation and the accuracy (R 2 ) are shown next to each curve

    Techniques Used: Competitive ELISA, Concentration Assay, Indirect ELISA, Incubation, Enzyme-linked Immunosorbent Assay

    Designing of the sf GFP-AhR fusion protein. a Schematic representation of the hAhR gene and the two recombinant proteins; sfGFP-AhR and sfGFP, used in this study. The different domains of the AhR gene are shown and the cloned LBD domain (231–428 aa) is indicated. The theoretical molecular size (kDa) and molecular weight (pMoles/μg) are shown to the right of each recombinant construct. Positions of the different elements; 6 × His tag, GGSSSG linker and glycosylation sites, are indicated using specific symbols ●, ♦ and *, respectively. b Cartoon representation of the modelled 3D structure of the sfGFP-AhR fusion, where TCDD binding cavity and the N-terminal 6 × His tag are shown. Structure simulation was predicted using Phyre2 server [ 47 ]
    Figure Legend Snippet: Designing of the sf GFP-AhR fusion protein. a Schematic representation of the hAhR gene and the two recombinant proteins; sfGFP-AhR and sfGFP, used in this study. The different domains of the AhR gene are shown and the cloned LBD domain (231–428 aa) is indicated. The theoretical molecular size (kDa) and molecular weight (pMoles/μg) are shown to the right of each recombinant construct. Positions of the different elements; 6 × His tag, GGSSSG linker and glycosylation sites, are indicated using specific symbols ●, ♦ and *, respectively. b Cartoon representation of the modelled 3D structure of the sfGFP-AhR fusion, where TCDD binding cavity and the N-terminal 6 × His tag are shown. Structure simulation was predicted using Phyre2 server [ 47 ]

    Techniques Used: Recombinant, Clone Assay, Molecular Weight, Construct, Binding Assay

    Construction of the pRSET- sf GFP-AhR plasmid. a PCR amplification was performed on the cDNA of HepG2 cells and the products were separated on a 1 % agarose gel; a fragment (265 bp) of actin gene as a control (lane 1), small domain from the AhR gene using internal primers (lane 2) and the full-length of LBD (lane 3). Expected sizes of the amplified bands are shown to the right. b 1 % agarose gel containing the pRSET- sf GFP-AhR (lanes 1 2) and pRSET- sf GFP (lanes 3 4); undigested (lanes 1 3) or digested with Bam HI/ Eco RI restriction enzymes (lanes 2 4). The extracted AhR insert is indicated with an arrow to the right
    Figure Legend Snippet: Construction of the pRSET- sf GFP-AhR plasmid. a PCR amplification was performed on the cDNA of HepG2 cells and the products were separated on a 1 % agarose gel; a fragment (265 bp) of actin gene as a control (lane 1), small domain from the AhR gene using internal primers (lane 2) and the full-length of LBD (lane 3). Expected sizes of the amplified bands are shown to the right. b 1 % agarose gel containing the pRSET- sf GFP-AhR (lanes 1 2) and pRSET- sf GFP (lanes 3 4); undigested (lanes 1 3) or digested with Bam HI/ Eco RI restriction enzymes (lanes 2 4). The extracted AhR insert is indicated with an arrow to the right

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    Optimization of the in-vitro sf GFP-AhR refolding. a Several additives were added at their indicated optimal concentration to solubilize a pellet (0.1 mg in 1 ml) of sf GFP-AhR inclusion bodies (soluble fraction). Remaining insoluble precipitates were recovered by centrifugation and solubilized in 8 M urea (insoluble fraction). Serial molar concentrations of Lauroylsarcosine ( b ) and Arginine ( c ) used for solubilizing the pellet of sf GFP-AhR inclusion bodies. The RFU was measured (EX: 485/EM: 538) in soluble and insoluble fractions for each additive and for the different concentrations of the last two additives
    Figure Legend Snippet: Optimization of the in-vitro sf GFP-AhR refolding. a Several additives were added at their indicated optimal concentration to solubilize a pellet (0.1 mg in 1 ml) of sf GFP-AhR inclusion bodies (soluble fraction). Remaining insoluble precipitates were recovered by centrifugation and solubilized in 8 M urea (insoluble fraction). Serial molar concentrations of Lauroylsarcosine ( b ) and Arginine ( c ) used for solubilizing the pellet of sf GFP-AhR inclusion bodies. The RFU was measured (EX: 485/EM: 538) in soluble and insoluble fractions for each additive and for the different concentrations of the last two additives

    Techniques Used: In Vitro, Concentration Assay, Centrifugation

    Expression of the fluorescent sf GFP-AhR. a SDS-PAGE (acrylamide 12 %) separation of protein samples obtained after the expression of the sf GFP-AhR, showing total cell extract before (lane 1) and after 16 h (lane 2) of IPTG induction, soluble fraction (lane 3) and the lysate of the inclusion bodies in 8 M urea (lane 4). Detection of migrated proteins was done by a coomassie blue staining. b Fluorescence of serial concentrations of the sf GFP and sf GFP-AhR was measured at the wavelength 485 nm for excitation (EX) and 538 nm for emission (EM). The values were expressed as a relative fluorescent unit (RFU) and the accuracy is shown next to each curve (R 2 ). (Inset) Fluorescence spectra of the different proteins (30 μg/ml) and the fluorophore fluorescein (1 μg/ml) were determined by measuring RFU at available pairs of wavelengths on the Fluoroskan Ascent® microplate reader. Blank conditions represent the fluorescence of PBS-containing wells
    Figure Legend Snippet: Expression of the fluorescent sf GFP-AhR. a SDS-PAGE (acrylamide 12 %) separation of protein samples obtained after the expression of the sf GFP-AhR, showing total cell extract before (lane 1) and after 16 h (lane 2) of IPTG induction, soluble fraction (lane 3) and the lysate of the inclusion bodies in 8 M urea (lane 4). Detection of migrated proteins was done by a coomassie blue staining. b Fluorescence of serial concentrations of the sf GFP and sf GFP-AhR was measured at the wavelength 485 nm for excitation (EX) and 538 nm for emission (EM). The values were expressed as a relative fluorescent unit (RFU) and the accuracy is shown next to each curve (R 2 ). (Inset) Fluorescence spectra of the different proteins (30 μg/ml) and the fluorophore fluorescein (1 μg/ml) were determined by measuring RFU at available pairs of wavelengths on the Fluoroskan Ascent® microplate reader. Blank conditions represent the fluorescence of PBS-containing wells

    Techniques Used: Expressing, SDS Page, Staining, Fluorescence

    16) Product Images from "Importance of the Cyanobacterial Gun4 Protein for Chlorophyll Metabolism and Assembly of Photosynthetic Complexes *"

    Article Title: Importance of the Cyanobacterial Gun4 Protein for Chlorophyll Metabolism and Assembly of Photosynthetic Complexes *

    Journal:

    doi: 10.1074/jbc.M803787200

    Analysis of membrane protein complexes and their assembly in wild-type and gun4 mutant cells. A , membrane protein complexes from wild-type and gun4 mutant labeled in vivo with 35 [S]Met/Cys were separated in 4–16% BN gel as described under
    Figure Legend Snippet: Analysis of membrane protein complexes and their assembly in wild-type and gun4 mutant cells. A , membrane protein complexes from wild-type and gun4 mutant labeled in vivo with 35 [S]Met/Cys were separated in 4–16% BN gel as described under

    Techniques Used: Mutagenesis, Labeling, In Vivo

    Transmission electron microscopy of Synechocystis 6803 cells. A , wild-type cells grown aerobically under medium light conditions. B, gun4 mutant cells grown aerobically under medium light intensity. C, gun4 mutant cells grown aerobically under low
    Figure Legend Snippet: Transmission electron microscopy of Synechocystis 6803 cells. A , wild-type cells grown aerobically under medium light conditions. B, gun4 mutant cells grown aerobically under medium light intensity. C, gun4 mutant cells grown aerobically under low

    Techniques Used: Transmission Assay, Electron Microscopy, Mutagenesis

    Localization, quantification, and activities of both chelatases. A , subcellular localization of Gun4, ferrochelatase ( FeCH ), and the magnesium chelatase ChlH and ChlD subunits. Membranes have been washed 2 (m2) or 3 (m3) times in thylakoid buffer followed
    Figure Legend Snippet: Localization, quantification, and activities of both chelatases. A , subcellular localization of Gun4, ferrochelatase ( FeCH ), and the magnesium chelatase ChlH and ChlD subunits. Membranes have been washed 2 (m2) or 3 (m3) times in thylakoid buffer followed

    Techniques Used:

    Phenotype of gun4 mutant cells and affinity purification of Gun4. A , absorption spectra of Synechocystis 6803 wild-type ( WT ) and mutant cells lacking Gun4 ( gun4 – ) or expressing a FLAG-tagged version of Gun4 (Δ gun4 :gun4FLAG). Cells
    Figure Legend Snippet: Phenotype of gun4 mutant cells and affinity purification of Gun4. A , absorption spectra of Synechocystis 6803 wild-type ( WT ) and mutant cells lacking Gun4 ( gun4 – ) or expressing a FLAG-tagged version of Gun4 (Δ gun4 :gun4FLAG). Cells

    Techniques Used: Mutagenesis, Affinity Purification, Expressing

    Inactivation of the gun4 gene in Synechocystis 6803 (GT strain). Wild-type and mutant gene copies were amplified using total chromosomal DNA from wild-type and gun4 – strains as templates. PCR amplification of the gun4 gene yielded a correct
    Figure Legend Snippet: Inactivation of the gun4 gene in Synechocystis 6803 (GT strain). Wild-type and mutant gene copies were amplified using total chromosomal DNA from wild-type and gun4 – strains as templates. PCR amplification of the gun4 gene yielded a correct

    Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction

    Semiquantitative immunoblot analysis of PSII and PSI subunits in wild type and gun4 mutant. Wild-type and gun4 mutant membrane fractions were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Different amounts of Chl corresponding
    Figure Legend Snippet: Semiquantitative immunoblot analysis of PSII and PSI subunits in wild type and gun4 mutant. Wild-type and gun4 mutant membrane fractions were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Different amounts of Chl corresponding

    Techniques Used: Mutagenesis, SDS Page

    17) Product Images from "Importance of the Cyanobacterial Gun4 Protein for Chlorophyll Metabolism and Assembly of Photosynthetic Complexes *"

    Article Title: Importance of the Cyanobacterial Gun4 Protein for Chlorophyll Metabolism and Assembly of Photosynthetic Complexes *

    Journal:

    doi: 10.1074/jbc.M803787200

    Localization, quantification, and activities of both chelatases. A , subcellular localization of Gun4, ferrochelatase ( FeCH ), and the magnesium chelatase ChlH and ChlD subunits. Membranes have been washed 2 (m2) or 3 (m3) times in thylakoid buffer followed
    Figure Legend Snippet: Localization, quantification, and activities of both chelatases. A , subcellular localization of Gun4, ferrochelatase ( FeCH ), and the magnesium chelatase ChlH and ChlD subunits. Membranes have been washed 2 (m2) or 3 (m3) times in thylakoid buffer followed

    Techniques Used:

    18) Product Images from "Redundant Cooperative Interactions for Assembly of a Human U6 Transcription Initiation Complex"

    Article Title: Redundant Cooperative Interactions for Assembly of a Human U6 Transcription Initiation Complex

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.22.8067-8078.2002

    Composition of various SNAP c subcomplexes. (A) Map of the known subunit-subunit interactions within SNAP c . Within SNAP190, aa 84 to 133 associate with SNAP19 and aa 164 to 268 of SNAP43, aa 263 to 503 correspond to the Myb domain with the Rh, Ra, Rb, Rc, and Rd repeats, aa 869 to 912 correspond to the Oct-1-interacting region (OIR), and aa 1281 to 1393 associate with SNAP45. Within SNAP43, aa 1 to 164 associate with SNAP50. Mini-SNAP c contains SNAP190 aa 1 to 514, SNAP43, and SNAP50, as outlined in red. (B) The composition of each stmSNAP c is indicated. All stmSNAP c s including stmSNAP c #23-26 contain a SNAP190 protein truncated after aa 505.
    Figure Legend Snippet: Composition of various SNAP c subcomplexes. (A) Map of the known subunit-subunit interactions within SNAP c . Within SNAP190, aa 84 to 133 associate with SNAP19 and aa 164 to 268 of SNAP43, aa 263 to 503 correspond to the Myb domain with the Rh, Ra, Rb, Rc, and Rd repeats, aa 869 to 912 correspond to the Oct-1-interacting region (OIR), and aa 1281 to 1393 associate with SNAP45. Within SNAP43, aa 1 to 164 associate with SNAP50. Mini-SNAP c contains SNAP190 aa 1 to 514, SNAP43, and SNAP50, as outlined in red. (B) The composition of each stmSNAP c is indicated. All stmSNAP c s including stmSNAP c #23-26 contain a SNAP190 protein truncated after aa 505.

    Techniques Used:

    19) Product Images from "Transcription factor Sp2 potentiates binding of the TALE homeoproteins Pbx1:Prep1 and the histone-fold domain protein Nf-y to composite genomic sites"

    Article Title: Transcription factor Sp2 potentiates binding of the TALE homeoproteins Pbx1:Prep1 and the histone-fold domain protein Nf-y to composite genomic sites

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.005341

    Sp2 binds directly to DNA-bound Pbx1:Prep1 via its N-terminal domain. A , sequence of the biotinylated double-stranded oligonucleotide used in DAPA. B , recombinant Sp2(1–525), Sp2(94–525), Pbx1:Prep1, and Nf-ya:b:c were incubated with the biotinylated double-stranded oligonucleotide as indicated. Bound proteins were detected by Western blotting. IN indicates input 10%; C indicates nonspecific binding control (magnetic beads); and P indicates probe (biotinylated oligonucleotide bound to magnetic beads). C , model diagram depicting the recruitment of Sp2 to its target promoters. Pbx1:Prep1 and Nf-ya:b:c bind to composite motifs where the Pbx1:Prep1 and Nf-y recognition sequences are separated by 11 bp. Sp2 interacts directly with Pbx1:Prep1 via its Sp-box and bridges Pbx1:Prep1 with Nf-y.
    Figure Legend Snippet: Sp2 binds directly to DNA-bound Pbx1:Prep1 via its N-terminal domain. A , sequence of the biotinylated double-stranded oligonucleotide used in DAPA. B , recombinant Sp2(1–525), Sp2(94–525), Pbx1:Prep1, and Nf-ya:b:c were incubated with the biotinylated double-stranded oligonucleotide as indicated. Bound proteins were detected by Western blotting. IN indicates input 10%; C indicates nonspecific binding control (magnetic beads); and P indicates probe (biotinylated oligonucleotide bound to magnetic beads). C , model diagram depicting the recruitment of Sp2 to its target promoters. Pbx1:Prep1 and Nf-ya:b:c bind to composite motifs where the Pbx1:Prep1 and Nf-y recognition sequences are separated by 11 bp. Sp2 interacts directly with Pbx1:Prep1 via its Sp-box and bridges Pbx1:Prep1 with Nf-y.

    Techniques Used: Sequencing, Recombinant, Incubation, Western Blot, Binding Assay, Magnetic Beads

    20) Product Images from "Crystal Structure of ?-Barrel Assembly Machinery BamCD Protein Complex *"

    Article Title: Crystal Structure of ?-Barrel Assembly Machinery BamCD Protein Complex *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.298166

    Structure of the BamCD complex and conformational changes in BamD upon binding. a , domain organization of BamC and BamD. Each TPR motif of BamD is shown in a different color. b , structure of the BamCD complex. Ribbon ( upper ) and surface ( lower ) diagrams
    Figure Legend Snippet: Structure of the BamCD complex and conformational changes in BamD upon binding. a , domain organization of BamC and BamD. Each TPR motif of BamD is shown in a different color. b , structure of the BamCD complex. Ribbon ( upper ) and surface ( lower ) diagrams

    Techniques Used: Binding Assay

    BamC-BamD interface. a , ribbon diagram of BamC ( red ) with ribbon and semitransparent surface diagrams of BamD ( gray ). The interfacing residues are colored yellow (BamC) and purple (BamD). b , conserved residues mapped onto BamD with absolutely conserved
    Figure Legend Snippet: BamC-BamD interface. a , ribbon diagram of BamC ( red ) with ribbon and semitransparent surface diagrams of BamD ( gray ). The interfacing residues are colored yellow (BamC) and purple (BamD). b , conserved residues mapped onto BamD with absolutely conserved

    Techniques Used:

    21) Product Images from "Arabidopsis Membrane Steroid Binding Protein 1 Is Involved in Inhibition of Cell Elongation W⃞"

    Article Title: Arabidopsis Membrane Steroid Binding Protein 1 Is Involved in Inhibition of Cell Elongation W⃞

    Journal: The Plant Cell

    doi: 10.1105/tpc.104.028381

    Steoid Binding Assays for MSBP1. (A) Expression and purification of recombinant MSBP1. M. mass, molecular mass marker; lanes 1, 2, and 3, uninduced or induced crude extract and purified recombinant control proteins (AtIPK2α); lanes 5, 6, and 7, uninduced or induced crude extract and purified MSBP1. Induction was performed with the supplementation of IPTG (1 mM in final concentration) for 2.5 h. Arrow shows the position of recombinant MSBP1. (B) Recombinant MSBP1 binds progesterone in vitro. The K d value and B max were calculated from data of three independent experiments. AtIPK2α, which was used as control protein, showed no binding to labeled progesterone. (C) Binding of MSBP1 to [ 3 H]-progesterone was competitively inhibited by unlabeled progesterone or steroid molecules 5α-DHT, 24-eBL, and stigmasterol but not by ABA. IC 50 showed the different binding affinity of MSBP1 to various steroids.
    Figure Legend Snippet: Steoid Binding Assays for MSBP1. (A) Expression and purification of recombinant MSBP1. M. mass, molecular mass marker; lanes 1, 2, and 3, uninduced or induced crude extract and purified recombinant control proteins (AtIPK2α); lanes 5, 6, and 7, uninduced or induced crude extract and purified MSBP1. Induction was performed with the supplementation of IPTG (1 mM in final concentration) for 2.5 h. Arrow shows the position of recombinant MSBP1. (B) Recombinant MSBP1 binds progesterone in vitro. The K d value and B max were calculated from data of three independent experiments. AtIPK2α, which was used as control protein, showed no binding to labeled progesterone. (C) Binding of MSBP1 to [ 3 H]-progesterone was competitively inhibited by unlabeled progesterone or steroid molecules 5α-DHT, 24-eBL, and stigmasterol but not by ABA. IC 50 showed the different binding affinity of MSBP1 to various steroids.

    Techniques Used: Binding Assay, Expressing, Purification, Recombinant, Marker, Concentration Assay, In Vitro, Labeling

    Expression Pattern Analysis of MSBP1 in Various Tissues and Differential Expression of which under Light and Darkness. (A) Semiquantitative RT-PCR analysis revealed constitutive expression of MSBP1 in various tissues, including cotyledon, stem, root, leaf, and flower. Actin2 was used as a positive internal control. (B) Promoter-reporter gene fusion studies indicated that MSBP1 was expressed in Arabidopsis seedlings and root tip (a), leaf (b), stem (c), petal (d), base of flower (e), and stigma (f). (C) Semiquantitative RT-PCR analysis revealed that expression of MSBP1 was not altered after treatment of seedlings with osmotic stresses and plant hormones, including auxin, BR, gibberellin, ABA, and cytokinin, but was severely suppressed in darkness (top panel). Detailed studies of MSBP1 expression indicated normal transcript levels under light and very low levels in darkness and induction when seedlings were transferred to light. By contrast, MSBP1 expression was suppressed when seedlings were transferred from light to darkness (bottom panel). M, DNA marker; C, untreated control; L, light; D, darkness; L-D, the seedlings were transferred from light to darkness for 24, 48, and 72 h; D-L, the seedlings were transferred from darkness to light for 24, 48, and 72 h. The Actin2 gene was used as a positive internal control. (D) Differential expression of MSBP1 -GUS in hypocotyls under dark and light conditions. After 2 d of germination, MSBP1 -GUS was strongly expressed in light and obviously suppressed by dark. Bar = 1 mm. (E) MSBP1 expression was not detected in hypocotyls after germination for 5, 6, and 7 d under darkness but was induced by transferring 4-d-old dark-grown seedlings into the light condition for 1 (Day 5, Dark→light), 2 (Day 6, Dark→light), and 3 d (Day 7, Dark→light). By contrast, expression of MSBP1 (L) was suppressed when 4-d-old light-grown seedlings were transferred to darkness for 1 (Day 5, Light→dark), 2 (Day 6, Light→dark), and 3 d (Day 7, Light→dark). Bar = 1 mm. (F) Expression of MSBP1-GUS under various light conditions (continuous blue light, red light, and far-red light). Seedlings were grown in each light condition for 6 d.
    Figure Legend Snippet: Expression Pattern Analysis of MSBP1 in Various Tissues and Differential Expression of which under Light and Darkness. (A) Semiquantitative RT-PCR analysis revealed constitutive expression of MSBP1 in various tissues, including cotyledon, stem, root, leaf, and flower. Actin2 was used as a positive internal control. (B) Promoter-reporter gene fusion studies indicated that MSBP1 was expressed in Arabidopsis seedlings and root tip (a), leaf (b), stem (c), petal (d), base of flower (e), and stigma (f). (C) Semiquantitative RT-PCR analysis revealed that expression of MSBP1 was not altered after treatment of seedlings with osmotic stresses and plant hormones, including auxin, BR, gibberellin, ABA, and cytokinin, but was severely suppressed in darkness (top panel). Detailed studies of MSBP1 expression indicated normal transcript levels under light and very low levels in darkness and induction when seedlings were transferred to light. By contrast, MSBP1 expression was suppressed when seedlings were transferred from light to darkness (bottom panel). M, DNA marker; C, untreated control; L, light; D, darkness; L-D, the seedlings were transferred from light to darkness for 24, 48, and 72 h; D-L, the seedlings were transferred from darkness to light for 24, 48, and 72 h. The Actin2 gene was used as a positive internal control. (D) Differential expression of MSBP1 -GUS in hypocotyls under dark and light conditions. After 2 d of germination, MSBP1 -GUS was strongly expressed in light and obviously suppressed by dark. Bar = 1 mm. (E) MSBP1 expression was not detected in hypocotyls after germination for 5, 6, and 7 d under darkness but was induced by transferring 4-d-old dark-grown seedlings into the light condition for 1 (Day 5, Dark→light), 2 (Day 6, Dark→light), and 3 d (Day 7, Dark→light). By contrast, expression of MSBP1 (L) was suppressed when 4-d-old light-grown seedlings were transferred to darkness for 1 (Day 5, Light→dark), 2 (Day 6, Light→dark), and 3 d (Day 7, Light→dark). Bar = 1 mm. (F) Expression of MSBP1-GUS under various light conditions (continuous blue light, red light, and far-red light). Seedlings were grown in each light condition for 6 d.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Transferring

    MSBP1 Localizes to the Plasma Membrane. Green fluorescence in transgenic plants harboring mock vector (CaMV35S:mGFP5) shows that mGFP5 is ubiquitously distributed in the cell cytoplasm ( [A] , hypocotyl cells; [C] , root-tip cells), whereas that in transgenic plants expressing CaMV35S:MSBP1:mGFP5 is localized to the plasma membrane ( [B] , hypocotyl cells; [D] , root-tip cells). Bars = 20 μm.
    Figure Legend Snippet: MSBP1 Localizes to the Plasma Membrane. Green fluorescence in transgenic plants harboring mock vector (CaMV35S:mGFP5) shows that mGFP5 is ubiquitously distributed in the cell cytoplasm ( [A] , hypocotyl cells; [C] , root-tip cells), whereas that in transgenic plants expressing CaMV35S:MSBP1:mGFP5 is localized to the plasma membrane ( [B] , hypocotyl cells; [D] , root-tip cells). Bars = 20 μm.

    Techniques Used: Fluorescence, Transgenic Assay, Plasmid Preparation, Expressing

    22) Product Images from "Arginine Residues on the Opposite Side of the Active Site Stimulate the Catalysis of Ribosome Depurination by Ricin A Chain by Interacting with the P-protein Stalk *"

    Article Title: Arginine Residues on the Opposite Side of the Active Site Stimulate the Catalysis of Ribosome Depurination by Ricin A Chain by Interacting with the P-protein Stalk *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.510966

    Interaction of RTA variants with the ribosomal stalk pentamer and with monomeric ribosomes. A , N-terminal His 10 -tagged RTA variants were captured on an NTA chip at 2300 RU, and the same amount of EGFP was captured on the reference channel as control.
    Figure Legend Snippet: Interaction of RTA variants with the ribosomal stalk pentamer and with monomeric ribosomes. A , N-terminal His 10 -tagged RTA variants were captured on an NTA chip at 2300 RU, and the same amount of EGFP was captured on the reference channel as control.

    Techniques Used: Chromatin Immunoprecipitation

    23) Product Images from "?B-Ras Is a Nuclear-Cytoplasmic Small GTPase That Inhibits NF-?B Activation through the Suppression of Transcriptional Activation of p65/RelA *"

    Article Title: ?B-Ras Is a Nuclear-Cytoplasmic Small GTPase That Inhibits NF-?B Activation through the Suppression of Transcriptional Activation of p65/RelA *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.117028

    Effect of κB-Ras on cytokine-induced NF-κB activation. A , HEK293T cells transfected with pNF-κB-luciferase plus FLAG-κB-Ras2 or T18N were stimulated with TNFα, and luciferase activity was measured. To verify the protein expression, immunoblot analysis was performed for κB-Ras2 and T18N mutant. B , HEK293T cells seeded on a 60-mm dish were transfected with κB-Ras2 (0.1 or 0.3 μg) or T18N (1 μg). Cells expressing κB-Ras2 (0.1 or 0.3 mg) or T18N were stimulated with TNFα. RT-PCR analysis for IL-8 and GAPDH was performed. The expression level of κB-Ras2 and T18N mutant was evaluated by immunoblot analysis. In the experiments in C and D , NIH-3T3 cells that stably harbor κB-responsive luciferase gene (KF-8 cells) were utilized. C , KF-8 cells were infected with retrovirus harboring shRNA for luciferase ( sh-Luc ) or murine κB-Ras2 ( sh -κ B-Ras2 ). Six days later, cells were harvested for immunoblot analysis to test the expressions of κB-Ras2, IκBα, IκBβ, and β-actin. D , KF-8 cells stably harboring the κB-luciferase gene were infected with control virus (sh-luciferase) or sh-κB-Ras2 virus. Cells were stimulated with TNFα (10 ng/ml) for 12 h. Cells were harvested, and luciferase activity was measured. In each experiment, error bars = S.D. ( n = 3; *, p
    Figure Legend Snippet: Effect of κB-Ras on cytokine-induced NF-κB activation. A , HEK293T cells transfected with pNF-κB-luciferase plus FLAG-κB-Ras2 or T18N were stimulated with TNFα, and luciferase activity was measured. To verify the protein expression, immunoblot analysis was performed for κB-Ras2 and T18N mutant. B , HEK293T cells seeded on a 60-mm dish were transfected with κB-Ras2 (0.1 or 0.3 μg) or T18N (1 μg). Cells expressing κB-Ras2 (0.1 or 0.3 mg) or T18N were stimulated with TNFα. RT-PCR analysis for IL-8 and GAPDH was performed. The expression level of κB-Ras2 and T18N mutant was evaluated by immunoblot analysis. In the experiments in C and D , NIH-3T3 cells that stably harbor κB-responsive luciferase gene (KF-8 cells) were utilized. C , KF-8 cells were infected with retrovirus harboring shRNA for luciferase ( sh-Luc ) or murine κB-Ras2 ( sh -κ B-Ras2 ). Six days later, cells were harvested for immunoblot analysis to test the expressions of κB-Ras2, IκBα, IκBβ, and β-actin. D , KF-8 cells stably harboring the κB-luciferase gene were infected with control virus (sh-luciferase) or sh-κB-Ras2 virus. Cells were stimulated with TNFα (10 ng/ml) for 12 h. Cells were harvested, and luciferase activity was measured. In each experiment, error bars = S.D. ( n = 3; *, p

    Techniques Used: Activation Assay, Transfection, Luciferase, Activity Assay, Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Infection, shRNA

    Effect of κB-Ras on TNFα-induced IκB degradation. A , FLAG-κB-Ras2 and T18N were ectopically expressed in HEK293T cells. Forty eight hours later, cells were stimulated with/without TNFα (10 ng/ml) for the indicated periods, and cells were then lysed with lysis buffer. Obtained lysates were analyzed by immunoblot analysis using anti-IκBα, IκBβ, and β-actin antibodies. CTL , control. To detect overexpressed κB-Ras2, anti-FLAG (M2) antibody was utilized. The degradation of IκBα and IκBβ was normalized with the protein of β-actin, and the quantified ratios of IκBα and IκBβ are shown in B and C , respectively. In each experiment, error bars = S.D. ( n = 3; *, p
    Figure Legend Snippet: Effect of κB-Ras on TNFα-induced IκB degradation. A , FLAG-κB-Ras2 and T18N were ectopically expressed in HEK293T cells. Forty eight hours later, cells were stimulated with/without TNFα (10 ng/ml) for the indicated periods, and cells were then lysed with lysis buffer. Obtained lysates were analyzed by immunoblot analysis using anti-IκBα, IκBβ, and β-actin antibodies. CTL , control. To detect overexpressed κB-Ras2, anti-FLAG (M2) antibody was utilized. The degradation of IκBα and IκBβ was normalized with the protein of β-actin, and the quantified ratios of IκBα and IκBβ are shown in B and C , respectively. In each experiment, error bars = S.D. ( n = 3; *, p

    Techniques Used: Lysis, CTL Assay

    24) Product Images from "Functional Characterization and Novel Rickettsiostatic Effects of a Kunitz-Type Serine Protease Inhibitor from the Tick Dermacentor variabilis ▿"

    Article Title: Functional Characterization and Novel Rickettsiostatic Effects of a Kunitz-Type Serine Protease Inhibitor from the Tick Dermacentor variabilis ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00866-08

    D. variabilis KPI limits rickettsial colonization of host cells. (A) Rickettsial burden is reduced in D. variabilis KPI-expressing cells compared to that in nontransfected and LacZ-transfected cells. Results are reported as the median. (A) The 95%
    Figure Legend Snippet: D. variabilis KPI limits rickettsial colonization of host cells. (A) Rickettsial burden is reduced in D. variabilis KPI-expressing cells compared to that in nontransfected and LacZ-transfected cells. Results are reported as the median. (A) The 95%

    Techniques Used: Expressing, Transfection

    D. variabilis KPI inhibits coagulation and trypsin activity. We tested D. variabilis KPI for its predicted activity as an anticoagulant and its role as a general trypsin inhibitor. (A) We used the aPTT test to determine if D. variabilis KPI caused a delay
    Figure Legend Snippet: D. variabilis KPI inhibits coagulation and trypsin activity. We tested D. variabilis KPI for its predicted activity as an anticoagulant and its role as a general trypsin inhibitor. (A) We used the aPTT test to determine if D. variabilis KPI caused a delay

    Techniques Used: Coagulation, Activity Assay

    D. variabilis KPI shares highly conserved cysteine with other tick Kunitz domain-bearing proteins. To determine the conservation of structure between D. variabilis KPI and other reported Kunitz-bearing protease inhibitors from ticks, we performed an alignment
    Figure Legend Snippet: D. variabilis KPI shares highly conserved cysteine with other tick Kunitz domain-bearing proteins. To determine the conservation of structure between D. variabilis KPI and other reported Kunitz-bearing protease inhibitors from ticks, we performed an alignment

    Techniques Used:

    D. variabilis KPI is highly expressed in the midgut in response to feeding. We performed real-time qPCR to determine tissue distribution and the effects of feeding on D. variabilis KPI expression. (A) Gene expression of D. variabilis KPI was greatest
    Figure Legend Snippet: D. variabilis KPI is highly expressed in the midgut in response to feeding. We performed real-time qPCR to determine tissue distribution and the effects of feeding on D. variabilis KPI expression. (A) Gene expression of D. variabilis KPI was greatest

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    R. montanensis induces D. variabilis KPI gene expression. Midguts from R. montanensis -challenged D. variabilis ticks shows sustained levels of D. variabilis KPI transcript over a 72-h time period. Results are reported as the median. The following 95%
    Figure Legend Snippet: R. montanensis induces D. variabilis KPI gene expression. Midguts from R. montanensis -challenged D. variabilis ticks shows sustained levels of D. variabilis KPI transcript over a 72-h time period. Results are reported as the median. The following 95%

    Techniques Used: Expressing

    25) Product Images from "GDSL LIPASE1 Modulates Plant Immunity through Feedback Regulation of Ethylene Signaling 1GDSL LIPASE1 Modulates Plant Immunity through Feedback Regulation of Ethylene Signaling 1 [W]"

    Article Title: GDSL LIPASE1 Modulates Plant Immunity through Feedback Regulation of Ethylene Signaling 1GDSL LIPASE1 Modulates Plant Immunity through Feedback Regulation of Ethylene Signaling 1 [W]

    Journal: Plant Physiology

    doi: 10.1104/pp.113.225649

    A model for GLIP1 function in the regulation of ethylene signaling and immunity. GLIP1 expression depends on ethylene signaling components and positively regulates both local and systemic pathogen resistance. GLIP1 constitutes feedback regulation loops and modulates ethylene signaling in two ways: by inducing ERF1 (positive) and by suppressing EIN3 (negative) via EBF1/EBF2. ERF1 /ethylene-regulated pathogen resistance, and EIN3 down-regulation elevates SID2 -regulated pathogen resistance. We propose that GLIP1-mediated feedback regulation of ethylene signaling is the underlying mechanism of GLIP1 function in induced systemic resistance to pathogens and that it operates through GLIP1-mediated production of a systemic signal(s).
    Figure Legend Snippet: A model for GLIP1 function in the regulation of ethylene signaling and immunity. GLIP1 expression depends on ethylene signaling components and positively regulates both local and systemic pathogen resistance. GLIP1 constitutes feedback regulation loops and modulates ethylene signaling in two ways: by inducing ERF1 (positive) and by suppressing EIN3 (negative) via EBF1/EBF2. ERF1 /ethylene-regulated pathogen resistance, and EIN3 down-regulation elevates SID2 -regulated pathogen resistance. We propose that GLIP1-mediated feedback regulation of ethylene signaling is the underlying mechanism of GLIP1 function in induced systemic resistance to pathogens and that it operates through GLIP1-mediated production of a systemic signal(s).

    Techniques Used: Expressing

    26) Product Images from "Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3"

    Article Title: Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00826-16

    Effect of depleting MmpL3 on the growth, viability, and ability of M. tuberculosis to export mycolic acids. (A) mmpL3 is essential for the growth of M. tuberculosis on agar plates. The M. tuberculosis mc 2 6206 WT and MmpL3-DUC strains were plated on 7H11-OADC
    Figure Legend Snippet: Effect of depleting MmpL3 on the growth, viability, and ability of M. tuberculosis to export mycolic acids. (A) mmpL3 is essential for the growth of M. tuberculosis on agar plates. The M. tuberculosis mc 2 6206 WT and MmpL3-DUC strains were plated on 7H11-OADC

    Techniques Used:

    Titration of MmpL3 in the M. tuberculosis conditional knockdown and impact on the mycolic acid content of the cells. (A) (Left) MmpL3 levels at day 2 in the MmpL3-DUC cultures grown in the presence of different concentrations of ATc (shown in
    Figure Legend Snippet: Titration of MmpL3 in the M. tuberculosis conditional knockdown and impact on the mycolic acid content of the cells. (A) (Left) MmpL3 levels at day 2 in the MmpL3-DUC cultures grown in the presence of different concentrations of ATc (shown in

    Techniques Used: Titration

    Effect of depleting MmpL3 on the susceptibility of M. tuberculosis to MmpL3 inhibitors. The zones of inhibition on 7H11-OADC agar plates for each drug were measured versus MmpL3-DUC and the M. tuberculosis H37Rv parent strain in the presence and absence
    Figure Legend Snippet: Effect of depleting MmpL3 on the susceptibility of M. tuberculosis to MmpL3 inhibitors. The zones of inhibition on 7H11-OADC agar plates for each drug were measured versus MmpL3-DUC and the M. tuberculosis H37Rv parent strain in the presence and absence

    Techniques Used: Inhibition

    Effect of depleting MmpL3 on the viability of nonreplicating hypoxic M. tuberculosis bacilli. Shown is survival of M. tuberculosis ATCC 25618 MmpL3-DUC during hypoxia upon addition of ATc (100 ng/ml) to the culture medium 9 and 15 days after the culture
    Figure Legend Snippet: Effect of depleting MmpL3 on the viability of nonreplicating hypoxic M. tuberculosis bacilli. Shown is survival of M. tuberculosis ATCC 25618 MmpL3-DUC during hypoxia upon addition of ATc (100 ng/ml) to the culture medium 9 and 15 days after the culture

    Techniques Used:

    Construction of conditional mmpL3 knockdown strains of M. tuberculosis . Allelic replacement at the mmpL3 locus of M. tuberculosis H37Rv strain ATCC 25618 rescued with mmpL3-DAS + 4 expressed from pGMCH-T38S38-P750-mmpL3-DAS + 4 under the control of an
    Figure Legend Snippet: Construction of conditional mmpL3 knockdown strains of M. tuberculosis . Allelic replacement at the mmpL3 locus of M. tuberculosis H37Rv strain ATCC 25618 rescued with mmpL3-DAS + 4 expressed from pGMCH-T38S38-P750-mmpL3-DAS + 4 under the control of an

    Techniques Used:

    MmpL3 is required for the replication and persistence of M. tuberculosis in mice. (A) Growth and survival and WT M. tuberculosis H37Rv ATCC 25618 (blue line and circles) and MmpL3-DUC (triangles) in C57BL/6 mouse lungs (left) and spleens (right). Mice
    Figure Legend Snippet: MmpL3 is required for the replication and persistence of M. tuberculosis in mice. (A) Growth and survival and WT M. tuberculosis H37Rv ATCC 25618 (blue line and circles) and MmpL3-DUC (triangles) in C57BL/6 mouse lungs (left) and spleens (right). Mice

    Techniques Used: Mouse Assay

    27) Product Images from "Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis"

    Article Title: Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.02130-16

    Pyrazinamide susceptibility of Mycobacterium bovis BCG strains ectopically expressing the Mycobacterium tuberculosis PncA at acidic pH. M. bovis BCG/pJT6a:: pncA (A) and M. bovis BCG fadD2 :: kan /pJT6a:: pncA (B) were diluted to an OD 600 of 0.01 in 7H9 medium
    Figure Legend Snippet: Pyrazinamide susceptibility of Mycobacterium bovis BCG strains ectopically expressing the Mycobacterium tuberculosis PncA at acidic pH. M. bovis BCG/pJT6a:: pncA (A) and M. bovis BCG fadD2 :: kan /pJT6a:: pncA (B) were diluted to an OD 600 of 0.01 in 7H9 medium

    Techniques Used: Expressing

    FadD2 loss of function confers hypersusceptibility to PZA.
    Figure Legend Snippet: FadD2 loss of function confers hypersusceptibility to PZA.

    Techniques Used:

    Disruption of FadD2 expression increases susceptibility to certain short-chain fatty acids.
    Figure Legend Snippet: Disruption of FadD2 expression increases susceptibility to certain short-chain fatty acids.

    Techniques Used: Expressing

    Enzymatic characterization of the M. tuberculosis FadD2. (A) Fatty acid substrate specificity of FadD2; (B) additional assays conducted to assess the allosteric modulation of FadD2 and use of POA as the substrate; (C) kinetic profile of FadD2 with oleate
    Figure Legend Snippet: Enzymatic characterization of the M. tuberculosis FadD2. (A) Fatty acid substrate specificity of FadD2; (B) additional assays conducted to assess the allosteric modulation of FadD2 and use of POA as the substrate; (C) kinetic profile of FadD2 with oleate

    Techniques Used:

    Intracellular CoA levels of Mycobacterium bovis BCG strains. (A) Intracellular CoA levels were measured from wild-type M. bovis BCG (BCG), M. bovis BCG fadD2 :: hyg (ΔfadD2), and M. bovis BCG fadD2 :: hyg /pTIC6a:: fadD2 (ΔfadD2-comp) that were
    Figure Legend Snippet: Intracellular CoA levels of Mycobacterium bovis BCG strains. (A) Intracellular CoA levels were measured from wild-type M. bovis BCG (BCG), M. bovis BCG fadD2 :: hyg (ΔfadD2), and M. bovis BCG fadD2 :: hyg /pTIC6a:: fadD2 (ΔfadD2-comp) that were

    Techniques Used:

    Bactericidal activity of pyrazinoic acid against Mycobacterium bovis BCG strains at pH 6.8. Wild-type M. bovis BCG (A), M. bovis BCG fadD2 :: kan /pJT6a (B), and M. bovis BCG fadD2 :: kan /pJT6a:: fadD2 (C) cells in mid-logarithmic phase were subcultured into
    Figure Legend Snippet: Bactericidal activity of pyrazinoic acid against Mycobacterium bovis BCG strains at pH 6.8. Wild-type M. bovis BCG (A), M. bovis BCG fadD2 :: kan /pJT6a (B), and M. bovis BCG fadD2 :: kan /pJT6a:: fadD2 (C) cells in mid-logarithmic phase were subcultured into

    Techniques Used: Activity Assay

    Effects of fadD2 disruption on CoA metabolism.
    Figure Legend Snippet: Effects of fadD2 disruption on CoA metabolism.

    Techniques Used:

    28) Product Images from "Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation"

    Article Title: Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00015-15

    Microtubule binding analysis of Ca Kar3 and Ca Cik1. (A) Diagrams of constructs used to give monomeric forms of the Ca Kar3 motor domain (MD) and the Ca Cik1 motor homology domain (MHD). (B) Representative SDS gels of supernatant and pellet fractions recovered
    Figure Legend Snippet: Microtubule binding analysis of Ca Kar3 and Ca Cik1. (A) Diagrams of constructs used to give monomeric forms of the Ca Kar3 motor domain (MD) and the Ca Cik1 motor homology domain (MHD). (B) Representative SDS gels of supernatant and pellet fractions recovered

    Techniques Used: Binding Assay, Construct

    Loss of Ca Kar3 or Ca Cik1 affects growth and viability. (A) Left panels show microcolony morphologies of wild-type (CF027), cik1 Δ/Δ (CF016), cik1 Δ/Δ + vector (CF126), cik1 Δ/Δ + CIK1 + (CF129), kar3 Δ/Δ
    Figure Legend Snippet: Loss of Ca Kar3 or Ca Cik1 affects growth and viability. (A) Left panels show microcolony morphologies of wild-type (CF027), cik1 Δ/Δ (CF016), cik1 Δ/Δ + vector (CF126), cik1 Δ/Δ + CIK1 + (CF129), kar3 Δ/Δ

    Techniques Used: Plasmid Preparation

    Time-lapse microscopy reveals that Ca Cik1 is involved in bipolar spindle formation. (A) cik1 Δ/Δ Tub2-GFP (CF174) cells that failed to establish a bipolar spindle. Each frame is shown at two different contrast levels to help visualize both
    Figure Legend Snippet: Time-lapse microscopy reveals that Ca Cik1 is involved in bipolar spindle formation. (A) cik1 Δ/Δ Tub2-GFP (CF174) cells that failed to establish a bipolar spindle. Each frame is shown at two different contrast levels to help visualize both

    Techniques Used: Time-lapse Microscopy

    Loss of Ca Cik1 disrupts formation of mature hyphae. (A) Cells lacking Ca Cik1 and/or Ca Kar3 are unable to form complex hyphal structures on solid media. Cells of the indicated genotypes (strains CF027, CF024, CF016, and CF019) were plated onto Spider medium,
    Figure Legend Snippet: Loss of Ca Cik1 disrupts formation of mature hyphae. (A) Cells lacking Ca Cik1 and/or Ca Kar3 are unable to form complex hyphal structures on solid media. Cells of the indicated genotypes (strains CF027, CF024, CF016, and CF019) were plated onto Spider medium,

    Techniques Used:

    Ca Cik1 and Ca Kar3 localize to SPBs in a mutually dependent manner. Ca Cik1-YFP (A) and Ca Kar3-GFP (B) localize to spindle pole bodies in both early and late anaphase, as well as in cells that have completed DNA segregation and initiated budding (M/G1).
    Figure Legend Snippet: Ca Cik1 and Ca Kar3 localize to SPBs in a mutually dependent manner. Ca Cik1-YFP (A) and Ca Kar3-GFP (B) localize to spindle pole bodies in both early and late anaphase, as well as in cells that have completed DNA segregation and initiated budding (M/G1).

    Techniques Used:

    Structural and functional analyses of Ca Kar3 / Cik1 complexes. (A) Domain architectures and boundaries of the full-length Ca Kar3 and Ca Cik1 constructs used for the study. (B) SDS-PAGE analysis of elution fractions following Ca Kar3/Cik1 Ni-NTA copurification.
    Figure Legend Snippet: Structural and functional analyses of Ca Kar3 / Cik1 complexes. (A) Domain architectures and boundaries of the full-length Ca Kar3 and Ca Cik1 constructs used for the study. (B) SDS-PAGE analysis of elution fractions following Ca Kar3/Cik1 Ni-NTA copurification.

    Techniques Used: Functional Assay, Construct, SDS Page, Copurification

    Ca Cik1 binds microtubules but lacks ATPase activity.
    Figure Legend Snippet: Ca Cik1 binds microtubules but lacks ATPase activity.

    Techniques Used: Activity Assay

    Short spindles and dissociated spindle structures in cells lacking Ca Cik1 are monopolar. (A) Logarithmically growing C. albicans Spc98-GFP Tub2-RFP (CF160) cells in SDC at 25°C show that the putative Spc98 homolog encoded by orf19.2600 localizes
    Figure Legend Snippet: Short spindles and dissociated spindle structures in cells lacking Ca Cik1 are monopolar. (A) Logarithmically growing C. albicans Spc98-GFP Tub2-RFP (CF160) cells in SDC at 25°C show that the putative Spc98 homolog encoded by orf19.2600 localizes

    Techniques Used:

    Loss of Ca Cik1 or Ca Kar3 causes spindle defects. (A) Blastoconidia of the wild-type (CF027), kar3 Δ/Δ (CF024), and cik1 Δ/Δ (CF016) strains were scored based on nuclear position. Data represent mean values for three independent
    Figure Legend Snippet: Loss of Ca Cik1 or Ca Kar3 causes spindle defects. (A) Blastoconidia of the wild-type (CF027), kar3 Δ/Δ (CF024), and cik1 Δ/Δ (CF016) strains were scored based on nuclear position. Data represent mean values for three independent

    Techniques Used:

    29) Product Images from "Characterization of Interactions between RTA and the Promoter of Polyadenylated Nuclear RNA in Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8"

    Article Title: Characterization of Interactions between RTA and the Promoter of Polyadenylated Nuclear RNA in Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.10.5000-5013.2002

    Expression of RTA and Rdbd. (A) Schematic diagram of recombinant RTA proteins. Full-length RTA protein (amino acids 1 to 691) was tagged with FLAG at the N terminus. Rdbd (amino acids 1 to 320) includes a putative DBD as well as a leucine zipper domain (LZ) tagged with FLAG at the N terminus and six histidine residues at the C terminus. AD, activation domain. (B) Representative Coomassie blue-stained gel of purified Rdbd protein. Rdbd protein was expressed in bacteria and purified with a Ni-NTA column. Eluates (E1 to E3) were analyzed by SDS-PAGE and stained with Coomassie blue. The arrow indicates purified Rdbd protein. M, molecular mass markers. (C) RTA and Rdbd expression in 293T cells and E. coli . Nuclear extracts were prepared from 293T cells transfected with either vector alone (lanes 1 and 5) or a FLAG-tagged RTA-expressing plasmid (pFLAG/RTA; lanes 2 and 6). Bacterially expressed recombinant full-length RTA (lanes 3 and 7) and Rdbd (lanes 4 and 8) were purified and separated on 10% gels with nuclear extracts from transfected 293T cells. Monoclonal anti-FLAG antibody (left) and polyclonal anti-RTA rabbit serum (right) were used for Western analysis.
    Figure Legend Snippet: Expression of RTA and Rdbd. (A) Schematic diagram of recombinant RTA proteins. Full-length RTA protein (amino acids 1 to 691) was tagged with FLAG at the N terminus. Rdbd (amino acids 1 to 320) includes a putative DBD as well as a leucine zipper domain (LZ) tagged with FLAG at the N terminus and six histidine residues at the C terminus. AD, activation domain. (B) Representative Coomassie blue-stained gel of purified Rdbd protein. Rdbd protein was expressed in bacteria and purified with a Ni-NTA column. Eluates (E1 to E3) were analyzed by SDS-PAGE and stained with Coomassie blue. The arrow indicates purified Rdbd protein. M, molecular mass markers. (C) RTA and Rdbd expression in 293T cells and E. coli . Nuclear extracts were prepared from 293T cells transfected with either vector alone (lanes 1 and 5) or a FLAG-tagged RTA-expressing plasmid (pFLAG/RTA; lanes 2 and 6). Bacterially expressed recombinant full-length RTA (lanes 3 and 7) and Rdbd (lanes 4 and 8) were purified and separated on 10% gels with nuclear extracts from transfected 293T cells. Monoclonal anti-FLAG antibody (left) and polyclonal anti-RTA rabbit serum (right) were used for Western analysis.

    Techniques Used: Expressing, Recombinant, Activation Assay, Staining, Purification, SDS Page, Transfection, Plasmid Preparation, Western Blot

    30) Product Images from "Methylation by NSun2 Represses the Levels and Function of MicroRNA 125b"

    Article Title: Methylation by NSun2 Represses the Levels and Function of MicroRNA 125b

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00243-14

    NSun2 methylates miR-125b in vivo . (A) Forty-eight hours after transfection of HeLa cells with a vector expressing NSun2 (pNSun2) or with an empty vector (V) or after transfection with an siRNA targeting NSun2 (siNSun2) or a control siRNA (Ctrl), NSun2
    Figure Legend Snippet: NSun2 methylates miR-125b in vivo . (A) Forty-eight hours after transfection of HeLa cells with a vector expressing NSun2 (pNSun2) or with an empty vector (V) or after transfection with an siRNA targeting NSun2 (siNSun2) or a control siRNA (Ctrl), NSun2

    Techniques Used: In Vivo, Transfection, Plasmid Preparation, Expressing

    Methylation by NSun2 enhances the expression of the targets of miR-125b. (A) HeLa cells were transfected with a vector expressing NSun2 (pNSun2) or an empty vector (V) or with an siRNA targeting NSun2 (siNSun2) or a control siRNA (Ctrl). Forty-eight hours
    Figure Legend Snippet: Methylation by NSun2 enhances the expression of the targets of miR-125b. (A) HeLa cells were transfected with a vector expressing NSun2 (pNSun2) or an empty vector (V) or with an siRNA targeting NSun2 (siNSun2) or a control siRNA (Ctrl). Forty-eight hours

    Techniques Used: Methylation, Expressing, Transfection, Plasmid Preparation

    NSun2 regulates miR-125b levels and function in response to oxidative stress. (A) Twenty-four hours after HeLa cells were exposed to H 2 O 2 (50 μM), the levels of proteins NSun2, p53, Bak1, CDC25C, and ppp1ca were analyzed by Western blotting. (B)
    Figure Legend Snippet: NSun2 regulates miR-125b levels and function in response to oxidative stress. (A) Twenty-four hours after HeLa cells were exposed to H 2 O 2 (50 μM), the levels of proteins NSun2, p53, Bak1, CDC25C, and ppp1ca were analyzed by Western blotting. (B)

    Techniques Used: Western Blot

    Methylation by NSun2 represses the processing of in vitro -methylated pri-miR-125b2 and pre-miR-125b2. (A) In vitro -methylated or unmethylated pre-miR-125b1 and pre-miR-125b2 (0.15 μg) were subjected to processing assays using cytoplasmic extracts
    Figure Legend Snippet: Methylation by NSun2 represses the processing of in vitro -methylated pri-miR-125b2 and pre-miR-125b2. (A) In vitro -methylated or unmethylated pre-miR-125b1 and pre-miR-125b2 (0.15 μg) were subjected to processing assays using cytoplasmic extracts

    Techniques Used: Methylation, In Vitro

    Methylation by NSun2 represses the gene-silencing function of miR-125b. (A to D) Schematic representation of the pGL3-derived reporters bearing the long (A) or short (B) 3′ UTR fragments and the corresponding fragments with mutated miR-125b sites
    Figure Legend Snippet: Methylation by NSun2 represses the gene-silencing function of miR-125b. (A to D) Schematic representation of the pGL3-derived reporters bearing the long (A) or short (B) 3′ UTR fragments and the corresponding fragments with mutated miR-125b sites

    Techniques Used: Methylation, Derivative Assay

    NSun2 methylates miR-125b in vitro . (A) (Left) Incorporation of 3 H-labeled S -adenosyl- l -methionine into miR-125, miR-125bΔ1, miR-125bΔ2, miR-125bΔ3 (see Fig. S1A in the supplemental material, schematic), miR-125a, miR-30s (a, b,
    Figure Legend Snippet: NSun2 methylates miR-125b in vitro . (A) (Left) Incorporation of 3 H-labeled S -adenosyl- l -methionine into miR-125, miR-125bΔ1, miR-125bΔ2, miR-125bΔ3 (see Fig. S1A in the supplemental material, schematic), miR-125a, miR-30s (a, b,

    Techniques Used: In Vitro, Labeling

    Methylation by NSun2 attenuates the recruitment of RISC by miR-125b. (A) Forty-eight hours after transfection of HeLa cells with an siRNA targeting NSun2 (+) or a control siRNA (−), cell lysates were prepared and subjected to RNA pulldown assays
    Figure Legend Snippet: Methylation by NSun2 attenuates the recruitment of RISC by miR-125b. (A) Forty-eight hours after transfection of HeLa cells with an siRNA targeting NSun2 (+) or a control siRNA (−), cell lysates were prepared and subjected to RNA pulldown assays

    Techniques Used: Methylation, Transfection

    Methylation by NSun2 represses the processing of miR-125b. (A) Forty-eight hours after transfection of IDH4 cells with an siRNA targeting NSun2, the protein levels of NSun2 and GAPDH were assessed by Western blotting (left). The levels of miR-125b (middle)
    Figure Legend Snippet: Methylation by NSun2 represses the processing of miR-125b. (A) Forty-eight hours after transfection of IDH4 cells with an siRNA targeting NSun2, the protein levels of NSun2 and GAPDH were assessed by Western blotting (left). The levels of miR-125b (middle)

    Techniques Used: Methylation, Transfection, Western Blot

    31) Product Images from "Immunologic study and optimization of Salmonella delivery strains expressing adhesin and toxin antigens for protection against progressive atrophic rhinitis in a murine model"

    Article Title: Immunologic study and optimization of Salmonella delivery strains expressing adhesin and toxin antigens for protection against progressive atrophic rhinitis in a murine model

    Journal: Canadian Journal of Veterinary Research

    doi:

    Serum IgG and vaginal mucosal IgA antibody titers against FimA, CP39, PtfA, ToxA, and F1P2 antigens in mice intranasally inoculated with the vaccine candidate (groups A to C) or phosphate-buffered saline as a control (group D). Refer to for
    Figure Legend Snippet: Serum IgG and vaginal mucosal IgA antibody titers against FimA, CP39, PtfA, ToxA, and F1P2 antigens in mice intranasally inoculated with the vaccine candidate (groups A to C) or phosphate-buffered saline as a control (group D). Refer to for

    Techniques Used: Mouse Assay

    Relative levels of interferon-gamma (INF-γ) and interleukin-4 (IL-4) mRNA induced by PtfA and F1P2 in the splenocytes of the 16-week-old mice, as determined by real-time polymerase chain reaction. Asterisks indicate a significant difference (
    Figure Legend Snippet: Relative levels of interferon-gamma (INF-γ) and interleukin-4 (IL-4) mRNA induced by PtfA and F1P2 in the splenocytes of the 16-week-old mice, as determined by real-time polymerase chain reaction. Asterisks indicate a significant difference (

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction

    32) Product Images from "Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis *"

    Article Title: Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.634840

    Schematic showing key residues and their ionization states in the photoactive site of light-adapted BR and Ca ChR1. A ). The green arrow indicates the role of Asp-85
    Figure Legend Snippet: Schematic showing key residues and their ionization states in the photoactive site of light-adapted BR and Ca ChR1. A ). The green arrow indicates the role of Asp-85

    Techniques Used:

    FTIR difference spectra in the 800–1800-cm −1 region of Ca ChR1 recorded using the photoreversal methods at 270 K (see “Experimental Procedures”). y axis markers are ∼0.2 mOD for all spectra.
    Figure Legend Snippet: FTIR difference spectra in the 800–1800-cm −1 region of Ca ChR1 recorded using the photoreversal methods at 270 K (see “Experimental Procedures”). y axis markers are ∼0.2 mOD for all spectra.

    Techniques Used:

    Three-dimensional structure of C1C2 chimera from Kato et al. ) (Protein Data Bank ID 3UG9 ) showing the position of the residues homologous to the SB counterions Glu-169 and Asp-299 along with the positively charged residue Arg-166 in Ca ChR1. The residue
    Figure Legend Snippet: Three-dimensional structure of C1C2 chimera from Kato et al. ) (Protein Data Bank ID 3UG9 ) showing the position of the residues homologous to the SB counterions Glu-169 and Asp-299 along with the positively charged residue Arg-166 in Ca ChR1. The residue

    Techniques Used:

    FTIR difference spectra in the 1680–1800-cm −1 region of Ca ChR1 WT ( first and third spectra ) and the mutant D299N ( second and fourth spectra ) recorded at 270 K in H 2 O using methods identical to those described in the legend for .
    Figure Legend Snippet: FTIR difference spectra in the 1680–1800-cm −1 region of Ca ChR1 WT ( first and third spectra ) and the mutant D299N ( second and fourth spectra ) recorded at 270 K in H 2 O using methods identical to those described in the legend for .

    Techniques Used: Mutagenesis

    FTIR difference spectra of Ca ChR1 WT recorded at 270 K over the 1680–1800-cm −1 region in both H 2 O ( first and third spectra ) and D 2 O ( second and fourth spectra ). Spectra were acquired using static methods, and differences shown are the
    Figure Legend Snippet: FTIR difference spectra of Ca ChR1 WT recorded at 270 K over the 1680–1800-cm −1 region in both H 2 O ( first and third spectra ) and D 2 O ( second and fourth spectra ). Spectra were acquired using static methods, and differences shown are the

    Techniques Used:

    Schematic model showing the proposed protonation of the ground state, P 1 and P 2 380 intermediates in Ca ChR1 along with proposed proton transfers. The large green arrow indicates the photon absorption by the unphotolyzed Ca ChR1. The smaller arrows indicate
    Figure Legend Snippet: Schematic model showing the proposed protonation of the ground state, P 1 and P 2 380 intermediates in Ca ChR1 along with proposed proton transfers. The large green arrow indicates the photon absorption by the unphotolyzed Ca ChR1. The smaller arrows indicate

    Techniques Used:

    Comparison of FTIR difference spectra of Ca ChR1 for static recorded at 270 K and rapid scan recorded at both 278 K and 293 K over the 1000–1800-cm −1 region (see “Experimental Procedures”). y axis markers indicate 0.2 mOD
    Figure Legend Snippet: Comparison of FTIR difference spectra of Ca ChR1 for static recorded at 270 K and rapid scan recorded at both 278 K and 293 K over the 1000–1800-cm −1 region (see “Experimental Procedures”). y axis markers indicate 0.2 mOD

    Techniques Used:

    FTIR difference spectra in the 1680–1800-cm −1 region of Ca ChR1 WT ( first and third spectra ) and the mutant E169Q ( second and fourth spectra ) recorded at 270 K in H 2 O using methods identical to those described in the legend for .
    Figure Legend Snippet: FTIR difference spectra in the 1680–1800-cm −1 region of Ca ChR1 WT ( first and third spectra ) and the mutant E169Q ( second and fourth spectra ) recorded at 270 K in H 2 O using methods identical to those described in the legend for .

    Techniques Used: Mutagenesis

    FTIR difference spectra in the 1680–1800-cm −1 region of Ca ChR1 WT ( first and third spectra ) and the mutant F139K ( second and fourth spectra ) recorded at 270 K in H 2 O using methods identical to those described in the legend for .
    Figure Legend Snippet: FTIR difference spectra in the 1680–1800-cm −1 region of Ca ChR1 WT ( first and third spectra ) and the mutant F139K ( second and fourth spectra ) recorded at 270 K in H 2 O using methods identical to those described in the legend for .

    Techniques Used: Mutagenesis

    FTIR difference spectra in the 1680–1800-cm −1 region of Ca ChR1 WT ( first and third spectra ) and the mutant D299E ( second and fourth spectra ) recorded at 270 K in H 2 O using methods identical to those described in the legend for .
    Figure Legend Snippet: FTIR difference spectra in the 1680–1800-cm −1 region of Ca ChR1 WT ( first and third spectra ) and the mutant D299E ( second and fourth spectra ) recorded at 270 K in H 2 O using methods identical to those described in the legend for .

    Techniques Used: Mutagenesis

    FTIR difference spectra in the 800–1800-cm −1 region of Ca ChR1 and mutants recorded at 270 K in H 2 O using methods identical to shown in . The y axis tick marks are ∼0.5, 0.3, 0.8, 0.7, and 0.5 mOD for the spectra from top
    Figure Legend Snippet: FTIR difference spectra in the 800–1800-cm −1 region of Ca ChR1 and mutants recorded at 270 K in H 2 O using methods identical to shown in . The y axis tick marks are ∼0.5, 0.3, 0.8, 0.7, and 0.5 mOD for the spectra from top

    Techniques Used:

    33) Product Images from "Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70"

    Article Title: Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

    Journal: Cell Stress & Chaperones

    doi: 10.1379/CSC-26R.1

    Determination of a mutually exclusive interaction of heat shock protein (Hsp)90 and heat shock cognate (Hsc)70 with cyclophilin 40 (CyP40). Ni-NTA agarose was charged with His-tagged Hsp90β and mixed 1:1 with Sepharose 4B. Bacterial lysate (150 μL) for wild-type bCyP40 was added to the diluted gel (40 μL), and after dilution to 500 μL total volume with binding buffer containing 35 mM imidazole and 0.2% Triton X-100, the mixture was rotated at 4°C for 3 hours. After removal of unbound proteins by successive washing, the gel containing Hsp90-bound CyP40 was exposed to 0, 100, 200, and 400 μL of induced Hsc70 bacterial lysate. Reaction volumes were brought to 500 μL with binding buffer containing imidazole and Triton X-100, and incubation was continued with rotation at 4°C for 3 hours. Gel-retained proteins were recovered with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and analyzed by SDS-PAGE with Coomassie blue staining. Glutathione-agarose containing immobilized GST-bCyP40 was mixed 1:5 with Sepharose 4B. The diluted gel (40 μL) was incubated as described above with 400 μL of Hsc70 lysate and gel-retained proteins were analyzed by SDS-PAGE. GST, glutathione S-transferase
    Figure Legend Snippet: Determination of a mutually exclusive interaction of heat shock protein (Hsp)90 and heat shock cognate (Hsc)70 with cyclophilin 40 (CyP40). Ni-NTA agarose was charged with His-tagged Hsp90β and mixed 1:1 with Sepharose 4B. Bacterial lysate (150 μL) for wild-type bCyP40 was added to the diluted gel (40 μL), and after dilution to 500 μL total volume with binding buffer containing 35 mM imidazole and 0.2% Triton X-100, the mixture was rotated at 4°C for 3 hours. After removal of unbound proteins by successive washing, the gel containing Hsp90-bound CyP40 was exposed to 0, 100, 200, and 400 μL of induced Hsc70 bacterial lysate. Reaction volumes were brought to 500 μL with binding buffer containing imidazole and Triton X-100, and incubation was continued with rotation at 4°C for 3 hours. Gel-retained proteins were recovered with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and analyzed by SDS-PAGE with Coomassie blue staining. Glutathione-agarose containing immobilized GST-bCyP40 was mixed 1:5 with Sepharose 4B. The diluted gel (40 μL) was incubated as described above with 400 μL of Hsc70 lysate and gel-retained proteins were analyzed by SDS-PAGE. GST, glutathione S-transferase

    Techniques Used: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining

    34) Product Images from "The Chloroplastic GrpE Homolog of Chlamydomonas"

    Article Title: The Chloroplastic GrpE Homolog of Chlamydomonas

    Journal: The Plant Cell

    doi: 10.1105/tpc.010202

    Complementation of the E. coli grpE Deletion Strain OD212 with CGE1 and Analysis of CGE1–DnaK Interactions. (A) Temperature-sensitive E. coli strain OD212 carrying a deletion of its grpE gene was transformed with a plasmid vector for the expression of CGE1 (CGE1-1 and CGE1-2) or the same vector expressing an unrelated gene (control). Dilutions of transformant cultures were spotted onto Luria-Bertani plates and incubated overnight at 25, 37, or 43°C. (B) Comparison of the expression of CGE1 in the OD212 transformants described in (A) with Chlamydomonas (Chlamy) CGE1 by protein gel blot analysis and detection with CGE1 antiserum (α-CGE1). Each lane contained 15 μg of total soluble protein. (C) Expression of hexahistidine-tagged versions of CGE1 and an unrelated 30-kD protein (control) from plasmid vectors was induced in E. coli strain M15 by isopropylthio-β-galactoside. Cells were lysed under native conditions (crude lysate; lanes 1 and 2) and incubated with nickel–nitrilotriacetic acid agarose (Ni-NTA). After washing, proteins bound to Ni-NTA were eluted by incubation with 250 mM imidazole (lanes 3 and 4). In a parallel experiment, Ni-NTA beads binding CGE1 were first incubated for 10 min at 24°C with a buffer containing 20 mM 3-( N -morpholino)-propanesulfonic acid (Mops)-KOH, pH 7.4, 80 mM KCl, 5 mM MgCl 2 , and either 5 mM ATP or no nucleotide (mock) (lanes 5 and 6). The proteins that had remained on the resin then were eluted with 250 mM imidazole (lanes 7 and 8). Eluted proteins were precipitated with trichloroacetic acid, separated on an SDS–10% polyacrylamide gel, and visualized by Coomassie blue staining (top) or transferred to nitrocellulose and immunodetected with an antiserum (α) against DnaK using enhanced chemiluminescence (bottom).
    Figure Legend Snippet: Complementation of the E. coli grpE Deletion Strain OD212 with CGE1 and Analysis of CGE1–DnaK Interactions. (A) Temperature-sensitive E. coli strain OD212 carrying a deletion of its grpE gene was transformed with a plasmid vector for the expression of CGE1 (CGE1-1 and CGE1-2) or the same vector expressing an unrelated gene (control). Dilutions of transformant cultures were spotted onto Luria-Bertani plates and incubated overnight at 25, 37, or 43°C. (B) Comparison of the expression of CGE1 in the OD212 transformants described in (A) with Chlamydomonas (Chlamy) CGE1 by protein gel blot analysis and detection with CGE1 antiserum (α-CGE1). Each lane contained 15 μg of total soluble protein. (C) Expression of hexahistidine-tagged versions of CGE1 and an unrelated 30-kD protein (control) from plasmid vectors was induced in E. coli strain M15 by isopropylthio-β-galactoside. Cells were lysed under native conditions (crude lysate; lanes 1 and 2) and incubated with nickel–nitrilotriacetic acid agarose (Ni-NTA). After washing, proteins bound to Ni-NTA were eluted by incubation with 250 mM imidazole (lanes 3 and 4). In a parallel experiment, Ni-NTA beads binding CGE1 were first incubated for 10 min at 24°C with a buffer containing 20 mM 3-( N -morpholino)-propanesulfonic acid (Mops)-KOH, pH 7.4, 80 mM KCl, 5 mM MgCl 2 , and either 5 mM ATP or no nucleotide (mock) (lanes 5 and 6). The proteins that had remained on the resin then were eluted with 250 mM imidazole (lanes 7 and 8). Eluted proteins were precipitated with trichloroacetic acid, separated on an SDS–10% polyacrylamide gel, and visualized by Coomassie blue staining (top) or transferred to nitrocellulose and immunodetected with an antiserum (α) against DnaK using enhanced chemiluminescence (bottom).

    Techniques Used: Transformation Assay, Plasmid Preparation, Expressing, Incubation, Western Blot, Binding Assay, Staining

    35) Product Images from "Abscisic Acid-Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1"

    Article Title: Abscisic Acid-Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1

    Journal: The Plant Cell

    doi: 10.1105/tpc.005272

    Mobility Shift of TRAB1 on SDS-PAGE Caused by ABA-Induced Phosphorylation. (A) Nuclear extracts of rice suspension-cultured cells (Oc cells) treated with ABA for the indicated times (0 indicates untreated cells) were analyzed by immunoblotting with anti-TRAB1 antibody. The arrow and asterisks indicate TRAB1-specific and nonspecific bands, respectively. Although the expression of the nonspecific bands was affected by ABA, the nature of these polypeptides is unknown. (B) TRAB1-dHA/His expressed in transgenic cells treated without (−) or with (+) ABA for 30 min was recovered with nickel–nitrilotriacetic acid agarose resin, incubated without (−) or with (+) CIAP, and analyzed by immunoblotting with anti-HA antibody. The signals seen at the top of the +CIAP lanes are parts of bulky CIAP bands that reacted nonspecifically with the anti-HA antibody.
    Figure Legend Snippet: Mobility Shift of TRAB1 on SDS-PAGE Caused by ABA-Induced Phosphorylation. (A) Nuclear extracts of rice suspension-cultured cells (Oc cells) treated with ABA for the indicated times (0 indicates untreated cells) were analyzed by immunoblotting with anti-TRAB1 antibody. The arrow and asterisks indicate TRAB1-specific and nonspecific bands, respectively. Although the expression of the nonspecific bands was affected by ABA, the nature of these polypeptides is unknown. (B) TRAB1-dHA/His expressed in transgenic cells treated without (−) or with (+) ABA for 30 min was recovered with nickel–nitrilotriacetic acid agarose resin, incubated without (−) or with (+) CIAP, and analyzed by immunoblotting with anti-HA antibody. The signals seen at the top of the +CIAP lanes are parts of bulky CIAP bands that reacted nonspecifically with the anti-HA antibody.

    Techniques Used: Mobility Shift, SDS Page, Cell Culture, Expressing, Transgenic Assay, Incubation

    36) Product Images from "Iridoid-specific Glucosyltransferase from Gardenia jasminoides *"

    Article Title: Iridoid-specific Glucosyltransferase from Gardenia jasminoides *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.242586

    Analysis of the glucosyltransferase activity of recombinant UGT85A24 expressed in E. coli . A , results from SDS-PAGE analysis of the crude protein from E. coli JM109 harboring pQE-30-GjUGT2 ( left lane: C ) and the recombinant enzyme (UGT85A24) purified using a nickel-nitrilotriacetic acid resin column ( right: P ). B , time course changes in genipin glucosylation by incubation with recombinant UGT85A24.
    Figure Legend Snippet: Analysis of the glucosyltransferase activity of recombinant UGT85A24 expressed in E. coli . A , results from SDS-PAGE analysis of the crude protein from E. coli JM109 harboring pQE-30-GjUGT2 ( left lane: C ) and the recombinant enzyme (UGT85A24) purified using a nickel-nitrilotriacetic acid resin column ( right: P ). B , time course changes in genipin glucosylation by incubation with recombinant UGT85A24.

    Techniques Used: Activity Assay, Recombinant, SDS Page, Purification, Incubation

    37) Product Images from "Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling *"

    Article Title: Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M115.054080

    Identification of super-complexes assembled from VDACs/Porins and TOM complexes. A, relative abundance-mass profiles of VDAC1–3 and TOM20 proteins. Color-coding and the Pearson correlation coefficient r of profile peaks are shown on the right. B, interaction of yeast Porin1 and the TOM complex detected by affinity purification. Mitochondria isolated from wild-type ( WT ) or yeast strain expressing HA-tagged VDAC homolog Porin1 ( Por1HA ) were subjected to affinity purification via anti-HA chromatography. Load (0.75%) and elution (100%) fractions were separated by SDS-PAGE, and proteins were detected by Western blotting with the indicated antisera. The Porin1 interactor OM14 and several TOM proteins ( upper panel ) were specifically co-purified, whereas three unrelated control proteins ( lower panel ) were not detected in the eluates. C, upper panel, affinity purifications as in B eluted under non-denaturing conditions and resolved by BN-PAGE. Western blot detection with anti-TOM40 ( left ) and anti-TOM22 ( right ) antibodies identified full-size TOM complex (around 450 kDa) specifically co-purified with HA-Porin1. Lower left panel, wild-type ( WT ) and Tom22His mitochondria subjected to nickel in complex with nitrilotriacetic acid purification followed by separation via BN-PAGE and Western blotting. Porin complexes were detected using anti-Porin1 antibodies (load 0.75%, elution 100%). Lower right panel, wild-type ( WT ) and Por1HA mitochondria were incubated with 35 S-labeled Oxa1 in the absence of a membrane potential to arrest the precursor at the TOM complex. Subsequently, samples were subjected to anti-HA chromatography, and protein complexes were separated by BN-PAGE. 35 S-Labeled Oxa1 arrested at the TOM complex was visualized with digital autoradiography. Note the increase in apparent mass of TOM complexes due to the stalled Oxa1 substrate.
    Figure Legend Snippet: Identification of super-complexes assembled from VDACs/Porins and TOM complexes. A, relative abundance-mass profiles of VDAC1–3 and TOM20 proteins. Color-coding and the Pearson correlation coefficient r of profile peaks are shown on the right. B, interaction of yeast Porin1 and the TOM complex detected by affinity purification. Mitochondria isolated from wild-type ( WT ) or yeast strain expressing HA-tagged VDAC homolog Porin1 ( Por1HA ) were subjected to affinity purification via anti-HA chromatography. Load (0.75%) and elution (100%) fractions were separated by SDS-PAGE, and proteins were detected by Western blotting with the indicated antisera. The Porin1 interactor OM14 and several TOM proteins ( upper panel ) were specifically co-purified, whereas three unrelated control proteins ( lower panel ) were not detected in the eluates. C, upper panel, affinity purifications as in B eluted under non-denaturing conditions and resolved by BN-PAGE. Western blot detection with anti-TOM40 ( left ) and anti-TOM22 ( right ) antibodies identified full-size TOM complex (around 450 kDa) specifically co-purified with HA-Porin1. Lower left panel, wild-type ( WT ) and Tom22His mitochondria subjected to nickel in complex with nitrilotriacetic acid purification followed by separation via BN-PAGE and Western blotting. Porin complexes were detected using anti-Porin1 antibodies (load 0.75%, elution 100%). Lower right panel, wild-type ( WT ) and Por1HA mitochondria were incubated with 35 S-labeled Oxa1 in the absence of a membrane potential to arrest the precursor at the TOM complex. Subsequently, samples were subjected to anti-HA chromatography, and protein complexes were separated by BN-PAGE. 35 S-Labeled Oxa1 arrested at the TOM complex was visualized with digital autoradiography. Note the increase in apparent mass of TOM complexes due to the stalled Oxa1 substrate.

    Techniques Used: Affinity Purification, Isolation, Expressing, Chromatography, SDS Page, Western Blot, Purification, Polyacrylamide Gel Electrophoresis, Incubation, Labeling, Autoradiography

    38) Product Images from "Abscisic Acid-Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1"

    Article Title: Abscisic Acid-Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1

    Journal: The Plant Cell

    doi: 10.1105/tpc.005272

    Mobility Shift of TRAB1 on SDS-PAGE Caused by ABA-Induced Phosphorylation. (A) Nuclear extracts of rice suspension-cultured cells (Oc cells) treated with ABA for the indicated times (0 indicates untreated cells) were analyzed by immunoblotting with anti-TRAB1 antibody. The arrow and asterisks indicate TRAB1-specific and nonspecific bands, respectively. Although the expression of the nonspecific bands was affected by ABA, the nature of these polypeptides is unknown. (B) TRAB1-dHA/His expressed in transgenic cells treated without (−) or with (+) ABA for 30 min was recovered with nickel–nitrilotriacetic acid agarose resin, incubated without (−) or with (+) CIAP, and analyzed by immunoblotting with anti-HA antibody. The signals seen at the top of the +CIAP lanes are parts of bulky CIAP bands that reacted nonspecifically with the anti-HA antibody.
    Figure Legend Snippet: Mobility Shift of TRAB1 on SDS-PAGE Caused by ABA-Induced Phosphorylation. (A) Nuclear extracts of rice suspension-cultured cells (Oc cells) treated with ABA for the indicated times (0 indicates untreated cells) were analyzed by immunoblotting with anti-TRAB1 antibody. The arrow and asterisks indicate TRAB1-specific and nonspecific bands, respectively. Although the expression of the nonspecific bands was affected by ABA, the nature of these polypeptides is unknown. (B) TRAB1-dHA/His expressed in transgenic cells treated without (−) or with (+) ABA for 30 min was recovered with nickel–nitrilotriacetic acid agarose resin, incubated without (−) or with (+) CIAP, and analyzed by immunoblotting with anti-HA antibody. The signals seen at the top of the +CIAP lanes are parts of bulky CIAP bands that reacted nonspecifically with the anti-HA antibody.

    Techniques Used: Mobility Shift, SDS Page, Cell Culture, Expressing, Transgenic Assay, Incubation

    39) Product Images from "Two Drosophila Ada2 Homologues Function in Different Multiprotein Complexes"

    Article Title: Two Drosophila Ada2 Homologues Function in Different Multiprotein Complexes

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.9.3305-3319.2003

    VP16 and Dmp53 interact with dAda2b-containing GNAT complexes. (A) GST pull-down assays were performed with GST-VP16 or GST alone bound to GSH-Sepharose beads and 300 μg of S2 nuclear extract. The precipitates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes for immunodetection assays with antisera against dGcn5, dAda2b, and dAda2a. Input, 30 μg of nuclear extract. (B) GST pull-down assays using a GST-Dmp53 fusion protein. (C) Coimmunoprecipitation assays were performed with anti-Dmp53 antibodies conjugated to protein A-Sepharose and 300 μg of S2 nuclear extract. The immunoprecipitated (IP) proteins were separated by SDS-PAGE prior to Western blotting analyses using antisera against dGcn5, dAda2b, and dAda2a. i, input; be, beads.
    Figure Legend Snippet: VP16 and Dmp53 interact with dAda2b-containing GNAT complexes. (A) GST pull-down assays were performed with GST-VP16 or GST alone bound to GSH-Sepharose beads and 300 μg of S2 nuclear extract. The precipitates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes for immunodetection assays with antisera against dGcn5, dAda2b, and dAda2a. Input, 30 μg of nuclear extract. (B) GST pull-down assays using a GST-Dmp53 fusion protein. (C) Coimmunoprecipitation assays were performed with anti-Dmp53 antibodies conjugated to protein A-Sepharose and 300 μg of S2 nuclear extract. The immunoprecipitated (IP) proteins were separated by SDS-PAGE prior to Western blotting analyses using antisera against dGcn5, dAda2b, and dAda2a. i, input; be, beads.

    Techniques Used: SDS Page, Immunodetection, Immunoprecipitation, Western Blot

    Characterization of the Drosophila GNAT subunits dAda2a, dAda2b, dAda3, dSpt3, and dTra1. (A) The antisera (is, immune serum) recognize proteins with the predicted molecular masses of dGcn5, dAda2b, dAda2a, dAda3, dSpt3, and dTra1 (arrowheads). Each lane contains 25 μg of nuclear extract. pre, preimmunization bleeds. Crude antisera against dAda2a and Spt3 detect a cross-reactive band of similar molecular mass, which is not recognized by affinity-purified antibodies (ap). (B) Developmental expression of dGcn5, dSpt3, dAda2a, and dAda2b. Lysates from Oregon R animals were electrophoresed on an SDS-10% polyacrylamide gel and analyzed by Western blotting. Embryonic lysates: 0-3 (blastoderm), 0-7 (gastrulation), 7-13 (germ band retraction), and 13-24 (differentiation). Larval stages: first, second, and third instars. Antibodies against tubulin served as loading controls. (C) HAT activity of the immunoprecipitates from 300 μg of S2 nuclear extract on nucleosomes (nuc) and core histones (ch), respectively. dAda3, dAda2a, dAda2b, and dSpt3 are associated with a dGcn5-specific HAT activity. Bacterially expressed rdGcn5 was used as a control. Top, fluorogram; bottom, photography of the Coomassie brilliant blue-stained SDS-polyacrylamide gel. (D) Western blot probed for dAda3 and dGcn5 (arrowheads). Input, 30 μg of nuclear extract; lane 1, immunoprecipitates from anti-dAda2b antibodies; lane 2, immunoprecipitates from anti-dAda2a; lane 3, immunoprecipitates from anti-dSpt3. hc, IgG heavy chain.
    Figure Legend Snippet: Characterization of the Drosophila GNAT subunits dAda2a, dAda2b, dAda3, dSpt3, and dTra1. (A) The antisera (is, immune serum) recognize proteins with the predicted molecular masses of dGcn5, dAda2b, dAda2a, dAda3, dSpt3, and dTra1 (arrowheads). Each lane contains 25 μg of nuclear extract. pre, preimmunization bleeds. Crude antisera against dAda2a and Spt3 detect a cross-reactive band of similar molecular mass, which is not recognized by affinity-purified antibodies (ap). (B) Developmental expression of dGcn5, dSpt3, dAda2a, and dAda2b. Lysates from Oregon R animals were electrophoresed on an SDS-10% polyacrylamide gel and analyzed by Western blotting. Embryonic lysates: 0-3 (blastoderm), 0-7 (gastrulation), 7-13 (germ band retraction), and 13-24 (differentiation). Larval stages: first, second, and third instars. Antibodies against tubulin served as loading controls. (C) HAT activity of the immunoprecipitates from 300 μg of S2 nuclear extract on nucleosomes (nuc) and core histones (ch), respectively. dAda3, dAda2a, dAda2b, and dSpt3 are associated with a dGcn5-specific HAT activity. Bacterially expressed rdGcn5 was used as a control. Top, fluorogram; bottom, photography of the Coomassie brilliant blue-stained SDS-polyacrylamide gel. (D) Western blot probed for dAda3 and dGcn5 (arrowheads). Input, 30 μg of nuclear extract; lane 1, immunoprecipitates from anti-dAda2b antibodies; lane 2, immunoprecipitates from anti-dAda2a; lane 3, immunoprecipitates from anti-dSpt3. hc, IgG heavy chain.

    Techniques Used: Affinity Purification, Expressing, Western Blot, HAT Assay, Activity Assay, Staining

    dAda2b, but not dAda2a, associates with dmTaf5, dmTaf9, dmTaf10, dSpt3, and dTra1. Affinity-purified antibodies were conjugated to protein A-Sepharose beads and were used in immunoprecipitation assays of 300 μg of nuclear extract from S2 cells. Input, 30 μg of nuclear extract. (A) Western blots immunoprobed for dSpt3 (left) and dTra1 (right). Lane 1, immunoprecipitates from anti-dAda2a (αdAda2a); lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dAda2b. (B) Western blots probed for dAda2a (left) and dAda2b (right). Lane 1, immunoprecipitates from anti-dSpt3; lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dSpt3; lane 7, immunoprecipitates from anti-dGcn5; lane 8, immunoprecipitates from anti-dAda3. hc, IgG heavy chain. (C) Western blots immunolabeled with antibodies against dmTaf5, dmTaf9, and dmTaf10. Lane 1, immunoprecipitates from anti-dAda2b; lane 2, immunoprecipitates from anti-dSpt3; lane 3, immunoprecipitates from anti-dAda3; lane 4, immunoprecipitates from anti-dAda2a.
    Figure Legend Snippet: dAda2b, but not dAda2a, associates with dmTaf5, dmTaf9, dmTaf10, dSpt3, and dTra1. Affinity-purified antibodies were conjugated to protein A-Sepharose beads and were used in immunoprecipitation assays of 300 μg of nuclear extract from S2 cells. Input, 30 μg of nuclear extract. (A) Western blots immunoprobed for dSpt3 (left) and dTra1 (right). Lane 1, immunoprecipitates from anti-dAda2a (αdAda2a); lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dAda2b. (B) Western blots probed for dAda2a (left) and dAda2b (right). Lane 1, immunoprecipitates from anti-dSpt3; lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dSpt3; lane 7, immunoprecipitates from anti-dGcn5; lane 8, immunoprecipitates from anti-dAda3. hc, IgG heavy chain. (C) Western blots immunolabeled with antibodies against dmTaf5, dmTaf9, and dmTaf10. Lane 1, immunoprecipitates from anti-dAda2b; lane 2, immunoprecipitates from anti-dSpt3; lane 3, immunoprecipitates from anti-dAda3; lane 4, immunoprecipitates from anti-dAda2a.

    Techniques Used: Affinity Purification, Immunoprecipitation, Western Blot, Immunolabeling

    dAda2b associates with dSAGA-specific subunits in biochemical fractionation assays. (A) Elution profiles of dGcn5, dAda3, dSpt3, dAda2b, and dAda2a from a Sephacryl S400 gel filtration column; 5 mg of S2 nuclear extract was separated on the column, and 15 μl of each fraction was subjected to SDS-PAGE, followed by Western blotting (fraction numbers indicated at the top). dGcn5 and dAda3 coelute in a range from ∼2.2 MDa to 300 kDa. dAda2b coelutes with dSpt3 in a range of ∼2 MDa to 800 kDa. dAda2a elutes in two peaks around 2.5 MDa and 440 kDa. i, input (30 μg of embryonic nuclear extract). (B) dSpt3, and dTra1 cofractionate with dAda2b, but not dAda2a, in anion-exchange chromatography. Bound proteins were eluted in a linear gradient of 100 to 450 mM sodium chloride from a 1-ml MonoQ anion-exchange column. The salt concentrations of the peak fractions are indicated above the blot. Fifteen microliters of each fraction was subjected to SDS-PAGE, followed by Western blotting (fraction numbers indicated at the top; f1 to f3, flowthrough). (C) MonoQ (MQ) fractions (frcts.) 16 to 20 (top) and 24 to 28 (bottom) of the anion-exchange chromatography were concentrated and subjected to size exclusion chromatography. dAda2b and dGcn5 coelute in a range of ∼1.8 MDa. dAda2a elutes in fractions of > 2 MDa and ∼450 kDa. dGcn5 is detectable only in fractions containing the smaller dAda2a complex. nuc., nuclear. (D) Gel filtration analysis of nuclear extract from 0- to 12-h-old embryos as described for panel A. A third dAda2a peak is observed in a molecular-mass range of ∼1.8 MDa. (E) Pooled dAda2a peak fractions from embryonic extracts (panel D) were subjected to immunoprecipitation assays with affinity-purified anti-dAda2a antibodies. The immunoprecipitates (IP) were separated by SDS-PAGE and were probed for dGcn5. Lane 1, fractions 58 to 61; lane 2, fractions 65 to 68; lane 3, fractions 81 to 84.
    Figure Legend Snippet: dAda2b associates with dSAGA-specific subunits in biochemical fractionation assays. (A) Elution profiles of dGcn5, dAda3, dSpt3, dAda2b, and dAda2a from a Sephacryl S400 gel filtration column; 5 mg of S2 nuclear extract was separated on the column, and 15 μl of each fraction was subjected to SDS-PAGE, followed by Western blotting (fraction numbers indicated at the top). dGcn5 and dAda3 coelute in a range from ∼2.2 MDa to 300 kDa. dAda2b coelutes with dSpt3 in a range of ∼2 MDa to 800 kDa. dAda2a elutes in two peaks around 2.5 MDa and 440 kDa. i, input (30 μg of embryonic nuclear extract). (B) dSpt3, and dTra1 cofractionate with dAda2b, but not dAda2a, in anion-exchange chromatography. Bound proteins were eluted in a linear gradient of 100 to 450 mM sodium chloride from a 1-ml MonoQ anion-exchange column. The salt concentrations of the peak fractions are indicated above the blot. Fifteen microliters of each fraction was subjected to SDS-PAGE, followed by Western blotting (fraction numbers indicated at the top; f1 to f3, flowthrough). (C) MonoQ (MQ) fractions (frcts.) 16 to 20 (top) and 24 to 28 (bottom) of the anion-exchange chromatography were concentrated and subjected to size exclusion chromatography. dAda2b and dGcn5 coelute in a range of ∼1.8 MDa. dAda2a elutes in fractions of > 2 MDa and ∼450 kDa. dGcn5 is detectable only in fractions containing the smaller dAda2a complex. nuc., nuclear. (D) Gel filtration analysis of nuclear extract from 0- to 12-h-old embryos as described for panel A. A third dAda2a peak is observed in a molecular-mass range of ∼1.8 MDa. (E) Pooled dAda2a peak fractions from embryonic extracts (panel D) were subjected to immunoprecipitation assays with affinity-purified anti-dAda2a antibodies. The immunoprecipitates (IP) were separated by SDS-PAGE and were probed for dGcn5. Lane 1, fractions 58 to 61; lane 2, fractions 65 to 68; lane 3, fractions 81 to 84.

    Techniques Used: Fractionation, Filtration, SDS Page, Western Blot, Multiple Displacement Amplification, Chromatography, Size-exclusion Chromatography, Immunoprecipitation, Affinity Purification

    Localization studies of dAda2b and dAda2a on polytene chromosomes from third-instar larvae. Polytene chromosome spreads were double-labeled with the indicated affinity-purified antibodies. The pictures were taken at different magnifications; the bars correspond to 20 μm. (A to D) Merged red and green channels. (A) Costaining using rabbit anti-dAda3 antibodies (red) and rat anti-dAda2b antibodies (green). The arrowheads indicate selected regions exclusively staining for dAda3. (B) Costaining with rabbit anti-dAda2a (red) and rat anti-dAda2b (green). The arrowheads indicate selected regions of signal overlap. The arrows indicate puffs strongly staining for dAda2a. (C) Costaining with rat anti-dAda2a (red) and rabbit anti-dAda3 (green). The arrowheads indicate selected regions of signal overlap. The arrows indicate puffs strongly staining for dAda2a. (D) Costaining with rabbit anti-dAda2a (red) and rat anti-dGcn5 (green). The arrowheads indicate selected regions of overlap. The arrows indicate puffs strongly staining for dAda2a. (E) Magnification of the boxed region in panel C. DNA was stained with DAPI. dAda2a strongly stains decondensed euchromatic regions (compare the white bars). dAda3 and dAda2a colocalize in less condensed euchromatic regions. (F) dAda2a and phosphorylated RNA polymerase II colocalize on polytene chromosomes from third-instar larvae. Top, staining with rabbit anti-dAda2a antibodies; middle, staining with mouse anti-phosphorylated C-terminal domain of RNA polymerase II antibodies; bottom, overlap of green and red stains appears in the merge of panels A and B as yellow, yellow-green, and orange.
    Figure Legend Snippet: Localization studies of dAda2b and dAda2a on polytene chromosomes from third-instar larvae. Polytene chromosome spreads were double-labeled with the indicated affinity-purified antibodies. The pictures were taken at different magnifications; the bars correspond to 20 μm. (A to D) Merged red and green channels. (A) Costaining using rabbit anti-dAda3 antibodies (red) and rat anti-dAda2b antibodies (green). The arrowheads indicate selected regions exclusively staining for dAda3. (B) Costaining with rabbit anti-dAda2a (red) and rat anti-dAda2b (green). The arrowheads indicate selected regions of signal overlap. The arrows indicate puffs strongly staining for dAda2a. (C) Costaining with rat anti-dAda2a (red) and rabbit anti-dAda3 (green). The arrowheads indicate selected regions of signal overlap. The arrows indicate puffs strongly staining for dAda2a. (D) Costaining with rabbit anti-dAda2a (red) and rat anti-dGcn5 (green). The arrowheads indicate selected regions of overlap. The arrows indicate puffs strongly staining for dAda2a. (E) Magnification of the boxed region in panel C. DNA was stained with DAPI. dAda2a strongly stains decondensed euchromatic regions (compare the white bars). dAda3 and dAda2a colocalize in less condensed euchromatic regions. (F) dAda2a and phosphorylated RNA polymerase II colocalize on polytene chromosomes from third-instar larvae. Top, staining with rabbit anti-dAda2a antibodies; middle, staining with mouse anti-phosphorylated C-terminal domain of RNA polymerase II antibodies; bottom, overlap of green and red stains appears in the merge of panels A and B as yellow, yellow-green, and orange.

    Techniques Used: Labeling, Affinity Purification, Staining

    40) Product Images from "Two Drosophila Ada2 Homologues Function in Different Multiprotein Complexes"

    Article Title: Two Drosophila Ada2 Homologues Function in Different Multiprotein Complexes

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.9.3305-3319.2003

    Characterization of the Drosophila GNAT subunits dAda2a, dAda2b, dAda3, dSpt3, and dTra1. (A) The antisera (is, immune serum) recognize proteins with the predicted molecular masses of dGcn5, dAda2b, dAda2a, dAda3, dSpt3, and dTra1 (arrowheads). Each lane contains 25 μg of nuclear extract. pre, preimmunization bleeds. Crude antisera against dAda2a and Spt3 detect a cross-reactive band of similar molecular mass, which is not recognized by affinity-purified antibodies (ap). (B) Developmental expression of dGcn5, dSpt3, dAda2a, and dAda2b. Lysates from Oregon R animals were electrophoresed on an SDS-10% polyacrylamide gel and analyzed by Western blotting. Embryonic lysates: 0-3 (blastoderm), 0-7 (gastrulation), 7-13 (germ band retraction), and 13-24 (differentiation). Larval stages: first, second, and third instars. Antibodies against tubulin served as loading controls. (C) HAT activity of the immunoprecipitates from 300 μg of S2 nuclear extract on nucleosomes (nuc) and core histones (ch), respectively. dAda3, dAda2a, dAda2b, and dSpt3 are associated with a dGcn5-specific HAT activity. Bacterially expressed rdGcn5 was used as a control. Top, fluorogram; bottom, photography of the Coomassie brilliant blue-stained SDS-polyacrylamide gel. (D) Western blot probed for dAda3 and dGcn5 (arrowheads). Input, 30 μg of nuclear extract; lane 1, immunoprecipitates from anti-dAda2b antibodies; lane 2, immunoprecipitates from anti-dAda2a; lane 3, immunoprecipitates from anti-dSpt3. hc, IgG heavy chain.
    Figure Legend Snippet: Characterization of the Drosophila GNAT subunits dAda2a, dAda2b, dAda3, dSpt3, and dTra1. (A) The antisera (is, immune serum) recognize proteins with the predicted molecular masses of dGcn5, dAda2b, dAda2a, dAda3, dSpt3, and dTra1 (arrowheads). Each lane contains 25 μg of nuclear extract. pre, preimmunization bleeds. Crude antisera against dAda2a and Spt3 detect a cross-reactive band of similar molecular mass, which is not recognized by affinity-purified antibodies (ap). (B) Developmental expression of dGcn5, dSpt3, dAda2a, and dAda2b. Lysates from Oregon R animals were electrophoresed on an SDS-10% polyacrylamide gel and analyzed by Western blotting. Embryonic lysates: 0-3 (blastoderm), 0-7 (gastrulation), 7-13 (germ band retraction), and 13-24 (differentiation). Larval stages: first, second, and third instars. Antibodies against tubulin served as loading controls. (C) HAT activity of the immunoprecipitates from 300 μg of S2 nuclear extract on nucleosomes (nuc) and core histones (ch), respectively. dAda3, dAda2a, dAda2b, and dSpt3 are associated with a dGcn5-specific HAT activity. Bacterially expressed rdGcn5 was used as a control. Top, fluorogram; bottom, photography of the Coomassie brilliant blue-stained SDS-polyacrylamide gel. (D) Western blot probed for dAda3 and dGcn5 (arrowheads). Input, 30 μg of nuclear extract; lane 1, immunoprecipitates from anti-dAda2b antibodies; lane 2, immunoprecipitates from anti-dAda2a; lane 3, immunoprecipitates from anti-dSpt3. hc, IgG heavy chain.

    Techniques Used: Affinity Purification, Expressing, Western Blot, HAT Assay, Activity Assay, Staining

    dAda2b, but not dAda2a, associates with dmTaf5, dmTaf9, dmTaf10, dSpt3, and dTra1. Affinity-purified antibodies were conjugated to protein A-Sepharose beads and were used in immunoprecipitation assays of 300 μg of nuclear extract from S2 cells. Input, 30 μg of nuclear extract. (A) Western blots immunoprobed for dSpt3 (left) and dTra1 (right). Lane 1, immunoprecipitates from anti-dAda2a (αdAda2a); lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dAda2b. (B) Western blots probed for dAda2a (left) and dAda2b (right). Lane 1, immunoprecipitates from anti-dSpt3; lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dSpt3; lane 7, immunoprecipitates from anti-dGcn5; lane 8, immunoprecipitates from anti-dAda3. hc, IgG heavy chain. (C) Western blots immunolabeled with antibodies against dmTaf5, dmTaf9, and dmTaf10. Lane 1, immunoprecipitates from anti-dAda2b; lane 2, immunoprecipitates from anti-dSpt3; lane 3, immunoprecipitates from anti-dAda3; lane 4, immunoprecipitates from anti-dAda2a.
    Figure Legend Snippet: dAda2b, but not dAda2a, associates with dmTaf5, dmTaf9, dmTaf10, dSpt3, and dTra1. Affinity-purified antibodies were conjugated to protein A-Sepharose beads and were used in immunoprecipitation assays of 300 μg of nuclear extract from S2 cells. Input, 30 μg of nuclear extract. (A) Western blots immunoprobed for dSpt3 (left) and dTra1 (right). Lane 1, immunoprecipitates from anti-dAda2a (αdAda2a); lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dAda2b. (B) Western blots probed for dAda2a (left) and dAda2b (right). Lane 1, immunoprecipitates from anti-dSpt3; lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dSpt3; lane 7, immunoprecipitates from anti-dGcn5; lane 8, immunoprecipitates from anti-dAda3. hc, IgG heavy chain. (C) Western blots immunolabeled with antibodies against dmTaf5, dmTaf9, and dmTaf10. Lane 1, immunoprecipitates from anti-dAda2b; lane 2, immunoprecipitates from anti-dSpt3; lane 3, immunoprecipitates from anti-dAda3; lane 4, immunoprecipitates from anti-dAda2a.

    Techniques Used: Affinity Purification, Immunoprecipitation, Western Blot, Immunolabeling

    dAda2b associates with dSAGA-specific subunits in biochemical fractionation assays. (A) Elution profiles of dGcn5, dAda3, dSpt3, dAda2b, and dAda2a from a Sephacryl S400 gel filtration column; 5 mg of S2 nuclear extract was separated on the column, and 15 μl of each fraction was subjected to SDS-PAGE, followed by Western blotting (fraction numbers indicated at the top). dGcn5 and dAda3 coelute in a range from ∼2.2 MDa to 300 kDa. dAda2b coelutes with dSpt3 in a range of ∼2 MDa to 800 kDa. dAda2a elutes in two peaks around 2.5 MDa and 440 kDa. i, input (30 μg of embryonic nuclear extract). (B) dSpt3, and dTra1 cofractionate with dAda2b, but not dAda2a, in anion-exchange chromatography. Bound proteins were eluted in a linear gradient of 100 to 450 mM sodium chloride from a 1-ml MonoQ anion-exchange column. The salt concentrations of the peak fractions are indicated above the blot. Fifteen microliters of each fraction was subjected to SDS-PAGE, followed by Western blotting (fraction numbers indicated at the top; f1 to f3, flowthrough). (C) MonoQ (MQ) fractions (frcts.) 16 to 20 (top) and 24 to 28 (bottom) of the anion-exchange chromatography were concentrated and subjected to size exclusion chromatography. dAda2b and dGcn5 coelute in a range of ∼1.8 MDa. dAda2a elutes in fractions of > 2 MDa and ∼450 kDa. dGcn5 is detectable only in fractions containing the smaller dAda2a complex. nuc., nuclear. (D) Gel filtration analysis of nuclear extract from 0- to 12-h-old embryos as described for panel A. A third dAda2a peak is observed in a molecular-mass range of ∼1.8 MDa. (E) Pooled dAda2a peak fractions from embryonic extracts (panel D) were subjected to immunoprecipitation assays with affinity-purified anti-dAda2a antibodies. The immunoprecipitates (IP) were separated by SDS-PAGE and were probed for dGcn5. Lane 1, fractions 58 to 61; lane 2, fractions 65 to 68; lane 3, fractions 81 to 84.
    Figure Legend Snippet: dAda2b associates with dSAGA-specific subunits in biochemical fractionation assays. (A) Elution profiles of dGcn5, dAda3, dSpt3, dAda2b, and dAda2a from a Sephacryl S400 gel filtration column; 5 mg of S2 nuclear extract was separated on the column, and 15 μl of each fraction was subjected to SDS-PAGE, followed by Western blotting (fraction numbers indicated at the top). dGcn5 and dAda3 coelute in a range from ∼2.2 MDa to 300 kDa. dAda2b coelutes with dSpt3 in a range of ∼2 MDa to 800 kDa. dAda2a elutes in two peaks around 2.5 MDa and 440 kDa. i, input (30 μg of embryonic nuclear extract). (B) dSpt3, and dTra1 cofractionate with dAda2b, but not dAda2a, in anion-exchange chromatography. Bound proteins were eluted in a linear gradient of 100 to 450 mM sodium chloride from a 1-ml MonoQ anion-exchange column. The salt concentrations of the peak fractions are indicated above the blot. Fifteen microliters of each fraction was subjected to SDS-PAGE, followed by Western blotting (fraction numbers indicated at the top; f1 to f3, flowthrough). (C) MonoQ (MQ) fractions (frcts.) 16 to 20 (top) and 24 to 28 (bottom) of the anion-exchange chromatography were concentrated and subjected to size exclusion chromatography. dAda2b and dGcn5 coelute in a range of ∼1.8 MDa. dAda2a elutes in fractions of > 2 MDa and ∼450 kDa. dGcn5 is detectable only in fractions containing the smaller dAda2a complex. nuc., nuclear. (D) Gel filtration analysis of nuclear extract from 0- to 12-h-old embryos as described for panel A. A third dAda2a peak is observed in a molecular-mass range of ∼1.8 MDa. (E) Pooled dAda2a peak fractions from embryonic extracts (panel D) were subjected to immunoprecipitation assays with affinity-purified anti-dAda2a antibodies. The immunoprecipitates (IP) were separated by SDS-PAGE and were probed for dGcn5. Lane 1, fractions 58 to 61; lane 2, fractions 65 to 68; lane 3, fractions 81 to 84.

    Techniques Used: Fractionation, Filtration, SDS Page, Western Blot, Multiple Displacement Amplification, Chromatography, Size-exclusion Chromatography, Immunoprecipitation, Affinity Purification

    Related Articles

    Western Blot:

    Article Title: Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-?: Application to Environmental Analysis
    Article Snippet: .. In order to purify recombinant ER-α, 2.5 mL of Ni-NTA-agarose phase (Qiagen, Courtaboeuf, France) was washed with washing buffer [WB: 20 mM Tris HCl, pH 7.5, 300 mM NaCl, 20% glycerol, 0.1 mg/mL charcoal-treated bovine serum albumin (BSA), and 10 mM imidazole] and incubated in a column with 100-mL recombinant receptor solution. .. After rolling for 16 hr, agarose phase was washed with WB, and the receptor was eluted with 7 mL eluting buffer (EB: 20 mM Tris HCl, pH 7.5, 300 mM NaCl, 20% glycerol, 0.1 mg/mL charcoal-treated BSA, and 100 mM imidazole).

    Incubation:

    Article Title: Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-?: Application to Environmental Analysis
    Article Snippet: .. In order to purify recombinant ER-α, 2.5 mL of Ni-NTA-agarose phase (Qiagen, Courtaboeuf, France) was washed with washing buffer [WB: 20 mM Tris HCl, pH 7.5, 300 mM NaCl, 20% glycerol, 0.1 mg/mL charcoal-treated bovine serum albumin (BSA), and 10 mM imidazole] and incubated in a column with 100-mL recombinant receptor solution. .. After rolling for 16 hr, agarose phase was washed with WB, and the receptor was eluted with 7 mL eluting buffer (EB: 20 mM Tris HCl, pH 7.5, 300 mM NaCl, 20% glycerol, 0.1 mg/mL charcoal-treated BSA, and 100 mM imidazole).

    Recombinant:

    Article Title: Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-?: Application to Environmental Analysis
    Article Snippet: .. In order to purify recombinant ER-α, 2.5 mL of Ni-NTA-agarose phase (Qiagen, Courtaboeuf, France) was washed with washing buffer [WB: 20 mM Tris HCl, pH 7.5, 300 mM NaCl, 20% glycerol, 0.1 mg/mL charcoal-treated bovine serum albumin (BSA), and 10 mM imidazole] and incubated in a column with 100-mL recombinant receptor solution. .. After rolling for 16 hr, agarose phase was washed with WB, and the receptor was eluted with 7 mL eluting buffer (EB: 20 mM Tris HCl, pH 7.5, 300 mM NaCl, 20% glycerol, 0.1 mg/mL charcoal-treated BSA, and 100 mM imidazole).

    Purification:

    Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
    Article Snippet: .. The His-tagged FumF protein was expressed and purified using Ni-NTA agarose resin (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. .. The protein concentration was determined by a Bio-Rad protein assay kit using bovine serum albumin as the standard.

    Article Title: Natural compound Oblongifolin C confers gemcitabine resistance in pancreatic cancer by downregulating Src/MAPK/ERK pathways
    Article Snippet: .. His-tagged Src was purified with Ni-NTA Agarose beads according to the manufacturers’ protocols (Qiagen). .. The ubiquitination levels and the amount of Src-his-tagged protein in the immunoprecipitation product were detected by western blotting.

    Article Title: A Versatile Approach to Transform Low-Affinity Peptides into Protein Probes with Co-Translationally Expressed Chemical Cross-Linker 1
    Article Snippet: .. SH3-His, wtTOP1-GFP, and TOP1-DOPA-GFP were purified using Ni-NTA agarose beads (Qiagen). ..

    Article Title: The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome
    Article Snippet: .. His Usp4 and His Sart3 were also purified from baculovirus-infected insect cells on NiNTA-agarose (Qiagen) as described above. ..

    Article Title: Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment
    Article Snippet: .. After these wash steps, purification of rSmLy6A was performed under denaturing conditions using Ni-NTA agarose beads (Qiagen) according to the manufacturer’s instructions. .. The resultant purified protein had urea removed by stepwise dialysis against ≥20 vols of 100mM NaH2 PO4 , 10mM Tris-HCl, pH 6.3 buffer containing 6M, 3 M, 1 M, then two buffer changes without urea.

    Article Title: Regulatory control of DNA end resection by Sae2 phosphorylation
    Article Snippet: .. Finally, pSae2 was purified using NiNTA agarose (Qiagen). .. We modified our original procedure to preserve maximal phosphorylation of the protein.

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    Qiagen nickel nitrilotriacetic acid agarose
    Complementation of the E. coli grpE Deletion Strain OD212 with CGE1 and Analysis of CGE1–DnaK Interactions. (A) Temperature-sensitive E. coli strain OD212 carrying a deletion of its grpE gene was transformed with a plasmid vector for the expression of CGE1 (CGE1-1 and CGE1-2) or the same vector expressing an unrelated gene (control). Dilutions of transformant cultures were spotted onto Luria-Bertani plates and incubated overnight at 25, 37, or 43°C. (B) Comparison of the expression of CGE1 in the OD212 transformants described in (A) with Chlamydomonas (Chlamy) CGE1 by protein gel blot analysis and detection with CGE1 antiserum (α-CGE1). Each lane contained 15 μg of total soluble protein. (C) Expression of hexahistidine-tagged versions of CGE1 and an unrelated 30-kD protein (control) from plasmid vectors was induced in E. coli strain M15 by isopropylthio-β-galactoside. Cells were lysed under native conditions (crude lysate; lanes 1 and 2) and incubated with <t>nickel–nitrilotriacetic</t> acid agarose (Ni-NTA). After washing, proteins bound to Ni-NTA were eluted by incubation with 250 mM imidazole (lanes 3 and 4). In a parallel experiment, Ni-NTA beads binding CGE1 were first incubated for 10 min at 24°C with a buffer containing 20 mM 3-( N -morpholino)-propanesulfonic acid (Mops)-KOH, pH 7.4, 80 mM KCl, 5 mM MgCl 2 , and either 5 mM ATP or no nucleotide (mock) (lanes 5 and 6). The proteins that had remained on the resin then were eluted with 250 mM imidazole (lanes 7 and 8). Eluted proteins were precipitated with trichloroacetic acid, separated on an SDS–10% polyacrylamide gel, and visualized by Coomassie blue staining (top) or transferred to nitrocellulose and immunodetected with an antiserum (α) against DnaK using enhanced chemiluminescence (bottom).
    Nickel Nitrilotriacetic Acid Agarose, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mobility Shift of TRAB1 on SDS-PAGE Caused by ABA-Induced Phosphorylation. (A) Nuclear extracts of rice suspension-cultured cells (Oc cells) treated with ABA for the indicated times (0 indicates untreated cells) were analyzed by immunoblotting with anti-TRAB1 antibody. The arrow and asterisks indicate TRAB1-specific and nonspecific bands, respectively. Although the expression of the nonspecific bands was affected by ABA, the nature of these polypeptides is unknown. (B) TRAB1-dHA/His expressed in transgenic cells treated without (−) or with (+) ABA for 30 min was recovered with <t>nickel–nitrilotriacetic</t> acid agarose resin, incubated without (−) or with (+) CIAP, and analyzed by immunoblotting with anti-HA antibody. The signals seen at the top of the +CIAP lanes are parts of bulky CIAP bands that reacted nonspecifically with the anti-HA antibody.
    Nickel Nitrilotriacetic Acid Agarose Resin, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of the glucosyltransferase activity of recombinant UGT85A24 expressed in E. coli . A , results from SDS-PAGE analysis of the crude protein from E. coli JM109 harboring pQE-30-GjUGT2 ( left lane: C ) and the recombinant enzyme (UGT85A24) purified using a <t>nickel-nitrilotriacetic</t> acid resin column ( right: P ). B , time course changes in genipin glucosylation by incubation with recombinant UGT85A24.
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    Identification of super-complexes assembled from VDACs/Porins and TOM complexes. A, relative abundance-mass profiles of VDAC1–3 and TOM20 proteins. Color-coding and the Pearson correlation coefficient r of profile peaks are shown on the right. B, interaction of yeast Porin1 and the TOM complex detected by affinity purification. Mitochondria isolated from wild-type ( WT ) or yeast strain expressing HA-tagged VDAC homolog Porin1 ( Por1HA ) were subjected to affinity purification via anti-HA chromatography. Load (0.75%) and elution (100%) fractions were separated by SDS-PAGE, and proteins were detected by Western blotting with the indicated antisera. The Porin1 interactor OM14 and several TOM proteins ( upper panel ) were specifically co-purified, whereas three unrelated control proteins ( lower panel ) were not detected in the eluates. C, upper panel, affinity purifications as in B eluted under non-denaturing conditions and resolved by BN-PAGE. Western blot detection with anti-TOM40 ( left ) and anti-TOM22 ( right ) antibodies identified full-size TOM complex (around 450 kDa) specifically co-purified with HA-Porin1. Lower left panel, wild-type ( WT ) and Tom22His mitochondria subjected to nickel in complex with <t>nitrilotriacetic</t> acid purification followed by separation via BN-PAGE and Western blotting. Porin complexes were detected using anti-Porin1 antibodies (load 0.75%, elution 100%). Lower right panel, wild-type ( WT ) and Por1HA mitochondria were incubated with 35 S-labeled Oxa1 in the absence of a membrane potential to arrest the precursor at the TOM complex. Subsequently, samples were subjected to anti-HA chromatography, and protein complexes were separated by BN-PAGE. 35 S-Labeled Oxa1 arrested at the TOM complex was visualized with digital autoradiography. Note the increase in apparent mass of TOM complexes due to the stalled Oxa1 substrate.
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    Complementation of the E. coli grpE Deletion Strain OD212 with CGE1 and Analysis of CGE1–DnaK Interactions. (A) Temperature-sensitive E. coli strain OD212 carrying a deletion of its grpE gene was transformed with a plasmid vector for the expression of CGE1 (CGE1-1 and CGE1-2) or the same vector expressing an unrelated gene (control). Dilutions of transformant cultures were spotted onto Luria-Bertani plates and incubated overnight at 25, 37, or 43°C. (B) Comparison of the expression of CGE1 in the OD212 transformants described in (A) with Chlamydomonas (Chlamy) CGE1 by protein gel blot analysis and detection with CGE1 antiserum (α-CGE1). Each lane contained 15 μg of total soluble protein. (C) Expression of hexahistidine-tagged versions of CGE1 and an unrelated 30-kD protein (control) from plasmid vectors was induced in E. coli strain M15 by isopropylthio-β-galactoside. Cells were lysed under native conditions (crude lysate; lanes 1 and 2) and incubated with nickel–nitrilotriacetic acid agarose (Ni-NTA). After washing, proteins bound to Ni-NTA were eluted by incubation with 250 mM imidazole (lanes 3 and 4). In a parallel experiment, Ni-NTA beads binding CGE1 were first incubated for 10 min at 24°C with a buffer containing 20 mM 3-( N -morpholino)-propanesulfonic acid (Mops)-KOH, pH 7.4, 80 mM KCl, 5 mM MgCl 2 , and either 5 mM ATP or no nucleotide (mock) (lanes 5 and 6). The proteins that had remained on the resin then were eluted with 250 mM imidazole (lanes 7 and 8). Eluted proteins were precipitated with trichloroacetic acid, separated on an SDS–10% polyacrylamide gel, and visualized by Coomassie blue staining (top) or transferred to nitrocellulose and immunodetected with an antiserum (α) against DnaK using enhanced chemiluminescence (bottom).

    Journal: The Plant Cell

    Article Title: The Chloroplastic GrpE Homolog of Chlamydomonas

    doi: 10.1105/tpc.010202

    Figure Lengend Snippet: Complementation of the E. coli grpE Deletion Strain OD212 with CGE1 and Analysis of CGE1–DnaK Interactions. (A) Temperature-sensitive E. coli strain OD212 carrying a deletion of its grpE gene was transformed with a plasmid vector for the expression of CGE1 (CGE1-1 and CGE1-2) or the same vector expressing an unrelated gene (control). Dilutions of transformant cultures were spotted onto Luria-Bertani plates and incubated overnight at 25, 37, or 43°C. (B) Comparison of the expression of CGE1 in the OD212 transformants described in (A) with Chlamydomonas (Chlamy) CGE1 by protein gel blot analysis and detection with CGE1 antiserum (α-CGE1). Each lane contained 15 μg of total soluble protein. (C) Expression of hexahistidine-tagged versions of CGE1 and an unrelated 30-kD protein (control) from plasmid vectors was induced in E. coli strain M15 by isopropylthio-β-galactoside. Cells were lysed under native conditions (crude lysate; lanes 1 and 2) and incubated with nickel–nitrilotriacetic acid agarose (Ni-NTA). After washing, proteins bound to Ni-NTA were eluted by incubation with 250 mM imidazole (lanes 3 and 4). In a parallel experiment, Ni-NTA beads binding CGE1 were first incubated for 10 min at 24°C with a buffer containing 20 mM 3-( N -morpholino)-propanesulfonic acid (Mops)-KOH, pH 7.4, 80 mM KCl, 5 mM MgCl 2 , and either 5 mM ATP or no nucleotide (mock) (lanes 5 and 6). The proteins that had remained on the resin then were eluted with 250 mM imidazole (lanes 7 and 8). Eluted proteins were precipitated with trichloroacetic acid, separated on an SDS–10% polyacrylamide gel, and visualized by Coomassie blue staining (top) or transferred to nitrocellulose and immunodetected with an antiserum (α) against DnaK using enhanced chemiluminescence (bottom).

    Article Snippet: Recombinant proteins were overexpressed in Escherichia coli and purified using nickel-nitrilotriacetic acid agarose according to the protocol of the manufacturer (Qiagen, Hilden, Germany).

    Techniques: Transformation Assay, Plasmid Preparation, Expressing, Incubation, Western Blot, Binding Assay, Staining

    Mobility Shift of TRAB1 on SDS-PAGE Caused by ABA-Induced Phosphorylation. (A) Nuclear extracts of rice suspension-cultured cells (Oc cells) treated with ABA for the indicated times (0 indicates untreated cells) were analyzed by immunoblotting with anti-TRAB1 antibody. The arrow and asterisks indicate TRAB1-specific and nonspecific bands, respectively. Although the expression of the nonspecific bands was affected by ABA, the nature of these polypeptides is unknown. (B) TRAB1-dHA/His expressed in transgenic cells treated without (−) or with (+) ABA for 30 min was recovered with nickel–nitrilotriacetic acid agarose resin, incubated without (−) or with (+) CIAP, and analyzed by immunoblotting with anti-HA antibody. The signals seen at the top of the +CIAP lanes are parts of bulky CIAP bands that reacted nonspecifically with the anti-HA antibody.

    Journal: The Plant Cell

    Article Title: Abscisic Acid-Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1

    doi: 10.1105/tpc.005272

    Figure Lengend Snippet: Mobility Shift of TRAB1 on SDS-PAGE Caused by ABA-Induced Phosphorylation. (A) Nuclear extracts of rice suspension-cultured cells (Oc cells) treated with ABA for the indicated times (0 indicates untreated cells) were analyzed by immunoblotting with anti-TRAB1 antibody. The arrow and asterisks indicate TRAB1-specific and nonspecific bands, respectively. Although the expression of the nonspecific bands was affected by ABA, the nature of these polypeptides is unknown. (B) TRAB1-dHA/His expressed in transgenic cells treated without (−) or with (+) ABA for 30 min was recovered with nickel–nitrilotriacetic acid agarose resin, incubated without (−) or with (+) CIAP, and analyzed by immunoblotting with anti-HA antibody. The signals seen at the top of the +CIAP lanes are parts of bulky CIAP bands that reacted nonspecifically with the anti-HA antibody.

    Article Snippet: TRAB1-dHA/His protein in the nuclear or protoplast extracts was recovered with nickel-nitrilotriacetic acid agarose resin (Qiagen).

    Techniques: Mobility Shift, SDS Page, Cell Culture, Expressing, Transgenic Assay, Incubation

    Analysis of the glucosyltransferase activity of recombinant UGT85A24 expressed in E. coli . A , results from SDS-PAGE analysis of the crude protein from E. coli JM109 harboring pQE-30-GjUGT2 ( left lane: C ) and the recombinant enzyme (UGT85A24) purified using a nickel-nitrilotriacetic acid resin column ( right: P ). B , time course changes in genipin glucosylation by incubation with recombinant UGT85A24.

    Journal: The Journal of Biological Chemistry

    Article Title: Iridoid-specific Glucosyltransferase from Gardenia jasminoides *

    doi: 10.1074/jbc.M111.242586

    Figure Lengend Snippet: Analysis of the glucosyltransferase activity of recombinant UGT85A24 expressed in E. coli . A , results from SDS-PAGE analysis of the crude protein from E. coli JM109 harboring pQE-30-GjUGT2 ( left lane: C ) and the recombinant enzyme (UGT85A24) purified using a nickel-nitrilotriacetic acid resin column ( right: P ). B , time course changes in genipin glucosylation by incubation with recombinant UGT85A24.

    Article Snippet: The recombinant protein was affinity-purified on a nickel-nitrilotriacetic acid-agarose matrix (Qiagen) according to the manufacturer's instructions.

    Techniques: Activity Assay, Recombinant, SDS Page, Purification, Incubation

    Identification of super-complexes assembled from VDACs/Porins and TOM complexes. A, relative abundance-mass profiles of VDAC1–3 and TOM20 proteins. Color-coding and the Pearson correlation coefficient r of profile peaks are shown on the right. B, interaction of yeast Porin1 and the TOM complex detected by affinity purification. Mitochondria isolated from wild-type ( WT ) or yeast strain expressing HA-tagged VDAC homolog Porin1 ( Por1HA ) were subjected to affinity purification via anti-HA chromatography. Load (0.75%) and elution (100%) fractions were separated by SDS-PAGE, and proteins were detected by Western blotting with the indicated antisera. The Porin1 interactor OM14 and several TOM proteins ( upper panel ) were specifically co-purified, whereas three unrelated control proteins ( lower panel ) were not detected in the eluates. C, upper panel, affinity purifications as in B eluted under non-denaturing conditions and resolved by BN-PAGE. Western blot detection with anti-TOM40 ( left ) and anti-TOM22 ( right ) antibodies identified full-size TOM complex (around 450 kDa) specifically co-purified with HA-Porin1. Lower left panel, wild-type ( WT ) and Tom22His mitochondria subjected to nickel in complex with nitrilotriacetic acid purification followed by separation via BN-PAGE and Western blotting. Porin complexes were detected using anti-Porin1 antibodies (load 0.75%, elution 100%). Lower right panel, wild-type ( WT ) and Por1HA mitochondria were incubated with 35 S-labeled Oxa1 in the absence of a membrane potential to arrest the precursor at the TOM complex. Subsequently, samples were subjected to anti-HA chromatography, and protein complexes were separated by BN-PAGE. 35 S-Labeled Oxa1 arrested at the TOM complex was visualized with digital autoradiography. Note the increase in apparent mass of TOM complexes due to the stalled Oxa1 substrate.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling *

    doi: 10.1074/mcp.M115.054080

    Figure Lengend Snippet: Identification of super-complexes assembled from VDACs/Porins and TOM complexes. A, relative abundance-mass profiles of VDAC1–3 and TOM20 proteins. Color-coding and the Pearson correlation coefficient r of profile peaks are shown on the right. B, interaction of yeast Porin1 and the TOM complex detected by affinity purification. Mitochondria isolated from wild-type ( WT ) or yeast strain expressing HA-tagged VDAC homolog Porin1 ( Por1HA ) were subjected to affinity purification via anti-HA chromatography. Load (0.75%) and elution (100%) fractions were separated by SDS-PAGE, and proteins were detected by Western blotting with the indicated antisera. The Porin1 interactor OM14 and several TOM proteins ( upper panel ) were specifically co-purified, whereas three unrelated control proteins ( lower panel ) were not detected in the eluates. C, upper panel, affinity purifications as in B eluted under non-denaturing conditions and resolved by BN-PAGE. Western blot detection with anti-TOM40 ( left ) and anti-TOM22 ( right ) antibodies identified full-size TOM complex (around 450 kDa) specifically co-purified with HA-Porin1. Lower left panel, wild-type ( WT ) and Tom22His mitochondria subjected to nickel in complex with nitrilotriacetic acid purification followed by separation via BN-PAGE and Western blotting. Porin complexes were detected using anti-Porin1 antibodies (load 0.75%, elution 100%). Lower right panel, wild-type ( WT ) and Por1HA mitochondria were incubated with 35 S-labeled Oxa1 in the absence of a membrane potential to arrest the precursor at the TOM complex. Subsequently, samples were subjected to anti-HA chromatography, and protein complexes were separated by BN-PAGE. 35 S-Labeled Oxa1 arrested at the TOM complex was visualized with digital autoradiography. Note the increase in apparent mass of TOM complexes due to the stalled Oxa1 substrate.

    Article Snippet: Tom22His -containing protein complexes were purified by nickel in complex with nitrilotriacetic acid-agarose (Qiagen, Germany) ( ).

    Techniques: Affinity Purification, Isolation, Expressing, Chromatography, SDS Page, Western Blot, Purification, Polyacrylamide Gel Electrophoresis, Incubation, Labeling, Autoradiography