Structured Review

Qiagen his tagged trx target complexes
In vivo pull-down assay showing interaction between <t>Trx</t> m and STN7/PetC. Protein complexes from Wt and His-tagged o/exTrxm, o/exTrxf, and o/exTrxm-mut cross-linked chloroplasts were pulled-down with <t>Ni-NTA</t> resin. After washing the beads, bound proteins were eluted by boiling and analyzed, together with input fractions, by western blot using anti-STN7, anti-PetC, anti-2-Cys Prx, and anti-Lhcb1 antibodies.
His Tagged Trx Target Complexes, supplied by Qiagen, used in various techniques. Bioz Stars score: 93/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/his tagged trx target complexes/product/Qiagen
Average 93 stars, based on 255 article reviews
Price from $9.99 to $1999.99
his tagged trx target complexes - by Bioz Stars, 2020-09
93/100 stars

Images

1) Product Images from "Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance"

Article Title: Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/ery415

In vivo pull-down assay showing interaction between Trx m and STN7/PetC. Protein complexes from Wt and His-tagged o/exTrxm, o/exTrxf, and o/exTrxm-mut cross-linked chloroplasts were pulled-down with Ni-NTA resin. After washing the beads, bound proteins were eluted by boiling and analyzed, together with input fractions, by western blot using anti-STN7, anti-PetC, anti-2-Cys Prx, and anti-Lhcb1 antibodies.
Figure Legend Snippet: In vivo pull-down assay showing interaction between Trx m and STN7/PetC. Protein complexes from Wt and His-tagged o/exTrxm, o/exTrxf, and o/exTrxm-mut cross-linked chloroplasts were pulled-down with Ni-NTA resin. After washing the beads, bound proteins were eluted by boiling and analyzed, together with input fractions, by western blot using anti-STN7, anti-PetC, anti-2-Cys Prx, and anti-Lhcb1 antibodies.

Techniques Used: In Vivo, Pull Down Assay, Western Blot

2) Product Images from "HrpE, the major component of the Xanthomonas type three protein secretion pilus, elicits plant immunity responses"

Article Title: HrpE, the major component of the Xanthomonas type three protein secretion pilus, elicits plant immunity responses

Journal: Scientific Reports

doi: 10.1038/s41598-018-27869-1

Analysis of citrus, tomato and pepper leaves responses to Xcc HrpE. ( a ) Representative photographs of leaves responses to the infiltration of pure HrpE-Trx- 6 His (HrpE), ranging from 0.5 µM to 5 µM, and 5 µM Trx- 6 His (Trx) (control) 1 dpi. Bar indicates 0.5 cm. ( b ) Representative fluorescence microscopy photographs of aniline blue staining of callose deposition in leaves infiltrated with 2.5 µM HrpE and Trx (control) 8 hpi (tomato and pepper) and 16 hpi (citrus). Bar indicates 20 μm. The right panel shows the quantification of callose intensities in citrus (C), tomato (T) and pepper (P) tissues infiltrated with HrpE (black bars) relative to Trx (grey bars). ( c ) Representative photographs of DAB stained leaves infiltrated as in ( b ) (Bar indicates 1 mm). In citrus, H 2 O 2 production is observed as brown precipitates in leaf tissues and in tomato and pepper, the brown precipitates are observed near to the leaf veins. The right panel shows the quantification of DAB staining in infiltrated C, T and P tissues with HrpE (black bars) relative to Trx (grey bars). For both, callose and DAB intensities quantifications, the means were calculated from 25 photographs obtained from different treated leaves from three independent experiments. Error bars indicate standard deviations. Asterisks represent significant differences based on one-way ANOVA ( p
Figure Legend Snippet: Analysis of citrus, tomato and pepper leaves responses to Xcc HrpE. ( a ) Representative photographs of leaves responses to the infiltration of pure HrpE-Trx- 6 His (HrpE), ranging from 0.5 µM to 5 µM, and 5 µM Trx- 6 His (Trx) (control) 1 dpi. Bar indicates 0.5 cm. ( b ) Representative fluorescence microscopy photographs of aniline blue staining of callose deposition in leaves infiltrated with 2.5 µM HrpE and Trx (control) 8 hpi (tomato and pepper) and 16 hpi (citrus). Bar indicates 20 μm. The right panel shows the quantification of callose intensities in citrus (C), tomato (T) and pepper (P) tissues infiltrated with HrpE (black bars) relative to Trx (grey bars). ( c ) Representative photographs of DAB stained leaves infiltrated as in ( b ) (Bar indicates 1 mm). In citrus, H 2 O 2 production is observed as brown precipitates in leaf tissues and in tomato and pepper, the brown precipitates are observed near to the leaf veins. The right panel shows the quantification of DAB staining in infiltrated C, T and P tissues with HrpE (black bars) relative to Trx (grey bars). For both, callose and DAB intensities quantifications, the means were calculated from 25 photographs obtained from different treated leaves from three independent experiments. Error bars indicate standard deviations. Asterisks represent significant differences based on one-way ANOVA ( p

Techniques Used: Fluorescence, Microscopy, Staining

3) Product Images from "Mycobacterium tuberculosis Rv3463 induces mycobactericidal activity in macrophages by enhancing phagolysosomal fusion and exhibits therapeutic potential"

Article Title: Mycobacterium tuberculosis Rv3463 induces mycobactericidal activity in macrophages by enhancing phagolysosomal fusion and exhibits therapeutic potential

Journal: Scientific Reports

doi: 10.1038/s41598-019-38982-0

Rv3463 induces macrophage activation via TLR2 and TLR4. Bone marrow-derived macrophages (BMDMs) derived from wild-type (WT), TLR2 −/− , TLR4 −/− , and TLR2/4 −/− mice were treated with Rv3463 (5 μg/ml), lipopolysaccharide (LPS, 100 ng/ml), or Pam3CSK4 (100 ng/ml) for 24 h. ( A ) The production of TNF-α, IL-6, and IL-12p70 in the culture supernatants was determined by ELISA. All data are expressed as mean ± SD ( n = 3). ( B ) Expression of CD80 and MHC class II molecules on BMDMs stimulated with each antigen was determined by staining and flow cytometry. The bar graphs show the mean percentage ± SEM of each surface molecule on F4/80 + cells across three independent experiments. * p
Figure Legend Snippet: Rv3463 induces macrophage activation via TLR2 and TLR4. Bone marrow-derived macrophages (BMDMs) derived from wild-type (WT), TLR2 −/− , TLR4 −/− , and TLR2/4 −/− mice were treated with Rv3463 (5 μg/ml), lipopolysaccharide (LPS, 100 ng/ml), or Pam3CSK4 (100 ng/ml) for 24 h. ( A ) The production of TNF-α, IL-6, and IL-12p70 in the culture supernatants was determined by ELISA. All data are expressed as mean ± SD ( n = 3). ( B ) Expression of CD80 and MHC class II molecules on BMDMs stimulated with each antigen was determined by staining and flow cytometry. The bar graphs show the mean percentage ± SEM of each surface molecule on F4/80 + cells across three independent experiments. * p

Techniques Used: Activation Assay, Derivative Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Flow Cytometry, Cytometry

4) 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

5) Product Images from "M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines"

Article Title: M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines

Journal: Current HIV Research

doi: 10.2174/1570162X17666181206111859

Delivery efficiency of Nef and Hsp20-Nef proteins using M918 at a molar ratio of 1:20 (Nef/M918 and Hsp20-Nef/M918) in HEK-293 T cells for 3 h post-transfection: Western blot analysis showed delivery of the full-length Nef protein (~27 kDa, Lane 1) and Hsp20-Nef protein (~ 47 kDa, Lane 3) in transfected cells by M918 peptide at 3 h after transfection as compared to the un-transfected cells (Lane 2). The corresponding bands were not also detected in the transfected cells with Nef or Hsp20-Nef proteins alone similar to the un-transfected cells (data not shown). MW is the molecular weight marker (10-180 kDa, Fermentas).
Figure Legend Snippet: Delivery efficiency of Nef and Hsp20-Nef proteins using M918 at a molar ratio of 1:20 (Nef/M918 and Hsp20-Nef/M918) in HEK-293 T cells for 3 h post-transfection: Western blot analysis showed delivery of the full-length Nef protein (~27 kDa, Lane 1) and Hsp20-Nef protein (~ 47 kDa, Lane 3) in transfected cells by M918 peptide at 3 h after transfection as compared to the un-transfected cells (Lane 2). The corresponding bands were not also detected in the transfected cells with Nef or Hsp20-Nef proteins alone similar to the un-transfected cells (data not shown). MW is the molecular weight marker (10-180 kDa, Fermentas).

Techniques Used: Transfection, Western Blot, Molecular Weight, Marker

Expression and purification of Hsp20-Nef protein in E. coli expression system as shown in SDS-PAGE ( A ) and western blotting ( B ); A ) Lane 1: Before induction, Lane 2: After induction, Lane 3: Purified protein using affinity chromatography, Lane 4-6: Further purification using reverse staining; B ) Lane 1: Before induction, Lane 2: After induction, Lane 3: Purified protein; MW is the molecular weight marker (10-180 kDa, Fermentas).
Figure Legend Snippet: Expression and purification of Hsp20-Nef protein in E. coli expression system as shown in SDS-PAGE ( A ) and western blotting ( B ); A ) Lane 1: Before induction, Lane 2: After induction, Lane 3: Purified protein using affinity chromatography, Lane 4-6: Further purification using reverse staining; B ) Lane 1: Before induction, Lane 2: After induction, Lane 3: Purified protein; MW is the molecular weight marker (10-180 kDa, Fermentas).

Techniques Used: Expressing, Purification, SDS Page, Western Blot, Affinity Chromatography, Staining, Molecular Weight, Marker

Cell viability of the Nef, Hsp20-Nef, M918, M918/Nef and M918/Hsp20-Nef treatments: HEK-293T cells were treated with 1µg of the purified proteins, the complexes at different molar ratios of 2:1, 5:1, 10:1, 15:1, 20:1 and 30:1 (peptide: protein), M918 peptide (2, 5, 10, 15, 20 and 30 µM) as well as 70% ethanol as a positive control. The MTT assay was used to assess cytotoxicity. Data were presented as mean ± standard deviations from two independent experiments.
Figure Legend Snippet: Cell viability of the Nef, Hsp20-Nef, M918, M918/Nef and M918/Hsp20-Nef treatments: HEK-293T cells were treated with 1µg of the purified proteins, the complexes at different molar ratios of 2:1, 5:1, 10:1, 15:1, 20:1 and 30:1 (peptide: protein), M918 peptide (2, 5, 10, 15, 20 and 30 µM) as well as 70% ethanol as a positive control. The MTT assay was used to assess cytotoxicity. Data were presented as mean ± standard deviations from two independent experiments.

Techniques Used: Purification, Positive Control, MTT Assay

The SEM micrograph of the spherical nanoparticles: A ) M918 peptide, B ) Hsp20-Nef, C ) Hsp20-Nef/M918, D ) Nef/M918, E ) Nef; A size of ~ 200-250 nm was observed for M918/Nef nanoparticles and ~ 50-80 nm for M918/Hsp20-Nef nanoparticles at 25˚C.
Figure Legend Snippet: The SEM micrograph of the spherical nanoparticles: A ) M918 peptide, B ) Hsp20-Nef, C ) Hsp20-Nef/M918, D ) Nef/M918, E ) Nef; A size of ~ 200-250 nm was observed for M918/Nef nanoparticles and ~ 50-80 nm for M918/Hsp20-Nef nanoparticles at 25˚C.

Techniques Used:

A ) Analysis of M918/Nef ( A ) and M918/Hsp20-Nef ( B ) complexes at different molar ratios in SDS-PAGE: Lane 1) purified Nef or Hsp20-Nef protein as a control, Lane 2) M918 peptide, Lane 3) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 2:1, Lane 4) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 5:1, Lane 5) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 10:1, Lane 6) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 15:1, Lane 7) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 20:1, Lane 8) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 30:1; MW is the molecular weight marker (10-180 kDa, Fermentas). The lanes 2-8 describe a chemical dissociation detected as a dominant band of ~ 27 kDa and ~ 47 kDa related to Nef and Hsp20-Nef proteins along with M918 peptide band in SDS-PAGE indicating the formation of protein/ peptide complexes.
Figure Legend Snippet: A ) Analysis of M918/Nef ( A ) and M918/Hsp20-Nef ( B ) complexes at different molar ratios in SDS-PAGE: Lane 1) purified Nef or Hsp20-Nef protein as a control, Lane 2) M918 peptide, Lane 3) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 2:1, Lane 4) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 5:1, Lane 5) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 10:1, Lane 6) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 15:1, Lane 7) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 20:1, Lane 8) M918/ Nef or M918/Hsp20-Nef complexes at ratio of 30:1; MW is the molecular weight marker (10-180 kDa, Fermentas). The lanes 2-8 describe a chemical dissociation detected as a dominant band of ~ 27 kDa and ~ 47 kDa related to Nef and Hsp20-Nef proteins along with M918 peptide band in SDS-PAGE indicating the formation of protein/ peptide complexes.

Techniques Used: SDS Page, Purification, Molecular Weight, Marker

6) Product Images from "Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L."

Article Title: Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L.

Journal: BMB Reports

doi: 10.5483/BMBRep.2018.51.11.122

OsTRFL1 interacts with rice DS-TBPs in yeast cells and in vitro . (A) Structural comparison of OsTRFL1, RTBP1, and OsTRBF1. The UBL domain, Myb DNA-binding motif, and SMH domain are indicated. (B) Yeast-two hybrid assay. OsTRFL1 was cloned into the pGBK T7 vector, and OsTRBF1 , RTBP1 , OsTRFL1 , OsKu70 , and T-antigen were cloned separately into the pGAD T7 vector. Each combination of the indicated plasmids was co-transformed into yeast AH109 cells. To test for protein-protein interactions, yeast cells were plated onto two-minus (−Leu/−Trp; left panel) or three-minus (−Leu/−Trp/−His) medium containing 10 mM 3-AT (right panel) and grown at 30°C for 4 days. T-antigen was used as a negative control. (C) In vitro pull-down assay. OsTRFL1, OsTRBF1, and RTBP1 DS-TBPs were expressed as MBP-or (His) 6 -fusion proteins in E. coli . The purified fusion proteins were co-incubated as indicated in the presence of His-affinity matrix. The bound protein was then eluted from the resin and immunoblotted with either anti-MBP or anti-His antibody.
Figure Legend Snippet: OsTRFL1 interacts with rice DS-TBPs in yeast cells and in vitro . (A) Structural comparison of OsTRFL1, RTBP1, and OsTRBF1. The UBL domain, Myb DNA-binding motif, and SMH domain are indicated. (B) Yeast-two hybrid assay. OsTRFL1 was cloned into the pGBK T7 vector, and OsTRBF1 , RTBP1 , OsTRFL1 , OsKu70 , and T-antigen were cloned separately into the pGAD T7 vector. Each combination of the indicated plasmids was co-transformed into yeast AH109 cells. To test for protein-protein interactions, yeast cells were plated onto two-minus (−Leu/−Trp; left panel) or three-minus (−Leu/−Trp/−His) medium containing 10 mM 3-AT (right panel) and grown at 30°C for 4 days. T-antigen was used as a negative control. (C) In vitro pull-down assay. OsTRFL1, OsTRBF1, and RTBP1 DS-TBPs were expressed as MBP-or (His) 6 -fusion proteins in E. coli . The purified fusion proteins were co-incubated as indicated in the presence of His-affinity matrix. The bound protein was then eluted from the resin and immunoblotted with either anti-MBP or anti-His antibody.

Techniques Used: In Vitro, Binding Assay, Y2H Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Negative Control, Pull Down Assay, Purification, Incubation

7) Product Images from "Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L."

Article Title: Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L.

Journal: BMB Reports

doi: 10.5483/BMBRep.2018.51.11.122

OsTRFL1 is associated with double-stranded telomeric repeats in planta . (A) Schematic representation of rice OsTRFL1. The C-terminal Myb DNA-binding motif is indicated. (B) Gel retardation assay. Different amounts (0, 0.5, and 1.0 μg) of bacterially-expressed Myc-(His) 6 -OsTRFL1 486–623 , (His) 6 -OsTRBF1 1–128 , and Myc-(His) 6 -RTBP1 506–620 fusion proteins, all of which possess the Myb DNA-binding domain, were incubated with 32 P-labeled (TTTAGGG) 4 telomere repeats. After incubating for 10 min on ice, the reaction mixtures were loaded on an 8% non-denaturing polyacrylamide gel. (C) ChIP assay of OsTRFL1. The 35S:OsTRFL1-sGFP , 35S:OsTRBF1-sGFP , 35S:RTBP1-sGFP , and 35S:OsRAD51D-sGFP constructs were introduced into tobacco leaves by Agrobacterium -mediated infiltration. After 2 days of infiltration, nuclear genomic DNA-protein complexes were isolated and subjected to immunoprecipitation with an anti-GFP antibody. The pull-downed DNA was hybridized with a 32 P-labeled (TTTAGGG) 70 telomere repeat probe. IP, immunoprecipitation.
Figure Legend Snippet: OsTRFL1 is associated with double-stranded telomeric repeats in planta . (A) Schematic representation of rice OsTRFL1. The C-terminal Myb DNA-binding motif is indicated. (B) Gel retardation assay. Different amounts (0, 0.5, and 1.0 μg) of bacterially-expressed Myc-(His) 6 -OsTRFL1 486–623 , (His) 6 -OsTRBF1 1–128 , and Myc-(His) 6 -RTBP1 506–620 fusion proteins, all of which possess the Myb DNA-binding domain, were incubated with 32 P-labeled (TTTAGGG) 4 telomere repeats. After incubating for 10 min on ice, the reaction mixtures were loaded on an 8% non-denaturing polyacrylamide gel. (C) ChIP assay of OsTRFL1. The 35S:OsTRFL1-sGFP , 35S:OsTRBF1-sGFP , 35S:RTBP1-sGFP , and 35S:OsRAD51D-sGFP constructs were introduced into tobacco leaves by Agrobacterium -mediated infiltration. After 2 days of infiltration, nuclear genomic DNA-protein complexes were isolated and subjected to immunoprecipitation with an anti-GFP antibody. The pull-downed DNA was hybridized with a 32 P-labeled (TTTAGGG) 70 telomere repeat probe. IP, immunoprecipitation.

Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Chromatin Immunoprecipitation, Construct, Isolation, Immunoprecipitation

OsTRFL1 interacts with rice DS-TBPs in yeast cells and in vitro . (A) Structural comparison of OsTRFL1, RTBP1, and OsTRBF1. The UBL domain, Myb DNA-binding motif, and SMH domain are indicated. (B) Yeast-two hybrid assay. OsTRFL1 was cloned into the pGBK T7 vector, and OsTRBF1 , RTBP1 , OsTRFL1 , OsKu70 , and T-antigen were cloned separately into the pGAD T7 vector. Each combination of the indicated plasmids was co-transformed into yeast AH109 cells. To test for protein-protein interactions, yeast cells were plated onto two-minus (−Leu/−Trp; left panel) or three-minus (−Leu/−Trp/−His) medium containing 10 mM 3-AT (right panel) and grown at 30°C for 4 days. T-antigen was used as a negative control. (C) In vitro pull-down assay. OsTRFL1, OsTRBF1, and RTBP1 DS-TBPs were expressed as MBP-or (His) 6 -fusion proteins in E. coli . The purified fusion proteins were co-incubated as indicated in the presence of His-affinity matrix. The bound protein was then eluted from the resin and immunoblotted with either anti-MBP or anti-His antibody.
Figure Legend Snippet: OsTRFL1 interacts with rice DS-TBPs in yeast cells and in vitro . (A) Structural comparison of OsTRFL1, RTBP1, and OsTRBF1. The UBL domain, Myb DNA-binding motif, and SMH domain are indicated. (B) Yeast-two hybrid assay. OsTRFL1 was cloned into the pGBK T7 vector, and OsTRBF1 , RTBP1 , OsTRFL1 , OsKu70 , and T-antigen were cloned separately into the pGAD T7 vector. Each combination of the indicated plasmids was co-transformed into yeast AH109 cells. To test for protein-protein interactions, yeast cells were plated onto two-minus (−Leu/−Trp; left panel) or three-minus (−Leu/−Trp/−His) medium containing 10 mM 3-AT (right panel) and grown at 30°C for 4 days. T-antigen was used as a negative control. (C) In vitro pull-down assay. OsTRFL1, OsTRBF1, and RTBP1 DS-TBPs were expressed as MBP-or (His) 6 -fusion proteins in E. coli . The purified fusion proteins were co-incubated as indicated in the presence of His-affinity matrix. The bound protein was then eluted from the resin and immunoblotted with either anti-MBP or anti-His antibody.

Techniques Used: In Vitro, Binding Assay, Y2H Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Negative Control, Pull Down Assay, Purification, Incubation

OsTRFL1 interacts with rice DB-TBPs in nuclear speckle-like structures. (A) The 35S:OsTRFL1-sGFP , 35S:RTBP1-sGFP , and 35S:OsTRBF1-sGFP constructs were co-expressed with 35: NLS-mRFP in tobacco leaves by Agrobacterium -mediated infiltration. After two days of infection, protoplasts were prepared from the infected leaves, and fluorescent protein signals were visualized by fluorescence microscopy. NLS-mRFP was used as a nuclear marker protein. Scale bars = 5 μm. (B) 35S:OsTRBF1-sGFP or 35S:RTBP1-sGFP was co-infiltrated with 35S:mRFP-OsTRFL1 into tobacco leaves. Fluorescent signals in the protoplasts were visualized by fluorescence microscopy. Scale bars = 5 μm. (C) BiFC assay. OsTRFL1-YFP N + RTBP1-YFP C , OsTRFL1-YFP N + OsTRBF1-YFP C , and OsTRFL1-YFP N + OsTRFL1-YFP C proteins were transiently expressed in three-week-old tobacco leaf cells. After 3 days of infiltration, the fluorescent signals were detected by fluorescence microscopy. OsKu70-YFP N + OsTRFL1-YFP C was included as a negative control. NLS-mRFP was used as a nuclei-localized marker protein. Scale bars = 5 μm.
Figure Legend Snippet: OsTRFL1 interacts with rice DB-TBPs in nuclear speckle-like structures. (A) The 35S:OsTRFL1-sGFP , 35S:RTBP1-sGFP , and 35S:OsTRBF1-sGFP constructs were co-expressed with 35: NLS-mRFP in tobacco leaves by Agrobacterium -mediated infiltration. After two days of infection, protoplasts were prepared from the infected leaves, and fluorescent protein signals were visualized by fluorescence microscopy. NLS-mRFP was used as a nuclear marker protein. Scale bars = 5 μm. (B) 35S:OsTRBF1-sGFP or 35S:RTBP1-sGFP was co-infiltrated with 35S:mRFP-OsTRFL1 into tobacco leaves. Fluorescent signals in the protoplasts were visualized by fluorescence microscopy. Scale bars = 5 μm. (C) BiFC assay. OsTRFL1-YFP N + RTBP1-YFP C , OsTRFL1-YFP N + OsTRBF1-YFP C , and OsTRFL1-YFP N + OsTRFL1-YFP C proteins were transiently expressed in three-week-old tobacco leaf cells. After 3 days of infiltration, the fluorescent signals were detected by fluorescence microscopy. OsKu70-YFP N + OsTRFL1-YFP C was included as a negative control. NLS-mRFP was used as a nuclei-localized marker protein. Scale bars = 5 μm.

Techniques Used: Construct, Infection, Fluorescence, Microscopy, Marker, Bimolecular Fluorescence Complementation Assay, Negative Control

8) Product Images from "Clec16a, Nrdp1, and USP8 Form a Ubiquitin-Dependent Tripartite Complex That Regulates β-Cell Mitophagy"

Article Title: Clec16a, Nrdp1, and USP8 Form a Ubiquitin-Dependent Tripartite Complex That Regulates β-Cell Mitophagy

Journal: Diabetes

doi: 10.2337/db17-0321

Clec16a possesses E3 ligase activity and directly ubiquitinates Nrdp1 via a non-K48 linkage. A : Representative α-Flag Western blot (WB) from purified Clec16a-6xHis-Flag protein after incubation in vitro with ATP, ubiquitin, E1, and 26 unique E2-conjugating enzymes at 37°C for 1 h. Arrows demarcate both the expected and novel high–molecular weight Clec16a proteins identified by Western blot. n = 3/group. B and C : Representative Western blot of in vitro ubiquitination assay of recombinant GST-Clec16a-Flag ( B ) or Clec16a-6xHis-Flag ( C ) protein after incubation with ATP, HA-ubiquitin (HA-Ub), and E1 in the presence or absence of E2 conjugation enzymes UbE2D3 and/or UbE2C at 37°C for 1 h. n = 3/group. D : Representative Western blot of in vitro ubiquitination assay of recombinant GST-Nrdp1-CSHQ after incubation with ATP, HA-ubiquitin, and E1 in the presence or absence of UbE2D3 and recombinant Clec16a-6xHis-Flag at 37°C for 1 h. n = 3/group. E : Representative WB of in vivo ubiquitination assay of overexpressed Myc-tagged Nrdp1 (Myc-Nrdp1) performed in HEK293T cells transfected with Flag–empty vector or Flag-Clec16a in the presence of HA-WT ubiquitin as well as ubiquitin mutants with all lysines converted to arginines (KO), ubiquitin only capable of K48-linked ubiquitination (K48), ubiquitin incapable of K48-linked ubiquitination (K48R), or ubiquitin only capable of K63-linked ubiquitination (K63) (as well as whole-cell lysate Myc-Nrdp1 and Flag-Clec16a levels). n = 3/group. IP, immunoprecipitation.
Figure Legend Snippet: Clec16a possesses E3 ligase activity and directly ubiquitinates Nrdp1 via a non-K48 linkage. A : Representative α-Flag Western blot (WB) from purified Clec16a-6xHis-Flag protein after incubation in vitro with ATP, ubiquitin, E1, and 26 unique E2-conjugating enzymes at 37°C for 1 h. Arrows demarcate both the expected and novel high–molecular weight Clec16a proteins identified by Western blot. n = 3/group. B and C : Representative Western blot of in vitro ubiquitination assay of recombinant GST-Clec16a-Flag ( B ) or Clec16a-6xHis-Flag ( C ) protein after incubation with ATP, HA-ubiquitin (HA-Ub), and E1 in the presence or absence of E2 conjugation enzymes UbE2D3 and/or UbE2C at 37°C for 1 h. n = 3/group. D : Representative Western blot of in vitro ubiquitination assay of recombinant GST-Nrdp1-CSHQ after incubation with ATP, HA-ubiquitin, and E1 in the presence or absence of UbE2D3 and recombinant Clec16a-6xHis-Flag at 37°C for 1 h. n = 3/group. E : Representative WB of in vivo ubiquitination assay of overexpressed Myc-tagged Nrdp1 (Myc-Nrdp1) performed in HEK293T cells transfected with Flag–empty vector or Flag-Clec16a in the presence of HA-WT ubiquitin as well as ubiquitin mutants with all lysines converted to arginines (KO), ubiquitin only capable of K48-linked ubiquitination (K48), ubiquitin incapable of K48-linked ubiquitination (K48R), or ubiquitin only capable of K63-linked ubiquitination (K63) (as well as whole-cell lysate Myc-Nrdp1 and Flag-Clec16a levels). n = 3/group. IP, immunoprecipitation.

Techniques Used: Activity Assay, Western Blot, Purification, Incubation, In Vitro, Molecular Weight, Ubiquitin Assay, Recombinant, Conjugation Assay, In Vivo, Transfection, Plasmid Preparation, Immunoprecipitation

9) Product Images from "Isolation of MLL1 Inhibitory RNA Aptamers"

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2018.157

3D-structure predictions for APT1-MLL1 interactions. (A) Predicted 3D-structure of APT1 was constructed. The numbers represent the order of nucleotide residues. Some nucleotide residues are indicated. (B) 3D-structure of the complex was calculated using NPDock web server and illustrated in two different views. MLL1 SET domain is represented as subdomains with different colors such as the N-flanking region in red, SET-N in dark yellow, SET-I in green, SET-C in cyan, and post-SET in blue. (C) Hydrogen bonds formed between APT1 and MLL1 protein in two regions (dotted lines). The bases interacting with MLL1 amino acids are illustrated.
Figure Legend Snippet: 3D-structure predictions for APT1-MLL1 interactions. (A) Predicted 3D-structure of APT1 was constructed. The numbers represent the order of nucleotide residues. Some nucleotide residues are indicated. (B) 3D-structure of the complex was calculated using NPDock web server and illustrated in two different views. MLL1 SET domain is represented as subdomains with different colors such as the N-flanking region in red, SET-N in dark yellow, SET-I in green, SET-C in cyan, and post-SET in blue. (C) Hydrogen bonds formed between APT1 and MLL1 protein in two regions (dotted lines). The bases interacting with MLL1 amino acids are illustrated.

Techniques Used: Construct

10) Product Images from "GFP-Fragment Reassembly Screens for the Functional Characterization of Variants of Uncertain Significance in Protein Interaction Domains of the BRCA1 and BRCA2 Genes"

Article Title: GFP-Fragment Reassembly Screens for the Functional Characterization of Variants of Uncertain Significance in Protein Interaction Domains of the BRCA1 and BRCA2 Genes

Journal: Cancers

doi: 10.3390/cancers11020151

Expression of BRCA1-CfrGFP ( A ) and NfrGFP-BRCA2HD/OB1 ( B ) wild-type (wt) and mutant constructs. The molecular masses are indicated on the left.
Figure Legend Snippet: Expression of BRCA1-CfrGFP ( A ) and NfrGFP-BRCA2HD/OB1 ( B ) wild-type (wt) and mutant constructs. The molecular masses are indicated on the left.

Techniques Used: Expressing, Mutagenesis, Construct

11) Product Images from "The Murine G+C-Rich Promoter Binding Protein mGPBP Is Required for Promoter-Specific Transcription"

Article Title: The Murine G+C-Rich Promoter Binding Protein mGPBP Is Required for Promoter-Specific Transcription

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.23.8773-8785.2003

The purified recombinant mGPBP can bind specifically to the mouse Ada gene's G+C-rich promoter in EMSA. (A) The DNA probe 4C′ is four copies of the MSPE C′ that were end ligated. Purified recombinant mGPBP (rmGPBP) bound specifically to the probe and caused a shift in probe electrophoretic mobility from the free-probe location (lane 1) to the bound-probe location (lane 2). This binding can be specifically competed out by adding 35-fold (lane 3) and 175-fold (lane 4) excess unlabeled probes but cannot be competed out by adding similar amounts of unlabeled E2F binding motifs (lanes 5 and 6) or a 200-bp plasmid sequence (lanes 7 and 8). (B) A single copy of the fragment C′ in the context of the labeled 236-bp mouse Ada gene promoter can also bind to and be electrophoretically retarded by the purified rmGPBP (lanes 1 and 2). This binding can be competed out by excess unlabeled probe (lanes 3 and 4) or the 4C′ probe used in panel A (lanes 5 and 6). This binding is again not competed out by unlabeled E2F binding sequences (lanes 7 and 8) or the 200-bp plasmid sequences (lanes 9 and 10).
Figure Legend Snippet: The purified recombinant mGPBP can bind specifically to the mouse Ada gene's G+C-rich promoter in EMSA. (A) The DNA probe 4C′ is four copies of the MSPE C′ that were end ligated. Purified recombinant mGPBP (rmGPBP) bound specifically to the probe and caused a shift in probe electrophoretic mobility from the free-probe location (lane 1) to the bound-probe location (lane 2). This binding can be specifically competed out by adding 35-fold (lane 3) and 175-fold (lane 4) excess unlabeled probes but cannot be competed out by adding similar amounts of unlabeled E2F binding motifs (lanes 5 and 6) or a 200-bp plasmid sequence (lanes 7 and 8). (B) A single copy of the fragment C′ in the context of the labeled 236-bp mouse Ada gene promoter can also bind to and be electrophoretically retarded by the purified rmGPBP (lanes 1 and 2). This binding can be competed out by excess unlabeled probe (lanes 3 and 4) or the 4C′ probe used in panel A (lanes 5 and 6). This binding is again not competed out by unlabeled E2F binding sequences (lanes 7 and 8) or the 200-bp plasmid sequences (lanes 9 and 10).

Techniques Used: Purification, Recombinant, Binding Assay, Plasmid Preparation, Sequencing, Labeling

12) Product Images from "Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes"

Article Title: Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes

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

doi: 10.1073/pnas.192206699

Stereoview of the superposition of C α atoms of CyPA-CsA-CN (red) over unligated CNA (green) and FKBP-FK506-CN (yellow). The complex structures were overlaid with optimal overlap of the catalytic domain of CNA.
Figure Legend Snippet: Stereoview of the superposition of C α atoms of CyPA-CsA-CN (red) over unligated CNA (green) and FKBP-FK506-CN (yellow). The complex structures were overlaid with optimal overlap of the catalytic domain of CNA.

Techniques Used:

Interfacial interactions between CN and immunophilins–immunosuppressants. CNA and CNB are shown as yellow and cyan ribbons. ( A ) The CN composite surface for CyPA-CsA binding. A total of 25 residues of CN are involved in interaction with CyPA-CsA: Arg-122, Tyr-159, Phe-160, Leu-312, Val-314, Tyr-315, Tyr-341, Trp-342, Pro-344, Asn-345, Trp-352, Ser-353, Pro-355, Phe-356, and Glu-359 of CNA, and Glu-47, Gln-50, Met-118, Val-119, Asn-122, Leu-123, Lys-124, and Lys-164 of CNB. Red balls represent residues interacting with CyPA and green balls represent residues interacting with CsA (green sticks). The residues from CNB are marked with the letter “B” attached to the residue number. ( B ) The CN surface for binding of CyPA-CsA and FKBP12-FK506. CN residues in red interact with both CyPA-CsA and FKBP12-FK506. Green residues are unique for CyPA-CsA binding and blue residues are unique for FKBP-FK506. CsA and FK506 are shown as green and blue sticks. ( C ) CyPA residues for recognition of CN (gold) and CsA (blue). CsA is shown in green sticks.
Figure Legend Snippet: Interfacial interactions between CN and immunophilins–immunosuppressants. CNA and CNB are shown as yellow and cyan ribbons. ( A ) The CN composite surface for CyPA-CsA binding. A total of 25 residues of CN are involved in interaction with CyPA-CsA: Arg-122, Tyr-159, Phe-160, Leu-312, Val-314, Tyr-315, Tyr-341, Trp-342, Pro-344, Asn-345, Trp-352, Ser-353, Pro-355, Phe-356, and Glu-359 of CNA, and Glu-47, Gln-50, Met-118, Val-119, Asn-122, Leu-123, Lys-124, and Lys-164 of CNB. Red balls represent residues interacting with CyPA and green balls represent residues interacting with CsA (green sticks). The residues from CNB are marked with the letter “B” attached to the residue number. ( B ) The CN surface for binding of CyPA-CsA and FKBP12-FK506. CN residues in red interact with both CyPA-CsA and FKBP12-FK506. Green residues are unique for CyPA-CsA binding and blue residues are unique for FKBP-FK506. CsA and FK506 are shown as green and blue sticks. ( C ) CyPA residues for recognition of CN (gold) and CsA (blue). CsA is shown in green sticks.

Techniques Used: Binding Assay

Ribbon representation of CyPA-CsA-CN. Color codes are CNA, gold; CNB, cyan; CsA, green; CyPA, red; Zn 2+ and Fe 3+ , pink; and calcium, blue. The residues from CN involved in binding of CyPA-CsA are shown as blue balls.
Figure Legend Snippet: Ribbon representation of CyPA-CsA-CN. Color codes are CNA, gold; CNB, cyan; CsA, green; CyPA, red; Zn 2+ and Fe 3+ , pink; and calcium, blue. The residues from CN involved in binding of CyPA-CsA are shown as blue balls.

Techniques Used: Binding Assay

13) Product Images from "Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes"

Article Title: Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes

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

doi: 10.1073/pnas.192206699

Stereoview of the superposition of C α atoms of CyPA-CsA-CN (red) over unligated CNA (green) and FKBP-FK506-CN (yellow). The complex structures were overlaid with optimal overlap of the catalytic domain of CNA.
Figure Legend Snippet: Stereoview of the superposition of C α atoms of CyPA-CsA-CN (red) over unligated CNA (green) and FKBP-FK506-CN (yellow). The complex structures were overlaid with optimal overlap of the catalytic domain of CNA.

Techniques Used:

Interfacial interactions between CN and immunophilins–immunosuppressants. CNA and CNB are shown as yellow and cyan ribbons. ( A ) The CN composite surface for CyPA-CsA binding. A total of 25 residues of CN are involved in interaction with CyPA-CsA: Arg-122, Tyr-159, Phe-160, Leu-312, Val-314, Tyr-315, Tyr-341, Trp-342, Pro-344, Asn-345, Trp-352, Ser-353, Pro-355, Phe-356, and Glu-359 of CNA, and Glu-47, Gln-50, Met-118, Val-119, Asn-122, Leu-123, Lys-124, and Lys-164 of CNB. Red balls represent residues interacting with CyPA and green balls represent residues interacting with CsA (green sticks). The residues from CNB are marked with the letter “B” attached to the residue number. ( B ) The CN surface for binding of CyPA-CsA and FKBP12-FK506. CN residues in red interact with both CyPA-CsA and FKBP12-FK506. Green residues are unique for CyPA-CsA binding and blue residues are unique for FKBP-FK506. CsA and FK506 are shown as green and blue sticks. ( C ) CyPA residues for recognition of CN (gold) and CsA (blue). CsA is shown in green sticks.
Figure Legend Snippet: Interfacial interactions between CN and immunophilins–immunosuppressants. CNA and CNB are shown as yellow and cyan ribbons. ( A ) The CN composite surface for CyPA-CsA binding. A total of 25 residues of CN are involved in interaction with CyPA-CsA: Arg-122, Tyr-159, Phe-160, Leu-312, Val-314, Tyr-315, Tyr-341, Trp-342, Pro-344, Asn-345, Trp-352, Ser-353, Pro-355, Phe-356, and Glu-359 of CNA, and Glu-47, Gln-50, Met-118, Val-119, Asn-122, Leu-123, Lys-124, and Lys-164 of CNB. Red balls represent residues interacting with CyPA and green balls represent residues interacting with CsA (green sticks). The residues from CNB are marked with the letter “B” attached to the residue number. ( B ) The CN surface for binding of CyPA-CsA and FKBP12-FK506. CN residues in red interact with both CyPA-CsA and FKBP12-FK506. Green residues are unique for CyPA-CsA binding and blue residues are unique for FKBP-FK506. CsA and FK506 are shown as green and blue sticks. ( C ) CyPA residues for recognition of CN (gold) and CsA (blue). CsA is shown in green sticks.

Techniques Used: Binding Assay

Ribbon representation of CyPA-CsA-CN. Color codes are CNA, gold; CNB, cyan; CsA, green; CyPA, red; Zn 2+ and Fe 3+ , pink; and calcium, blue. The residues from CN involved in binding of CyPA-CsA are shown as blue balls.
Figure Legend Snippet: Ribbon representation of CyPA-CsA-CN. Color codes are CNA, gold; CNB, cyan; CsA, green; CyPA, red; Zn 2+ and Fe 3+ , pink; and calcium, blue. The residues from CN involved in binding of CyPA-CsA are shown as blue balls.

Techniques Used: Binding Assay

14) Product Images from "Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro"

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

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

doi: 10.1073/pnas.1619473114

The Mlph subunit of the tripartite complex is specifically phosphorylated by PKA. ( A ) Schematic illustration of the MyoVa-dependent tripartite transport complex on the melanosome surface. The Rab27a GTPase resides in the melanosome membrane and recruits Mlph in a GTP-dependent manner. Mlph in turn recruits the MyoVa motor to form the tripartite transport complex. ( B ). ( C ) Tripartite complex reconstituted with 6×His-SNAP–tagged Rab27a S-H , FLAG-tagged MyoVa F , and full-length (lane I) or C-terminally truncated (lanes II and III) FLAG-tagged Mlph F purified by Ni-NTA affinity purification. As a control for nonspecific binding, FLAG-tagged MyoVa F for details). MW, molecular mass marker. ( D ) The individually expressed full-length subunits of the tripartite complex along with C-terminally truncated Mlph and MyoVa HMM constructs were treated with PKA and radiolabeled ATP. Autoradiography showed specific phosphorylation of Mlph. Deletion of the C terminus of Mlph significantly decreased the phosphorylation levels. A FLAG-mock purification was included to control for unspecific phosphorylation.
Figure Legend Snippet: The Mlph subunit of the tripartite complex is specifically phosphorylated by PKA. ( A ) Schematic illustration of the MyoVa-dependent tripartite transport complex on the melanosome surface. The Rab27a GTPase resides in the melanosome membrane and recruits Mlph in a GTP-dependent manner. Mlph in turn recruits the MyoVa motor to form the tripartite transport complex. ( B ). ( C ) Tripartite complex reconstituted with 6×His-SNAP–tagged Rab27a S-H , FLAG-tagged MyoVa F , and full-length (lane I) or C-terminally truncated (lanes II and III) FLAG-tagged Mlph F purified by Ni-NTA affinity purification. As a control for nonspecific binding, FLAG-tagged MyoVa F for details). MW, molecular mass marker. ( D ) The individually expressed full-length subunits of the tripartite complex along with C-terminally truncated Mlph and MyoVa HMM constructs were treated with PKA and radiolabeled ATP. Autoradiography showed specific phosphorylation of Mlph. Deletion of the C terminus of Mlph significantly decreased the phosphorylation levels. A FLAG-mock purification was included to control for unspecific phosphorylation.

Techniques Used: Purification, Affinity Purification, Binding Assay, Marker, Construct, Autoradiography

15) Product Images from "The Gene for the Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Small Subunit Relocated to the Plastid Genome of Tobacco Directs the Synthesis of Small Subunits That Assemble into Rubisco"

Article Title: The Gene for the Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Small Subunit Relocated to the Plastid Genome of Tobacco Directs the Synthesis of Small Subunits That Assemble into Rubisco

Journal: The Plant Cell

doi:

Scheme of the Synthesis and Assembly of Rubisco in Tobacco Transformants with a Plastid Transgene Expressing the Small Subunit Precursor (pre-SSu). The dashed arrows indicate steps that may be rate limiting for the production of chloroplast-synthesized small subunits. Arrow 1 indicates the rates of engagement of the Rbc ); LSu and SSu, large and small subunits of Rubisco; N α ); tp, transit peptide.
Figure Legend Snippet: Scheme of the Synthesis and Assembly of Rubisco in Tobacco Transformants with a Plastid Transgene Expressing the Small Subunit Precursor (pre-SSu). The dashed arrows indicate steps that may be rate limiting for the production of chloroplast-synthesized small subunits. Arrow 1 indicates the rates of engagement of the Rbc ); LSu and SSu, large and small subunits of Rubisco; N α ); tp, transit peptide.

Techniques Used: Expressing, Synthesized

Expression of Plastid-Encoded Rbc S Transgenes and Assembly of the Products into the Rubisco Holoenzyme. (A) Coomassie blue–stained SDS gel of protein extracted from young leaves of nontransformed tobacco (NT), the T 1 transformants tpSSuH4 and SSuH2, and Escherichia ( E. coli ) BL21 (DE3) expressing the plasmids pETSSuH and pETtpSSuH. The insoluble protein (P) was pelleted, and His-tagged protein complexes (E) were isolated from the supernatant fraction as described in Methods. For the leaf extracts, each lane was loaded with protein (pelleted or His tagged) that had been derived from 0.5 cm 2 of leaf. For the Escherichia extracts, the His-tagged polypeptides in the soluble fractions constituted 5.4% (pETtpSSuH) and 2% (pETSSuH) of the total soluble protein as determined by the dye binding method (Pierce Coomassie Plus). (B) Immunoblot of an analogous gel, loaded with leaf samples only, probed with PentaHis antibody that recognized the His tag. (C) Immunoblot probed with tobacco Rubisco antibody. The positions corresponding to the 14.6-kD mature small subunit (SSu), the 15.6-kD hepta-His–tagged mature small subunit (SSuH), and the 21.3-kD hepta-His–tagged small subunit precursor with the N-terminal transit peptide (tpSSuH) are indicated. M, molecular mass marker lane showing soybean trypsin inhibitor (20.1 kD) and α-lactalbumin (14.4 kD).
Figure Legend Snippet: Expression of Plastid-Encoded Rbc S Transgenes and Assembly of the Products into the Rubisco Holoenzyme. (A) Coomassie blue–stained SDS gel of protein extracted from young leaves of nontransformed tobacco (NT), the T 1 transformants tpSSuH4 and SSuH2, and Escherichia ( E. coli ) BL21 (DE3) expressing the plasmids pETSSuH and pETtpSSuH. The insoluble protein (P) was pelleted, and His-tagged protein complexes (E) were isolated from the supernatant fraction as described in Methods. For the leaf extracts, each lane was loaded with protein (pelleted or His tagged) that had been derived from 0.5 cm 2 of leaf. For the Escherichia extracts, the His-tagged polypeptides in the soluble fractions constituted 5.4% (pETtpSSuH) and 2% (pETSSuH) of the total soluble protein as determined by the dye binding method (Pierce Coomassie Plus). (B) Immunoblot of an analogous gel, loaded with leaf samples only, probed with PentaHis antibody that recognized the His tag. (C) Immunoblot probed with tobacco Rubisco antibody. The positions corresponding to the 14.6-kD mature small subunit (SSu), the 15.6-kD hepta-His–tagged mature small subunit (SSuH), and the 21.3-kD hepta-His–tagged small subunit precursor with the N-terminal transit peptide (tpSSuH) are indicated. M, molecular mass marker lane showing soybean trypsin inhibitor (20.1 kD) and α-lactalbumin (14.4 kD).

Techniques Used: Expressing, Staining, SDS-Gel, Isolation, Derivative Assay, Binding Assay, Marker

Turnover of Rubisco Subunits and the D1 Subunit of Photosystem II Measured by 35 S Pulse–Chase Labeling. (A) Autoradiographs of SDS gels loaded with total leaf protein and isolated His-tagged Rubisco (see Methods), equivalent to 1.8 and 14.5 cm 2 of leaf, respectively. Bands corresponding to the D1 protein and the Rubisco large (LSu), small (SSu), and His-tagged small (SSuH) subunits are indicated. (B) Densitometric measurement of 35 S labeling in the various subunits during the chase period. LSu (closed circles), SSu (closed squares), and D1 (closed triangles) band densities were measured in the total protein lanes. Because the SSuH bands in these lanes were too faint for accurate measurement, they were measured in the His-tagged Rubisco lanes, in which a lower background allowed a 10-fold greater amplification of the intensities of the labeled bands. The LSu (open circles) and SSu (open squares) bands also were measured in these lanes.
Figure Legend Snippet: Turnover of Rubisco Subunits and the D1 Subunit of Photosystem II Measured by 35 S Pulse–Chase Labeling. (A) Autoradiographs of SDS gels loaded with total leaf protein and isolated His-tagged Rubisco (see Methods), equivalent to 1.8 and 14.5 cm 2 of leaf, respectively. Bands corresponding to the D1 protein and the Rubisco large (LSu), small (SSu), and His-tagged small (SSuH) subunits are indicated. (B) Densitometric measurement of 35 S labeling in the various subunits during the chase period. LSu (closed circles), SSu (closed squares), and D1 (closed triangles) band densities were measured in the total protein lanes. Because the SSuH bands in these lanes were too faint for accurate measurement, they were measured in the His-tagged Rubisco lanes, in which a lower background allowed a 10-fold greater amplification of the intensities of the labeled bands. The LSu (open circles) and SSu (open squares) bands also were measured in these lanes.

Techniques Used: Pulse Chase, Labeling, Isolation, Amplification

16) Product Images from "The acidic C-terminal domain and A-box of HMGB-1 regulates p53-mediated transcription"

Article Title: The acidic C-terminal domain and A-box of HMGB-1 regulates p53-mediated transcription

Journal: Nucleic Acids Research

doi:

The C-terminal acidic domain and A-box of HMGB-1 is essential for p53-mediated transcriptional activation from chromatin template. ( A ) DNA supercoiling assays for in vitro assembled chromatin. Chromatin was assembled using the ATP regeneration system without ATP (lane 3) or with ATP (lane 4). Lane 1, supercoiled DNA used for chromatin assembly; lane 2, relaxed DNA after recombinant topoisomerase I treatment of the DNA of lane 1. ( B ) MNase digestion analysis of assembled chromatin. The chromatin was treated with varying concentrations of MNase for 6 min (lanes 2–5) and fixed concentrations of MNase with varying times of 5 (lane 6) and 10 min (lane 7) at room temperature. After deproteinization, the resulting DNA was resolved on a 1.25% agarose gel and stained with ethidium bromide. ( C ) Schematic diagram of in vitro transcription from chromatin template. ( D ) In vitro transcription from chromatin template. Freshly assembled chromatin template subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without p53 (lane 1) or with 50 ng of p53 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of p53 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6). ( E ) HMGB-1 and its truncated form cannot activate transcription of Gal4VP16 from the chromatin template. Freshly assembled chromatin template was subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without Gal4VP16 (lane 1) or with 50 ng of Gal4VP16 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of Gal4VP16 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6).
Figure Legend Snippet: The C-terminal acidic domain and A-box of HMGB-1 is essential for p53-mediated transcriptional activation from chromatin template. ( A ) DNA supercoiling assays for in vitro assembled chromatin. Chromatin was assembled using the ATP regeneration system without ATP (lane 3) or with ATP (lane 4). Lane 1, supercoiled DNA used for chromatin assembly; lane 2, relaxed DNA after recombinant topoisomerase I treatment of the DNA of lane 1. ( B ) MNase digestion analysis of assembled chromatin. The chromatin was treated with varying concentrations of MNase for 6 min (lanes 2–5) and fixed concentrations of MNase with varying times of 5 (lane 6) and 10 min (lane 7) at room temperature. After deproteinization, the resulting DNA was resolved on a 1.25% agarose gel and stained with ethidium bromide. ( C ) Schematic diagram of in vitro transcription from chromatin template. ( D ) In vitro transcription from chromatin template. Freshly assembled chromatin template subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without p53 (lane 1) or with 50 ng of p53 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of p53 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6). ( E ) HMGB-1 and its truncated form cannot activate transcription of Gal4VP16 from the chromatin template. Freshly assembled chromatin template was subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without Gal4VP16 (lane 1) or with 50 ng of Gal4VP16 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of Gal4VP16 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6).

Techniques Used: Activation Assay, In Vitro, Recombinant, Agarose Gel Electrophoresis, Staining

Acidic C-terminal domain and A-box of HMGB-1 specifically stimulates transactivation by p53. ( A ) H1299 cells were transiently transfected with p53 (250 ng) and HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA (2.5 µg) either alone or in combination. The amount of transfected DNA was normalized with equivalent amounts of control parental vectors. The reporter constructs used was PG13Luc (1 µg). CMV-βgal (1 µg) was used as internal control for all the transfection experiments. Relative luciferase activity is plotted ( y -axis) after normalization of luciferase activity with β-galactosidase activity. The relative luciferase activity is an average of three experiments. ( B ) H1299 cells were transiently transfected with Gal4VP16 (100 ng) and HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA (1 µg) either alone or in combination. The amount of transfected DNA was normalized with the equivalent amount of control parental vectors. The reporter construct used was G10Luc (500 ng). CMV-βgal (500 ng) was used as an internal control for all the transfection experiments. Relative luciferase activity is plotted ( y -axis) after normalization of luciferase activity with β-galactosidase activity. The relative luciferase activity is an average of three experiments. VP16, Gal4 Vp16.
Figure Legend Snippet: Acidic C-terminal domain and A-box of HMGB-1 specifically stimulates transactivation by p53. ( A ) H1299 cells were transiently transfected with p53 (250 ng) and HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA (2.5 µg) either alone or in combination. The amount of transfected DNA was normalized with equivalent amounts of control parental vectors. The reporter constructs used was PG13Luc (1 µg). CMV-βgal (1 µg) was used as internal control for all the transfection experiments. Relative luciferase activity is plotted ( y -axis) after normalization of luciferase activity with β-galactosidase activity. The relative luciferase activity is an average of three experiments. ( B ) H1299 cells were transiently transfected with Gal4VP16 (100 ng) and HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA (1 µg) either alone or in combination. The amount of transfected DNA was normalized with the equivalent amount of control parental vectors. The reporter construct used was G10Luc (500 ng). CMV-βgal (500 ng) was used as an internal control for all the transfection experiments. Relative luciferase activity is plotted ( y -axis) after normalization of luciferase activity with β-galactosidase activity. The relative luciferase activity is an average of three experiments. VP16, Gal4 Vp16.

Techniques Used: Transfection, Construct, Luciferase, Activity Assay

Effect of C-terminal domain and A-box of HMGB-1 in p53-mediated apoptosis. H1299 cells were transfected with 2.5 µg of p53 or 5 µg of HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA either alone or in combination. Hoechst stain cells were observed under a fluorescence microscope using a blind approach. Apoptotic nuclei are indicated (arrows). The percentage of apoptotic nuclei refers to the average number of apoptotic cells present in 300 nuclei over three independent experiments ( I ).
Figure Legend Snippet: Effect of C-terminal domain and A-box of HMGB-1 in p53-mediated apoptosis. H1299 cells were transfected with 2.5 µg of p53 or 5 µg of HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA either alone or in combination. Hoechst stain cells were observed under a fluorescence microscope using a blind approach. Apoptotic nuclei are indicated (arrows). The percentage of apoptotic nuclei refers to the average number of apoptotic cells present in 300 nuclei over three independent experiments ( I ).

Techniques Used: Transfection, Staining, Fluorescence, Microscopy

In vitro transcription from naked DNA template by p53 in the presence of HMGB-1FL and its truncated forms. ( A ) Schematic representation of the plasmid template (used for transcription) containing five p53 binding sites upstream of adenovirus major late core promoter (MLP) and G-less cassette. The transcription start site is represented as +1. ( B ) The newly constructed template P (p53) 5 ML subjected to an in vitro transcription experiment using HeLa nuclear extract and without or with increasing concentrations of p53: lane 1, without p53; lane 2, 30 ng of p53; lane 3, 50 ng of p53; lane 4, 100 ng of p53; lane 5, 200 ng of p53. ( C ) Schematic representation of transcription protocol. ( D ) Effect of C-terminal domain on p53-mediated transcription. The DNA template (100 ng) was subjected to transcription according to the protocol in (C) with (lanes 2–8) or without (lane 1), 50 ng of p53 (lane 2) and the indicated amount of HMGB-1 and its truncated forms. Fifty, 100 and 200 ng of HMGB-1ΔC (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8) were added along with p53 (50 ng). ( E ) The effect of A-box of HMGB-1 on p53-mediated transcription from p (p53) 5 ML naked DNA template. Transcription reactions were performed according to the protocol in (C), without p53 (lane 1) or with 50 ng of p53 (lane 2) and 50, 100 and 200 ng of HMGB-1ΔA (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8) in the presence of p53 (50 ng). ( F ) Effect of C-terminal domain on Gal4VP16-mediated transcription. The DNA template (100 ng) was subjected to transcription according to protocol (C). The transcription reaction was performed with Gal4VP16 (lanes 2–8) or without Gal4VP16 (lane 1). Fifty nanograms of Gal4VP16 (lane 2) were incubated with 50, 100 and 200 ng of HMGB-1ΔC (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8). ( G ) The effect of A-box of Gal4VP16-mediated transcription. The DNA template (100 ng) was subjected to transcription according to the protocol in (C). The transcription reaction was performed with Gal4VP16 (lanes 2–8) or without Gal4VP16 (lane 1). Fifty nanograms of Gal4VP16 (lane 2) were incubated with 50, 100 and 200 ng of HMGB-1ΔA (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8).
Figure Legend Snippet: In vitro transcription from naked DNA template by p53 in the presence of HMGB-1FL and its truncated forms. ( A ) Schematic representation of the plasmid template (used for transcription) containing five p53 binding sites upstream of adenovirus major late core promoter (MLP) and G-less cassette. The transcription start site is represented as +1. ( B ) The newly constructed template P (p53) 5 ML subjected to an in vitro transcription experiment using HeLa nuclear extract and without or with increasing concentrations of p53: lane 1, without p53; lane 2, 30 ng of p53; lane 3, 50 ng of p53; lane 4, 100 ng of p53; lane 5, 200 ng of p53. ( C ) Schematic representation of transcription protocol. ( D ) Effect of C-terminal domain on p53-mediated transcription. The DNA template (100 ng) was subjected to transcription according to the protocol in (C) with (lanes 2–8) or without (lane 1), 50 ng of p53 (lane 2) and the indicated amount of HMGB-1 and its truncated forms. Fifty, 100 and 200 ng of HMGB-1ΔC (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8) were added along with p53 (50 ng). ( E ) The effect of A-box of HMGB-1 on p53-mediated transcription from p (p53) 5 ML naked DNA template. Transcription reactions were performed according to the protocol in (C), without p53 (lane 1) or with 50 ng of p53 (lane 2) and 50, 100 and 200 ng of HMGB-1ΔA (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8) in the presence of p53 (50 ng). ( F ) Effect of C-terminal domain on Gal4VP16-mediated transcription. The DNA template (100 ng) was subjected to transcription according to protocol (C). The transcription reaction was performed with Gal4VP16 (lanes 2–8) or without Gal4VP16 (lane 1). Fifty nanograms of Gal4VP16 (lane 2) were incubated with 50, 100 and 200 ng of HMGB-1ΔC (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8). ( G ) The effect of A-box of Gal4VP16-mediated transcription. The DNA template (100 ng) was subjected to transcription according to the protocol in (C). The transcription reaction was performed with Gal4VP16 (lanes 2–8) or without Gal4VP16 (lane 1). Fifty nanograms of Gal4VP16 (lane 2) were incubated with 50, 100 and 200 ng of HMGB-1ΔA (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8).

Techniques Used: In Vitro, Plasmid Preparation, Binding Assay, Construct, Incubation

Recombinant and native proteins used in the different experiments. ( A ) Diagrammatic representation of HMGB-1 and its truncated forms, HMGB-1ΔC and HMGB-1ΔA. ( B ) 200 ng of His 6 -tagged HMGB-1FL (lane 1), 100 ng of HMGB-1ΔC (lane 2) and 200 ng of HMGB-1ΔA (lane 3) were analyzed by SDS–PAGE (18%). ( C ) The authenticity of these proteins was analyzed by western blot using HMGB-2 rabbit polyclonal antibody. HMG proteins were separated by 12% SDS–PAGE followed by western blot: HMGB-1FL (lane 1), HMGB-1ΔC (lane 2) and HMGB-1ΔA (lane 3). ( D ) FLAG-tagged full-length human p53 purified from E.coli and analyzed by SDS–PAGE (10%). The authenticity of the p53 protein was confirmed by western blot using p53 monoclonal antibody, DO1 (Oncogene). ( E ) Analysis of the recombinant Drosophila ACF complex by SDS–PAGE (8%) which was purified from FLAG-tagged ACF and ISWI baculovirus infected Sf21 cells. ( F ) Recombinant His 6 -tagged mouse NAP1 purified from E.coli. ( G ) Native core histone purified from HeLa nuclear pellet and analyzed by SDS–PAGE (15%).
Figure Legend Snippet: Recombinant and native proteins used in the different experiments. ( A ) Diagrammatic representation of HMGB-1 and its truncated forms, HMGB-1ΔC and HMGB-1ΔA. ( B ) 200 ng of His 6 -tagged HMGB-1FL (lane 1), 100 ng of HMGB-1ΔC (lane 2) and 200 ng of HMGB-1ΔA (lane 3) were analyzed by SDS–PAGE (18%). ( C ) The authenticity of these proteins was analyzed by western blot using HMGB-2 rabbit polyclonal antibody. HMG proteins were separated by 12% SDS–PAGE followed by western blot: HMGB-1FL (lane 1), HMGB-1ΔC (lane 2) and HMGB-1ΔA (lane 3). ( D ) FLAG-tagged full-length human p53 purified from E.coli and analyzed by SDS–PAGE (10%). The authenticity of the p53 protein was confirmed by western blot using p53 monoclonal antibody, DO1 (Oncogene). ( E ) Analysis of the recombinant Drosophila ACF complex by SDS–PAGE (8%) which was purified from FLAG-tagged ACF and ISWI baculovirus infected Sf21 cells. ( F ) Recombinant His 6 -tagged mouse NAP1 purified from E.coli. ( G ) Native core histone purified from HeLa nuclear pellet and analyzed by SDS–PAGE (15%).

Techniques Used: Recombinant, SDS Page, Western Blot, Purification, Infection

The C-terminal acidic domain and A-box of HMGB-1 is essential to enhance sequence-specific DNA binding of p53. The effect of the C-terminal domain of HMGB-1 on p53-mediated DNA binding is shown. Human p53 (50 ng) was incubated with 3 ng of a 32 P-labeled p53 binding oligonucleotide in the absence (lane 1) or presence (lane 2) of 100, 200 and 300 ng of HMGB-1ΔA (lanes 3–5), HMGB-1ΔC (lanes 6–8) and HMGB-1FL (lanes 9–11). The 32 P-labeled probe was also incubated with only 100, 200 and 300 ng of HMGB-1ΔA (lanes 12–14), HMGB-1ΔC (lanes 15–17) and HMGB-1FL (lanes 18–20). Lane 1 contains only probe without any protein.
Figure Legend Snippet: The C-terminal acidic domain and A-box of HMGB-1 is essential to enhance sequence-specific DNA binding of p53. The effect of the C-terminal domain of HMGB-1 on p53-mediated DNA binding is shown. Human p53 (50 ng) was incubated with 3 ng of a 32 P-labeled p53 binding oligonucleotide in the absence (lane 1) or presence (lane 2) of 100, 200 and 300 ng of HMGB-1ΔA (lanes 3–5), HMGB-1ΔC (lanes 6–8) and HMGB-1FL (lanes 9–11). The 32 P-labeled probe was also incubated with only 100, 200 and 300 ng of HMGB-1ΔA (lanes 12–14), HMGB-1ΔC (lanes 15–17) and HMGB-1FL (lanes 18–20). Lane 1 contains only probe without any protein.

Techniques Used: Sequencing, Binding Assay, Incubation, Labeling

17) 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

18) Product Images from "Control of Multicellular Development by the Physically Interacting Deneddylases DEN1/DenA and COP9 Signalosome"

Article Title: Control of Multicellular Development by the Physically Interacting Deneddylases DEN1/DenA and COP9 Signalosome

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003275

Fungal DenA developmental functions are independent of Nedd8 processing activity. (A) Yeast-2-hybrid interaction between A. nidulans DenA and the precursor (nedd8pc) or the mature form (nedd8m) of Nedd8. (B) Western analysis with α-Cdc53 and α-Rub1/Nedd8 to visualize yeast cullin neddylation. Deletion of yuh1 prevents cleavage of the Rub1 precursor, therefore neddylated protein species can neither be recognized with α-Cdc53 nor with α-Rub1/Nedd8. DenA expression driven by the inducible GAL1 promoter was applied to test the processing ability of the protease towards the Rub1 precursor. Expression of DenA was visualized with α-V5. DenA expression was not sufficient to restore Rub1 processing in a yuh1 deficient S. cerevisiae strain [44] . (C) An A. nidulans strain expressing a mature nedd8 construct ( nedd8m ) instead of normal nedd8 (wt) displayed a wild type like phenotype. Asexual structures (co) displayed on agar surface after 2 days of growth in the presence of light (scale bar = 20 µm) and after 7 days (scale bar = 500 µm). Sexual structures (cl) were only formed in the dark, but not in the light after 7 days incubation at 37°C (scale bar = 500 µm). Quantification of asexual spores from both strains after 2 days of incubation at 37°C in light. (D) The ΔdenA/nedd8m strain (mature Nedd8) and the ΔdenA (precursor Nedd8) are unresponsive to light-dependent inhibition of sexual development (compare rows 2 and 3; scale bar = 50 µm, first row; scale bar = 225 µm, second and third row) and impaired in asexual development (conidiation). (E) Human DEN1 cleaved human Nedd8 C-terminally fused with GFP while A. nidulans DenA did not. Nedd8-GFP substrate was combined with decreasing amounts of recombinant DEN1 or DenA, respectively (8–0.5 µM). The reaction mixture was incubated for 30 min at 37°C, immediately denatured, separated by SDS-PAGE and subjected to western blot analysis. α-GFP was applied to monitor cleavage of the Nedd8-GFP substrate. Samples were separated by additional SDS-PAGE and silver stained to prove for the presence of the respective deneddylase.
Figure Legend Snippet: Fungal DenA developmental functions are independent of Nedd8 processing activity. (A) Yeast-2-hybrid interaction between A. nidulans DenA and the precursor (nedd8pc) or the mature form (nedd8m) of Nedd8. (B) Western analysis with α-Cdc53 and α-Rub1/Nedd8 to visualize yeast cullin neddylation. Deletion of yuh1 prevents cleavage of the Rub1 precursor, therefore neddylated protein species can neither be recognized with α-Cdc53 nor with α-Rub1/Nedd8. DenA expression driven by the inducible GAL1 promoter was applied to test the processing ability of the protease towards the Rub1 precursor. Expression of DenA was visualized with α-V5. DenA expression was not sufficient to restore Rub1 processing in a yuh1 deficient S. cerevisiae strain [44] . (C) An A. nidulans strain expressing a mature nedd8 construct ( nedd8m ) instead of normal nedd8 (wt) displayed a wild type like phenotype. Asexual structures (co) displayed on agar surface after 2 days of growth in the presence of light (scale bar = 20 µm) and after 7 days (scale bar = 500 µm). Sexual structures (cl) were only formed in the dark, but not in the light after 7 days incubation at 37°C (scale bar = 500 µm). Quantification of asexual spores from both strains after 2 days of incubation at 37°C in light. (D) The ΔdenA/nedd8m strain (mature Nedd8) and the ΔdenA (precursor Nedd8) are unresponsive to light-dependent inhibition of sexual development (compare rows 2 and 3; scale bar = 50 µm, first row; scale bar = 225 µm, second and third row) and impaired in asexual development (conidiation). (E) Human DEN1 cleaved human Nedd8 C-terminally fused with GFP while A. nidulans DenA did not. Nedd8-GFP substrate was combined with decreasing amounts of recombinant DEN1 or DenA, respectively (8–0.5 µM). The reaction mixture was incubated for 30 min at 37°C, immediately denatured, separated by SDS-PAGE and subjected to western blot analysis. α-GFP was applied to monitor cleavage of the Nedd8-GFP substrate. Samples were separated by additional SDS-PAGE and silver stained to prove for the presence of the respective deneddylase.

Techniques Used: Activity Assay, Western Blot, Expressing, Construct, Incubation, Inhibition, Recombinant, SDS Page, Staining

19) Product Images from "Ligand Binding Reduces SUMOylation of the Peroxisome Proliferator-activated Receptor ? (PPAR?) Activation Function 1 (AF1) Domain"

Article Title: Ligand Binding Reduces SUMOylation of the Peroxisome Proliferator-activated Receptor ? (PPAR?) Activation Function 1 (AF1) Domain

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066947

Transcriptional activity of PPARγ mutants. (A) HeLa (top) and RAW264.7 (bottom) cells were transfected with the Aox-tk luciferase reporter construct along with the indicated PPARγ1 lysine mutants. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (+) or the vehicle (-), and incubated for additional 24 hours. The reporter activity in the absence of PPARγ was arbitrarily set to 1. Error bars are mean +/− SD. Statistical significance of activation by PPARγ mutants compared to wild type PPARγ in the absence (*) or presence ( + ) of rosiglitazone was calculated using the Student´s t-test. * and + , p
Figure Legend Snippet: Transcriptional activity of PPARγ mutants. (A) HeLa (top) and RAW264.7 (bottom) cells were transfected with the Aox-tk luciferase reporter construct along with the indicated PPARγ1 lysine mutants. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (+) or the vehicle (-), and incubated for additional 24 hours. The reporter activity in the absence of PPARγ was arbitrarily set to 1. Error bars are mean +/− SD. Statistical significance of activation by PPARγ mutants compared to wild type PPARγ in the absence (*) or presence ( + ) of rosiglitazone was calculated using the Student´s t-test. * and + , p

Techniques Used: Activity Assay, Transfection, Luciferase, Construct, Incubation, Activation Assay

Transrepression activity of PPARγ mutants. (A) RAW264.7 macrophages were transfected with the iNOS luciferase reporter plasmid along with PPARγ mutants. Forty-two hours after transfection, cells were treated for 6 hours with 1 µg/ml LPS and 1 µM rosiglitazone (Rosi) as indicated. The reporter activities in the presence of LPS were set to 100% promoter activity. (B) Hela cells were transfected with the 3xNF-κB luciferase reporter plasmid along with PPARγ mutants. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (Rosi). Four hours prior lysis, 10 ng/ml interleukin-1β (IL-1ß) was added as indicated. The reporter activities obtained by interleukin-1ß stimulation were set to 100% promoter activity. Error bars are mean +/− SD. Statistics was performed using Student´s t-test. *, p
Figure Legend Snippet: Transrepression activity of PPARγ mutants. (A) RAW264.7 macrophages were transfected with the iNOS luciferase reporter plasmid along with PPARγ mutants. Forty-two hours after transfection, cells were treated for 6 hours with 1 µg/ml LPS and 1 µM rosiglitazone (Rosi) as indicated. The reporter activities in the presence of LPS were set to 100% promoter activity. (B) Hela cells were transfected with the 3xNF-κB luciferase reporter plasmid along with PPARγ mutants. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (Rosi). Four hours prior lysis, 10 ng/ml interleukin-1β (IL-1ß) was added as indicated. The reporter activities obtained by interleukin-1ß stimulation were set to 100% promoter activity. Error bars are mean +/− SD. Statistics was performed using Student´s t-test. *, p

Techniques Used: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Lysis

20) Product Images from "Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular ?-tubulin"

Article Title: Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular ?-tubulin

Journal: Scientific Reports

doi: 10.1038/srep03199

Identification of the TgRON4-binding region in TUBB2C. (a), (b) Upper: Schematic representation of TUBB2C and its N- and C-truncated mutants. Lower: The indicated HA-tagged TUBB2C proteins were coexpressed with 3xFLAG-tagged TgRON4B. The cell lysates were immunoprecipitated with an anti-HA antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. Owing to low expression, transient transfection of TUBB2C FR3 was scaled up ( see Methods ).
Figure Legend Snippet: Identification of the TgRON4-binding region in TUBB2C. (a), (b) Upper: Schematic representation of TUBB2C and its N- and C-truncated mutants. Lower: The indicated HA-tagged TUBB2C proteins were coexpressed with 3xFLAG-tagged TgRON4B. The cell lysates were immunoprecipitated with an anti-HA antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. Owing to low expression, transient transfection of TUBB2C FR3 was scaled up ( see Methods ).

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Expressing, Transfection

The C-terminal half of TgRON4 binds to host β-tubulin. (a) Schematic representation of TgRON4 and its deletion mutants. (b), (c) Coimmunoprecipitation of β-tubulin with deletion mutants of 3xFLAG-tagged TgRON4. 293 T (b) and Vero (c) cells were transiently transfected with the indicated TgRON4 expression plasmids. Membrane fractions were immunoprecipitated with an anti-β-tubulin antibody. The membrane fractions (3.3% input) and immunoprecipitates (IP) were analyzed by Western blotting with anti-FLAG and anti-β-tubulin antibodies.
Figure Legend Snippet: The C-terminal half of TgRON4 binds to host β-tubulin. (a) Schematic representation of TgRON4 and its deletion mutants. (b), (c) Coimmunoprecipitation of β-tubulin with deletion mutants of 3xFLAG-tagged TgRON4. 293 T (b) and Vero (c) cells were transiently transfected with the indicated TgRON4 expression plasmids. Membrane fractions were immunoprecipitated with an anti-β-tubulin antibody. The membrane fractions (3.3% input) and immunoprecipitates (IP) were analyzed by Western blotting with anti-FLAG and anti-β-tubulin antibodies.

Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot

Identification of the TUBB2C-binding region in TgRON4. (a) Schematic representation of TgRON4B and its deletion mutants. (b), (c) The indicated 3xFLAG-tagged RON4 proteins were coexpressed with HA-tagged TUBB2C F3R in Vero and CHO cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies.
Figure Legend Snippet: Identification of the TUBB2C-binding region in TgRON4. (a) Schematic representation of TgRON4B and its deletion mutants. (b), (c) The indicated 3xFLAG-tagged RON4 proteins were coexpressed with HA-tagged TUBB2C F3R in Vero and CHO cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies.

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot

21) Product Images from "Protein Arginine Methyltransferase 1 Interacts with and Activates p38? to Facilitate Erythroid Differentiation"

Article Title: Protein Arginine Methyltransferase 1 Interacts with and Activates p38? to Facilitate Erythroid Differentiation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056715

PRMT1 stimulated EPO-induced erythroid differentiation in primary human CD34 + hematopoietic progenitor cells which required the activation of p38 MAPK. (A) The PRMT1 methyltransferase activity in the homogenates of human CD34 + cells was assayed by in vitro methylation using hnRNP K (2 µg) as a substrate and visualized by fluorography. (B) Upon EPO treatment, PRMT1 activity was rapidly stimulated and reached its peak at 2 hr, as shown with three different substrates: hnRNP K, hnRNP A1 and hnRNP A2. A representative fluorograph from Donor A is shown. (C) The results from four individuals were expressed as fold stimulation at 2 hr after EPO treatment. Methyl incorporation into protein substrates was quantified by liquid scintillation counting. (D) PRMT1 protein levels did not change after EPO treatment. (E) The recombinant TAT-conjugated HA-PRMT1 proteins were introduced into CD34 + cells through the protein transduction method. HA-GFP recombinant proteins served as a negative control. (F, G) TAT-PRMT1 (0.1 µM) significantly stimulated the EPO-induced erythroid differentiation of CD34 + cells, as measured by benzidine staining and the surface expression of glycophorin A. Experiments were performed with cells from at least three different donors. Data are presented as means ± S.E. of at least three independent experiments; *, p
Figure Legend Snippet: PRMT1 stimulated EPO-induced erythroid differentiation in primary human CD34 + hematopoietic progenitor cells which required the activation of p38 MAPK. (A) The PRMT1 methyltransferase activity in the homogenates of human CD34 + cells was assayed by in vitro methylation using hnRNP K (2 µg) as a substrate and visualized by fluorography. (B) Upon EPO treatment, PRMT1 activity was rapidly stimulated and reached its peak at 2 hr, as shown with three different substrates: hnRNP K, hnRNP A1 and hnRNP A2. A representative fluorograph from Donor A is shown. (C) The results from four individuals were expressed as fold stimulation at 2 hr after EPO treatment. Methyl incorporation into protein substrates was quantified by liquid scintillation counting. (D) PRMT1 protein levels did not change after EPO treatment. (E) The recombinant TAT-conjugated HA-PRMT1 proteins were introduced into CD34 + cells through the protein transduction method. HA-GFP recombinant proteins served as a negative control. (F, G) TAT-PRMT1 (0.1 µM) significantly stimulated the EPO-induced erythroid differentiation of CD34 + cells, as measured by benzidine staining and the surface expression of glycophorin A. Experiments were performed with cells from at least three different donors. Data are presented as means ± S.E. of at least three independent experiments; *, p

Techniques Used: Activation Assay, Activity Assay, In Vitro, Methylation, Recombinant, Transduction, Negative Control, Staining, Expressing

22) Product Images from "High-level production of human interleukin-10 fusions in tobacco cell suspension cultures"

Article Title: High-level production of human interleukin-10 fusions in tobacco cell suspension cultures

Journal: Plant Biotechnology Journal

doi: 10.1111/pbi.12041

Schematic representation of the constructs used in tobacco BY-2 cell transformation. NPT II, neomycin phosphotransferase II gene under the control of the nopaline synthase promoter and terminator; cauliflower mosaic virus (CaMV)-35S, double-enhanced cauliflower mosaic virus 35S promoter. NSP, human IL-10 native signal peptide (54 bp); IL-10, human IL-10 coding sequence (480 bp); Thr, Thombin protease recognition sequence (27 bp); HIS, six histidine tag; KDEL, ER-retrieval tetrapeptide; T NOS , nopaline synthase terminator; tobacco etch virus (TEV), tobacco etch virus protease recognition site (21 bp); green fluorescent protein (GFP), smGFP coding sequence (714 bp); TE, translational enhancer from the tCUP promoter (88 bp) (Wu et al ., 2001 ); Pr1b, signal peptide from the tobacco pathogenesis-related 1b gene (90 bp); ELP, elastin-like polypeptide (420 bp); SII, StrepII purification tag (24 bp); RB, LB, Agrobacterium tumefaciens Ti plasmid right border and left border. Constructs are not drawn to scale.
Figure Legend Snippet: Schematic representation of the constructs used in tobacco BY-2 cell transformation. NPT II, neomycin phosphotransferase II gene under the control of the nopaline synthase promoter and terminator; cauliflower mosaic virus (CaMV)-35S, double-enhanced cauliflower mosaic virus 35S promoter. NSP, human IL-10 native signal peptide (54 bp); IL-10, human IL-10 coding sequence (480 bp); Thr, Thombin protease recognition sequence (27 bp); HIS, six histidine tag; KDEL, ER-retrieval tetrapeptide; T NOS , nopaline synthase terminator; tobacco etch virus (TEV), tobacco etch virus protease recognition site (21 bp); green fluorescent protein (GFP), smGFP coding sequence (714 bp); TE, translational enhancer from the tCUP promoter (88 bp) (Wu et al ., 2001 ); Pr1b, signal peptide from the tobacco pathogenesis-related 1b gene (90 bp); ELP, elastin-like polypeptide (420 bp); SII, StrepII purification tag (24 bp); RB, LB, Agrobacterium tumefaciens Ti plasmid right border and left border. Constructs are not drawn to scale.

Techniques Used: Construct, Transformation Assay, Sequencing, Purification, Plasmid Preparation

23) Product Images from "Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling"

Article Title: Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling

Journal: Febs Letters

doi: 10.1016/j.febslet.2012.09.027

Histaminylated GTPases Cdc42 and Gαo1 show increased effector binding and decreased GTP hydrolysis. (a) Histaminylation of G proteins prevents GTP hydrolysis. Thin layer chromatography followed by autoradiography of [α- 32 P]-GTP incubated with native Gαo1, histaminylated (TGM2 exposed) Gαo1, and buffer. (b) Histaminylation stabilizes a Gαo1/RGS4 complex . 6×His-Gαo1, or TGM2/HA-exposed 6×His-Gαo1, was loaded with GMP-PNP, GDP and GTP and incubated with 2 μM GST-RGS4. The complex was immunoprecipitated ( n = 5) and analyzed densitometrically. ∗ p
Figure Legend Snippet: Histaminylated GTPases Cdc42 and Gαo1 show increased effector binding and decreased GTP hydrolysis. (a) Histaminylation of G proteins prevents GTP hydrolysis. Thin layer chromatography followed by autoradiography of [α- 32 P]-GTP incubated with native Gαo1, histaminylated (TGM2 exposed) Gαo1, and buffer. (b) Histaminylation stabilizes a Gαo1/RGS4 complex . 6×His-Gαo1, or TGM2/HA-exposed 6×His-Gαo1, was loaded with GMP-PNP, GDP and GTP and incubated with 2 μM GST-RGS4. The complex was immunoprecipitated ( n = 5) and analyzed densitometrically. ∗ p

Techniques Used: Binding Assay, Thin Layer Chromatography, Autoradiography, Incubation, Immunoprecipitation

24) Product Images from "Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: solubility challenges"

Article Title: Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: solubility challenges

Journal: Scientific Reports

doi: 10.1038/srep01609

Cloning and expression of full-length DGK alpha in pT71myc. (a) Top, schematic of full-length S. s crofa DGK alpha in pT71myc. Bottom, a magnification of the epitope tag region of alpha in pT71myc. pT71myc adds to the N-terminus of the protein a fusion tag consisting of a hexahistidine, a thrombin proteolytic site, a myc tag, and a TEV protease site. The total mass of this protein construct (including the fusion tag) and pI are predicted to be 87.5 kD and 6.10, respectively. The epitope-tagged region of the construct is magnified for clarity. (b) SDS-PAGE of 8% acrylamide gels followed by: left, Coomassie staining; right, immunoblotting against hexahistidine; top, induced at 37°C for three hours; bottom, induced at 16°C overnight. L, lysate; S, supernatant (after removing insoluble from lysate).
Figure Legend Snippet: Cloning and expression of full-length DGK alpha in pT71myc. (a) Top, schematic of full-length S. s crofa DGK alpha in pT71myc. Bottom, a magnification of the epitope tag region of alpha in pT71myc. pT71myc adds to the N-terminus of the protein a fusion tag consisting of a hexahistidine, a thrombin proteolytic site, a myc tag, and a TEV protease site. The total mass of this protein construct (including the fusion tag) and pI are predicted to be 87.5 kD and 6.10, respectively. The epitope-tagged region of the construct is magnified for clarity. (b) SDS-PAGE of 8% acrylamide gels followed by: left, Coomassie staining; right, immunoblotting against hexahistidine; top, induced at 37°C for three hours; bottom, induced at 16°C overnight. L, lysate; S, supernatant (after removing insoluble from lysate).

Techniques Used: Clone Assay, Expressing, Construct, SDS Page, Staining

Expression, purification, and analytical gel filtration of alphacat in pT71myc coexpressed with bacterial chaperones. (a) SDS-PAGE of 12% acrylamide gels followed by: left, Coomassie staining; right, immunoblot against DGK alpha C-terminus; top, induced at 37°C for three hours; bottom, induced at 16°C overnight. U, uninduced; L, lysate; S, supernatant (after removing insoluble from lysate). (b) SDS-PAGE of an 8% acrylamide gel followed by immunoblotting against DGK alpha C-terminus. (c) SDS-PAGE of a 10% acrylamide gel followed by Coomassie staining. (d) Top, elution volumes of molecular weight standards and of purified alphacat from a Sephadex® G-200 column. Left vertical axis, A620 of blue dextran elution and A280 of protein standard elutions. Right vertical axis, quantification of the immunoblot signal from the blot shown at bottom, mean ± standard deviation (SD) (arbitrary units (AU)). Densitometry of the ~50 kD band was measured three times from the same film using ImageJ. Bottom, SDS-PAGE of a 10% acrylamide gel followed by immunoblotting against DGK alpha C-terminus. (e) SDS-PAGE of a 10% acrylamide gel followed by Coomassie staining. The lanes marked “+” were purified in the presence of 1 mM ATP.
Figure Legend Snippet: Expression, purification, and analytical gel filtration of alphacat in pT71myc coexpressed with bacterial chaperones. (a) SDS-PAGE of 12% acrylamide gels followed by: left, Coomassie staining; right, immunoblot against DGK alpha C-terminus; top, induced at 37°C for three hours; bottom, induced at 16°C overnight. U, uninduced; L, lysate; S, supernatant (after removing insoluble from lysate). (b) SDS-PAGE of an 8% acrylamide gel followed by immunoblotting against DGK alpha C-terminus. (c) SDS-PAGE of a 10% acrylamide gel followed by Coomassie staining. (d) Top, elution volumes of molecular weight standards and of purified alphacat from a Sephadex® G-200 column. Left vertical axis, A620 of blue dextran elution and A280 of protein standard elutions. Right vertical axis, quantification of the immunoblot signal from the blot shown at bottom, mean ± standard deviation (SD) (arbitrary units (AU)). Densitometry of the ~50 kD band was measured three times from the same film using ImageJ. Bottom, SDS-PAGE of a 10% acrylamide gel followed by immunoblotting against DGK alpha C-terminus. (e) SDS-PAGE of a 10% acrylamide gel followed by Coomassie staining. The lanes marked “+” were purified in the presence of 1 mM ATP.

Techniques Used: Expressing, Purification, Filtration, SDS Page, Staining, Acrylamide Gel Assay, Molecular Weight, Standard Deviation

Cloning, expression, refolding, purification, and analytical gel filtration of alphacat in pT71myc. (a) Top, a schematic of S. scrofa DGK alpha. Middle, a schematic of “alphacat”, which consists of residues 333–733 of DGK alpha, including the LCB5 domain, but missing the C-terminal cysteine. Bottom, a magnification of the epitope tag region of alphacat in pT71myc. pT71myc adds to the N-terminus of the protein a fusion tag consisting of a hexahistidine, a thrombin proteolytic site, a myc tag, and a TEV protease site. The total mass of this protein construct (including the fusion tag) and pI are predicted to be 49.8 kD and 6.99, respectively. (b) SDS-PAGE of 12% acrylamide gels followed by: left, Coomassie staining; right, immunoblot against histidine. IB, immunoblot. (c) SDS-PAGE of a 12% acrylamide gel followed by Coomassie staining. DNAse I was added to the lysate to 10 U per mL. TX-100, Triton X-100. (d) SDS-PAGE of 12% acrylamide gels followed by: top, Coomassie staining; bottom, immunoblot against DGK alpha C-terminus. (e) Elution volumes of molecular weight standards and of purified alphacat from a Sephadex® G-200 column. Left vertical axis, the absorbance at 620 nm (A620) of blue dextran elution and the absorbance at 280 nm (A280) of protein standard elutions. Right vertical axis, protein concentration of eluted alphacat, as measured by the Bio-Rad Protein Assay.
Figure Legend Snippet: Cloning, expression, refolding, purification, and analytical gel filtration of alphacat in pT71myc. (a) Top, a schematic of S. scrofa DGK alpha. Middle, a schematic of “alphacat”, which consists of residues 333–733 of DGK alpha, including the LCB5 domain, but missing the C-terminal cysteine. Bottom, a magnification of the epitope tag region of alphacat in pT71myc. pT71myc adds to the N-terminus of the protein a fusion tag consisting of a hexahistidine, a thrombin proteolytic site, a myc tag, and a TEV protease site. The total mass of this protein construct (including the fusion tag) and pI are predicted to be 49.8 kD and 6.99, respectively. (b) SDS-PAGE of 12% acrylamide gels followed by: left, Coomassie staining; right, immunoblot against histidine. IB, immunoblot. (c) SDS-PAGE of a 12% acrylamide gel followed by Coomassie staining. DNAse I was added to the lysate to 10 U per mL. TX-100, Triton X-100. (d) SDS-PAGE of 12% acrylamide gels followed by: top, Coomassie staining; bottom, immunoblot against DGK alpha C-terminus. (e) Elution volumes of molecular weight standards and of purified alphacat from a Sephadex® G-200 column. Left vertical axis, the absorbance at 620 nm (A620) of blue dextran elution and the absorbance at 280 nm (A280) of protein standard elutions. Right vertical axis, protein concentration of eluted alphacat, as measured by the Bio-Rad Protein Assay.

Techniques Used: Clone Assay, Expressing, Purification, Filtration, Construct, SDS Page, Staining, Acrylamide Gel Assay, Molecular Weight, Protein Concentration

25) Product Images from "Ataxia and peripheral nerve hypomyelination in ADAM22-deficient mice"

Article Title: Ataxia and peripheral nerve hypomyelination in ADAM22-deficient mice

Journal: BMC Neuroscience

doi: 10.1186/1471-2202-6-33

Hypomyelination of peripheral nerves in ADAM22-deficient mice. Epon embedded semithin cross-sections of the sciatic nerves (A, B), the trigeminal nerves (C, D) and the spinal cord [lateral funiculus] (E, F) of the indicated genotypes at postnatal day 10 were stained with toluidine blue. Note that the ADAM22-deficient mouse shows thin myelin or lack of myelin in the peripheral nerve fibres (B, D), but the spinal myelinated fibres are intact (F).
Figure Legend Snippet: Hypomyelination of peripheral nerves in ADAM22-deficient mice. Epon embedded semithin cross-sections of the sciatic nerves (A, B), the trigeminal nerves (C, D) and the spinal cord [lateral funiculus] (E, F) of the indicated genotypes at postnatal day 10 were stained with toluidine blue. Note that the ADAM22-deficient mouse shows thin myelin or lack of myelin in the peripheral nerve fibres (B, D), but the spinal myelinated fibres are intact (F).

Techniques Used: Mouse Assay, Staining

Adam22 gene structure and tissue specific transcripts. (A) RT-PCR analysis. Amplified DNA fragments were analysed by 1 % agarose gel electrophoresis. Lanes 1. 100 bp DNA ladder; 2. cerebellum; 3. spinal cord; 4. dorsal root ganglion; 5. sciatic nerve; 6. cultured Schwann cells; 7. distilled water (B) Exon organization of the mouse Adam22 gene is illustrated. Boxes indicate exons. The G01 transcript (orthologous to the human ADAM22 isoform 1 transcript) is composed of grey boxes. Boxes in black indicate non-coding region. (C) Summary of the isolated clones. The nucleotide sequence data have been deposited with the DDBJ/EMBL/GenBank Data Libraries under the accession numbers described in the table. (D) Number of clones isolated from each tissue is summarized. Cb; cerebellum, Sp; spinal cord, DRG; dorsal root ganglion, SN; sciatic nerve, cSC; cultured Schwann cells.
Figure Legend Snippet: Adam22 gene structure and tissue specific transcripts. (A) RT-PCR analysis. Amplified DNA fragments were analysed by 1 % agarose gel electrophoresis. Lanes 1. 100 bp DNA ladder; 2. cerebellum; 3. spinal cord; 4. dorsal root ganglion; 5. sciatic nerve; 6. cultured Schwann cells; 7. distilled water (B) Exon organization of the mouse Adam22 gene is illustrated. Boxes indicate exons. The G01 transcript (orthologous to the human ADAM22 isoform 1 transcript) is composed of grey boxes. Boxes in black indicate non-coding region. (C) Summary of the isolated clones. The nucleotide sequence data have been deposited with the DDBJ/EMBL/GenBank Data Libraries under the accession numbers described in the table. (D) Number of clones isolated from each tissue is summarized. Cb; cerebellum, Sp; spinal cord, DRG; dorsal root ganglion, SN; sciatic nerve, cSC; cultured Schwann cells.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Cell Culture, Isolation, Clone Assay, Sequencing

Uncoordinated movements in the Adam22 (-/-) mice at postnatal day 18 (P18). (A) Adam22 (-/-) mice were smaller than their wild-type (+/+) littermates. (B, C) Adam22 (-/-) mice at P18 were unable to support themselves on their hindlimbs.
Figure Legend Snippet: Uncoordinated movements in the Adam22 (-/-) mice at postnatal day 18 (P18). (A) Adam22 (-/-) mice were smaller than their wild-type (+/+) littermates. (B, C) Adam22 (-/-) mice at P18 were unable to support themselves on their hindlimbs.

Techniques Used: Mouse Assay

Neuronal ADAM22 mRNA expression in the CNS. To determine the ADAM22 mRNA distribution, in situ hybridisation analysis using 35 S-labeled probe was performed. Coronal (A, B) and sagittal (C, D) sections of the mouse brain and spinal cord (E) were shown. Using the antisense probe (A, C), strong signals were obtained, especially in the hippocampus and the cerebellum, while no signals was detected by the sense probe (B, D). In the spinal cord, autoradiograms of ADAM22 mRNA was detected in the grey matter (E).
Figure Legend Snippet: Neuronal ADAM22 mRNA expression in the CNS. To determine the ADAM22 mRNA distribution, in situ hybridisation analysis using 35 S-labeled probe was performed. Coronal (A, B) and sagittal (C, D) sections of the mouse brain and spinal cord (E) were shown. Using the antisense probe (A, C), strong signals were obtained, especially in the hippocampus and the cerebellum, while no signals was detected by the sense probe (B, D). In the spinal cord, autoradiograms of ADAM22 mRNA was detected in the grey matter (E).

Techniques Used: Expressing, In Situ, Hybridization, Labeling

Normal neurodevelopment in the CNS of ADAM22-deficient mice. Sagittal sections of the cerebellum from wild-type mice (A, C, E, G) and homozygous mutants (B, D, F, H) at postnatal day 13 were stained for calbindin (C and D; green) or MBP (G and H; green), and were counterstained with DAPI (A, B, E, F; blue). Significant abnormalities were not observed in the homozygotes. Hippocampal neurons were stained by anti-Neu N antibody (I and J). Spinal myelin was analysed by MBP staining (K and L). These analyses showed no obvious differences between homozygotes (J, L) and wild-type littermates (I, K). Bar: (A, B, E, F) 100 μm, (K, L) 250 μm.
Figure Legend Snippet: Normal neurodevelopment in the CNS of ADAM22-deficient mice. Sagittal sections of the cerebellum from wild-type mice (A, C, E, G) and homozygous mutants (B, D, F, H) at postnatal day 13 were stained for calbindin (C and D; green) or MBP (G and H; green), and were counterstained with DAPI (A, B, E, F; blue). Significant abnormalities were not observed in the homozygotes. Hippocampal neurons were stained by anti-Neu N antibody (I and J). Spinal myelin was analysed by MBP staining (K and L). These analyses showed no obvious differences between homozygotes (J, L) and wild-type littermates (I, K). Bar: (A, B, E, F) 100 μm, (K, L) 250 μm.

Techniques Used: Mouse Assay, Staining

Electron microscopic analysis of sciatic nerves. Electron micrographs of the sciatic nerves from Adam22 +/- (A) and Adam22 -/- (B) mice at postnatal day 10 are shown. In the heterozygote (A), thick myelin was formed, while no myelin was formed in the ADAM22-deficient mouse (B). The axons looked normal in each genotype.
Figure Legend Snippet: Electron microscopic analysis of sciatic nerves. Electron micrographs of the sciatic nerves from Adam22 +/- (A) and Adam22 -/- (B) mice at postnatal day 10 are shown. In the heterozygote (A), thick myelin was formed, while no myelin was formed in the ADAM22-deficient mouse (B). The axons looked normal in each genotype.

Techniques Used: Mouse Assay

Targeted mutation of Adam22 . (A) The genomic structure of the wild-type Adam22 allele (top), the targeting construct (middle) and the disrupted allele (bottom) are shown. ADAM22 expression was disrupted by the insertion of a termination codon and a PGKneo cassette into exon 8. An MC1/TK cassette at the end of the targeting vector allows for negative selection. The 3' probe represents the position of the external probe used for Southern blot analysis, and expected Bam HI [B] fragments are indicated by arrows. (B) Southern blot analysis of mouse genomic DNA. The expected DNA fragments for the wild-type allele and disrupted allele are 7.5-kb and 2.5-kb, respectively. +/+, wild-type; +/-, heterozygote; -/-, homozygote. (C) Western blot analysis of ADAM22 expression in the mouse cerebellum. Absence of ADAM22 protein in the Adam22 (-/-) mutant cerebellum was shown using anti-ADAM22-cp (cytoplasmic domain) polyclonal antibody.
Figure Legend Snippet: Targeted mutation of Adam22 . (A) The genomic structure of the wild-type Adam22 allele (top), the targeting construct (middle) and the disrupted allele (bottom) are shown. ADAM22 expression was disrupted by the insertion of a termination codon and a PGKneo cassette into exon 8. An MC1/TK cassette at the end of the targeting vector allows for negative selection. The 3' probe represents the position of the external probe used for Southern blot analysis, and expected Bam HI [B] fragments are indicated by arrows. (B) Southern blot analysis of mouse genomic DNA. The expected DNA fragments for the wild-type allele and disrupted allele are 7.5-kb and 2.5-kb, respectively. +/+, wild-type; +/-, heterozygote; -/-, homozygote. (C) Western blot analysis of ADAM22 expression in the mouse cerebellum. Absence of ADAM22 protein in the Adam22 (-/-) mutant cerebellum was shown using anti-ADAM22-cp (cytoplasmic domain) polyclonal antibody.

Techniques Used: Mutagenesis, Construct, Expressing, Plasmid Preparation, Selection, Southern Blot, Western Blot

26) Product Images from "Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding"

Article Title: Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding

Journal: Cell

doi: 10.1016/j.cell.2013.03.043

Experimental Validation of CLASH Identified miR-92a Targets, Related to Figure 5 Changes in mRNA abundance upon miR-92a depletion in PTH-AGO1-HEK293 cells, measured by Affymetrix microarrays. The performance of various classes of miR-92a targets identified in CLASH analyses, and targets containing the miR-92a motif, are compared to transcripts containing a match to the miR-92a 7-mer seed sequence (positive control), to random transcripts, and to targets lacking a match to the miR-92a 7-mer seed (negative control). The left and right edge of the box represent 25th and 75th percentile, respectively. The ends of the whiskers show the minimum and maximum values of the data.
Figure Legend Snippet: Experimental Validation of CLASH Identified miR-92a Targets, Related to Figure 5 Changes in mRNA abundance upon miR-92a depletion in PTH-AGO1-HEK293 cells, measured by Affymetrix microarrays. The performance of various classes of miR-92a targets identified in CLASH analyses, and targets containing the miR-92a motif, are compared to transcripts containing a match to the miR-92a 7-mer seed sequence (positive control), to random transcripts, and to targets lacking a match to the miR-92a 7-mer seed (negative control). The left and right edge of the box represent 25th and 75th percentile, respectively. The ends of the whiskers show the minimum and maximum values of the data.

Techniques Used: Sequencing, Positive Control, Negative Control

Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.
Figure Legend Snippet: Overview of Experimental and Bioinformatic Procedures (A) Growing cells were UV irradiated, and PTH-AGO1 was purified. RNA fragmentation, ligation, cDNA synthesis, and sequencing of AGO1-associated RNAs allowed the identification of sites of AGO1 binding (as single reads) and RNA-RNA interactions at AGO1-binding sites (as chimeric reads). (B) Sequencing reads were mapped to a database of human transcripts using BLAST ( Altschul et al., 1990 ). Sequences reliably mapped to two different sites were folded in silico using UNAFold ( Markham and Zuker, 2008 ) to identify the interaction site of the RNA molecules that gave rise to the chimeric cDNA. (C) Example interaction between miR-196a/b and HOXC8 that was supported by chimeric reads (red), and a cluster of nonchimeric reads (green). The blue dashed line represents the location of the miRNA bit of chimera, and the red dashed line shows the 25 nt mRNA extension added during the analysis. The interaction was previously shown experimentally ( Li et al., 2010 ) and can be predicted by RNAhybrid ( Rehmsmeier et al., 2004 ). (D) Distribution of all miRNA interactions among various classes of RNAs. The main miRNA targets are mRNAs and are represented by 18,514 interactions. See also Figure S1 and Tables S1 and S2 A–S2C.

Techniques Used: Irradiation, Purification, Ligation, Sequencing, Binding Assay, In Silico

27) Product Images from "A Novel Candidate Vaccine for Cytauxzoonosis Inferred from Comparative Apicomplexan Genomics"

Article Title: A Novel Candidate Vaccine for Cytauxzoonosis Inferred from Comparative Apicomplexan Genomics

Journal: PLoS ONE

doi: 10.1371/journal.pone.0071233

Conserved gene synteny between T. parva p67 and C. felis cf76. cf76 is identified in silico within a highly conserved syntenic block of genes similarly to the leading vaccine candidate for T. parva , p67.
Figure Legend Snippet: Conserved gene synteny between T. parva p67 and C. felis cf76. cf76 is identified in silico within a highly conserved syntenic block of genes similarly to the leading vaccine candidate for T. parva , p67.

Techniques Used: In Silico, Blocking Assay

Assessment of feline sero-reactivity to cf76 and cf76 fragments by western blot. Purified full length cf76 (1), the N-terminal region (2), the central region (3), and the C-terminal region (4) were probed with pooled sera (1∶500) from cats surviving C. felis infection (A) or naive cats (B).
Figure Legend Snippet: Assessment of feline sero-reactivity to cf76 and cf76 fragments by western blot. Purified full length cf76 (1), the N-terminal region (2), the central region (3), and the C-terminal region (4) were probed with pooled sera (1∶500) from cats surviving C. felis infection (A) or naive cats (B).

Techniques Used: Western Blot, Purification, Infection

In situ hybridization to identify transcription of cf76 in C. felis-infected lung tissue. A. Hematoxylin and eosin stained lung tissue demonstrating schizonts forming a parasitic thrombus within a pulmonary vessel, 20X, B. Negative sense riboprobe, hematoxylin and eosin counterstain, numerous positive cells (brown) are demonstrating intracytoplasmic presence of cf76 antigen, 20X, C. Irrelevant negative sense riboprobe, hematoxylin and eosin counterstain, 20X.
Figure Legend Snippet: In situ hybridization to identify transcription of cf76 in C. felis-infected lung tissue. A. Hematoxylin and eosin stained lung tissue demonstrating schizonts forming a parasitic thrombus within a pulmonary vessel, 20X, B. Negative sense riboprobe, hematoxylin and eosin counterstain, numerous positive cells (brown) are demonstrating intracytoplasmic presence of cf76 antigen, 20X, C. Irrelevant negative sense riboprobe, hematoxylin and eosin counterstain, 20X.

Techniques Used: In Situ Hybridization, Infection, Staining

Assessment of purified cf76 and cf76 fragments by western blot. Purified full length cf76 (1), the N-terminal region (2), the central region (3), and the C-terminal region (4) were probed with anti-HIS N-terminal tag (A) and anti-HA C-terminal tag antibodies (B).
Figure Legend Snippet: Assessment of purified cf76 and cf76 fragments by western blot. Purified full length cf76 (1), the N-terminal region (2), the central region (3), and the C-terminal region (4) were probed with anti-HIS N-terminal tag (A) and anti-HA C-terminal tag antibodies (B).

Techniques Used: Purification, Western Blot

28) Product Images from "Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase"

Article Title: Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

Journal: Scientific Reports

doi: 10.1038/srep03362

The putative Sec7 region of Mon2 does not promote guanine nucleotide exchange of Arl1 in vitro. (a) Coomassie stained gel showing the Mon2-N protein purified from insect cells. M, molecular weight marker (kd). (b–f) Typical exchange kinetic traces during in vitro guanine nucleotide exchange of Arl1. His-Δ14Arl1 (1.0 μM) was incubated with Mon2-N (0.7 μM) (b), GST-Big1-Sec7 (0.7 μM) (c), GST (0.7 μM) (d), EDTA (0.7 μM) (e) or buffer (f), respectively, in the presence of Mant-GMPPNP. The experimental data (gray squares) were fitted to a single exponential decay function y = y 0 + A*exp[−(x − x 0 )/τ] (red curve) and normalized. The time constant τ and adjusted R 2 are labeled in each plot. (g) The column graph showing the guanine nucleotide exchange activities (1/τ) of various factors on Arl1 or Arf1. n denotes the number of independent experiments. Error bar indicates standard error of mean. The p values (by t-test) of selected pair of data are indicated.
Figure Legend Snippet: The putative Sec7 region of Mon2 does not promote guanine nucleotide exchange of Arl1 in vitro. (a) Coomassie stained gel showing the Mon2-N protein purified from insect cells. M, molecular weight marker (kd). (b–f) Typical exchange kinetic traces during in vitro guanine nucleotide exchange of Arl1. His-Δ14Arl1 (1.0 μM) was incubated with Mon2-N (0.7 μM) (b), GST-Big1-Sec7 (0.7 μM) (c), GST (0.7 μM) (d), EDTA (0.7 μM) (e) or buffer (f), respectively, in the presence of Mant-GMPPNP. The experimental data (gray squares) were fitted to a single exponential decay function y = y 0 + A*exp[−(x − x 0 )/τ] (red curve) and normalized. The time constant τ and adjusted R 2 are labeled in each plot. (g) The column graph showing the guanine nucleotide exchange activities (1/τ) of various factors on Arl1 or Arf1. n denotes the number of independent experiments. Error bar indicates standard error of mean. The p values (by t-test) of selected pair of data are indicated.

Techniques Used: In Vitro, Staining, Purification, Molecular Weight, Marker, Incubation, Labeling

29) Product Images from "The Membrane-Associated Transcription Factor NAC089 Controls ER-Stress-Induced Programmed Cell Death in Plants"

Article Title: The Membrane-Associated Transcription Factor NAC089 Controls ER-Stress-Induced Programmed Cell Death in Plants

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004243

Hypothetical working model of cell survival and cell death pathways in plant UPR. In response to ER stress, the ER-localized bZIP28P is activated through S1P/S2P sequential cleavage. The precursor bZIP60P is also ER membrane-associated under normal growth condition; ER stress activates the ER-localized IRE1, which splices the bZIP60 mRNA to produce a shorter bZIP60 mRNA encoding the activated form bZIP60S. Both the activated bZIP28D and bZIP60S enter the nucleus and up-regulate downstream genes such as molecular chaperones and ERAD components to ensure cell survival. The activated bZIP60S also induces its own transcription and another transcription factor NAC103 to amplify the cell survival signal. IRE1 could also protect cells through RIDD to reduce the entry of secretory proteins into the ER lumen. On the other hand, the ER membrane-localized NAC089P is activated to produce the nuclear form NAC089D when the ER stress is severe. NAC089D activates several downstream PCD-related genes to promote cell death. Both bZIP28D and bZIP60S control the up-regulation of NAC089 under ER stress condition and may also control the expression of negative regulators for NAC089 activation. Thus, both pro-survival and pro-death signals are controlled by bZIP28 and bZIP60, and the balance between their outputs probably decides the life-or-death cell fate in Arabidopsis plants under ER stress conditions.
Figure Legend Snippet: Hypothetical working model of cell survival and cell death pathways in plant UPR. In response to ER stress, the ER-localized bZIP28P is activated through S1P/S2P sequential cleavage. The precursor bZIP60P is also ER membrane-associated under normal growth condition; ER stress activates the ER-localized IRE1, which splices the bZIP60 mRNA to produce a shorter bZIP60 mRNA encoding the activated form bZIP60S. Both the activated bZIP28D and bZIP60S enter the nucleus and up-regulate downstream genes such as molecular chaperones and ERAD components to ensure cell survival. The activated bZIP60S also induces its own transcription and another transcription factor NAC103 to amplify the cell survival signal. IRE1 could also protect cells through RIDD to reduce the entry of secretory proteins into the ER lumen. On the other hand, the ER membrane-localized NAC089P is activated to produce the nuclear form NAC089D when the ER stress is severe. NAC089D activates several downstream PCD-related genes to promote cell death. Both bZIP28D and bZIP60S control the up-regulation of NAC089 under ER stress condition and may also control the expression of negative regulators for NAC089 activation. Thus, both pro-survival and pro-death signals are controlled by bZIP28 and bZIP60, and the balance between their outputs probably decides the life-or-death cell fate in Arabidopsis plants under ER stress conditions.

Techniques Used: Expressing, Activation Assay

30) Product Images from "N-glycosylation is required for secretion and enzymatic activity of human hyaluronidase1"

Article Title: N-glycosylation is required for secretion and enzymatic activity of human hyaluronidase1

Journal: FEBS Open Bio

doi: 10.1016/j.fob.2014.06.001

N -glycosylation is essential for HYAL1 secretion. (A and B) Exponentially growing HT1080-neo (neo), HT1080-HYAL1-MH (wt), HT1080-HYAL1-MH/N99Q (N99Q), HT1080-HYAL1-MH/N216Q (N216Q), and HT1080-HYAL1-MH/N350Q (N350Q) cells were lysed, and each cell lysate was electrophoresed and immunoblotted with the indicated antibodies (A). Total RNAs were isolated from neo, wt, N99Q, N216Q, and N350Q cells, and semi-quantitative RT-PCR was performed (B). (C) Exponentially growing neo, wt, N99Q, N216Q, and N350Q cells were cultured in serum-free DMEM for 24 h before the conditioned media and cell lysates were collected. Conditioned media were incubated with Ni-NTA agarose. Ni-NTA-bound HYAL1 was washed and eluted with 300 mM imidazole. The eluted samples and cell lysates were electrophoresed and immunoblotted with the indicated antibodies.
Figure Legend Snippet: N -glycosylation is essential for HYAL1 secretion. (A and B) Exponentially growing HT1080-neo (neo), HT1080-HYAL1-MH (wt), HT1080-HYAL1-MH/N99Q (N99Q), HT1080-HYAL1-MH/N216Q (N216Q), and HT1080-HYAL1-MH/N350Q (N350Q) cells were lysed, and each cell lysate was electrophoresed and immunoblotted with the indicated antibodies (A). Total RNAs were isolated from neo, wt, N99Q, N216Q, and N350Q cells, and semi-quantitative RT-PCR was performed (B). (C) Exponentially growing neo, wt, N99Q, N216Q, and N350Q cells were cultured in serum-free DMEM for 24 h before the conditioned media and cell lysates were collected. Conditioned media were incubated with Ni-NTA agarose. Ni-NTA-bound HYAL1 was washed and eluted with 300 mM imidazole. The eluted samples and cell lysates were electrophoresed and immunoblotted with the indicated antibodies.

Techniques Used: Isolation, Quantitative RT-PCR, Cell Culture, Incubation

N -glycosylation of HYAL1 at Asn 99 , Asn 216 , and Asn 350 demonstrated by MALDI-TOF MS analysis. (A) Exponentially growing HT1080-HYAL1-MH cells were cultured in serum-free DMEM for 24 h before the conditioned medium was collected. The conditioned medium was concentrated by ultrafiltration membrane and incubated with Ni-NTA agarose for 2 h. The Ni-NTA agarose was washed 5 times and eluted with 500 mM imidazole. The obtained proteins were electrophoresed on an SDS–polyacrylamide gel and visualized by CBB staining. (B–D) Purified HYAL1 was treated with ( lower ) or without ( upper ) PNGase F and was subjected to an SDS-polyacrylamide gel. Samples were digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF MS. The fragments converting Asn 99 (B), Asn 216 (C), and Asn 350 (D) with Asp residues by PNGase F had the expected masses of 4431.8, 3581.0, and 3397.9, respectively. Underlined “D”s indicate Asp residues converted from glycosylated Asn residues after treatment with PNGase F.
Figure Legend Snippet: N -glycosylation of HYAL1 at Asn 99 , Asn 216 , and Asn 350 demonstrated by MALDI-TOF MS analysis. (A) Exponentially growing HT1080-HYAL1-MH cells were cultured in serum-free DMEM for 24 h before the conditioned medium was collected. The conditioned medium was concentrated by ultrafiltration membrane and incubated with Ni-NTA agarose for 2 h. The Ni-NTA agarose was washed 5 times and eluted with 500 mM imidazole. The obtained proteins were electrophoresed on an SDS–polyacrylamide gel and visualized by CBB staining. (B–D) Purified HYAL1 was treated with ( lower ) or without ( upper ) PNGase F and was subjected to an SDS-polyacrylamide gel. Samples were digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF MS. The fragments converting Asn 99 (B), Asn 216 (C), and Asn 350 (D) with Asp residues by PNGase F had the expected masses of 4431.8, 3581.0, and 3397.9, respectively. Underlined “D”s indicate Asp residues converted from glycosylated Asn residues after treatment with PNGase F.

Techniques Used: Mass Spectrometry, Cell Culture, Incubation, Staining, Purification

31) Product Images from "N-glycosylation is required for secretion and enzymatic activity of human hyaluronidase1"

Article Title: N-glycosylation is required for secretion and enzymatic activity of human hyaluronidase1

Journal: FEBS Open Bio

doi: 10.1016/j.fob.2014.06.001

N -glycosylation is essential for HYAL1 secretion. (A and B) Exponentially growing HT1080-neo (neo), HT1080-HYAL1-MH (wt), HT1080-HYAL1-MH/N99Q (N99Q), HT1080-HYAL1-MH/N216Q (N216Q), and HT1080-HYAL1-MH/N350Q (N350Q) cells were lysed, and each cell lysate was electrophoresed and immunoblotted with the indicated antibodies (A). Total RNAs were isolated from neo, wt, N99Q, N216Q, and N350Q cells, and semi-quantitative RT-PCR was performed (B). (C) Exponentially growing neo, wt, N99Q, N216Q, and N350Q cells were cultured in serum-free DMEM for 24 h before the conditioned media and cell lysates were collected. Conditioned media were incubated with Ni-NTA agarose. Ni-NTA-bound HYAL1 was washed and eluted with 300 mM imidazole. The eluted samples and cell lysates were electrophoresed and immunoblotted with the indicated antibodies.
Figure Legend Snippet: N -glycosylation is essential for HYAL1 secretion. (A and B) Exponentially growing HT1080-neo (neo), HT1080-HYAL1-MH (wt), HT1080-HYAL1-MH/N99Q (N99Q), HT1080-HYAL1-MH/N216Q (N216Q), and HT1080-HYAL1-MH/N350Q (N350Q) cells were lysed, and each cell lysate was electrophoresed and immunoblotted with the indicated antibodies (A). Total RNAs were isolated from neo, wt, N99Q, N216Q, and N350Q cells, and semi-quantitative RT-PCR was performed (B). (C) Exponentially growing neo, wt, N99Q, N216Q, and N350Q cells were cultured in serum-free DMEM for 24 h before the conditioned media and cell lysates were collected. Conditioned media were incubated with Ni-NTA agarose. Ni-NTA-bound HYAL1 was washed and eluted with 300 mM imidazole. The eluted samples and cell lysates were electrophoresed and immunoblotted with the indicated antibodies.

Techniques Used: Isolation, Quantitative RT-PCR, Cell Culture, Incubation

N -glycosylation of HYAL1 at Asn 99 , Asn 216 , and Asn 350 demonstrated by MALDI-TOF MS analysis. (A) Exponentially growing HT1080-HYAL1-MH cells were cultured in serum-free DMEM for 24 h before the conditioned medium was collected. The conditioned medium was concentrated by ultrafiltration membrane and incubated with Ni-NTA agarose for 2 h. The Ni-NTA agarose was washed 5 times and eluted with 500 mM imidazole. The obtained proteins were electrophoresed on an SDS–polyacrylamide gel and visualized by CBB staining. (B–D) Purified HYAL1 was treated with ( lower ) or without ( upper ) PNGase F and was subjected to an SDS-polyacrylamide gel. Samples were digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF MS. The fragments converting Asn 99 (B), Asn 216 (C), and Asn 350 (D) with Asp residues by PNGase F had the expected masses of 4431.8, 3581.0, and 3397.9, respectively. Underlined “D”s indicate Asp residues converted from glycosylated Asn residues after treatment with PNGase F.
Figure Legend Snippet: N -glycosylation of HYAL1 at Asn 99 , Asn 216 , and Asn 350 demonstrated by MALDI-TOF MS analysis. (A) Exponentially growing HT1080-HYAL1-MH cells were cultured in serum-free DMEM for 24 h before the conditioned medium was collected. The conditioned medium was concentrated by ultrafiltration membrane and incubated with Ni-NTA agarose for 2 h. The Ni-NTA agarose was washed 5 times and eluted with 500 mM imidazole. The obtained proteins were electrophoresed on an SDS–polyacrylamide gel and visualized by CBB staining. (B–D) Purified HYAL1 was treated with ( lower ) or without ( upper ) PNGase F and was subjected to an SDS-polyacrylamide gel. Samples were digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF MS. The fragments converting Asn 99 (B), Asn 216 (C), and Asn 350 (D) with Asp residues by PNGase F had the expected masses of 4431.8, 3581.0, and 3397.9, respectively. Underlined “D”s indicate Asp residues converted from glycosylated Asn residues after treatment with PNGase F.

Techniques Used: Mass Spectrometry, Cell Culture, Incubation, Staining, Purification

32) Product Images from "Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1"

Article Title: Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0000386

Histone demethylase activity of TAP tagged Swm1 and Swm2 complexes (A C), recombinant human LSD1 (B), and GST-Swm1 (D), with various methylated histone substrates. The substrates: calf thymus bulk histones (BH), chicken polynucleosomes (Nuc), calf thymus histone H3 (H3) and recombinant H3 (rH3) along with their sites of lysine (K) methylation are indicated below the panels. Control indicates a mock TAP-tag purification from the wild type strain. hLSD1 was recombinant E. coli expressed human LSD1 protein and GST-Swm1 was recombinant E. coli expressed Swm1.
Figure Legend Snippet: Histone demethylase activity of TAP tagged Swm1 and Swm2 complexes (A C), recombinant human LSD1 (B), and GST-Swm1 (D), with various methylated histone substrates. The substrates: calf thymus bulk histones (BH), chicken polynucleosomes (Nuc), calf thymus histone H3 (H3) and recombinant H3 (rH3) along with their sites of lysine (K) methylation are indicated below the panels. Control indicates a mock TAP-tag purification from the wild type strain. hLSD1 was recombinant E. coli expressed human LSD1 protein and GST-Swm1 was recombinant E. coli expressed Swm1.

Techniques Used: Activity Assay, Recombinant, Methylation, Purification

33) Product Images from "Archaeal MCM has separable processivity, substrate choice and helicase domains"

Article Title: Archaeal MCM has separable processivity, substrate choice and helicase domains

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl1117

( A ) Determination of the polarity of Sso MCM helicase activity. 25, 50 or 100 ng of Sso MCM or 100 ng of Mth MCM were incubated with substrates designed to have 3′, 5 or 3′ and 5′ single–stranded extensions. The positions of substrate (S) and product (P) are indicated. ( B ) Figure 5 B shows the helicase activity of the different deletion constructs at various concentrations, N-half and HTH are omitted as no activity was detected. ( C ) Figure 5 C compares the activity of the AAA+ core with an equivalent construct possessing a K > A mutation in the Walker A motif.
Figure Legend Snippet: ( A ) Determination of the polarity of Sso MCM helicase activity. 25, 50 or 100 ng of Sso MCM or 100 ng of Mth MCM were incubated with substrates designed to have 3′, 5 or 3′ and 5′ single–stranded extensions. The positions of substrate (S) and product (P) are indicated. ( B ) Figure 5 B shows the helicase activity of the different deletion constructs at various concentrations, N-half and HTH are omitted as no activity was detected. ( C ) Figure 5 C compares the activity of the AAA+ core with an equivalent construct possessing a K > A mutation in the Walker A motif.

Techniques Used: Activity Assay, Incubation, Construct, Mutagenesis

EMSA assays were performed as described ( 15 ), ( A ) Representative EMSA assay with 0, 50, 100, 150, 200, 250, 300, 350, 400, 500 and 600 nM wild-type MCM ( B ) Figure 3 B demonstrates the percentage of DNA bound at the indicated concentration for constructs: full length, ▵A, ▵HTH and ▵A/HTH. ( C ) Figure 3 C demonstrates the results for N-half, C-half, AAA+ core, HTH and an equimolar mix of N-half and AAA+ core. ( D ) EMSA assays employing 6 µM Core and/or N-half protein. The positions of free DNA and the relevant complexes are indicated.
Figure Legend Snippet: EMSA assays were performed as described ( 15 ), ( A ) Representative EMSA assay with 0, 50, 100, 150, 200, 250, 300, 350, 400, 500 and 600 nM wild-type MCM ( B ) Figure 3 B demonstrates the percentage of DNA bound at the indicated concentration for constructs: full length, ▵A, ▵HTH and ▵A/HTH. ( C ) Figure 3 C demonstrates the results for N-half, C-half, AAA+ core, HTH and an equimolar mix of N-half and AAA+ core. ( D ) EMSA assays employing 6 µM Core and/or N-half protein. The positions of free DNA and the relevant complexes are indicated.

Techniques Used: Concentration Assay, Construct

( A ) Figure 2 A shows the UV 280 nm trace from gel filtration over a Superose 6 column using 25 µM of each of the indicated proteins. ( B ) Figure 2 B shows the UV 280 nm trace from gel filtration over a Superdex 200 column using two concentrations for each protein. N-half: 200 µM, 25 µM. C-half 25 µM, 2.5 µM. AAA+ Core 25 µM, 2.5 µM. HTH 60 µM, 1 µM. The absorbance of each trace was normalised using the UNICORN program. ( C ) 25 µM Core domain, N-half or a mixture of both were incubated in the presence (+) or absence (−) of glutaraldehyde prior to SDS-PAGE on a 4–12% polyacrylamide gradient gel. ( D ) 25 µM AAA+ Core were run alone or mixed with 200 µM N-half on a Superdex 200 column. 80 µl samples were collected and electrophoresed on a 10% polyacrylamide gel. ( E ) His-tagged AAA+ domain was used in pull-down experiments with untagged N-terminal domain. 2–1% N-terminal domain input, 20–10% N-terminal domain input, Bead—Ni-NTA beads incubated with N-terminus, Beads + Core – Ni-NTA beads bound to AAA+ domain incubated with N-terminus.
Figure Legend Snippet: ( A ) Figure 2 A shows the UV 280 nm trace from gel filtration over a Superose 6 column using 25 µM of each of the indicated proteins. ( B ) Figure 2 B shows the UV 280 nm trace from gel filtration over a Superdex 200 column using two concentrations for each protein. N-half: 200 µM, 25 µM. C-half 25 µM, 2.5 µM. AAA+ Core 25 µM, 2.5 µM. HTH 60 µM, 1 µM. The absorbance of each trace was normalised using the UNICORN program. ( C ) 25 µM Core domain, N-half or a mixture of both were incubated in the presence (+) or absence (−) of glutaraldehyde prior to SDS-PAGE on a 4–12% polyacrylamide gradient gel. ( D ) 25 µM AAA+ Core were run alone or mixed with 200 µM N-half on a Superdex 200 column. 80 µl samples were collected and electrophoresed on a 10% polyacrylamide gel. ( E ) His-tagged AAA+ domain was used in pull-down experiments with untagged N-terminal domain. 2–1% N-terminal domain input, 20–10% N-terminal domain input, Bead—Ni-NTA beads incubated with N-terminus, Beads + Core – Ni-NTA beads bound to AAA+ domain incubated with N-terminus.

Techniques Used: Filtration, Incubation, SDS Page

34) Product Images from "Two Coiled-Coil Domains of Chlamydia trachomatis IncA Affect Membrane Fusion Events during Infection"

Article Title: Two Coiled-Coil Domains of Chlamydia trachomatis IncA Affect Membrane Fusion Events during Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069769

SLD2 is required for oligomerization but both SNARE-like domains are necessary for homotypic fusion. (A) SLD2-containing IncA mutants co-elute with GST-TfR-IncA. 6xHis-tagged IncA mutants were co-expressed with GST-TfR-IncA in BL21(DE3) E. coli , and GST-containing complexes were purified over glutathione beads. Co-eluted IncA mutants were detected by western blot using anti-6xHis antibody. GST-TfR-IncA was visualized by Coomassie Blue staining. IncA mutants that contain SLD2 co-precipitated while the two truncated mutants lacking SLD2 did not. GST control shows basal levels of wildtype 6x-His-IncA binding. Results shown are typical of five independent experiments. (B) Transfection with wildtype- or Δ34-IncA leads to nonfusogenic inclusions. HeLa cells were transfected with plasmids expressing the indicated DsRed-IncA mutant or vector control (pDsRed-monomer-C1) and subsequently infected with C. trachomatis L2 for 24 hr. The location of each DsRed-IncA construct is shown on the left (red), while the inclusions are shown in the middle (blue, Hoechst staining). The right column shows the overlay. Arrows denote inclusions. Note the multiple inclusions in wildtype-IncA and Δ34-IncA transfected cells compared to the cells transfected with other IncA constructs. Scale bars represent 10 µm. Images are typical of four independent experiments. (C) Expression of either wildtype or Δ34-IncA inhibits subsequent inclusion development in HeLa cells. The number of inclusions/cell was determined by fluorescence microscopy. More than fifty infected cells per replicate per transfection were assessed for multiple inclusions. The number of infected cells carrying a single inclusion was divided by the total number of infected cells and expressed as a percent. Data are averages of four independent experiments. Error bars represent one standard deviation. Asterisks denote a significant difference compared to control-transfected cells (p
Figure Legend Snippet: SLD2 is required for oligomerization but both SNARE-like domains are necessary for homotypic fusion. (A) SLD2-containing IncA mutants co-elute with GST-TfR-IncA. 6xHis-tagged IncA mutants were co-expressed with GST-TfR-IncA in BL21(DE3) E. coli , and GST-containing complexes were purified over glutathione beads. Co-eluted IncA mutants were detected by western blot using anti-6xHis antibody. GST-TfR-IncA was visualized by Coomassie Blue staining. IncA mutants that contain SLD2 co-precipitated while the two truncated mutants lacking SLD2 did not. GST control shows basal levels of wildtype 6x-His-IncA binding. Results shown are typical of five independent experiments. (B) Transfection with wildtype- or Δ34-IncA leads to nonfusogenic inclusions. HeLa cells were transfected with plasmids expressing the indicated DsRed-IncA mutant or vector control (pDsRed-monomer-C1) and subsequently infected with C. trachomatis L2 for 24 hr. The location of each DsRed-IncA construct is shown on the left (red), while the inclusions are shown in the middle (blue, Hoechst staining). The right column shows the overlay. Arrows denote inclusions. Note the multiple inclusions in wildtype-IncA and Δ34-IncA transfected cells compared to the cells transfected with other IncA constructs. Scale bars represent 10 µm. Images are typical of four independent experiments. (C) Expression of either wildtype or Δ34-IncA inhibits subsequent inclusion development in HeLa cells. The number of inclusions/cell was determined by fluorescence microscopy. More than fifty infected cells per replicate per transfection were assessed for multiple inclusions. The number of infected cells carrying a single inclusion was divided by the total number of infected cells and expressed as a percent. Data are averages of four independent experiments. Error bars represent one standard deviation. Asterisks denote a significant difference compared to control-transfected cells (p

Techniques Used: Purification, Western Blot, Staining, Binding Assay, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Infection, Construct, Fluorescence, Microscopy, Standard Deviation

35) Product Images from "The Y271 and I274 Amino Acids in Reverse Transcriptase of Human Immunodeficiency Virus-1 Are Critical to Protein Stability"

Article Title: The Y271 and I274 Amino Acids in Reverse Transcriptase of Human Immunodeficiency Virus-1 Are Critical to Protein Stability

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006108

Structural models of naturally existent and artificially introduced substitutions of residues 271 and 274. We take p51 subunit as an example. (A) Locations of residues 271 and 274 in HIV-1 RT subunits p66 (grey) and p51 (green) (PDB coordinate 1RTH). The left panel shows a better view of residue 271 (blue); while the right panel reveals a better view of residue 274 (pink). (B) Structural models of naturally existent 271Y, 271F, 271H and 271C residues as well as the lethal 271A mutation of p51 subunit. The same area was highlighted above in the left panel of Fig. 6A . A big side chain (blue) is found in naturally occurring residues but not in 271A. (C) Structural models of naturally existing 274I, 274V and 274L residues as well as the lethal 274A mutation of p51 subunit. The same region was highlighted above in the right panel of Fig. 6A . A big side chain (pink) is found in naturally occurring residues I, V and L, but not in 274A.
Figure Legend Snippet: Structural models of naturally existent and artificially introduced substitutions of residues 271 and 274. We take p51 subunit as an example. (A) Locations of residues 271 and 274 in HIV-1 RT subunits p66 (grey) and p51 (green) (PDB coordinate 1RTH). The left panel shows a better view of residue 271 (blue); while the right panel reveals a better view of residue 274 (pink). (B) Structural models of naturally existent 271Y, 271F, 271H and 271C residues as well as the lethal 271A mutation of p51 subunit. The same area was highlighted above in the left panel of Fig. 6A . A big side chain (blue) is found in naturally occurring residues but not in 271A. (C) Structural models of naturally existing 274I, 274V and 274L residues as well as the lethal 274A mutation of p51 subunit. The same region was highlighted above in the right panel of Fig. 6A . A big side chain (pink) is found in naturally occurring residues I, V and L, but not in 274A.

Techniques Used: Mutagenesis

Detection of dimmer-formation of mutant RT. (A) RT was detected in wild type and mutant pseudoviruses generated in the presence and absence of EFV, the most potent dimerization enhancer, by Western blotting using mouse monoclonal anti-RT and anti-CA p24 antibodies, respectively. (B) Purified wild type and mutant RT subunit p66 were analyzed by native gel electrophoresis followed by Western blotting using mouse monoclonal anti-RT antibody in the presence or absence of β-mercaptoethanol (β-ME). Compared with wild type p66, which basically existed as homodimer, p66 subunit of the 271A and 274A mutants formed higher order oligomers, suggestive of conformational change.
Figure Legend Snippet: Detection of dimmer-formation of mutant RT. (A) RT was detected in wild type and mutant pseudoviruses generated in the presence and absence of EFV, the most potent dimerization enhancer, by Western blotting using mouse monoclonal anti-RT and anti-CA p24 antibodies, respectively. (B) Purified wild type and mutant RT subunit p66 were analyzed by native gel electrophoresis followed by Western blotting using mouse monoclonal anti-RT antibody in the presence or absence of β-mercaptoethanol (β-ME). Compared with wild type p66, which basically existed as homodimer, p66 subunit of the 271A and 274A mutants formed higher order oligomers, suggestive of conformational change.

Techniques Used: Mutagenesis, Generated, Western Blot, Purification, Nucleic Acid Electrophoresis

Detection of Gag-Pol polyprotein products in pseudoviral particles and the protease inhibition assay. (A) Cleavage products of Gag-Pol polyprotein, integrase (IN p32), protease (PR p11) and capsid protein p24 (CA p24) in wild type (WT) and mutant pseudoviruses were detected by Western blotting using mouse monoclonal anti-IN, anti-PR and anti-CA antibodies, respectively. (B) Pr160 Gag-pol , Pr55 Gag and CA p24 were detected in wild type (WT) as well as Y271A and I274A mutant pseudoviruses produced in the absence (0) or presence of 0.2 and 2 µM protease inhibitor (indinavir) by Western blotting using mouse monoclonal anti-CA. (C) Pr160 Gag-pol and its subunits RT p66 and p51 were detected in wild type (WT) as well as Y271A and I274A mutant pseudoviruses produced in the absence (0) or presence of 0.2 and 2 µM protease inhibitor (indinavir) by Western blotting using mouse monoclonal anti-RT antibody.
Figure Legend Snippet: Detection of Gag-Pol polyprotein products in pseudoviral particles and the protease inhibition assay. (A) Cleavage products of Gag-Pol polyprotein, integrase (IN p32), protease (PR p11) and capsid protein p24 (CA p24) in wild type (WT) and mutant pseudoviruses were detected by Western blotting using mouse monoclonal anti-IN, anti-PR and anti-CA antibodies, respectively. (B) Pr160 Gag-pol , Pr55 Gag and CA p24 were detected in wild type (WT) as well as Y271A and I274A mutant pseudoviruses produced in the absence (0) or presence of 0.2 and 2 µM protease inhibitor (indinavir) by Western blotting using mouse monoclonal anti-CA. (C) Pr160 Gag-pol and its subunits RT p66 and p51 were detected in wild type (WT) as well as Y271A and I274A mutant pseudoviruses produced in the absence (0) or presence of 0.2 and 2 µM protease inhibitor (indinavir) by Western blotting using mouse monoclonal anti-RT antibody.

Techniques Used: Inhibition, Mutagenesis, Western Blot, Produced, Protease Inhibitor

36) Product Images from "Deficiency of the tRNATyr:?35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery"

Article Title: Deficiency of the tRNATyr:?35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn1037

Test of the activity of the recombinant Pab and Sso Pus7-like enzymes on different Sso and Pab tRNAs and pre-tRNA substrates. ( A ) 2D structures of the P. abyssi tRNA Tyr (GUA) and tRNA Asp (GUC) and S. solfataricus pre-tRNA Tyr (GUA) and tRNA Asp (GUC). The UA dinucleotides present in these RNAs are indicated by arrows. The potential U35 target sites in mature and pre-tRNA Tyr (GUA) and the U13 target sites in the Pab tRNA Tyr (GUA) and the Sso and Pab tRNAs Asp (GUC) are circled. ( B ) Tests of the in vitro activity of the recombinant Sso Pus7-like and Pab Pus7-like enzymes on the Sso pre-tRNA Tyr (GUA) and Pab tRNA Tyr (GUA) and their respective U35C variants. All the transcripts were [α- 32 P]ATP-labelled and incubated for 90 min at 55°C or 80°C with the recombinant proteins (Pab Pus7-like or Sso Pus7-like) as indicated on the left of the panel. Modified RNAs were analysed as described in Figure 2 . The molar amounts of Ψ residue formed per mole of RNA in the experiments are given for each 2D-TLC autoradiogram. Reproducible results were obtained in three independent experiments. ( C ) In vitro activity tests with the Sso and Pab recombinant Pus7-like enzymes on the Sso and Pab tRNA Asp (GUC) and the U13C Pab tRNA Asp variant, same legend as for (B). ( D ) The Pab Pus7-like enzyme modifies only UA dinucleotides. The P. abyssi tRNA Tyr (GUA) was labelled by incorporation of [α- 32 P] ATP, CTP, UTP or GTP. Formation of Ψ residue by Pab aPus7 was tested after 90 min of incubation at 55°C.
Figure Legend Snippet: Test of the activity of the recombinant Pab and Sso Pus7-like enzymes on different Sso and Pab tRNAs and pre-tRNA substrates. ( A ) 2D structures of the P. abyssi tRNA Tyr (GUA) and tRNA Asp (GUC) and S. solfataricus pre-tRNA Tyr (GUA) and tRNA Asp (GUC). The UA dinucleotides present in these RNAs are indicated by arrows. The potential U35 target sites in mature and pre-tRNA Tyr (GUA) and the U13 target sites in the Pab tRNA Tyr (GUA) and the Sso and Pab tRNAs Asp (GUC) are circled. ( B ) Tests of the in vitro activity of the recombinant Sso Pus7-like and Pab Pus7-like enzymes on the Sso pre-tRNA Tyr (GUA) and Pab tRNA Tyr (GUA) and their respective U35C variants. All the transcripts were [α- 32 P]ATP-labelled and incubated for 90 min at 55°C or 80°C with the recombinant proteins (Pab Pus7-like or Sso Pus7-like) as indicated on the left of the panel. Modified RNAs were analysed as described in Figure 2 . The molar amounts of Ψ residue formed per mole of RNA in the experiments are given for each 2D-TLC autoradiogram. Reproducible results were obtained in three independent experiments. ( C ) In vitro activity tests with the Sso and Pab recombinant Pus7-like enzymes on the Sso and Pab tRNA Asp (GUC) and the U13C Pab tRNA Asp variant, same legend as for (B). ( D ) The Pab Pus7-like enzyme modifies only UA dinucleotides. The P. abyssi tRNA Tyr (GUA) was labelled by incorporation of [α- 32 P] ATP, CTP, UTP or GTP. Formation of Ψ residue by Pab aPus7 was tested after 90 min of incubation at 55°C.

Techniques Used: Activity Assay, Recombinant, In Vitro, Incubation, Modification, Thin Layer Chromatography, Variant Assay

Presence of unusual Pus7-like enzymes in Sulfolobales species and A. pernix . ( A ) Multiple sequence alignment of Pus7 proteins from various archaeal species. Only the regions of motif IIIa, and motif II which contains the catalytic D residue, are shown. Different colours are used to highlight each of the conserved residues in motifs IIIa and II. The names of the organisms are indicated on the left, red arrows show species that contain a pre-tRNA Tyr -specific H/ACA sRNA. ( B ) 3D structure modelization of the Pab aPus7 (left) and Sso aPus7 (right) active sites. Modelization was done by using the crystal structure of M. mazei TruD (PDB 1Z2Z). Only highly conserved amino acids in the catalytic sites of RNA:Ψ-synthases are represented in the Pab and Sso aPus7 models, the K27I, R90A and H91N substitutions in Sso aPus7 can be seen.
Figure Legend Snippet: Presence of unusual Pus7-like enzymes in Sulfolobales species and A. pernix . ( A ) Multiple sequence alignment of Pus7 proteins from various archaeal species. Only the regions of motif IIIa, and motif II which contains the catalytic D residue, are shown. Different colours are used to highlight each of the conserved residues in motifs IIIa and II. The names of the organisms are indicated on the left, red arrows show species that contain a pre-tRNA Tyr -specific H/ACA sRNA. ( B ) 3D structure modelization of the Pab aPus7 (left) and Sso aPus7 (right) active sites. Modelization was done by using the crystal structure of M. mazei TruD (PDB 1Z2Z). Only highly conserved amino acids in the catalytic sites of RNA:Ψ-synthases are represented in the Pab and Sso aPus7 models, the K27I, R90A and H91N substitutions in Sso aPus7 can be seen.

Techniques Used: Sequencing

Test of the activity of Pab aPus7 and Sso aPus7 recombinant WT or variant proteins on the [α- 32 P]ATP-labelled P. abyssi tRNA Asp (GUC). Enzymatic tests were performed as described in the legend to Figure 5 , except that we used point-mutated variants of the enzymes, Pab aPus7 K19I, R78A/H79N, and with the three R78A, H79N and K19I mutations and Sso aPus7 I127K, A90R/N91H and with the three I127K, A90R and N91H mutations. Incubations with the enzymes were performed both at 55°C ( A ) and at 80°C ( B ) and the molar amounts of Ψ residue formed per mole of tRNA Asp are given in a histogram. Error bars correspond to the standard deviations in three independent experiments.
Figure Legend Snippet: Test of the activity of Pab aPus7 and Sso aPus7 recombinant WT or variant proteins on the [α- 32 P]ATP-labelled P. abyssi tRNA Asp (GUC). Enzymatic tests were performed as described in the legend to Figure 5 , except that we used point-mutated variants of the enzymes, Pab aPus7 K19I, R78A/H79N, and with the three R78A, H79N and K19I mutations and Sso aPus7 I127K, A90R/N91H and with the three I127K, A90R and N91H mutations. Incubations with the enzymes were performed both at 55°C ( A ) and at 80°C ( B ) and the molar amounts of Ψ residue formed per mole of tRNA Asp are given in a histogram. Error bars correspond to the standard deviations in three independent experiments.

Techniques Used: Activity Assay, Recombinant, Variant Assay

Formation of Ψ35 in tRNA Tyr (GUA) is likely catalysed by Pab aPus7 in P. abyssi , while the Sso1 H/ACA sRNP might be the catalysts in S. solfataricus. ( A ) Modification of the [α- 32 P]ATP-labelled WT tRNA Tyr (GUA) and its [α- 32 P]GTP-labelled A36G variant by recombinant Pab aPus7 and in a Pab cellular extract was tested by the nearest neighbour approach. Incubations with the recombinant proteins were performed for 90 min at 55°C and with the extract for 90 min at 65°C. Additional spots on the TLC chromatograms corresponding to the formation of m 1 G37, Cm56, m 1 I57 and m 1 A58 are indicated. The molar amounts of Ψ residue formed per mole of RNA are given. ( B ) The activity at 65°C of the reconstituted Sso1 sRNP (Sso1/LCNG) and an Sso cellular extract was tested on the WT Sso pre-tRNA Tyr (GUA) and its U36G variant. The Sso1 H/ACA sRNP was reconstituted and its activity was tested as described in the legend to Figure 2 B.
Figure Legend Snippet: Formation of Ψ35 in tRNA Tyr (GUA) is likely catalysed by Pab aPus7 in P. abyssi , while the Sso1 H/ACA sRNP might be the catalysts in S. solfataricus. ( A ) Modification of the [α- 32 P]ATP-labelled WT tRNA Tyr (GUA) and its [α- 32 P]GTP-labelled A36G variant by recombinant Pab aPus7 and in a Pab cellular extract was tested by the nearest neighbour approach. Incubations with the recombinant proteins were performed for 90 min at 55°C and with the extract for 90 min at 65°C. Additional spots on the TLC chromatograms corresponding to the formation of m 1 G37, Cm56, m 1 I57 and m 1 A58 are indicated. The molar amounts of Ψ residue formed per mole of RNA are given. ( B ) The activity at 65°C of the reconstituted Sso1 sRNP (Sso1/LCNG) and an Sso cellular extract was tested on the WT Sso pre-tRNA Tyr (GUA) and its U36G variant. The Sso1 H/ACA sRNP was reconstituted and its activity was tested as described in the legend to Figure 2 B.

Techniques Used: Modification, Variant Assay, Recombinant, Thin Layer Chromatography, Activity Assay

37) Product Images from "Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein"

Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008381

C-terminal biotinylation using the Ssp GyrB S11 split-intein. A. The three precursor proteins (MI N C, EI N H, and GST-ACP-I N C) were incubated with (+) or without (−) the peptide I C -B in the presence of 0.1 mM TCEP for 18 h at room temperature, and the reaction products were analyzed by Western blotting using antibodies against biotin. From the three precursor proteins, the three target proteins for biotinylation were a maltose binding protein (M, 43 kDa), an enhanced green fluorecent protein (E, 27 kDa), and a glutathione-S-transferase/acyl carrier fusion protein (GST-ACP, 39 kDa), respectively. B. Efficiency of C-terminal biotinylation of the three target proteins as determined by densitometry analysis on Western blots using anti-C antibodies (for MI N C and GST-ACP-I N C) and anti-H antibodies (for EI N H). Error bars represent standard deviations from triplicate experiments.
Figure Legend Snippet: C-terminal biotinylation using the Ssp GyrB S11 split-intein. A. The three precursor proteins (MI N C, EI N H, and GST-ACP-I N C) were incubated with (+) or without (−) the peptide I C -B in the presence of 0.1 mM TCEP for 18 h at room temperature, and the reaction products were analyzed by Western blotting using antibodies against biotin. From the three precursor proteins, the three target proteins for biotinylation were a maltose binding protein (M, 43 kDa), an enhanced green fluorecent protein (E, 27 kDa), and a glutathione-S-transferase/acyl carrier fusion protein (GST-ACP, 39 kDa), respectively. B. Efficiency of C-terminal biotinylation of the three target proteins as determined by densitometry analysis on Western blots using anti-C antibodies (for MI N C and GST-ACP-I N C) and anti-H antibodies (for EI N H). Error bars represent standard deviations from triplicate experiments.

Techniques Used: Incubation, Western Blot, Binding Assay

38) Product Images from "Essential role of CIB1 in regulating PAK1 activation and cell migration"

Article Title: Essential role of CIB1 in regulating PAK1 activation and cell migration

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200502090

Identification of CIB1-binding sites within the PAK1 NH 2 terminus. (A) CIB1 binds to full-length wild-type GST-PAK1 (wtPAK1) and GST-PAK1 K298A (kdPAK1) but not the NH 2 -terminal deletion GST-N165-PAK1. Solid-phase binding assays were performed as in Fig. 1 C, and soluble GST-PAK1, GST-PAK1 K298A, and GST-N165-PAK1 were added to wells coated with CIB1. (B) Delineation of the CIB1-binding sites in the PAK1 NH 2 -terminal sequence by SPOT peptide method. Top and middle panels show two sets of CIB1 reactive spots labeled as sites I and II relative to the PBD- or Rac/Cdc42-binding sites (residues 70–113, dotted boxes). The bottom panel indicates the inhibitory switch (IS) and kinase inhibitor (KI) domains. Sequences of corresponding CIB1-binding sites are below. (C) Inhibition of CIB1 binding to PAK1 with site I and II peptides. Ni-NTA agarose beads loaded with purified His-PAK1 were added to recombinant CIB1 that was preincubated with and without PAK1 peptides corresponding to sites I and II and scrambled sites I and II. Peptide sequences of sites I, II, and IIΔ are shown below. The graph represents densitometry of affinity precipitates probed for CIB1 and normalized to His-PAK1 that was immunoblotted from the same membrane. Data represent SEM from two independent experiments (error bars). (D) Loss of CIB1 binding to site I and II PAK1 mutants. Clarified lysates from HEK293 cells cotransfected with CIB1 and myc wild-type (wt)PAK1, myc-PAK1 siteIAAA (myc-AAAPAK1), or myc-PAK1 siteIΔ/siteIIAAA (myc-Δ/AAA PAK1) were immunoprecipitated with a control IgG or anti-CIB1 antibody and with samples immunoblotted for myc (top) and CIB1 (bottom; n ≥ 4). Western blots of the input expression of CIB1 and myc-tagged wtPAK1 (closed arrowheads), AAAPAK (closed arrowheads), or Δ/AAAPAK1 (open arrowheads).
Figure Legend Snippet: Identification of CIB1-binding sites within the PAK1 NH 2 terminus. (A) CIB1 binds to full-length wild-type GST-PAK1 (wtPAK1) and GST-PAK1 K298A (kdPAK1) but not the NH 2 -terminal deletion GST-N165-PAK1. Solid-phase binding assays were performed as in Fig. 1 C, and soluble GST-PAK1, GST-PAK1 K298A, and GST-N165-PAK1 were added to wells coated with CIB1. (B) Delineation of the CIB1-binding sites in the PAK1 NH 2 -terminal sequence by SPOT peptide method. Top and middle panels show two sets of CIB1 reactive spots labeled as sites I and II relative to the PBD- or Rac/Cdc42-binding sites (residues 70–113, dotted boxes). The bottom panel indicates the inhibitory switch (IS) and kinase inhibitor (KI) domains. Sequences of corresponding CIB1-binding sites are below. (C) Inhibition of CIB1 binding to PAK1 with site I and II peptides. Ni-NTA agarose beads loaded with purified His-PAK1 were added to recombinant CIB1 that was preincubated with and without PAK1 peptides corresponding to sites I and II and scrambled sites I and II. Peptide sequences of sites I, II, and IIΔ are shown below. The graph represents densitometry of affinity precipitates probed for CIB1 and normalized to His-PAK1 that was immunoblotted from the same membrane. Data represent SEM from two independent experiments (error bars). (D) Loss of CIB1 binding to site I and II PAK1 mutants. Clarified lysates from HEK293 cells cotransfected with CIB1 and myc wild-type (wt)PAK1, myc-PAK1 siteIAAA (myc-AAAPAK1), or myc-PAK1 siteIΔ/siteIIAAA (myc-Δ/AAA PAK1) were immunoprecipitated with a control IgG or anti-CIB1 antibody and with samples immunoblotted for myc (top) and CIB1 (bottom; n ≥ 4). Western blots of the input expression of CIB1 and myc-tagged wtPAK1 (closed arrowheads), AAAPAK (closed arrowheads), or Δ/AAAPAK1 (open arrowheads).

Techniques Used: Binding Assay, Sequencing, Labeling, Inhibition, Purification, Recombinant, Immunoprecipitation, Western Blot, Expressing

CIB1 overexpression inhibits, and endogenous CIB1 depletion increases, cell migration. (A) Serum-starved REF52 cells transfected with GFP vector, control vector, or CIB1 ± kdPAK1 or kdLIMK1 were subjected to haptotactic transwell migration assays toward FN. Transfected cells on either the top membrane (nonmigrating cells) or bottom membrane (migrating cells) were visualized by staining for CIB1 expression (middle). Control migration was visualized by GFP fluorescence (right). Cells overexpressing vector CIB1 ± kdPAK1 or ± kdLIMK1 on the top and bottom membranes were also stained as described in migration assays and were counted. Migration is represented as the percentage of the total number of transfected cells from the upper and lower membranes (left). Data represent means ± SEM ( n = 3). (B) MEFs derived from wild-type (PAK +/+ ) and PAK1-null (PAK −/− ) mice were transfected with GFP vector or CIB1. Serum-starved cells were assayed for haptotactic migration toward 3 μg/ml FN, and migration was determined as in A (left). Data represent means ± SEM ( n = 4). Right panels show representative images of migrated transfected cells (green, top) and phalloidin staining from the same field (red, bottom). (A and B) Bars, 20 μm. (C) HeLaS3 (left) and REF52 (right) cells were mock transfected or transfected with control or specific CIB1 siRNA and subjected to haptotactic transwell migration assays toward FN (as in A). Data represent means ± SEM (error bars; n = 4 for each cell type). Inset blots show representative endogenous CIB1 protein expression and nonspecific (NS) band or PAK1 expression from the same blot as the loading control.
Figure Legend Snippet: CIB1 overexpression inhibits, and endogenous CIB1 depletion increases, cell migration. (A) Serum-starved REF52 cells transfected with GFP vector, control vector, or CIB1 ± kdPAK1 or kdLIMK1 were subjected to haptotactic transwell migration assays toward FN. Transfected cells on either the top membrane (nonmigrating cells) or bottom membrane (migrating cells) were visualized by staining for CIB1 expression (middle). Control migration was visualized by GFP fluorescence (right). Cells overexpressing vector CIB1 ± kdPAK1 or ± kdLIMK1 on the top and bottom membranes were also stained as described in migration assays and were counted. Migration is represented as the percentage of the total number of transfected cells from the upper and lower membranes (left). Data represent means ± SEM ( n = 3). (B) MEFs derived from wild-type (PAK +/+ ) and PAK1-null (PAK −/− ) mice were transfected with GFP vector or CIB1. Serum-starved cells were assayed for haptotactic migration toward 3 μg/ml FN, and migration was determined as in A (left). Data represent means ± SEM ( n = 4). Right panels show representative images of migrated transfected cells (green, top) and phalloidin staining from the same field (red, bottom). (A and B) Bars, 20 μm. (C) HeLaS3 (left) and REF52 (right) cells were mock transfected or transfected with control or specific CIB1 siRNA and subjected to haptotactic transwell migration assays toward FN (as in A). Data represent means ± SEM (error bars; n = 4 for each cell type). Inset blots show representative endogenous CIB1 protein expression and nonspecific (NS) band or PAK1 expression from the same blot as the loading control.

Techniques Used: Over Expression, Migration, Transfection, Plasmid Preparation, Staining, Expressing, Fluorescence, Derivative Assay, Mouse Assay

Cdc42 and Ca 2 + affect the CIB1–PAK interaction, and CIB1 stimulates PAK1 activity in vitro. (A) Activated Cdc42-GTPγS or CIB1, but not inactive Cdc42-GDP, competes with immobilized CIB1 for binding to soluble His-PAK1. Soluble His-PAK1 was incubated with increasing concentrations of soluble CIB1 or Cdc42 that was preloaded with GDP or GTPγS before incubation with immobilized CIB1. (B) Determination of Ca 2+ -dependent binding of His-PAK1 to CIB1. His-PAK1 was diluted in buffer containing 0–5 mM EGTA before addition to immobilized CIB1. Approximate free Ca 2+ concentrations were calculated using the MaxChelator program ( Bers et al., 1994 ). (C) Stimulation of recombinant His-PAK1 activity by recombinant CIB1 or Cdc42-GTPγS. His-PAK1 autophosphorylation was assayed in the absence (top) or presence of myelin basic protein (MBP) to detect active kinase (middle). White lines indicate that intervening lanes have been spliced out. Myelin basic protein phosphorylation ([ 32 P]MBP) from densitometry analysis induced by His-PAK1 alone was assigned a value of 1 (bar graph). Data represent means ± SEM (error bars; n = 3).
Figure Legend Snippet: Cdc42 and Ca 2 + affect the CIB1–PAK interaction, and CIB1 stimulates PAK1 activity in vitro. (A) Activated Cdc42-GTPγS or CIB1, but not inactive Cdc42-GDP, competes with immobilized CIB1 for binding to soluble His-PAK1. Soluble His-PAK1 was incubated with increasing concentrations of soluble CIB1 or Cdc42 that was preloaded with GDP or GTPγS before incubation with immobilized CIB1. (B) Determination of Ca 2+ -dependent binding of His-PAK1 to CIB1. His-PAK1 was diluted in buffer containing 0–5 mM EGTA before addition to immobilized CIB1. Approximate free Ca 2+ concentrations were calculated using the MaxChelator program ( Bers et al., 1994 ). (C) Stimulation of recombinant His-PAK1 activity by recombinant CIB1 or Cdc42-GTPγS. His-PAK1 autophosphorylation was assayed in the absence (top) or presence of myelin basic protein (MBP) to detect active kinase (middle). White lines indicate that intervening lanes have been spliced out. Myelin basic protein phosphorylation ([ 32 P]MBP) from densitometry analysis induced by His-PAK1 alone was assigned a value of 1 (bar graph). Data represent means ± SEM (error bars; n = 3).

Techniques Used: Activity Assay, In Vitro, Binding Assay, Incubation, Recombinant

CIB1 specifically stimulates PAK1 activity in vivo independently of small GTPases. (A) CIB1 specifically activates the PAK1 isoform. Endogenous PAK1 (bottom left) and PAK3 (bottom right) were immunoprecipitated from lysates prepared from vector- and CIB1-transfected REF52 cells either held in suspension or replated on FN for 20 min. Immunoprecipitated PAK was subjected to in vitro kinase assays using myelin basic protein (MBP) as substrate. The bar graph (top) depicts [ 32 P]MBP values after normalization for PAK immunoprecipitation. [ 32 P]MBP values from vector control cells were set as 1. Data represent SEM from two independent experiments (error bars). (B) GTPase-independent PAK1 activation. Serum-starved vector and CIB1-transfected REF52 cells held in suspension were treated with or without 100 ng/ml toxin B and either left in suspension or adhered to FN. Cells were lysed at the indicated times, and immunoprecipitated endogenous PAK1 was subjected to in vitro kinase assays using myelin basic protein as substrate. White lines indicate that intervening lanes have been spliced out; n = 3. (C) Efficacy of toxin B treatment was determined by assaying REF52 cell lysates for activated Rac1 and Cdc42 (see PAK1 kinase and Rac/Cdc42 activation assays). Affinity precipitates were analyzed by Western blotting for both Rac1 and Cdc42.
Figure Legend Snippet: CIB1 specifically stimulates PAK1 activity in vivo independently of small GTPases. (A) CIB1 specifically activates the PAK1 isoform. Endogenous PAK1 (bottom left) and PAK3 (bottom right) were immunoprecipitated from lysates prepared from vector- and CIB1-transfected REF52 cells either held in suspension or replated on FN for 20 min. Immunoprecipitated PAK was subjected to in vitro kinase assays using myelin basic protein (MBP) as substrate. The bar graph (top) depicts [ 32 P]MBP values after normalization for PAK immunoprecipitation. [ 32 P]MBP values from vector control cells were set as 1. Data represent SEM from two independent experiments (error bars). (B) GTPase-independent PAK1 activation. Serum-starved vector and CIB1-transfected REF52 cells held in suspension were treated with or without 100 ng/ml toxin B and either left in suspension or adhered to FN. Cells were lysed at the indicated times, and immunoprecipitated endogenous PAK1 was subjected to in vitro kinase assays using myelin basic protein as substrate. White lines indicate that intervening lanes have been spliced out; n = 3. (C) Efficacy of toxin B treatment was determined by assaying REF52 cell lysates for activated Rac1 and Cdc42 (see PAK1 kinase and Rac/Cdc42 activation assays). Affinity precipitates were analyzed by Western blotting for both Rac1 and Cdc42.

Techniques Used: Activity Assay, In Vivo, Immunoprecipitation, Plasmid Preparation, Transfection, In Vitro, Activation Assay, Western Blot

CIB1 modulates downstream signaling to cofilin. (A) Lysates were prepared from control or specific CIB1 siRNA-transfected REF52 cells either held in suspension or adhered to FN for the indicated times. Densitometry of p-cofilin levels from lysates that were prepared from control or specific CIB siRNA was normalized to ERK or PAK1 from the same blots. Error bars represent means ± SEM ( n = 2). (B) Representative membrane immunoblotted with antibodies against total or phosphorylated cofilin (p-cofilin). The top half of the membrane was also immunoblotted for PAK1 expression (bottom). (C) Lysates prepared from REF52 cells overexpressing empty vector or CIB1 ± kdPAK1 or kdLIMK1 were analyzed for cofilin phosphorylation as in A. The membrane was reprobed for total cofilin (bottom). Lysates from cells expressing control vector, CIB1, or CIB1 coexpressed with kdPAK1 or kdLIMK1 were immunoblotted using anti-CIB1, -PAK1, or -LIMK1 antibodies (middle and right). PAK1 immunoblots show both endogenous PAK1 and overexpressed kdPAK1 (top middle). Immunoblotting for LIMK1 also shows endogenous LIMK1 and overexpressed kdLIMK1 (top right). Middle blots show overexpressed CIB1. Membranes were also probed with an anti-ERK antibody as a loading control (bottom, middle and right). Data represent two separate experiments.
Figure Legend Snippet: CIB1 modulates downstream signaling to cofilin. (A) Lysates were prepared from control or specific CIB1 siRNA-transfected REF52 cells either held in suspension or adhered to FN for the indicated times. Densitometry of p-cofilin levels from lysates that were prepared from control or specific CIB siRNA was normalized to ERK or PAK1 from the same blots. Error bars represent means ± SEM ( n = 2). (B) Representative membrane immunoblotted with antibodies against total or phosphorylated cofilin (p-cofilin). The top half of the membrane was also immunoblotted for PAK1 expression (bottom). (C) Lysates prepared from REF52 cells overexpressing empty vector or CIB1 ± kdPAK1 or kdLIMK1 were analyzed for cofilin phosphorylation as in A. The membrane was reprobed for total cofilin (bottom). Lysates from cells expressing control vector, CIB1, or CIB1 coexpressed with kdPAK1 or kdLIMK1 were immunoblotted using anti-CIB1, -PAK1, or -LIMK1 antibodies (middle and right). PAK1 immunoblots show both endogenous PAK1 and overexpressed kdPAK1 (top middle). Immunoblotting for LIMK1 also shows endogenous LIMK1 and overexpressed kdLIMK1 (top right). Middle blots show overexpressed CIB1. Membranes were also probed with an anti-ERK antibody as a loading control (bottom, middle and right). Data represent two separate experiments.

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

CIB1 is required for adhesion-induced PAK1 activation, and loss of CIB1 disrupts PAK1/GTPase signaling. (A) Mock, control, or specific CIB1 siRNA-transfected REF52 cells were either held in suspension or replated onto FN-coated dishes and were lysed at the indicated times. Immunoprecipitated endogenous PAK1 was subjected to in vitro kinase assays ( n = 3). (B) Control or CIB1 siRNA-transfected REF52 cell lysates were assayed for activated and total Rac1 and Cdc42. CIB1 knockdown in REF52 cells was confirmed by immunoblotting cell lysates for endogenous CIB1 expression (right) with ERK as a loading control from the same membrane. (C) REF52 cells transfected with control (left) or CIB1 siRNA (right) were replated on FN for 3 h and were stained with phalloidin. Images are representative of two separate experiments. Bars, 20 μm.
Figure Legend Snippet: CIB1 is required for adhesion-induced PAK1 activation, and loss of CIB1 disrupts PAK1/GTPase signaling. (A) Mock, control, or specific CIB1 siRNA-transfected REF52 cells were either held in suspension or replated onto FN-coated dishes and were lysed at the indicated times. Immunoprecipitated endogenous PAK1 was subjected to in vitro kinase assays ( n = 3). (B) Control or CIB1 siRNA-transfected REF52 cell lysates were assayed for activated and total Rac1 and Cdc42. CIB1 knockdown in REF52 cells was confirmed by immunoblotting cell lysates for endogenous CIB1 expression (right) with ERK as a loading control from the same membrane. (C) REF52 cells transfected with control (left) or CIB1 siRNA (right) were replated on FN for 3 h and were stained with phalloidin. Images are representative of two separate experiments. Bars, 20 μm.

Techniques Used: Activation Assay, Transfection, Immunoprecipitation, In Vitro, Expressing, Staining

CIB1 binds to PAK1 in vivo and in vitro. (A) Immunoprecipitates from platelet lysates were immunoblotted with an antiphosphoserine (left) or anti-PAK (middle) antibody. Whole cell lysates (WCL) were probed with an anti-PAK antibody (right; n ≥ 3 experiments). (B) CIB1 and control IgY immunoprecipitates from lysates of REF52 cells in suspension or adhered to FN were immunoblotted for PAK (top) and CIB1 (bottom). (C) Solid-phase binding assays using immobilized CIB1 and soluble PAK proteins. Increasing concentrations of His-PAK1 were added to wells coated with and without immobilized CIB1. His-PAK1 binding was detected using an anti-His antibody. (D) PAK isozyme–binding specificity was determined by adding GST-PAK1 or GST-PAK2 to immobilized CIB1. PAK binding was detected with an anti-GST antibody. WB, Western blot; IP, immunoprecipitates. Error bars represent SEM.
Figure Legend Snippet: CIB1 binds to PAK1 in vivo and in vitro. (A) Immunoprecipitates from platelet lysates were immunoblotted with an antiphosphoserine (left) or anti-PAK (middle) antibody. Whole cell lysates (WCL) were probed with an anti-PAK antibody (right; n ≥ 3 experiments). (B) CIB1 and control IgY immunoprecipitates from lysates of REF52 cells in suspension or adhered to FN were immunoblotted for PAK (top) and CIB1 (bottom). (C) Solid-phase binding assays using immobilized CIB1 and soluble PAK proteins. Increasing concentrations of His-PAK1 were added to wells coated with and without immobilized CIB1. His-PAK1 binding was detected using an anti-His antibody. (D) PAK isozyme–binding specificity was determined by adding GST-PAK1 or GST-PAK2 to immobilized CIB1. PAK binding was detected with an anti-GST antibody. WB, Western blot; IP, immunoprecipitates. Error bars represent SEM.

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

39) Product Images from "Fyn and PTP-PEST-mediated Regulation of Wiskott-Aldrich Syndrome Protein (WASp) Tyrosine Phosphorylation Is Required for Coupling T Cell Antigen Receptor Engagement to WASp Effector Function and T Cell Activation"

Article Title: Fyn and PTP-PEST-mediated Regulation of Wiskott-Aldrich Syndrome Protein (WASp) Tyrosine Phosphorylation Is Required for Coupling T Cell Antigen Receptor Engagement to WASp Effector Function and T Cell Activation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20030976

Fyn and PTP-PEST modulate WASp effects on induction of actin polymerization and synapse formation. (A) Polymerization of 2.8 μM pyrene-labeled actin monomer was assayed in the presence of 20 nM Arp2/3 complex, 100 nM GST-WASp or GST-WASpΔGBD, and GST fusion proteins containing 50–500 nM either Fyn, PTP-PEST, PSTPIP, or cdc42-V12. Polymerization was monitored by the increase in prenyl-actin fluorescence. B. The pyrene actin assay was used to compare the WASp-Arp2/3–actin polymerizing activities of cdc42 at low (15 nM), medium (250 nM), or high (500 nM) concentration and Fyn at low (10 nM), medium (100 nM), or high (200 nM) concentration alone or in combination. (C) Fyn effects on synapse formation were evaluated by incubating lymphocytes from OT-II (a and b) and Fyn −/− /OT-II (c) mice with unpulsed (a) or OVA peptide–pulsed (b and c) LB27.4 cells followed by cell fixation, staining for WASp, Fyn, and actin, and visualization by immunofluorescent microscopy. Images on the far left of each panel represent a merge of the other three images within each panel. A computer-generated three-dimensional reconstruction of the synaptic region formed between wild-type T cells and APCs (d) shows the localization of Fyn in the central area of the synapse and the distribution of WASp in both the central and peripheral synaptic region. Synapses were quantified (e) by counting the numbers of T cell–APC conjugates showing clustered actin at the conjugation site. Values shown are the percent of conjugates with synapse formation and represent means (± SEM) of three independent experiments. (D) PTP-PEST effects on synapse formation were assessed using WAS −/− /OT-II lymphocytes transfected with pDSRED-WASp (a), pcDNA3-WASp and pDSRED-PSTPIP1 (b), or pcDNA3-WASp, pEGFP-PSTPIP1, and pDSRED-PTP-PEST (c). Cells were incubated with OVA peptide–pulsed LB27.4 B cells, fixed, and stained for actin and/or PKC-θ, and visualized by immunofluorescent microscopy. The image on the far right of each panel is a merged image of all other images in the panel. Synapses were quantified by counting the number of T cell–B cell conjugates showing clustered actin at the synaptic site. Values shown are the percent of conjugates with synapse formation and represent the means (± SEM) of three independent experiments.
Figure Legend Snippet: Fyn and PTP-PEST modulate WASp effects on induction of actin polymerization and synapse formation. (A) Polymerization of 2.8 μM pyrene-labeled actin monomer was assayed in the presence of 20 nM Arp2/3 complex, 100 nM GST-WASp or GST-WASpΔGBD, and GST fusion proteins containing 50–500 nM either Fyn, PTP-PEST, PSTPIP, or cdc42-V12. Polymerization was monitored by the increase in prenyl-actin fluorescence. B. The pyrene actin assay was used to compare the WASp-Arp2/3–actin polymerizing activities of cdc42 at low (15 nM), medium (250 nM), or high (500 nM) concentration and Fyn at low (10 nM), medium (100 nM), or high (200 nM) concentration alone or in combination. (C) Fyn effects on synapse formation were evaluated by incubating lymphocytes from OT-II (a and b) and Fyn −/− /OT-II (c) mice with unpulsed (a) or OVA peptide–pulsed (b and c) LB27.4 cells followed by cell fixation, staining for WASp, Fyn, and actin, and visualization by immunofluorescent microscopy. Images on the far left of each panel represent a merge of the other three images within each panel. A computer-generated three-dimensional reconstruction of the synaptic region formed between wild-type T cells and APCs (d) shows the localization of Fyn in the central area of the synapse and the distribution of WASp in both the central and peripheral synaptic region. Synapses were quantified (e) by counting the numbers of T cell–APC conjugates showing clustered actin at the conjugation site. Values shown are the percent of conjugates with synapse formation and represent means (± SEM) of three independent experiments. (D) PTP-PEST effects on synapse formation were assessed using WAS −/− /OT-II lymphocytes transfected with pDSRED-WASp (a), pcDNA3-WASp and pDSRED-PSTPIP1 (b), or pcDNA3-WASp, pEGFP-PSTPIP1, and pDSRED-PTP-PEST (c). Cells were incubated with OVA peptide–pulsed LB27.4 B cells, fixed, and stained for actin and/or PKC-θ, and visualized by immunofluorescent microscopy. The image on the far right of each panel is a merged image of all other images in the panel. Synapses were quantified by counting the number of T cell–B cell conjugates showing clustered actin at the synaptic site. Values shown are the percent of conjugates with synapse formation and represent the means (± SEM) of three independent experiments.

Techniques Used: Labeling, Fluorescence, Pyrene Actin Assay, Concentration Assay, Mouse Assay, Staining, Microscopy, Generated, Conjugation Assay, Transfection, Incubation

WASp inducibly associates and colocalizes with PSTPIP1 and PTP-PEST. (A) Jurkat T cells were stimulated for the indicated times with anti-CD3 and anti-CD28 antibodies and lysates were then prepared and immunoprecipitated with anti-PSTPIP1 antibody. The immune complexes were subjected to SDS-PAGE and sequentially immunoblotted with anti-PST-PEST, anti-WASp, and anti-PSTPIP1 antibodies. (B) Jurkat T cells were stimulated with anti-CD3 and anti-CD28 antibodies and lysates were then prepared and immunoprecipitated with anti-WASp antibody. Complexes were resolved by SDS-PAGE followed by sequential immunoblotting with anti–PTP-PEST and anti-WASp antibodies. (C) Lysates prepared from Jurkat T cells were incubated with GST, GST-PSTPIP1, full-length (FL), GST-PSTPIPCOIL, or GST-PSTPIPSH3 fusion proteins bound to glutathione- sepharose beads. Complexes were resolved by SDS-PAGE and immunoblotted using an anti–PTP-PEST antibody. (D) Cos-7 cells were transiently transfected with pEGFP-PSTPIP1 (a), pEGFP-PTP-PEST (b), pEGFP-WASp (c), pEGFP-PSTPIP1 and pcΔNA3-PTP-PEST (d), or pEGFP-PSTPIP1, DSRED-WASp, and pcDNA3-PTP-PEST (e). Cells were fixed, stained with rhodamine phalloidin for actin (a–c) or with anti–PTP-PEST antibody (d and e) and Cy5 anti–rabbit Ig (e), and then analyzed by confocal immunofluorescent microscopy. The images shown are representative of three independent experiments. (E) pcDNA3 constructs for expression of wild-type or catalytically inactive (C231S) PTP-PEST were cotransfected with pEGFP-WASp (WASp-GFP) into Jurkat cells. The cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies, lysed, and the lysate proteins were immunoprecipitated with anti-GFP antibodies. The complexes were subjected to SDS-PAGE and immunoblotted sequentially with anti–p-Tyr and anti-GFP antibodies.
Figure Legend Snippet: WASp inducibly associates and colocalizes with PSTPIP1 and PTP-PEST. (A) Jurkat T cells were stimulated for the indicated times with anti-CD3 and anti-CD28 antibodies and lysates were then prepared and immunoprecipitated with anti-PSTPIP1 antibody. The immune complexes were subjected to SDS-PAGE and sequentially immunoblotted with anti-PST-PEST, anti-WASp, and anti-PSTPIP1 antibodies. (B) Jurkat T cells were stimulated with anti-CD3 and anti-CD28 antibodies and lysates were then prepared and immunoprecipitated with anti-WASp antibody. Complexes were resolved by SDS-PAGE followed by sequential immunoblotting with anti–PTP-PEST and anti-WASp antibodies. (C) Lysates prepared from Jurkat T cells were incubated with GST, GST-PSTPIP1, full-length (FL), GST-PSTPIPCOIL, or GST-PSTPIPSH3 fusion proteins bound to glutathione- sepharose beads. Complexes were resolved by SDS-PAGE and immunoblotted using an anti–PTP-PEST antibody. (D) Cos-7 cells were transiently transfected with pEGFP-PSTPIP1 (a), pEGFP-PTP-PEST (b), pEGFP-WASp (c), pEGFP-PSTPIP1 and pcΔNA3-PTP-PEST (d), or pEGFP-PSTPIP1, DSRED-WASp, and pcDNA3-PTP-PEST (e). Cells were fixed, stained with rhodamine phalloidin for actin (a–c) or with anti–PTP-PEST antibody (d and e) and Cy5 anti–rabbit Ig (e), and then analyzed by confocal immunofluorescent microscopy. The images shown are representative of three independent experiments. (E) pcDNA3 constructs for expression of wild-type or catalytically inactive (C231S) PTP-PEST were cotransfected with pEGFP-WASp (WASp-GFP) into Jurkat cells. The cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies, lysed, and the lysate proteins were immunoprecipitated with anti-GFP antibodies. The complexes were subjected to SDS-PAGE and immunoblotted sequentially with anti–p-Tyr and anti-GFP antibodies.

Techniques Used: Immunoprecipitation, SDS Page, Incubation, Transfection, Staining, Microscopy, Construct, Expressing

40) Product Images from "Rasd1 interacts with Ear2 (Nr2f6) to regulate renin transcription"

Article Title: Rasd1 interacts with Ear2 (Nr2f6) to regulate renin transcription

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-12-4

Effects of wild-type and Rasd1 mutants on nuclear-cytoplasmic distribution of Ear2 . (A-G) Immunofluorescence staining of COS-7 cells transfected with pHisHA-Rasd1 (A1-3) or pGST-Ear2 (B1-3) or pHisHA-Rasd1 (wild type or mutants) + pGST-Ear2 (C1 to G4). HisHARasd1 (wild type or mutants) were detected with anti-HA antibody and visualized with AlexaFluor 568 (red); GST-Ear2 was labeled with anti-GST AlexaFluor 488 (green); nucleus was labeled with 4',6-diamidino-2-phenylindole (DAPI) (blue). Confocal imaging showed that HisHA-Rasd1 is present in both the nucleus and cytoplasm (A1-3). GST-Ear2 is located mainly in the nucleus (B1-3), but when co-transfected together with HisHA-Rasd1, GST-Ear2 is detected in both the nucleus and cytoplasm (C2).
Figure Legend Snippet: Effects of wild-type and Rasd1 mutants on nuclear-cytoplasmic distribution of Ear2 . (A-G) Immunofluorescence staining of COS-7 cells transfected with pHisHA-Rasd1 (A1-3) or pGST-Ear2 (B1-3) or pHisHA-Rasd1 (wild type or mutants) + pGST-Ear2 (C1 to G4). HisHARasd1 (wild type or mutants) were detected with anti-HA antibody and visualized with AlexaFluor 568 (red); GST-Ear2 was labeled with anti-GST AlexaFluor 488 (green); nucleus was labeled with 4',6-diamidino-2-phenylindole (DAPI) (blue). Confocal imaging showed that HisHA-Rasd1 is present in both the nucleus and cytoplasm (A1-3). GST-Ear2 is located mainly in the nucleus (B1-3), but when co-transfected together with HisHA-Rasd1, GST-Ear2 is detected in both the nucleus and cytoplasm (C2).

Techniques Used: Immunofluorescence, Staining, Transfection, Labeling, Imaging

Ear2 ligand binding domain interacts with Rasd1 . (A) Schematic diagram showing the truncated constructs of Ear2. Ear2 contains 4 main domains: an activator function I site, a DNA binding domain, a zinc finger domain and a ligand binding domain. Full length (FL) Ear2 has 390 amino acids and each of the six truncated Ear2 constructs is represented by their amino acid numbers on the left. (B) Immunoblot analysis showed that Rasd1 binds to Ear2 ligand binding domain. COS-7 cells were co-transfected with pHisHA-Rasd1 and the indicated pGST-Ear2 constructs. HisHA-Rasd1 from the cell lysates was immobilized on Ni-NTA beads and GST-Ear2 constructs bound to the complexes were eluted by heating at 95°C for 10 minutes, and detected by immunoblotting with anti-GST (lanes 1 and 5-7). GST-Ear2 constructs not co-precipitated with HisHA-Rasd1 were detected in the flow-through (lanes 2-4 and 8). (C) Luciferase study showed that Ear2 ligand binding domain and DNA binding domain are required for interacting with Rasd1 to modulate renin transcription. COS-7 cells were transfected with p4.1-Luc (2.0 μg), pGST-Ear2 (1.5 μg) or plasmids expressing Ear2 truncated constructs and pHisHA-Rasd1 (1.5 μg) as indicated, together with pSV-β-gal (0.5 μg). Total DNA transfected was held constant with the respective carrier vector plasmids. Controls are transfected with pGL3-basic (2.0 μg), pSV-β-gal (0.5 μg) and appropriate amounts of the respective carrier vectors. Relative luciferase activity was normalized against β-gal activity. Ear2 constructs that were missing their DNA binding domains (Ear2-N53, Ear2-C131, Ear2-C194) did not significantly repress renin transcription. Rasd1 alleviated Ear2-repressed renin transcription only if Ear2 ligand binding domain is present (Ear2-FL, Ear2-C54). * denotes p
Figure Legend Snippet: Ear2 ligand binding domain interacts with Rasd1 . (A) Schematic diagram showing the truncated constructs of Ear2. Ear2 contains 4 main domains: an activator function I site, a DNA binding domain, a zinc finger domain and a ligand binding domain. Full length (FL) Ear2 has 390 amino acids and each of the six truncated Ear2 constructs is represented by their amino acid numbers on the left. (B) Immunoblot analysis showed that Rasd1 binds to Ear2 ligand binding domain. COS-7 cells were co-transfected with pHisHA-Rasd1 and the indicated pGST-Ear2 constructs. HisHA-Rasd1 from the cell lysates was immobilized on Ni-NTA beads and GST-Ear2 constructs bound to the complexes were eluted by heating at 95°C for 10 minutes, and detected by immunoblotting with anti-GST (lanes 1 and 5-7). GST-Ear2 constructs not co-precipitated with HisHA-Rasd1 were detected in the flow-through (lanes 2-4 and 8). (C) Luciferase study showed that Ear2 ligand binding domain and DNA binding domain are required for interacting with Rasd1 to modulate renin transcription. COS-7 cells were transfected with p4.1-Luc (2.0 μg), pGST-Ear2 (1.5 μg) or plasmids expressing Ear2 truncated constructs and pHisHA-Rasd1 (1.5 μg) as indicated, together with pSV-β-gal (0.5 μg). Total DNA transfected was held constant with the respective carrier vector plasmids. Controls are transfected with pGL3-basic (2.0 μg), pSV-β-gal (0.5 μg) and appropriate amounts of the respective carrier vectors. Relative luciferase activity was normalized against β-gal activity. Ear2 constructs that were missing their DNA binding domains (Ear2-N53, Ear2-C131, Ear2-C194) did not significantly repress renin transcription. Rasd1 alleviated Ear2-repressed renin transcription only if Ear2 ligand binding domain is present (Ear2-FL, Ear2-C54). * denotes p

Techniques Used: Ligand Binding Assay, Construct, Binding Assay, Transfection, Flow Cytometry, Luciferase, Expressing, Plasmid Preparation, Activity Assay

Rasd1 and Ear2 interact in vitro and in living cells . (A) In vitro binding of GST-Ear2 and HisHA-Rasd1. COS-7 cells were transfected with plasmids expressing the indicated constructs. Cell lysates containing HisHA-Rasd1 was incubated with immobilized GST-Ear2 or GST, and specifically bound HisHA-Rasd1 was eluted by heating at 95°C for 10 minutes, and detected by Western blotting with Anti-HA antibody. (B) HisHA-Rasd1 and GST-Ear2 interact in intact mammalian cells. pHisHA-Rasd1 was co-transfected with plasmids expressing either GST-Ear2 or GST into COS-7 cells. GST and GST-Ear2 were captured from the cell lysates by GSH-linked beads and HisHA-Rasd1 bound to the beads were detected as above. (C) COS-7 cells were co-transfected with plasmids expressing HisHA-Rasd1 and GST-Ear2 or GST. Immunoprecipitations were performed with anti-GST antibody. Co-immunoprecipitated HisHA-Rasd1 was detected by Anti-HA antibody. (D and E) Rasd1 and Ear2 form a physiological complex in living cells. Endogenous Rasd1-Ear2 complexes were detected by immunoprecipitating Ear2 from HEK293T cell lysates (D) or mouse brain lysates (E) with goat polyclonal IgG anti-Ear2 antibody, and probing for Rasd1 (D and E, lanes 1). A non-relevant goat polyclonal IgG antibody was used as a negative control (D and E, lanes 2). IP and IB denote immunoprecipitation and immunoblot, respectively.
Figure Legend Snippet: Rasd1 and Ear2 interact in vitro and in living cells . (A) In vitro binding of GST-Ear2 and HisHA-Rasd1. COS-7 cells were transfected with plasmids expressing the indicated constructs. Cell lysates containing HisHA-Rasd1 was incubated with immobilized GST-Ear2 or GST, and specifically bound HisHA-Rasd1 was eluted by heating at 95°C for 10 minutes, and detected by Western blotting with Anti-HA antibody. (B) HisHA-Rasd1 and GST-Ear2 interact in intact mammalian cells. pHisHA-Rasd1 was co-transfected with plasmids expressing either GST-Ear2 or GST into COS-7 cells. GST and GST-Ear2 were captured from the cell lysates by GSH-linked beads and HisHA-Rasd1 bound to the beads were detected as above. (C) COS-7 cells were co-transfected with plasmids expressing HisHA-Rasd1 and GST-Ear2 or GST. Immunoprecipitations were performed with anti-GST antibody. Co-immunoprecipitated HisHA-Rasd1 was detected by Anti-HA antibody. (D and E) Rasd1 and Ear2 form a physiological complex in living cells. Endogenous Rasd1-Ear2 complexes were detected by immunoprecipitating Ear2 from HEK293T cell lysates (D) or mouse brain lysates (E) with goat polyclonal IgG anti-Ear2 antibody, and probing for Rasd1 (D and E, lanes 1). A non-relevant goat polyclonal IgG antibody was used as a negative control (D and E, lanes 2). IP and IB denote immunoprecipitation and immunoblot, respectively.

Techniques Used: In Vitro, Binding Assay, Transfection, Expressing, Construct, Incubation, Western Blot, Immunoprecipitation, Negative Control

Related Articles

Affinity Chromatography:

Article Title: M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines
Article Snippet: .. The recombinant Hsp20-Nef protein was purified by affinity chromatography using a Ni-NTA agarose column according to the manufacturer’s instructions (Qiagen). .. Next, the purified Hsp20-Nef protein was dialyzed against PBS1X, assessed by NanoDrop spectrophotometry, and stored at -70◦ C until used.

Article Title: Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L.
Article Snippet: .. Bacterially expressed MBP-OsTRFL1, MBP-OsTRBF1, (His)6 -OsTRFL1, (His)6 -OsTRBF1, and (His)6 -RTBP1 recombinant proteins were purified by affinity chromatography using Ni-NTA agarose (Qiagen) for (His)6 -tagged proteins and amylose resin (New England Biolabs) for MBP-tagged proteins. .. In vitro pull-down and immunoblot analyses were performed as described by Byun et al . ( ) with anti-His and anti-MBP antibodies (Applied Biological Materials, Richmond, BC, Canada).

Protease Inhibitor:

Article Title: Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance
Article Snippet: .. Cross-linked chloroplasts were lysed [50 mM BisTris, 6 N HCl, 50 mM NaCl, 10% (w/v) glycerol, 1% digitonin, and protease inhibitor cocktail], and His-tagged Trx target complexes were pulled down using Ni-NTA agarose (Qiagen, Hilden, Germany) as follows: chloroplast lysates were incubated with 200 µl of Ni-NTA resin for 3–4 h at 4 °C. .. After centrifugation at 600 g for 1 min, the supernatant was discarded and the pellet was washed five times with washing buffer (lysis buffer without digitonin).

Purification:

Article Title: HrpE, the major component of the Xanthomonas type three protein secretion pilus, elicits plant immunity responses
Article Snippet: .. HrpE-Trx-6 His (HrpE), NHrpE-Trx-6 His (N-HrpE), CHrpE-Trx-6 His (C-HrpE) and Trx-6 His (Trx) were purified with a Ni2+ -nitrilotriacetate (Ni-NTA) agarose column (Qiagen) and CsGRP-GST (CsGRP) and GST with a Glutathione Sepharose column (GE Healthcare). ..

Article Title: M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines
Article Snippet: .. The recombinant Hsp20-Nef protein was purified by affinity chromatography using a Ni-NTA agarose column according to the manufacturer’s instructions (Qiagen). .. Next, the purified Hsp20-Nef protein was dialyzed against PBS1X, assessed by NanoDrop spectrophotometry, and stored at -70◦ C until used.

Article Title: Clec16a, Nrdp1, and USP8 Form a Ubiquitin-Dependent Tripartite Complex That Regulates β-Cell Mitophagy
Article Snippet: .. Glutathione S-transferase (GST)-tagged and 6xHis-tagged proteins were purified utilizing glutathione matrix or nickel-charged resin (HiCap and Ni-NTA agarose; Qiagen) per the manufacturers’ protocols. .. In vitro transcribed/translated Clec16a and Nrdp1 were expressed in T7-driven mammalian expression systems (Promega) per the manufacturers’ protocols.

Article Title: A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues
Article Snippet: .. His-tagged EglaRR01 was expressed and purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen) according to the manufacturer’s instructions. .. Endoglucanase Activity Assay The enzyme’s ability to degrade CMC was determined by measuring the concentration of reducing sugars using the dinitrosalicylic acid method (DNS) [ ].

Article Title: Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L.
Article Snippet: .. Bacterially expressed MBP-OsTRFL1, MBP-OsTRBF1, (His)6 -OsTRFL1, (His)6 -OsTRBF1, and (His)6 -RTBP1 recombinant proteins were purified by affinity chromatography using Ni-NTA agarose (Qiagen) for (His)6 -tagged proteins and amylose resin (New England Biolabs) for MBP-tagged proteins. .. In vitro pull-down and immunoblot analyses were performed as described by Byun et al . ( ) with anti-His and anti-MBP antibodies (Applied Biological Materials, Richmond, BC, Canada).

Immunoprecipitation:

Article Title: Mycobacterium tuberculosis Rv3463 induces mycobactericidal activity in macrophages by enhancing phagolysosomal fusion and exhibits therapeutic potential
Article Snippet: .. The cell lysate and 20 μg His-tagged Rv3463 protein were mixed and incubated at 4 °C for 6 h, and then His-tagged Rv3463 (His)-, TLR2- and TLR4-associated proteins were immunoprecipitated by incubation with Ni-NTA Agarose (Qiagen, Hilden, Germany) or Dynabeads®Protein A (Thermo Fisher Scientific) for 24 h at 4 °C after incubation with an anti-mouse IgG Ab as a control Ab for anti-Rv3463 (His), anti-rabbit IgG Ab as a control Ab for the anti-TLR2 and anti-TLR4 for 4 h at 4 °C. ..

Incubation:

Article Title: Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance
Article Snippet: .. Cross-linked chloroplasts were lysed [50 mM BisTris, 6 N HCl, 50 mM NaCl, 10% (w/v) glycerol, 1% digitonin, and protease inhibitor cocktail], and His-tagged Trx target complexes were pulled down using Ni-NTA agarose (Qiagen, Hilden, Germany) as follows: chloroplast lysates were incubated with 200 µl of Ni-NTA resin for 3–4 h at 4 °C. .. After centrifugation at 600 g for 1 min, the supernatant was discarded and the pellet was washed five times with washing buffer (lysis buffer without digitonin).

Article Title: Mycobacterium tuberculosis Rv3463 induces mycobactericidal activity in macrophages by enhancing phagolysosomal fusion and exhibits therapeutic potential
Article Snippet: .. The cell lysate and 20 μg His-tagged Rv3463 protein were mixed and incubated at 4 °C for 6 h, and then His-tagged Rv3463 (His)-, TLR2- and TLR4-associated proteins were immunoprecipitated by incubation with Ni-NTA Agarose (Qiagen, Hilden, Germany) or Dynabeads®Protein A (Thermo Fisher Scientific) for 24 h at 4 °C after incubation with an anti-mouse IgG Ab as a control Ab for anti-Rv3463 (His), anti-rabbit IgG Ab as a control Ab for the anti-TLR2 and anti-TLR4 for 4 h at 4 °C. ..

Recombinant:

Article Title: M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines
Article Snippet: .. The recombinant Hsp20-Nef protein was purified by affinity chromatography using a Ni-NTA agarose column according to the manufacturer’s instructions (Qiagen). .. Next, the purified Hsp20-Nef protein was dialyzed against PBS1X, assessed by NanoDrop spectrophotometry, and stored at -70◦ C until used.

Article Title: Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L.
Article Snippet: .. Bacterially expressed MBP-OsTRFL1, MBP-OsTRBF1, (His)6 -OsTRFL1, (His)6 -OsTRBF1, and (His)6 -RTBP1 recombinant proteins were purified by affinity chromatography using Ni-NTA agarose (Qiagen) for (His)6 -tagged proteins and amylose resin (New England Biolabs) for MBP-tagged proteins. .. In vitro pull-down and immunoblot analyses were performed as described by Byun et al . ( ) with anti-His and anti-MBP antibodies (Applied Biological Materials, Richmond, BC, Canada).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Qiagen ni nitrilotriacetic acid nta sepharose resin
    Targeted gene insertion and expression in E. coli . ( A ) The gel electrophoresis of amplified PCR cellulose products from B. licheniformis ATCC 14580 (lane 1) and INP from P. syringae KCTC 1832 (lane 2). M: 1 kb DNA marker; ( B ) SDS-PAGE analysis of the recombinant cells; M: standard protein size marker (molecular biomasses in kilodaltons), lane 1: the supernatant fraction of recombinant cell culture medium, lane 2: the total cell lysates of recombinant cell; ( C ) The purified fusion proteins following <t>Ni-nitrilotriacetic</t> acid <t>(NTA)-sepharose</t> resin treatment; M: standard protein size marker (kDa), lane 1: imidazole concentration of 20 mM in the binding buffer, lane 2: imidazole concentration of 50 mM in the binding buffer, lane 3: imidazole concentration of 100 mM in the binding buffer; ( D ) Western blot analysis of the purified fusion protein from SDS-PAGE results probed with anti-His-tag antibody, respectively.
    Ni Nitrilotriacetic Acid Nta Sepharose Resin, supplied by Qiagen, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ni nitrilotriacetic acid nta sepharose resin/product/Qiagen
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    ni nitrilotriacetic acid nta sepharose resin - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Qiagen ninta agarose
    Phosphorylated C-terminus of Sae2 interacts with Rad50. a Full-length phosphorylated recombinant MBP-tagged <t>pSae2</t> was mock-treated or dephosphorylated with λ phosphatase upon binding to amylose resin and incubated with recombinant MRX complex. The eluates were visualized by silver staining. Prescission protease was added to all samples as a protein stabilizer and to cleave the MBP tag off pSae2. b The FLAG-tagged recombinant Rad50 protein was immobilized on anti-FLAG affinity resin and incubated with phosphorylated C-terminal domain of pSae2 (pSae2 ΔN169, residues 170–345), which had been either mock-treated or dephosphorylated with λ phosphatase. The bound proteins were eluted and detected by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. c The phosphorylated recombinant MBP-tagged C-terminal domain of pSae2 (residues 170–345) was bound to amylose resin, eluted, cleaved with prescission protease, and immobilized on <t>NiNTA</t> resin. The bound pSae2 ΔN169 was mock-treated or dephosphorylated with λ phosphatase and incubated with recombinant wild-type Rad50 or ATP binding-deficient Rad50 K40A. Proteins were eluted and visualized by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. d Assay as in c . Phosphorylated C-terminal domain of pSae2 was incubated with wild-type Rad50 or Rad50 K81I (representative Rad50S) mutant
    Ninta Agarose, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ninta agarose/product/Qiagen
    Average 99 stars, based on 275 article reviews
    Price from $9.99 to $1999.99
    ninta agarose - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Targeted gene insertion and expression in E. coli . ( A ) The gel electrophoresis of amplified PCR cellulose products from B. licheniformis ATCC 14580 (lane 1) and INP from P. syringae KCTC 1832 (lane 2). M: 1 kb DNA marker; ( B ) SDS-PAGE analysis of the recombinant cells; M: standard protein size marker (molecular biomasses in kilodaltons), lane 1: the supernatant fraction of recombinant cell culture medium, lane 2: the total cell lysates of recombinant cell; ( C ) The purified fusion proteins following Ni-nitrilotriacetic acid (NTA)-sepharose resin treatment; M: standard protein size marker (kDa), lane 1: imidazole concentration of 20 mM in the binding buffer, lane 2: imidazole concentration of 50 mM in the binding buffer, lane 3: imidazole concentration of 100 mM in the binding buffer; ( D ) Western blot analysis of the purified fusion protein from SDS-PAGE results probed with anti-His-tag antibody, respectively.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Bacillus Cellulase Molecular Cloning, Expression, and Surface Display on the Outer Membrane of Escherichia coli

    doi: 10.3390/molecules23020503

    Figure Lengend Snippet: Targeted gene insertion and expression in E. coli . ( A ) The gel electrophoresis of amplified PCR cellulose products from B. licheniformis ATCC 14580 (lane 1) and INP from P. syringae KCTC 1832 (lane 2). M: 1 kb DNA marker; ( B ) SDS-PAGE analysis of the recombinant cells; M: standard protein size marker (molecular biomasses in kilodaltons), lane 1: the supernatant fraction of recombinant cell culture medium, lane 2: the total cell lysates of recombinant cell; ( C ) The purified fusion proteins following Ni-nitrilotriacetic acid (NTA)-sepharose resin treatment; M: standard protein size marker (kDa), lane 1: imidazole concentration of 20 mM in the binding buffer, lane 2: imidazole concentration of 50 mM in the binding buffer, lane 3: imidazole concentration of 100 mM in the binding buffer; ( D ) Western blot analysis of the purified fusion protein from SDS-PAGE results probed with anti-His-tag antibody, respectively.

    Article Snippet: The (His) 6-tagged cellulase protein was bound to Ni-nitrilotriacetic acid (NTA)-sepharose resin (Qiagen, Hilden, Germany), pre-equilibrated with binding buffer, and washed with imidazole in a step-gradient manner range of 20 to 100 mM.

    Techniques: Expressing, Nucleic Acid Electrophoresis, Amplification, Polymerase Chain Reaction, Marker, SDS Page, Recombinant, Cell Culture, Purification, Concentration Assay, Binding Assay, Western Blot

    Phosphorylated C-terminus of Sae2 interacts with Rad50. a Full-length phosphorylated recombinant MBP-tagged pSae2 was mock-treated or dephosphorylated with λ phosphatase upon binding to amylose resin and incubated with recombinant MRX complex. The eluates were visualized by silver staining. Prescission protease was added to all samples as a protein stabilizer and to cleave the MBP tag off pSae2. b The FLAG-tagged recombinant Rad50 protein was immobilized on anti-FLAG affinity resin and incubated with phosphorylated C-terminal domain of pSae2 (pSae2 ΔN169, residues 170–345), which had been either mock-treated or dephosphorylated with λ phosphatase. The bound proteins were eluted and detected by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. c The phosphorylated recombinant MBP-tagged C-terminal domain of pSae2 (residues 170–345) was bound to amylose resin, eluted, cleaved with prescission protease, and immobilized on NiNTA resin. The bound pSae2 ΔN169 was mock-treated or dephosphorylated with λ phosphatase and incubated with recombinant wild-type Rad50 or ATP binding-deficient Rad50 K40A. Proteins were eluted and visualized by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. d Assay as in c . Phosphorylated C-terminal domain of pSae2 was incubated with wild-type Rad50 or Rad50 K81I (representative Rad50S) mutant

    Journal: Nature Communications

    Article Title: Regulatory control of DNA end resection by Sae2 phosphorylation

    doi: 10.1038/s41467-018-06417-5

    Figure Lengend Snippet: Phosphorylated C-terminus of Sae2 interacts with Rad50. a Full-length phosphorylated recombinant MBP-tagged pSae2 was mock-treated or dephosphorylated with λ phosphatase upon binding to amylose resin and incubated with recombinant MRX complex. The eluates were visualized by silver staining. Prescission protease was added to all samples as a protein stabilizer and to cleave the MBP tag off pSae2. b The FLAG-tagged recombinant Rad50 protein was immobilized on anti-FLAG affinity resin and incubated with phosphorylated C-terminal domain of pSae2 (pSae2 ΔN169, residues 170–345), which had been either mock-treated or dephosphorylated with λ phosphatase. The bound proteins were eluted and detected by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. c The phosphorylated recombinant MBP-tagged C-terminal domain of pSae2 (residues 170–345) was bound to amylose resin, eluted, cleaved with prescission protease, and immobilized on NiNTA resin. The bound pSae2 ΔN169 was mock-treated or dephosphorylated with λ phosphatase and incubated with recombinant wild-type Rad50 or ATP binding-deficient Rad50 K40A. Proteins were eluted and visualized by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. d Assay as in c . Phosphorylated C-terminal domain of pSae2 was incubated with wild-type Rad50 or Rad50 K81I (representative Rad50S) mutant

    Article Snippet: Finally, pSae2 was purified using NiNTA agarose (Qiagen).

    Techniques: Recombinant, Binding Assay, Incubation, Silver Staining, Staining, Western Blot, Avidin-Biotin Assay, Mutagenesis

    Expression of the molecular probe TOP1-DOPA-GFP. (A) A schematic diagram of 30.0 kDa TOP1-DOPA-GFP. (B) Total protein analyses of L-DOPA incorporation into TOP1-DOPA-GFP. TOP1-DOPA-GFP was expressed in the presence and absence of L-DOPA, purified with Ni-NTA resin, and resolved by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) Redox cycling staining of TOP1-DOPA-GFP. Proteins from a similar gel as (B) were blotted to a nitrocellulose membrane and stained with NBT reagent (2 M sodium glycinate, 0.24 mM NBT, pH 10). This method detects quino-proteins and confirmed the presence of L-DOPA/dopaquinone in TOP1-DOPA-GFP.

    Journal: Analytical biochemistry

    Article Title: A Versatile Approach to Transform Low-Affinity Peptides into Protein Probes with Co-Translationally Expressed Chemical Cross-Linker 1

    doi: 10.1016/j.ab.2010.05.026

    Figure Lengend Snippet: Expression of the molecular probe TOP1-DOPA-GFP. (A) A schematic diagram of 30.0 kDa TOP1-DOPA-GFP. (B) Total protein analyses of L-DOPA incorporation into TOP1-DOPA-GFP. TOP1-DOPA-GFP was expressed in the presence and absence of L-DOPA, purified with Ni-NTA resin, and resolved by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) Redox cycling staining of TOP1-DOPA-GFP. Proteins from a similar gel as (B) were blotted to a nitrocellulose membrane and stained with NBT reagent (2 M sodium glycinate, 0.24 mM NBT, pH 10). This method detects quino-proteins and confirmed the presence of L-DOPA/dopaquinone in TOP1-DOPA-GFP.

    Article Snippet: SH3-His, wtTOP1-GFP, and TOP1-DOPA-GFP were purified using Ni-NTA agarose beads (Qiagen).

    Techniques: Expressing, Purification, SDS Page, Staining

    OC promotes Src protein degradation by the ubiquitin-proteasome pathway. MIA-RES cells were treated with OC for the indicated dose (0–10 μM) a and indicated time periods (0–48 h) b , then the level of ubiquitination was detected by western blotting using an anti-ubiquitin antibody. c MIA-RES cells were treated with 50 μM cycloheximide (CHX) with or without 10 μM OC for the indicated time and the cell lysates were analyzed by immunoblotting. β -actin served as a loading control. d MIA-RES cells were co-treated with 15 μM MG132 with or without 10 μM OC and harvested at 0, 3, 6,12, and 24 h after treatment for western blotting analysis. β -actin served as a loading control. e MIA-RES cells were transfected with 2 μg Src plasmid for 24 h, and treated with 10 μM OC for 24 h. The cells were treated with 15 μM MG132 for 6 h before collected, and 6xHis-tagged proteins of Src were purified with Ni-NTA Agarose beads, followed by western blotting analyses to detect the level of Src ubiquitination.

    Journal: Cell Death & Disease

    Article Title: Natural compound Oblongifolin C confers gemcitabine resistance in pancreatic cancer by downregulating Src/MAPK/ERK pathways

    doi: 10.1038/s41419-018-0574-1

    Figure Lengend Snippet: OC promotes Src protein degradation by the ubiquitin-proteasome pathway. MIA-RES cells were treated with OC for the indicated dose (0–10 μM) a and indicated time periods (0–48 h) b , then the level of ubiquitination was detected by western blotting using an anti-ubiquitin antibody. c MIA-RES cells were treated with 50 μM cycloheximide (CHX) with or without 10 μM OC for the indicated time and the cell lysates were analyzed by immunoblotting. β -actin served as a loading control. d MIA-RES cells were co-treated with 15 μM MG132 with or without 10 μM OC and harvested at 0, 3, 6,12, and 24 h after treatment for western blotting analysis. β -actin served as a loading control. e MIA-RES cells were transfected with 2 μg Src plasmid for 24 h, and treated with 10 μM OC for 24 h. The cells were treated with 15 μM MG132 for 6 h before collected, and 6xHis-tagged proteins of Src were purified with Ni-NTA Agarose beads, followed by western blotting analyses to detect the level of Src ubiquitination.

    Article Snippet: His-tagged Src was purified with Ni-NTA Agarose beads according to the manufacturers’ protocols (Qiagen).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Purification