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.
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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 "Transmission Characteristics of Barley Yellow Striate Mosaic Virus in Its Planthopper Vector Laodelphax striatellus"

Article Title: Transmission Characteristics of Barley Yellow Striate Mosaic Virus in Its Planthopper Vector Laodelphax striatellus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01419

The propagation of BYSMV in L. striatellus . (A) The virus accumulations of infected hindguts and other tissues of the alimentary canals at 4-day and 8-day padp were verified by western blotting analysis with anti-N polyclonal antiserum. The whole alimentary canals of viruliferous SBPHs served as negative controls (Mock). The actin of L. striatellus was detected as the loading controls. (B) RT-qPCR assay detecting relative accumulation of BYSMV-N mRNA in the infected hindguts and other tissues of the alimentary canals at 4-day and 8-day padp, respectively. The viral expression level of 4-day-hindgut was normalized as the relative expression level, which was set to 1 data points are the mean value of three independent experiments. The significant difference was analyzed by Student’s t -test. ∗∗ P -value
Figure Legend Snippet: The propagation of BYSMV in L. striatellus . (A) The virus accumulations of infected hindguts and other tissues of the alimentary canals at 4-day and 8-day padp were verified by western blotting analysis with anti-N polyclonal antiserum. The whole alimentary canals of viruliferous SBPHs served as negative controls (Mock). The actin of L. striatellus was detected as the loading controls. (B) RT-qPCR assay detecting relative accumulation of BYSMV-N mRNA in the infected hindguts and other tissues of the alimentary canals at 4-day and 8-day padp, respectively. The viral expression level of 4-day-hindgut was normalized as the relative expression level, which was set to 1 data points are the mean value of three independent experiments. The significant difference was analyzed by Student’s t -test. ∗∗ P -value

Techniques Used: Infection, Western Blot, Quantitative RT-PCR, Expressing

3) 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

4) Product Images from "Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition"

Article Title: Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.00546

Cortical localization of CesA1p in dinoflagellates. (A) Western blot analysis of cell lysates from thecate and athecate dinoflagellates. (B) Fluorescence photomicrographs of whole cells labeled with anti-CesA1p antibody. (C) Confocal images showing the immunofluorescence-labeled cryosections prepared from L. polyedrum cells. Green fluorescence (Alexa Fluor 488 goat anti-rabbit IgG), corresponding to CesA1p, was localized to margins of overlapping CTPs; the continuity over one CTP was demarcated in enlarge image in (D) . CFW fluorescence (Blue) also localized to this layer. CTPs are not flattened but three-dimensional structures with different front and back side ( Figure 1A,B ). Red fluorescence corresponded to the auto-fluorescence of chloroplast pigments. (D) CesA1p immunofluorescent pattern in cortical region with higher magnification. Its continuity around a CTP was highlighted with arrow. Antigen-purified anti-CesA1p antibody was used throughout the experiments. Scale bars represent 10 μm.
Figure Legend Snippet: Cortical localization of CesA1p in dinoflagellates. (A) Western blot analysis of cell lysates from thecate and athecate dinoflagellates. (B) Fluorescence photomicrographs of whole cells labeled with anti-CesA1p antibody. (C) Confocal images showing the immunofluorescence-labeled cryosections prepared from L. polyedrum cells. Green fluorescence (Alexa Fluor 488 goat anti-rabbit IgG), corresponding to CesA1p, was localized to margins of overlapping CTPs; the continuity over one CTP was demarcated in enlarge image in (D) . CFW fluorescence (Blue) also localized to this layer. CTPs are not flattened but three-dimensional structures with different front and back side ( Figure 1A,B ). Red fluorescence corresponded to the auto-fluorescence of chloroplast pigments. (D) CesA1p immunofluorescent pattern in cortical region with higher magnification. Its continuity around a CTP was highlighted with arrow. Antigen-purified anti-CesA1p antibody was used throughout the experiments. Scale bars represent 10 μm.

Techniques Used: Western Blot, Fluorescence, Labeling, Immunofluorescence, Purification

LpCesA1 expression and cellulose synthesis (CS) during cyst-to-swarmer transition. (A) Fluorescence photomicrographs of L. polyedrum cells (stained with CFW) during cyst-to-swarmer transition (T C–S ). Control contained 0.0625% (v/v) of DMSO, the vehicle for DCB. Ecdysis resulted in a decrease in cellulose content (CFW fluorescence) from T = –2 to T = 0. (B) CFW (cellulose)-stained cells were distributed in R1 and R2 regions in relation to their relative CFW fluorescence intensity and cell size (forward scatter). R1, with less CFW staining should be the ecdysal cyst whereas the R2 should be the swarmer cells (and cyst with attached cell wall). However, these two populations were complicated with some shed cell-wall attaching to the ecdysal cyst. DCB served as a negative control for cells which did not increase in cellulose content, verifying sub-population of R2 cells had detached cell wall (some R2 cells had larger FSc). Almost all cells had higher level of CFW-staining by T = 8–12, suggesting continuous cell wall growth after regaining motility (between T = 4 and T = 8 in different cells). The 74.5% (R1) was thus an underestimate for ecdysal cyst, as no swarmer cell could be identified in DCB-treated cells; transfection involved lipofectamine and additional centrifugation, nor did visual observation reveal any motile cells in T = 0 sample. (C) Crystalline cellulose levels per cell during T C–S . (D) Levels of LpCesA1 transcripts during T C–S . (E) Anti-CesA1p immunoblot of cell lysates collected during T C–S . Control experiment: DCB (100 μM) completely inhibited the reformation of CTPs (A) and significantly ( P
Figure Legend Snippet: LpCesA1 expression and cellulose synthesis (CS) during cyst-to-swarmer transition. (A) Fluorescence photomicrographs of L. polyedrum cells (stained with CFW) during cyst-to-swarmer transition (T C–S ). Control contained 0.0625% (v/v) of DMSO, the vehicle for DCB. Ecdysis resulted in a decrease in cellulose content (CFW fluorescence) from T = –2 to T = 0. (B) CFW (cellulose)-stained cells were distributed in R1 and R2 regions in relation to their relative CFW fluorescence intensity and cell size (forward scatter). R1, with less CFW staining should be the ecdysal cyst whereas the R2 should be the swarmer cells (and cyst with attached cell wall). However, these two populations were complicated with some shed cell-wall attaching to the ecdysal cyst. DCB served as a negative control for cells which did not increase in cellulose content, verifying sub-population of R2 cells had detached cell wall (some R2 cells had larger FSc). Almost all cells had higher level of CFW-staining by T = 8–12, suggesting continuous cell wall growth after regaining motility (between T = 4 and T = 8 in different cells). The 74.5% (R1) was thus an underestimate for ecdysal cyst, as no swarmer cell could be identified in DCB-treated cells; transfection involved lipofectamine and additional centrifugation, nor did visual observation reveal any motile cells in T = 0 sample. (C) Crystalline cellulose levels per cell during T C–S . (D) Levels of LpCesA1 transcripts during T C–S . (E) Anti-CesA1p immunoblot of cell lysates collected during T C–S . Control experiment: DCB (100 μM) completely inhibited the reformation of CTPs (A) and significantly ( P

Techniques Used: Expressing, Fluorescence, Staining, Negative Control, Transfection, Centrifugation

5) 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

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 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

7) 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

8) 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

9) Product Images from "The Development and Validation of a Novel Nanobody-Based Competitive ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in Cattle"

Article Title: The Development and Validation of a Novel Nanobody-Based Competitive ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in Cattle

Journal: Frontiers in Veterinary Science

doi: 10.3389/fvets.2018.00250

Sera screening analysis of naïve and vaccinated calves' samples using the Nanobody (Nb94) based 3ABC competitive ELISA and PrioCHECK NSP test. A total of 72 calves were serially sampled three times; before first vaccination (naïve state), after first vaccination, and after second vaccination. The prevalence of FMDV NSP antibodies were determined by the Nb (Nb94) based 3ABC competitive ELISA (A) and PrioCHECK NSP test (B) . Cutoff for each assay is indicated by dash line.
Figure Legend Snippet: Sera screening analysis of naïve and vaccinated calves' samples using the Nanobody (Nb94) based 3ABC competitive ELISA and PrioCHECK NSP test. A total of 72 calves were serially sampled three times; before first vaccination (naïve state), after first vaccination, and after second vaccination. The prevalence of FMDV NSP antibodies were determined by the Nb (Nb94) based 3ABC competitive ELISA (A) and PrioCHECK NSP test (B) . Cutoff for each assay is indicated by dash line.

Techniques Used: Competitive ELISA

Binding affinity analysis of selected Nanobodies (Nbs) against FMDV 3ABC protein. (A) The binding affinity results of six anti-FMDV 3ABC Nbs, evaluated by indirect ELISA. During experiments Nb9 was used as a negative control. The results are presented in Relative Light Units (RLU). (B) . The immunorecognition performance of six selected anti-FMDV 3ABC Nbs in a competitive ELISA format using a set of 8 infected and noninfected control samples. Mean percentage of inhibition (PI) for each Nb was calculated using the formula: 100–(X Aver infected/X Aver noninfected) × 100.
Figure Legend Snippet: Binding affinity analysis of selected Nanobodies (Nbs) against FMDV 3ABC protein. (A) The binding affinity results of six anti-FMDV 3ABC Nbs, evaluated by indirect ELISA. During experiments Nb9 was used as a negative control. The results are presented in Relative Light Units (RLU). (B) . The immunorecognition performance of six selected anti-FMDV 3ABC Nbs in a competitive ELISA format using a set of 8 infected and noninfected control samples. Mean percentage of inhibition (PI) for each Nb was calculated using the formula: 100–(X Aver infected/X Aver noninfected) × 100.

Techniques Used: Binding Assay, Indirect ELISA, Negative Control, Competitive ELISA, Infection, Inhibition

Sera screening analysis of infected and noninfected cattle samples using the Nanobody (Nb94) based 3ABC competitive ELISA. Box plot representing 100 noninfected (negative) and 150 infected (confirmed by reverse transcription PCR or virus isolation) (positive) sera collected in Uganda. FMDV serotype of infected cattle, included serotype O, SAT1 and SAT2 (50 samples for each). Cutoff assay is indicated by dash line. Numbers in brackets indicate the total number of tested sera.
Figure Legend Snippet: Sera screening analysis of infected and noninfected cattle samples using the Nanobody (Nb94) based 3ABC competitive ELISA. Box plot representing 100 noninfected (negative) and 150 infected (confirmed by reverse transcription PCR or virus isolation) (positive) sera collected in Uganda. FMDV serotype of infected cattle, included serotype O, SAT1 and SAT2 (50 samples for each). Cutoff assay is indicated by dash line. Numbers in brackets indicate the total number of tested sera.

Techniques Used: Infection, Competitive ELISA, Polymerase Chain Reaction, Virus Isolation Assay

Selection of Nanobodies (Nbs) from an immune phage-displayed Nb library against FMDV 3ABC protein. Periplasmic Extract (PE) ELISA results demonstrating 19 Nb colonies analyzed for the immunorecognition to FMDV 3ABC protein and peptides (3A, 3B, 3C1, and 3C2). Luria-Bertani (LB) broth medium was used as negative control. Assay cutoff is indicated by dash line.
Figure Legend Snippet: Selection of Nanobodies (Nbs) from an immune phage-displayed Nb library against FMDV 3ABC protein. Periplasmic Extract (PE) ELISA results demonstrating 19 Nb colonies analyzed for the immunorecognition to FMDV 3ABC protein and peptides (3A, 3B, 3C1, and 3C2). Luria-Bertani (LB) broth medium was used as negative control. Assay cutoff is indicated by dash line.

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

10) Product Images from "A DNA prime-live vaccine boost strategy in mice can augment IFN-? responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis"

Article Title: A DNA prime-live vaccine boost strategy in mice can augment IFN-? responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

Journal: Immunology

doi: 10.1046/j.1365-2567.2003.01589.x

Interferon-γ-secreting cells in BALB/c mice 14 weeks after first vaccination. Mice were primed either with plasmid DNA expressing ESAT-6 and Ag85A or control plasmid before boosting with BCG only (a) or WAg520 (b). Some mice were given plasmid DNA or control plasmid only (no boost) or an attenuated M. bovis only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determinations are shown.
Figure Legend Snippet: Interferon-γ-secreting cells in BALB/c mice 14 weeks after first vaccination. Mice were primed either with plasmid DNA expressing ESAT-6 and Ag85A or control plasmid before boosting with BCG only (a) or WAg520 (b). Some mice were given plasmid DNA or control plasmid only (no boost) or an attenuated M. bovis only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determinations are shown.

Techniques Used: Mouse Assay, Plasmid Preparation, Expressing, Enzyme-linked Immunospot

Interferon-γ-secreting cells in C57BL/6 mice 14 weeks after first vaccination. Mice were primed with plasmid DNA (no boost) expressing ESAT-6 and Ag85A, or control plasmid before boosting with BCG (a) or WAg520 (b). Some mice were given plasmid DNA (no boost) or control plasmid only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determinations are shown.
Figure Legend Snippet: Interferon-γ-secreting cells in C57BL/6 mice 14 weeks after first vaccination. Mice were primed with plasmid DNA (no boost) expressing ESAT-6 and Ag85A, or control plasmid before boosting with BCG (a) or WAg520 (b). Some mice were given plasmid DNA (no boost) or control plasmid only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determinations are shown.

Techniques Used: Mouse Assay, Plasmid Preparation, Expressing, Enzyme-linked Immunospot

Interferon-γ-secreting cells in BALB/c mice 24 weeks after first vaccination. Mice were primed with plasmid DNA expressing ESAT-6 and Ag85A before boosting with BCG. Some mice were given only plasmid DNA (no boost) or BCG only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determination are shown.
Figure Legend Snippet: Interferon-γ-secreting cells in BALB/c mice 24 weeks after first vaccination. Mice were primed with plasmid DNA expressing ESAT-6 and Ag85A before boosting with BCG. Some mice were given only plasmid DNA (no boost) or BCG only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determination are shown.

Techniques Used: Mouse Assay, Plasmid Preparation, Expressing, Enzyme-linked Immunospot

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 "Stop codon selection in eukaryotic translation termination: comparison of the discriminating potential between human and ciliate eRF1s"

Article Title: Stop codon selection in eukaryotic translation termination: comparison of the discriminating potential between human and ciliate eRF1s

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg146

Fig. 1. Cross-linking patterns obtained with 42mer mRNA analogs containing stop codons (UAG, UAA or UAG) or a sense codon (UCA) in presence of C-terminally His-tagged human wild-type eRF1 (wt) or mutated human eRF1 containing either S64D or I35V-L126F substitutions. After irradiation, the reaction products were separated onto a 10% SDS–polyacrylamide gel and analyzed by autoradiography. A control reaction without eRF1 (No eRF1) is shown for each mRNA analog. The 68 kDa band corresponding to the eRF1–mRNA cross-link is indicated by an arrow. Molecular mass markers in kDa are indicated on the left.
Figure Legend Snippet: Fig. 1. Cross-linking patterns obtained with 42mer mRNA analogs containing stop codons (UAG, UAA or UAG) or a sense codon (UCA) in presence of C-terminally His-tagged human wild-type eRF1 (wt) or mutated human eRF1 containing either S64D or I35V-L126F substitutions. After irradiation, the reaction products were separated onto a 10% SDS–polyacrylamide gel and analyzed by autoradiography. A control reaction without eRF1 (No eRF1) is shown for each mRNA analog. The 68 kDa band corresponding to the eRF1–mRNA cross-link is indicated by an arrow. Molecular mass markers in kDa are indicated on the left.

Techniques Used: Irradiation, Autoradiography

Fig. 6. Schematic representation of plasmid pET21b derivatives expressing C-terminally His-tagged eRF1 from human (pET-Hs-eRF1-His 6 ) or Euplotes (pET-Eu-eRF1-His 6 ) under the control of the T7 promoter (T7p). The restrictions sites used for the construction of the recombinant eRF1s are indicated. The restriction sites indicated under pET-Hs-eRF1-His 6 were introduced by site-directed mutagenesis.
Figure Legend Snippet: Fig. 6. Schematic representation of plasmid pET21b derivatives expressing C-terminally His-tagged eRF1 from human (pET-Hs-eRF1-His 6 ) or Euplotes (pET-Eu-eRF1-His 6 ) under the control of the T7 promoter (T7p). The restrictions sites used for the construction of the recombinant eRF1s are indicated. The restriction sites indicated under pET-Hs-eRF1-His 6 were introduced by site-directed mutagenesis.

Techniques Used: Plasmid Preparation, Expressing, Positron Emission Tomography, Recombinant, Mutagenesis

Fig. 5. The structure of eRF1. ( A ) Crystal structure of human eRF1 with the ribbon representation of the secondary structure. Major domains and secondary structure elements are labeled. Functionally important motifs are indicated by an arrow with the one letter amino acid code (GGQ and NIKS). In domain 1, the different colors indicate the regions of human eRF1 substituted by regions from Euplotes eRF1: amino acids 1–34 are colored pink, amino acids 35–51 are in dark blue, amino acids 52–68 are in green, amino acids 69–94 are in yellow and amino acids 95–145 are in red. Domain 2 is colored light blue and domain 3 is in gray. The coordinate data were obtained from the Protein Data Bank (accession code ccss 1TD9). ( B ) Enlargement of domain 1. ( C ) Linear representation of domain 1 (residues 1–145). The regions of human eRF1 swapped for Euplotes eRF1 are represented in cylinders (α-helices) and large arrows (β-strands) using the same colors as in (A). The positions of the junctions (35, 52, 68, 94) are indicated.
Figure Legend Snippet: Fig. 5. The structure of eRF1. ( A ) Crystal structure of human eRF1 with the ribbon representation of the secondary structure. Major domains and secondary structure elements are labeled. Functionally important motifs are indicated by an arrow with the one letter amino acid code (GGQ and NIKS). In domain 1, the different colors indicate the regions of human eRF1 substituted by regions from Euplotes eRF1: amino acids 1–34 are colored pink, amino acids 35–51 are in dark blue, amino acids 52–68 are in green, amino acids 69–94 are in yellow and amino acids 95–145 are in red. Domain 2 is colored light blue and domain 3 is in gray. The coordinate data were obtained from the Protein Data Bank (accession code ccss 1TD9). ( B ) Enlargement of domain 1. ( C ) Linear representation of domain 1 (residues 1–145). The regions of human eRF1 swapped for Euplotes eRF1 are represented in cylinders (α-helices) and large arrows (β-strands) using the same colors as in (A). The positions of the junctions (35, 52, 68, 94) are indicated.

Techniques Used: Labeling

Fig. 3. Comparison of the cross-linking pattern of human eRF1 (Hs-eRF1) with recombinant Eu-eRF1(1–224). Eu-eRF1(1–224) contains residues 1–224 from E.aediculatus eRF1 and residues 225–435 from human eRF1. ( A ) Schematic representation of the amino acid sequences of Hs-eRF1 and recombinant Eu-eRF1(1–224). The approximate locations of the NIKS (domain 1) and GGQ (domain 2) motifs are indicated. The region of Euplotes eRF1 in Eu-eRF1(1–224) is shaded in light gray. ( B ) Cross-linking patterns of 42mer mRNA analogs containing UGA, UAA, UAG, UCA or UGG codons (as indicated below the autoradiogram) in the presence of Hs-eRF1 or Eu-eRF1(1–224) as indicated above the autoradiograms. The cross-linking pattern of the UGA mRNA analog in the absence of eRF1 is shown in lane 0. The irradiated reactions were analyzed by 7.5% SDS–PAGE. The region containing the eRF1–mRNA cross-links is boxed with broken line. ( C ) Enlargement views of regions boxed with broken lines in (B). Cross-links between mRNA analogs containing the canonical stop (UGA, UAA, UAG) or sense (UGG and UCA) codons as indicated below the autoradiograms and Hs-eRF1 (upper panel) or recombinant Eu-eRF1(1–224) (lower panel). The cross-linking pattern of the UGA mRNA analog in the absence of eRF1 is shown in lane 0. An asterisk indicates a Hs-eRF1–mRNA cross-link and a hash symbol indicates a Eu-eRF1(1–224)–mRNA cross-link.
Figure Legend Snippet: Fig. 3. Comparison of the cross-linking pattern of human eRF1 (Hs-eRF1) with recombinant Eu-eRF1(1–224). Eu-eRF1(1–224) contains residues 1–224 from E.aediculatus eRF1 and residues 225–435 from human eRF1. ( A ) Schematic representation of the amino acid sequences of Hs-eRF1 and recombinant Eu-eRF1(1–224). The approximate locations of the NIKS (domain 1) and GGQ (domain 2) motifs are indicated. The region of Euplotes eRF1 in Eu-eRF1(1–224) is shaded in light gray. ( B ) Cross-linking patterns of 42mer mRNA analogs containing UGA, UAA, UAG, UCA or UGG codons (as indicated below the autoradiogram) in the presence of Hs-eRF1 or Eu-eRF1(1–224) as indicated above the autoradiograms. The cross-linking pattern of the UGA mRNA analog in the absence of eRF1 is shown in lane 0. The irradiated reactions were analyzed by 7.5% SDS–PAGE. The region containing the eRF1–mRNA cross-links is boxed with broken line. ( C ) Enlargement views of regions boxed with broken lines in (B). Cross-links between mRNA analogs containing the canonical stop (UGA, UAA, UAG) or sense (UGG and UCA) codons as indicated below the autoradiograms and Hs-eRF1 (upper panel) or recombinant Eu-eRF1(1–224) (lower panel). The cross-linking pattern of the UGA mRNA analog in the absence of eRF1 is shown in lane 0. An asterisk indicates a Hs-eRF1–mRNA cross-link and a hash symbol indicates a Eu-eRF1(1–224)–mRNA cross-link.

Techniques Used: Recombinant, Irradiation, SDS Page

13) Product Images from "Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29 ▿Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29 ▿ †"

Article Title: Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29 ▿Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29 ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01210-10

RP and CP form salt-resistant complexes in not4Δ cells. Total cellular extracts from wild-type or not4Δ cells expressing Rpn11-ProtA (for RP purification) and Pre1-ProtA (for CP purification) were incubated with IgG beads. After the beads were washed with a high-salt buffer, RP and CP were eluted from the column by TEV cleavage (see Materials and Methods) and analyzed by SDS-PAGE (A) or by immunoblotting (B) for the presence of Rpt1 or CP subunits (α7 or α1–7). Numbers to the left are molecular masses (in kilodaltons). The asterisk in panel B marks a nonspecific band. (C) The same samples were analyzed on a 3.5% native gel and either stained with Coomassie blue (left), tested for activity (middle), or analyzed for the presence of Rpt1 and α1–7 by immunoblotting (two right-most blots). (D) Rpn11-ProtA was affinity purified in a high concentration of salt (0.5 M) from the not4Δ mutant expressing the indicated Myc-Not4 derivatives. Purified proteins were then separated on a 3.5% native gel and either tested for activity or transferred to nitrocellulose, probed with Ponceau S, and analyzed for the presence of Rpt1 or α1–7. The same purified proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against Rpt1 or α1–7 (bottom). (E) In vitro reconstitution of proteasomes. Separately purified RP and CP from wild-type and not4Δ cells (the same as those used for panel A) were mixed in the following combinations: wild-type RP (lane 1), wild-type RP plus wild-type CP (lane 2), wild-type RP plus CP not4Δ (lane 3), RP not4Δ plus wild-type CP (lane 4), wild-type CP (lane 5), RP not4Δ plus CP not4Δ (lane 6), CP not4Δ (lane 7), and RP not4Δ (lane 8). Samples were incubated at 30°C for 30 min, loaded onto a 3.5% native gel, and tested for activity or immunoblotted with anti-Rpt1, α1–7, or Rpn5 antibodies.
Figure Legend Snippet: RP and CP form salt-resistant complexes in not4Δ cells. Total cellular extracts from wild-type or not4Δ cells expressing Rpn11-ProtA (for RP purification) and Pre1-ProtA (for CP purification) were incubated with IgG beads. After the beads were washed with a high-salt buffer, RP and CP were eluted from the column by TEV cleavage (see Materials and Methods) and analyzed by SDS-PAGE (A) or by immunoblotting (B) for the presence of Rpt1 or CP subunits (α7 or α1–7). Numbers to the left are molecular masses (in kilodaltons). The asterisk in panel B marks a nonspecific band. (C) The same samples were analyzed on a 3.5% native gel and either stained with Coomassie blue (left), tested for activity (middle), or analyzed for the presence of Rpt1 and α1–7 by immunoblotting (two right-most blots). (D) Rpn11-ProtA was affinity purified in a high concentration of salt (0.5 M) from the not4Δ mutant expressing the indicated Myc-Not4 derivatives. Purified proteins were then separated on a 3.5% native gel and either tested for activity or transferred to nitrocellulose, probed with Ponceau S, and analyzed for the presence of Rpt1 or α1–7. The same purified proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against Rpt1 or α1–7 (bottom). (E) In vitro reconstitution of proteasomes. Separately purified RP and CP from wild-type and not4Δ cells (the same as those used for panel A) were mixed in the following combinations: wild-type RP (lane 1), wild-type RP plus wild-type CP (lane 2), wild-type RP plus CP not4Δ (lane 3), RP not4Δ plus wild-type CP (lane 4), wild-type CP (lane 5), RP not4Δ plus CP not4Δ (lane 6), CP not4Δ (lane 7), and RP not4Δ (lane 8). Samples were incubated at 30°C for 30 min, loaded onto a 3.5% native gel, and tested for activity or immunoblotted with anti-Rpt1, α1–7, or Rpn5 antibodies.

Techniques Used: Expressing, Purification, Incubation, SDS Page, Staining, Activity Assay, Affinity Purification, Concentration Assay, Mutagenesis, In Vitro

14) 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

15) Product Images from "Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1"

Article Title: Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1

Journal: EMBO Reports

doi: 10.1093/embo-reports/kvf178

Fig. 1. ( A ) by the WebLab ViewerLite program version 4.0 (Molecular Simulations). ( B ) Alignments of the eRF1 and aRF1 amino acid sequences for the NIKS and Y–C–F minidomains. Positions are numbered for human eRF1. Accession numbers in brackets are from the NCBI-Entrez-Proteins database. Amino acids are shaded according to their identity percentage (white letters, black shading, –95%; white letters, dark grey shading, –85%; black letters, light grey shading, –35%). Variant-code ciliates are in bold.
Figure Legend Snippet: Fig. 1. ( A ) by the WebLab ViewerLite program version 4.0 (Molecular Simulations). ( B ) Alignments of the eRF1 and aRF1 amino acid sequences for the NIKS and Y–C–F minidomains. Positions are numbered for human eRF1. Accession numbers in brackets are from the NCBI-Entrez-Proteins database. Amino acids are shaded according to their identity percentage (white letters, black shading, –95%; white letters, dark grey shading, –85%; black letters, light grey shading, –35%). Variant-code ciliates are in bold.

Techniques Used: Variant Assay

16) 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

17) Product Images from "Saccharomyces cerevisiae-Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography"

Article Title: Saccharomyces cerevisiae-Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076851

Purification of the Ptr2 transporter. Membranes from a 1 litre shake culture were solubilised in DDM and applied to a Ni-NTA column. Ptr2-GFP-His fusions were eluted from the column and digested with TEV protease. Purification of Ptr2 and removal of the released GFP-8His tag was carried out by reverse IMAC. Samples from the 3-step procedure were analysed by SDS-PAGE in a 10% gel. (A) Detection by in-gel fluorescence. (B) Detection by Coomassie staining. Lane 1: Ni-NTA elution. Lane 2: Cleavage by TEV protease. Lane 3: Flow-through of reverse Ni-NTA chromatography after cleavage with TEV protease. M: PageRuler prestained protein ladder (Fermentas). Positions of the Ptr2-GFP-His fusion, Ptr2 transporter, cleaved off GFP-8His and the TEV protease are indicated.
Figure Legend Snippet: Purification of the Ptr2 transporter. Membranes from a 1 litre shake culture were solubilised in DDM and applied to a Ni-NTA column. Ptr2-GFP-His fusions were eluted from the column and digested with TEV protease. Purification of Ptr2 and removal of the released GFP-8His tag was carried out by reverse IMAC. Samples from the 3-step procedure were analysed by SDS-PAGE in a 10% gel. (A) Detection by in-gel fluorescence. (B) Detection by Coomassie staining. Lane 1: Ni-NTA elution. Lane 2: Cleavage by TEV protease. Lane 3: Flow-through of reverse Ni-NTA chromatography after cleavage with TEV protease. M: PageRuler prestained protein ladder (Fermentas). Positions of the Ptr2-GFP-His fusion, Ptr2 transporter, cleaved off GFP-8His and the TEV protease are indicated.

Techniques Used: Purification, SDS Page, Fluorescence, Staining, Flow Cytometry, Chromatography

TEV protease digests of Ni-NTA purified transporter- and transceptor GFP-fusions. TEV protease digestions of the indicated GFP-fusions purified by Ni-NTA affinity chromatography. Digestions were analyzed by SDS-PAGE using in-gel fluorescence to visualize the GFP-fusions and the released GFP-8His. (−) undigested; (+) digested with TEV protease. Released GFP-8His is indicated with an arrow.
Figure Legend Snippet: TEV protease digests of Ni-NTA purified transporter- and transceptor GFP-fusions. TEV protease digestions of the indicated GFP-fusions purified by Ni-NTA affinity chromatography. Digestions were analyzed by SDS-PAGE using in-gel fluorescence to visualize the GFP-fusions and the released GFP-8His. (−) undigested; (+) digested with TEV protease. Released GFP-8His is indicated with an arrow.

Techniques Used: Purification, Affinity Chromatography, SDS Page, Fluorescence

18) Product Images from "The major thylakoid protein kinases STN7 and STN8 revisited: effects of altered STN8 levels and regulatory specificities of the STN kinases"

Article Title: The major thylakoid protein kinases STN7 and STN8 revisited: effects of altered STN8 levels and regulatory specificities of the STN kinases

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2013.00417

Redox sensitivity of STN7. (A) Redox titration of STN7 variants. Thylakoids of WT (Col-0) and STN7 C→S:65 + 70 were equilibrated at various redox potentials using the DTTred/DTTox redox couple. After separation by non-reducing SDS-PAGE, STN7 was detected by immunoblotting. DTTred/DTTox ratios are indicated above each lane. Oxidized (ox) and reduced (red) forms of the STN7 monomer are indicated by black arrowheads. (B) STN7—TRX-f interaction assayed by thioredoxin affinity chromatography. His-tagged recombinant thioredoxins f and m, with one cysteine replaced by serine (recΔ TRX-f, -m), were bound to Ni-NTA and incubated with thylakoids solubilized with 1.5% digitonin corresponding to 2 mg of Chl. After several washes, the eluates from recΔ TRX-f and -m resins were subjected to Western blot analysis using STN7-specific antibodies. Solubilized WT (Col-0) thylakoids (input; corresponding to 10 μg of Chl), the flow through off the recΔ TRX-f resin (flow; equivalent to 10 μg Chl), washes 1–3 of the recΔ TRX-f resin and the eluate from a similarly treated Ni-NTA resin not coupled to recΔ TRX (mock elution) were loaded as controls. (C) TRX-f mobility shift assay. Thylakoid membranes (10 μg Chl) from WT (Col-0), STN7 C→S:65 + 70 and stn7-1 mutants were solubilized with 0.2% DOC and incubated with either 25 μg of recΔ TRX-f or without additives. Proteins were separated by non-reducing SDS-PAGE and Western analysis was performed using STN7-specific antibodies.
Figure Legend Snippet: Redox sensitivity of STN7. (A) Redox titration of STN7 variants. Thylakoids of WT (Col-0) and STN7 C→S:65 + 70 were equilibrated at various redox potentials using the DTTred/DTTox redox couple. After separation by non-reducing SDS-PAGE, STN7 was detected by immunoblotting. DTTred/DTTox ratios are indicated above each lane. Oxidized (ox) and reduced (red) forms of the STN7 monomer are indicated by black arrowheads. (B) STN7—TRX-f interaction assayed by thioredoxin affinity chromatography. His-tagged recombinant thioredoxins f and m, with one cysteine replaced by serine (recΔ TRX-f, -m), were bound to Ni-NTA and incubated with thylakoids solubilized with 1.5% digitonin corresponding to 2 mg of Chl. After several washes, the eluates from recΔ TRX-f and -m resins were subjected to Western blot analysis using STN7-specific antibodies. Solubilized WT (Col-0) thylakoids (input; corresponding to 10 μg of Chl), the flow through off the recΔ TRX-f resin (flow; equivalent to 10 μg Chl), washes 1–3 of the recΔ TRX-f resin and the eluate from a similarly treated Ni-NTA resin not coupled to recΔ TRX (mock elution) were loaded as controls. (C) TRX-f mobility shift assay. Thylakoid membranes (10 μg Chl) from WT (Col-0), STN7 C→S:65 + 70 and stn7-1 mutants were solubilized with 0.2% DOC and incubated with either 25 μg of recΔ TRX-f or without additives. Proteins were separated by non-reducing SDS-PAGE and Western analysis was performed using STN7-specific antibodies.

Techniques Used: Titration, SDS Page, Affinity Chromatography, Recombinant, Incubation, Western Blot, Flow Cytometry, Mobility Shift

19) Product Images from "Selenocysteine insertion directed by the 3?-UTR SECIS element in Escherichia coli"

Article Title: Selenocysteine insertion directed by the 3?-UTR SECIS element in Escherichia coli

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki547

Opal suppression is a major readthrough event. ( A ) SDS–PAGE analysis of the recombinant GPx4 expressed from the construct carrying the functional 3′-UTR SECIS element. Extracts prepared from BL21(DE3) cells carrying pET21b and pSUABC (lane 1) and the GPx4 construct with the functional SECIS element and pSUABC (lane 2) were applied to a His-tag affinity column and proteins bound to the column were eluted and analyzed by SDS–PAGE. ( B ) Mass spectrometry analysis of the readthrough product. The recombinant protein shown in (A) was cut from the SDS–PAGE gel, digested with trypsin, alkylated with iodoacetamide and subjected to tandem mass spectrometry sequencing. A double-charged fragment at 833.4 m / z was identified, which corresponded to a tryptophan-containing readthrough fragment. The ms/ms spectrum of the 833.4 parent ion is shown and the major daughter ions and the parent ion are labeled. We identified these peaks as Y, B or A ions (not shown in this figure) resulting from fragmentation of the sequence GFVCIVTNVASQWGK. The black letters above each peak indicate the amino acid sequence of the corresponding daughter ion and the red letters indicate the amino acids lost in the fragmentation.
Figure Legend Snippet: Opal suppression is a major readthrough event. ( A ) SDS–PAGE analysis of the recombinant GPx4 expressed from the construct carrying the functional 3′-UTR SECIS element. Extracts prepared from BL21(DE3) cells carrying pET21b and pSUABC (lane 1) and the GPx4 construct with the functional SECIS element and pSUABC (lane 2) were applied to a His-tag affinity column and proteins bound to the column were eluted and analyzed by SDS–PAGE. ( B ) Mass spectrometry analysis of the readthrough product. The recombinant protein shown in (A) was cut from the SDS–PAGE gel, digested with trypsin, alkylated with iodoacetamide and subjected to tandem mass spectrometry sequencing. A double-charged fragment at 833.4 m / z was identified, which corresponded to a tryptophan-containing readthrough fragment. The ms/ms spectrum of the 833.4 parent ion is shown and the major daughter ions and the parent ion are labeled. We identified these peaks as Y, B or A ions (not shown in this figure) resulting from fragmentation of the sequence GFVCIVTNVASQWGK. The black letters above each peak indicate the amino acid sequence of the corresponding daughter ion and the red letters indicate the amino acids lost in the fragmentation.

Techniques Used: SDS Page, Recombinant, Construct, Functional Assay, Affinity Column, Mass Spectrometry, Sequencing, Labeling

20) Product Images from "Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1"

Article Title: Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066603

CD spectra of Bt-CBP21 and Cry1Ac toxin. Purified Bt-CBP21 and Cry1Ac (150 µg) were dissolved in 10 mM sodium-phosphate buffer, pH 7.0. Only Bt-CBP21 (Green curve), only Cry1Ac (Blue curve) and mixture of both the proteins (1∶1) (Brown curve) was analysed by CD spectra at 24°C. Samples were scanned five times at 50 nm/min with bandwidth of 1 nm and response time of 0.5 sec in a spectrophotometer (J-800, Jasco). An overlay of each spectrum is shown and spectrum of ‘buffer alone’ served as control. All measurements were conducted twice.
Figure Legend Snippet: CD spectra of Bt-CBP21 and Cry1Ac toxin. Purified Bt-CBP21 and Cry1Ac (150 µg) were dissolved in 10 mM sodium-phosphate buffer, pH 7.0. Only Bt-CBP21 (Green curve), only Cry1Ac (Blue curve) and mixture of both the proteins (1∶1) (Brown curve) was analysed by CD spectra at 24°C. Samples were scanned five times at 50 nm/min with bandwidth of 1 nm and response time of 0.5 sec in a spectrophotometer (J-800, Jasco). An overlay of each spectrum is shown and spectrum of ‘buffer alone’ served as control. All measurements were conducted twice.

Techniques Used: Purification, Size-exclusion Chromatography, Spectrophotometry

In vitro pull down assay of Cry1Ac and Bt-CBP21. Reaction mixture (100 µl) containing purified Cry1Ac protoxin (40 µg) and purified Bt-CBP21 (30 µg) were mixed with 1X binding buffer and incubated for 1 h at 25°C, as described in Materials and methods . Unbound proteins were removed upon centrifugation. The protein mixture was incubated with Ni-NTA matrix for 30 min at 4°C. The resin was washed with 1X binding buffer and bound proteins were eluted with 1X binding buffer containing 300 mM imidazole. All fractions were resolved on 10% SDS-PAGE. Lane M: Protein molecular marker (in kDa), Lane 1: Purified Cry1Ac, Lane 2: Purified Bt-CBP21, Lane 3: Flowthrough, Lanes 4–6: Wash fractions, Lane 7: elution fraction (300 mM Imidazole).
Figure Legend Snippet: In vitro pull down assay of Cry1Ac and Bt-CBP21. Reaction mixture (100 µl) containing purified Cry1Ac protoxin (40 µg) and purified Bt-CBP21 (30 µg) were mixed with 1X binding buffer and incubated for 1 h at 25°C, as described in Materials and methods . Unbound proteins were removed upon centrifugation. The protein mixture was incubated with Ni-NTA matrix for 30 min at 4°C. The resin was washed with 1X binding buffer and bound proteins were eluted with 1X binding buffer containing 300 mM imidazole. All fractions were resolved on 10% SDS-PAGE. Lane M: Protein molecular marker (in kDa), Lane 1: Purified Cry1Ac, Lane 2: Purified Bt-CBP21, Lane 3: Flowthrough, Lanes 4–6: Wash fractions, Lane 7: elution fraction (300 mM Imidazole).

Techniques Used: In Vitro, Pull Down Assay, Purification, Binding Assay, Incubation, Centrifugation, SDS Page, Marker

Binding of Cry1Ac protoxin to colloidal chitin. Cry1Ac protoxin was analyzed for its affinity towards colloidal chitin. Protoxin purified on Renograffin gradient was solubilized with 50 mM sodium-carbonate buffer, pH 10.5, passed through PD10 desalting column and bound proteins were eluted with 50 mM sodium-acetate pH 5.5; 50 mM Tris-HCl, pH 7.5 and 50 mM sodium-carbonate, pH 10.5. Proteins were incubated with colloidal chitin isolated from crab shells and pupae caracasses of H. armigera . Chitin bound proteins were recovered by boiling the matrix in SDS-sample buffer. All fractions were resolved on 10% SDS-PAGE. Lane M: Protein standard marker (in kilodaltons), Lane 1: Loaded sample, Lanes 2 and 3: Flowthrough and elution fractions at pH 10.5, Lanes 4 and 5: Flowthrough and elution fractions at pH 7.5, Lanes 6 and 7: Flowthrough and elution fractions at pH 5.5.
Figure Legend Snippet: Binding of Cry1Ac protoxin to colloidal chitin. Cry1Ac protoxin was analyzed for its affinity towards colloidal chitin. Protoxin purified on Renograffin gradient was solubilized with 50 mM sodium-carbonate buffer, pH 10.5, passed through PD10 desalting column and bound proteins were eluted with 50 mM sodium-acetate pH 5.5; 50 mM Tris-HCl, pH 7.5 and 50 mM sodium-carbonate, pH 10.5. Proteins were incubated with colloidal chitin isolated from crab shells and pupae caracasses of H. armigera . Chitin bound proteins were recovered by boiling the matrix in SDS-sample buffer. All fractions were resolved on 10% SDS-PAGE. Lane M: Protein standard marker (in kilodaltons), Lane 1: Loaded sample, Lanes 2 and 3: Flowthrough and elution fractions at pH 10.5, Lanes 4 and 5: Flowthrough and elution fractions at pH 7.5, Lanes 6 and 7: Flowthrough and elution fractions at pH 5.5.

Techniques Used: Binding Assay, Purification, Incubation, Isolation, SDS Page, Marker

21) 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

22) Product Images from "Annexin5 Plays a Vital Role in Arabidopsis Pollen Development via Ca2+-Dependent Membrane Trafficking"

Article Title: Annexin5 Plays a Vital Role in Arabidopsis Pollen Development via Ca2+-Dependent Membrane Trafficking

Journal: PLoS ONE

doi: 10.1371/journal.pone.0102407

Ann5 binds to negatively charged phospholipids and this association is stimulated by Ca 2+ . (A) Multiple sequence alignment of the deduced amino acid sequences of repeats I and IV from Arabidopsis thaliana AnnAt1 to 8. Ca 2+ -binding sites of type II GXGTD-(37 residues)-E/D are indicated by blue and red shadows. The residues with red asterisks indicate the locations of PCR-based site-directed mutagenesis. The mutants of repeats I and IV were named Ann5G26EG28E and Ann5G257EG259E, respectively, and the conserved glycine residues were replaced by glutamic acid residues. (B) Predicted three-dimensional structure of the Ann5 protein. The annexin core domain was composed of four homologous repeats that are colored in red (repeat I), green (repeat II), yellow (repeat III) and blue (repeat IV) and shaped as a slightly curved disc. The convex surface on which calcium ions bind (magenta spheres) participates in peripheral membrane binding. The type II Ca 2+ -binding sites are labeled with sticks, and the ribbon illustrates the highly α-helical structure. (C) Phospholipid-binding properties of the recombinant His6-Ann5, His6-Ann5G26EG28E, His6-Ann5G257EG259E and His6-Ann5G26EG28EG257EG259E proteins. The individual protein (50 µg) was incubated with liposomes (1∶1 PC/PS) in the presence of increasing Ca 2+ concentrations. (-) denotes that the reaction mixtures contained neither liposomes nor Ca 2+ . (D) Comparison of the Ca 2+ -dependent phospholipid binding abilities of Ann5 and its mutants using densitometry analysis of the signal intensities of blots as described in (C). The phospholipid binding amount of Ann5 in 200 µM Ca 2+ is normalized as 1 (control). The relative intensities are displayed as fold-binding over the control. Values represent mean ± SD (n = 6).
Figure Legend Snippet: Ann5 binds to negatively charged phospholipids and this association is stimulated by Ca 2+ . (A) Multiple sequence alignment of the deduced amino acid sequences of repeats I and IV from Arabidopsis thaliana AnnAt1 to 8. Ca 2+ -binding sites of type II GXGTD-(37 residues)-E/D are indicated by blue and red shadows. The residues with red asterisks indicate the locations of PCR-based site-directed mutagenesis. The mutants of repeats I and IV were named Ann5G26EG28E and Ann5G257EG259E, respectively, and the conserved glycine residues were replaced by glutamic acid residues. (B) Predicted three-dimensional structure of the Ann5 protein. The annexin core domain was composed of four homologous repeats that are colored in red (repeat I), green (repeat II), yellow (repeat III) and blue (repeat IV) and shaped as a slightly curved disc. The convex surface on which calcium ions bind (magenta spheres) participates in peripheral membrane binding. The type II Ca 2+ -binding sites are labeled with sticks, and the ribbon illustrates the highly α-helical structure. (C) Phospholipid-binding properties of the recombinant His6-Ann5, His6-Ann5G26EG28E, His6-Ann5G257EG259E and His6-Ann5G26EG28EG257EG259E proteins. The individual protein (50 µg) was incubated with liposomes (1∶1 PC/PS) in the presence of increasing Ca 2+ concentrations. (-) denotes that the reaction mixtures contained neither liposomes nor Ca 2+ . (D) Comparison of the Ca 2+ -dependent phospholipid binding abilities of Ann5 and its mutants using densitometry analysis of the signal intensities of blots as described in (C). The phospholipid binding amount of Ann5 in 200 µM Ca 2+ is normalized as 1 (control). The relative intensities are displayed as fold-binding over the control. Values represent mean ± SD (n = 6).

Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction, Mutagenesis, Labeling, Recombinant, Incubation

23) Product Images from "Geminin is partially localized to the centrosome and plays a role in proper centrosome duplication"

Article Title: Geminin is partially localized to the centrosome and plays a role in proper centrosome duplication

Journal: Biology of the Cell

doi: 10.1042/BC20080109

Disruption of the dynein–dynactin complex by overexpressed exogenous dynamitin/p50 impairs both geminin and Arp1 centrosomal localization GFP–dynamitin/p50 was introduced into HeLa cells using Lipofectamine™ 2000. At 24 h after transfection, the cells were fixed with ice-cold methanol and examined by immunofluorescence staining using antibodies against geminin and Arp1. Transfection of GFP was taken as a negative control. In GFP-expressing control HeLa cells, the centrosomal/spindle pole localization of both geminin and Arp1 was not affected, as indicated in the upper panels of ( A ) and ( B ). Similar results were obtained in low-level dynamitin/p50-expressing cells, in which both geminin and Arp1 were normally localized on centrosome/spindle poles [middle panels in ( A ) and ( B )]. High expression of dynamitin/p50 in HeLa cells did impair centrosomal localization of both geminin and Arp1, as indicated in the bottom panels of ( A ) and ( B ). Scale bar=10 μm.
Figure Legend Snippet: Disruption of the dynein–dynactin complex by overexpressed exogenous dynamitin/p50 impairs both geminin and Arp1 centrosomal localization GFP–dynamitin/p50 was introduced into HeLa cells using Lipofectamine™ 2000. At 24 h after transfection, the cells were fixed with ice-cold methanol and examined by immunofluorescence staining using antibodies against geminin and Arp1. Transfection of GFP was taken as a negative control. In GFP-expressing control HeLa cells, the centrosomal/spindle pole localization of both geminin and Arp1 was not affected, as indicated in the upper panels of ( A ) and ( B ). Similar results were obtained in low-level dynamitin/p50-expressing cells, in which both geminin and Arp1 were normally localized on centrosome/spindle poles [middle panels in ( A ) and ( B )]. High expression of dynamitin/p50 in HeLa cells did impair centrosomal localization of both geminin and Arp1, as indicated in the bottom panels of ( A ) and ( B ). Scale bar=10 μm.

Techniques Used: Transfection, Immunofluorescence, Staining, Negative Control, Expressing

Geminin regulates centrosome duplication in MDA-MB-231 cells ( A ) siRNA of geminin resulted in centrosome over-duplication in MDA-MB-231 cells. Cells were transfected with siRNA targeting geminin (Gem siRNA) or non-specific siRNA as a control. Immunofluorescence staining of γ-tubulin (red) showed that the centrosomes were over-duplicated in geminin siRNA cells. Scale bar=10 μm. The cells with supernumerary centrosomes were counted and are shown in ( B ). The efficiency of geminin-knockdown was detected by Western blot analysis and is shown on the top right panel in ( B ). ( C ) Centrosomes were over-duplicated after being treated with HU in MDA-MB-231 cells. MDA-MB-231 cells were treated with/without 4 mM HU for 48 h and then immunofluorescence staining of γ-tubulin was performed to observe centrosomes. Scale bar=10 μm. ( D and E ) The coiled-coil motif is required for geminin to inhibit centrosome over-duplication in HU-arrested MDA-MB-231 cells. MDA-MB-231 cells were treated with 4 mM HU for 12 h and then transfected with geminin and its mutants as a GFP-fusion protein and cultured for another 48 h in the presence of HU to maintain the cells in S-phase. Cells were then either processed for immunofluorescence staining of γ-tubulin to observe centrosomes or Western blotted using anti-geminin or anti-GFP antibodies to exam the level of GFP-fusion proteins of geminin and its mutants. Note that over-duplication of centrosomes induced by HU was inhibited by overexpression of mutant 94–160, which contains the coiled-coil motif, as well as wild-type geminin. These results indicated that the coiled-coil motif is required for geminin to inhibit centrosome over-duplication. Supernumerary centrosomes were counted ( E ) and error bars represent 1 S.D.
Figure Legend Snippet: Geminin regulates centrosome duplication in MDA-MB-231 cells ( A ) siRNA of geminin resulted in centrosome over-duplication in MDA-MB-231 cells. Cells were transfected with siRNA targeting geminin (Gem siRNA) or non-specific siRNA as a control. Immunofluorescence staining of γ-tubulin (red) showed that the centrosomes were over-duplicated in geminin siRNA cells. Scale bar=10 μm. The cells with supernumerary centrosomes were counted and are shown in ( B ). The efficiency of geminin-knockdown was detected by Western blot analysis and is shown on the top right panel in ( B ). ( C ) Centrosomes were over-duplicated after being treated with HU in MDA-MB-231 cells. MDA-MB-231 cells were treated with/without 4 mM HU for 48 h and then immunofluorescence staining of γ-tubulin was performed to observe centrosomes. Scale bar=10 μm. ( D and E ) The coiled-coil motif is required for geminin to inhibit centrosome over-duplication in HU-arrested MDA-MB-231 cells. MDA-MB-231 cells were treated with 4 mM HU for 12 h and then transfected with geminin and its mutants as a GFP-fusion protein and cultured for another 48 h in the presence of HU to maintain the cells in S-phase. Cells were then either processed for immunofluorescence staining of γ-tubulin to observe centrosomes or Western blotted using anti-geminin or anti-GFP antibodies to exam the level of GFP-fusion proteins of geminin and its mutants. Note that over-duplication of centrosomes induced by HU was inhibited by overexpression of mutant 94–160, which contains the coiled-coil motif, as well as wild-type geminin. These results indicated that the coiled-coil motif is required for geminin to inhibit centrosome over-duplication. Supernumerary centrosomes were counted ( E ) and error bars represent 1 S.D.

Techniques Used: Multiple Displacement Amplification, Transfection, Immunofluorescence, Staining, Western Blot, Cell Culture, Over Expression, Mutagenesis

The coiled-coil motif is required for geminin centrosomal localization and interaction with Arp1 ( A ) Schematic diagrams of geminin and its truncated mutants. ( B ) The coiled-coil motif is required for geminin to interact with Arp1. For in vitro pulldown assays, four mutants of GST–geminin were purified and coupled on to glutathione–Sepharose 4B beads and incubated with the lysate of HeLa cells for 2 h. The proteins on the beads were separated on SDS/PAGE (10% gel) and subjected to Western blot analysis using antibodies against Arp1. Cdt1 was immunoprobed as a positive control. Coomassie Blue staining of the proteins represents the protein loading in the pulldown assay (upper panel). The molecular mass in kDa is indicated on the left-hand side of the gel. ( C ) The coiled-coil motif is required for centrosomal localization of geminin. HeLa cells were transfected with geminin or its mutants in the form of GFP-fusion proteins, and subjected to an ATP-inhibitor assay as described in the Materials and methods section. Only mutant 94–160 which contains the coiled-coil motif of geminin presents centrosomal enrichment in a similar manner to wild-type geminin. γ-Tubulin (γ-tub; red) was immunostained as a marker of centrosomes. Scale bar=10 μm.
Figure Legend Snippet: The coiled-coil motif is required for geminin centrosomal localization and interaction with Arp1 ( A ) Schematic diagrams of geminin and its truncated mutants. ( B ) The coiled-coil motif is required for geminin to interact with Arp1. For in vitro pulldown assays, four mutants of GST–geminin were purified and coupled on to glutathione–Sepharose 4B beads and incubated with the lysate of HeLa cells for 2 h. The proteins on the beads were separated on SDS/PAGE (10% gel) and subjected to Western blot analysis using antibodies against Arp1. Cdt1 was immunoprobed as a positive control. Coomassie Blue staining of the proteins represents the protein loading in the pulldown assay (upper panel). The molecular mass in kDa is indicated on the left-hand side of the gel. ( C ) The coiled-coil motif is required for centrosomal localization of geminin. HeLa cells were transfected with geminin or its mutants in the form of GFP-fusion proteins, and subjected to an ATP-inhibitor assay as described in the Materials and methods section. Only mutant 94–160 which contains the coiled-coil motif of geminin presents centrosomal enrichment in a similar manner to wild-type geminin. γ-Tubulin (γ-tub; red) was immunostained as a marker of centrosomes. Scale bar=10 μm.

Techniques Used: In Vitro, Purification, Incubation, SDS Page, Western Blot, Positive Control, Staining, Transfection, Mutagenesis, Marker

Cell-cycle-dependent centrosomal localization of geminin ( A ) The centrosomal localization of geminin is accompanied with the progression of the cell cycle. Synchronized HeLa cells were fixed in ice-cold methanol and subjected to immunofluorescence staining using anti-geminin and anti-γ-tubulin antibodies. Geminin (Gem) locates on to centrosomes from late G 1 - to M-phase, and disassociates from centrosomes in early G 1 -phase (indicated with arrowheads). Scale bar=10 μm. ( B ) Centrosomes purified from synchronized HeLa cells were analysed by Western blotting. The absence of geminin (Gem) in purified early G 1 -phase centrosomes correlated with the results in ( A ), that geminin released from centrosomes in early G 1 phase. γ-tub, γ-tubulin. ( C ) Synchronized HeLa cells in ( A ) and ( B ) were analysed by FACS. HeLa cells were arrested at late G 1 -phase by double-thymidine treatment. Cells were then released into fresh medium for 4, 10 and 16 h to enter S-, G 2 - and early G 1 -phase. M-phase cells were obtained by 24 h thymidine treatment followed by 12 h nocodazole treatment.
Figure Legend Snippet: Cell-cycle-dependent centrosomal localization of geminin ( A ) The centrosomal localization of geminin is accompanied with the progression of the cell cycle. Synchronized HeLa cells were fixed in ice-cold methanol and subjected to immunofluorescence staining using anti-geminin and anti-γ-tubulin antibodies. Geminin (Gem) locates on to centrosomes from late G 1 - to M-phase, and disassociates from centrosomes in early G 1 -phase (indicated with arrowheads). Scale bar=10 μm. ( B ) Centrosomes purified from synchronized HeLa cells were analysed by Western blotting. The absence of geminin (Gem) in purified early G 1 -phase centrosomes correlated with the results in ( A ), that geminin released from centrosomes in early G 1 phase. γ-tub, γ-tubulin. ( C ) Synchronized HeLa cells in ( A ) and ( B ) were analysed by FACS. HeLa cells were arrested at late G 1 -phase by double-thymidine treatment. Cells were then released into fresh medium for 4, 10 and 16 h to enter S-, G 2 - and early G 1 -phase. M-phase cells were obtained by 24 h thymidine treatment followed by 12 h nocodazole treatment.

Techniques Used: Immunofluorescence, Staining, Purification, Western Blot, FACS

Arp1 as a potential centrosomal protein co-localized with geminin ( A ) Lysate from HeLa cells expressing exogenous GFP–geminin was immunoprecipitated (IP) using an antibody against geminin or rabbit IgG (rIgG) as a control. The proteins co-immunoprecipitated with geminin were separated on SDS/PAGE (10% gel) and stained with Coomassie Blue R-250. The protein bands were subjected to MS analysis. The bands indicated with a circle are (from the bottom to the top): dynactin 1 (p150Glued), Cdt1, GFP–geminin and Arp1 respectively. The molecular mass in kDa is indicated on the left-hand side of the gel. ( B ) Arp1 co-stained with γ-tubulin (γ-tub) in HeLa cells. HeLa cells were fixed and subjected to immunofluorescence staining using antibodies against Arp1 and γ-tubulin. The results showed that a fraction of Arp1 is located on centrosomes and Arp1 was well co-localized with γ-tubulin (indicated with arrowheads). Scale bar=10 μm. ( C ) Components of purified centrosomes from HeLa cells were examined for the co-existence of geminin (Gem) and Arp1. Fractions of centrosomes isolated from a discontinuous sucrose gradient were separated using SDS/PAGE (10% gel) and immunoblotted using antibodies against geminin and Arp1. γ-Tubulin (γ-tub) was immunoblotted as a positive control for centrosome purification. ( D ) Co-localization of endogenous geminin (Gem) and Arp1 was viewed by immunofluorescence staining using antibodies against geminin and Arp1 in HeLa cells fixed with ice-cold methanol. Both geminin and Arp1 were apparently co-localized in the centrosome in interphase and spindle poles in mitosis (indicated with arrowheads). Scale bar=10 μm.
Figure Legend Snippet: Arp1 as a potential centrosomal protein co-localized with geminin ( A ) Lysate from HeLa cells expressing exogenous GFP–geminin was immunoprecipitated (IP) using an antibody against geminin or rabbit IgG (rIgG) as a control. The proteins co-immunoprecipitated with geminin were separated on SDS/PAGE (10% gel) and stained with Coomassie Blue R-250. The protein bands were subjected to MS analysis. The bands indicated with a circle are (from the bottom to the top): dynactin 1 (p150Glued), Cdt1, GFP–geminin and Arp1 respectively. The molecular mass in kDa is indicated on the left-hand side of the gel. ( B ) Arp1 co-stained with γ-tubulin (γ-tub) in HeLa cells. HeLa cells were fixed and subjected to immunofluorescence staining using antibodies against Arp1 and γ-tubulin. The results showed that a fraction of Arp1 is located on centrosomes and Arp1 was well co-localized with γ-tubulin (indicated with arrowheads). Scale bar=10 μm. ( C ) Components of purified centrosomes from HeLa cells were examined for the co-existence of geminin (Gem) and Arp1. Fractions of centrosomes isolated from a discontinuous sucrose gradient were separated using SDS/PAGE (10% gel) and immunoblotted using antibodies against geminin and Arp1. γ-Tubulin (γ-tub) was immunoblotted as a positive control for centrosome purification. ( D ) Co-localization of endogenous geminin (Gem) and Arp1 was viewed by immunofluorescence staining using antibodies against geminin and Arp1 in HeLa cells fixed with ice-cold methanol. Both geminin and Arp1 were apparently co-localized in the centrosome in interphase and spindle poles in mitosis (indicated with arrowheads). Scale bar=10 μm.

Techniques Used: Expressing, Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Immunofluorescence, Purification, Isolation, Positive Control

Geminin directly interacts with Arp1 ( A ) Geminin and Arp1 interacted with each other. In the upper panels, Arp1 was co-immunoprecipitated with geminin. Lysates of HeLa cells were immunoprecipitated (IP) with antibodies against geminin or rabbit IgG (rIgG) as a control. The co-existence of Arp1 and geminin was examined by immunoblotting analysis. Cdt1 was immunoblotted as a positive control for binding proteins of geminin. In the bottom panels, geminin was co-immunoprecipitated with Arp1. Lysates from GFP–Arp1-overexpressing HeLa cells were tested for immunosedimentation of geminin by Arp1 using an antibody against Arp1 or rIgG as a control. ( B ) In vitro pulldown assay for testing the association between Arp1 and GST–geminin. Purified GST–geminin was coupled to glutathione–Sepharose 4B beads and incubated with the lysate of HeLa cells for 2 h. The proteins on the beads were separated on SDS/PAGE (10% gel) and transferred on to a nitrocellulose membrane and stained with Fast Green (upper panel). The nitrocellulose membrane was then immunoprobed for Arp1 with a specific antibody. Cdt1 was immunoblotted as a positive control of geminin-associated proteins (bottom panels). Gem, geminin.
Figure Legend Snippet: Geminin directly interacts with Arp1 ( A ) Geminin and Arp1 interacted with each other. In the upper panels, Arp1 was co-immunoprecipitated with geminin. Lysates of HeLa cells were immunoprecipitated (IP) with antibodies against geminin or rabbit IgG (rIgG) as a control. The co-existence of Arp1 and geminin was examined by immunoblotting analysis. Cdt1 was immunoblotted as a positive control for binding proteins of geminin. In the bottom panels, geminin was co-immunoprecipitated with Arp1. Lysates from GFP–Arp1-overexpressing HeLa cells were tested for immunosedimentation of geminin by Arp1 using an antibody against Arp1 or rIgG as a control. ( B ) In vitro pulldown assay for testing the association between Arp1 and GST–geminin. Purified GST–geminin was coupled to glutathione–Sepharose 4B beads and incubated with the lysate of HeLa cells for 2 h. The proteins on the beads were separated on SDS/PAGE (10% gel) and transferred on to a nitrocellulose membrane and stained with Fast Green (upper panel). The nitrocellulose membrane was then immunoprobed for Arp1 with a specific antibody. Cdt1 was immunoblotted as a positive control of geminin-associated proteins (bottom panels). Gem, geminin.

Techniques Used: Immunoprecipitation, Positive Control, Binding Assay, In Vitro, Purification, Incubation, SDS Page, Staining

Dynein–dynactin-mediated centrosomal targeting of geminin is dependent on microtubules ( A ) Geminin accumulation on centrosomes depends on microtubules. Before the cells were fixed, nocodazole (20 μM) was added to the medium and cells were further incubated for 30 min. Co-immunofluorescence staining of geminin (Gem; green) and α-tubulin (red) was performed to examine altered localization of endogenous geminin when microtubules were depolymerized by nocodazole. As indicated with the arrowheads, centrosomal enrichment of geminin was inhibited after nocodazole treatment. Scale bar=10 μm. ( B ) Geminin accumulated on centrosomes after cellular ATP reduction. An ATP-inhibitor assay was performed as described in the Material and methods section. HeLa cells were transfected with GFP–geminin (GFP-Gem) and treated with either saline/glucose or saline/glucose plus 5 mM Az and 1 mM DOG and processed for microscopy. As indicated with arrowheads, after incubation for 30 min in Az/DOG, the accumulation of GFP–geminin on centrosomes was enhanced. When microtubules were depolymerized with 20 μM nocodazole, centrosomal enrichment of geminin was inhibited even in the presence of Az/DOG. ( C ) Live-cell analysis of geminin on centrosomes after Az/DOG treatment. Cells were imaged at 0, 15 and 30 min after Az/DOG was added to the medium. As indicated with arrowheads, GFP–geminin gradually accumulated on centrosomes. For microtubule-depolymerization assays, 20 μM nocodazole was added to the medium 30 min before Az/DOG treatment. The centrosomal localization of GFP–geminin was disrupted in the present of Az/DOG. Scale bar=10 μm. ( D ) Co-immunofluorescence staining of geminin (Gem) and γ-tubulin (red) showed geminin enriched on centrosomes after cellular ATP induction. The altered localization of endogenous geminin (right-hand panels) was consistent with that of GFP–geminin (GFP-Gem; left-hand panels) under the same treatment. Scale bar=10 μm.
Figure Legend Snippet: Dynein–dynactin-mediated centrosomal targeting of geminin is dependent on microtubules ( A ) Geminin accumulation on centrosomes depends on microtubules. Before the cells were fixed, nocodazole (20 μM) was added to the medium and cells were further incubated for 30 min. Co-immunofluorescence staining of geminin (Gem; green) and α-tubulin (red) was performed to examine altered localization of endogenous geminin when microtubules were depolymerized by nocodazole. As indicated with the arrowheads, centrosomal enrichment of geminin was inhibited after nocodazole treatment. Scale bar=10 μm. ( B ) Geminin accumulated on centrosomes after cellular ATP reduction. An ATP-inhibitor assay was performed as described in the Material and methods section. HeLa cells were transfected with GFP–geminin (GFP-Gem) and treated with either saline/glucose or saline/glucose plus 5 mM Az and 1 mM DOG and processed for microscopy. As indicated with arrowheads, after incubation for 30 min in Az/DOG, the accumulation of GFP–geminin on centrosomes was enhanced. When microtubules were depolymerized with 20 μM nocodazole, centrosomal enrichment of geminin was inhibited even in the presence of Az/DOG. ( C ) Live-cell analysis of geminin on centrosomes after Az/DOG treatment. Cells were imaged at 0, 15 and 30 min after Az/DOG was added to the medium. As indicated with arrowheads, GFP–geminin gradually accumulated on centrosomes. For microtubule-depolymerization assays, 20 μM nocodazole was added to the medium 30 min before Az/DOG treatment. The centrosomal localization of GFP–geminin was disrupted in the present of Az/DOG. Scale bar=10 μm. ( D ) Co-immunofluorescence staining of geminin (Gem) and γ-tubulin (red) showed geminin enriched on centrosomes after cellular ATP induction. The altered localization of endogenous geminin (right-hand panels) was consistent with that of GFP–geminin (GFP-Gem; left-hand panels) under the same treatment. Scale bar=10 μm.

Techniques Used: Incubation, Immunofluorescence, Staining, Transfection, Microscopy

Geminin localizes on centrosomes ( A ) A fraction of GFP–geminin localized to ‘aster-like’ structures in a live cell. HeLa cells were transfected with pEGFPC2-geminin vector using a standard calcium phosphate transfection protocol. The culture dish was placed on to a heated sample stage within a heated chamber (37°C) at 5% CO 2 . GFP–geminin-expressing living cells were imaged by a Zeiss200M fluorescence microscope (Axiovert 200M) with the plan APO 63′/1.35 objective. GFP–geminin is expressed and partially localized to ‘aster-like’ structures in interphase cells as well as spindle fibres in cells undergoing mitosis as indicated with arrowheads (red). Scale bar=10 μm. ( B ) Centrosomal localization of both exogenous GFP–geminin and endogenous geminin. GFP–geminin-expressing HeLa cells were fixed with ice-cold methanol and immunostained with a γ-tubulin antibody. After incubation with a TRITC-conjugated secondary antibody, cells were mounted with mowiol containing DAPI for staining of DNA. Staining for γ-tubulin indicated the presence of centrosomes. Endogenous geminin was examined by immunostaining with an anti-geminin antibody. A clear centrosome co-staining of γ-tubulin and geminin (indicated with arrowheads) was achieved. Scale bar=10 μm. ( C ) The endogenous geminin from different cell lines was probed using the same antibody against human geminin as used in Western blot analysis, suggesting that the anti-geminin antibody specifically recognized geminin in these cells. α-Tubulin was used as a loading control. The molecular mass in kDa is indicated on the left-hand side of the gel. 293, HEK (human embryonic kidney)-293 cells; 231, MDA-MB-231 cells; 3T3, NIH 3T3 cells. ( D ) Co-localization of geminin and Arp1 on centrosomes in MDA-MB-231 cells and MEF cells. MDA-MB-231 and MEF cells were fixed with ice-cold methanol and co-immunostained using antibodies against geminin and γ-tubulin (γ-tub). The similar centrosomal localization of both geminin and γ-tubulin (indicated with arrowheads, red) was observed in these cell lines as in ( B ). Scale bar=10 μm. Gem, geminin.
Figure Legend Snippet: Geminin localizes on centrosomes ( A ) A fraction of GFP–geminin localized to ‘aster-like’ structures in a live cell. HeLa cells were transfected with pEGFPC2-geminin vector using a standard calcium phosphate transfection protocol. The culture dish was placed on to a heated sample stage within a heated chamber (37°C) at 5% CO 2 . GFP–geminin-expressing living cells were imaged by a Zeiss200M fluorescence microscope (Axiovert 200M) with the plan APO 63′/1.35 objective. GFP–geminin is expressed and partially localized to ‘aster-like’ structures in interphase cells as well as spindle fibres in cells undergoing mitosis as indicated with arrowheads (red). Scale bar=10 μm. ( B ) Centrosomal localization of both exogenous GFP–geminin and endogenous geminin. GFP–geminin-expressing HeLa cells were fixed with ice-cold methanol and immunostained with a γ-tubulin antibody. After incubation with a TRITC-conjugated secondary antibody, cells were mounted with mowiol containing DAPI for staining of DNA. Staining for γ-tubulin indicated the presence of centrosomes. Endogenous geminin was examined by immunostaining with an anti-geminin antibody. A clear centrosome co-staining of γ-tubulin and geminin (indicated with arrowheads) was achieved. Scale bar=10 μm. ( C ) The endogenous geminin from different cell lines was probed using the same antibody against human geminin as used in Western blot analysis, suggesting that the anti-geminin antibody specifically recognized geminin in these cells. α-Tubulin was used as a loading control. The molecular mass in kDa is indicated on the left-hand side of the gel. 293, HEK (human embryonic kidney)-293 cells; 231, MDA-MB-231 cells; 3T3, NIH 3T3 cells. ( D ) Co-localization of geminin and Arp1 on centrosomes in MDA-MB-231 cells and MEF cells. MDA-MB-231 and MEF cells were fixed with ice-cold methanol and co-immunostained using antibodies against geminin and γ-tubulin (γ-tub). The similar centrosomal localization of both geminin and γ-tubulin (indicated with arrowheads, red) was observed in these cell lines as in ( B ). Scale bar=10 μm. Gem, geminin.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Fluorescence, Microscopy, Incubation, Staining, Immunostaining, Western Blot, Multiple Displacement Amplification

24) Product Images from "Disappearance of the budding yeast Bub2-Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit"

Article Title: Disappearance of the budding yeast Bub2-Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200507162

Lack of Bub2 GAP activity leads to persistent symmetric localization of Bub2 on SPBs. (A, B, and D) Bacterially expressed GST-Bub2, MBP-Bfa1, and 6×His-Tem1 were used in in vitro GAP assays as previously described ( Geymonat et al., 2002 ). In brief, 240 nM of 6×His-Tem1 was loaded with γ-[ 32 P]GTP in either the absence or presence of 150 nM MBP-Bfa1 and incubated at 30°C for 10 min. The mixture was then added to 15 μM of GST-Bub2 or buffer alone, and kinetics of GTP hydrolysis and dissociation was followed by filter binding assays (see Materials and methods). (C) Increasing amounts of the indicated Bub2 variants (0:1, 1:1, 2:1, 2.6:1, 6.6:1, 13:1, 26:1, 53:1, and 80:1 molar ratio Bub2/Bfa1) were added to 605 nM of 6×His-Tem1– γ-[ 32 P]GTP in the presence of 378 nM MBP-Bfa1 (final concentrations), and the fraction of filter-bound radioactivity was measured after 8 min. (E) Localization of Bub2R85A-HA3 at anaphase SPBs was monitored by in situ immunostaining with anti-HA antibodies. (F) Localization of Bfa1-HA6 in asynchronous BUB2R85A-myc9 cells was monitored as in E. (G, top) Protein extracts from BUB2-myc9 (ySP710), bub2R85A-myc9 (ySP4696), BUB2-myc9 BFA1-HA6 (ySP5087), and two different bub2R85A-myc9 BFA1-HA6 strains (ySP5118 and ySP5119) were used for Western blot analysis of either the total levels of Bub2-myc9 and Bfa1-HA6 (total) or the amount of Bub2-myc9 coimmunoprecipitating with Bfa1-HA6 (anti-HA IP). (bottom) Protein extracts from BFA1-HA6 (ySP2035), BUB2-myc9 BFA1-HA6 (ySP5087), and two different bub2R85A-myc9 BFA1-HA6 strains (ySP5118 and ySP5119) were used for Western blot analysis of either the total levels of Bub2-myc9 and Bfa1-HA6 (total) or the amount of Bfa1-HA6 coimmunoprecipitating with Bub2-myc9 (anti-myc IP).
Figure Legend Snippet: Lack of Bub2 GAP activity leads to persistent symmetric localization of Bub2 on SPBs. (A, B, and D) Bacterially expressed GST-Bub2, MBP-Bfa1, and 6×His-Tem1 were used in in vitro GAP assays as previously described ( Geymonat et al., 2002 ). In brief, 240 nM of 6×His-Tem1 was loaded with γ-[ 32 P]GTP in either the absence or presence of 150 nM MBP-Bfa1 and incubated at 30°C for 10 min. The mixture was then added to 15 μM of GST-Bub2 or buffer alone, and kinetics of GTP hydrolysis and dissociation was followed by filter binding assays (see Materials and methods). (C) Increasing amounts of the indicated Bub2 variants (0:1, 1:1, 2:1, 2.6:1, 6.6:1, 13:1, 26:1, 53:1, and 80:1 molar ratio Bub2/Bfa1) were added to 605 nM of 6×His-Tem1– γ-[ 32 P]GTP in the presence of 378 nM MBP-Bfa1 (final concentrations), and the fraction of filter-bound radioactivity was measured after 8 min. (E) Localization of Bub2R85A-HA3 at anaphase SPBs was monitored by in situ immunostaining with anti-HA antibodies. (F) Localization of Bfa1-HA6 in asynchronous BUB2R85A-myc9 cells was monitored as in E. (G, top) Protein extracts from BUB2-myc9 (ySP710), bub2R85A-myc9 (ySP4696), BUB2-myc9 BFA1-HA6 (ySP5087), and two different bub2R85A-myc9 BFA1-HA6 strains (ySP5118 and ySP5119) were used for Western blot analysis of either the total levels of Bub2-myc9 and Bfa1-HA6 (total) or the amount of Bub2-myc9 coimmunoprecipitating with Bfa1-HA6 (anti-HA IP). (bottom) Protein extracts from BFA1-HA6 (ySP2035), BUB2-myc9 BFA1-HA6 (ySP5087), and two different bub2R85A-myc9 BFA1-HA6 strains (ySP5118 and ySP5119) were used for Western blot analysis of either the total levels of Bub2-myc9 and Bfa1-HA6 (total) or the amount of Bfa1-HA6 coimmunoprecipitating with Bub2-myc9 (anti-myc IP).

Techniques Used: Activity Assay, In Vitro, Incubation, Binding Assay, Radioactivity, In Situ, Immunostaining, Western Blot

25) Product Images from "A Novel Rab9 Effector Required for Endosome-to-TGN Transport"

Article Title: A Novel Rab9 Effector Required for Endosome-to-TGN Transport

Journal: The Journal of Cell Biology

doi:

Purified p40 binds Rab9–GTP in strong preference to Rab9–GDP. ( A ) An example of immunoblot binding data obtained is shown. Values shown in B were determined by PhosphorImager quantitation; Rab9 standards were included on the gels to permit determination of the nanogram amounts of Rab9 bound.
Figure Legend Snippet: Purified p40 binds Rab9–GTP in strong preference to Rab9–GDP. ( A ) An example of immunoblot binding data obtained is shown. Values shown in B were determined by PhosphorImager quantitation; Rab9 standards were included on the gels to permit determination of the nanogram amounts of Rab9 bound.

Techniques Used: Purification, Binding Assay, Quantitation Assay

26) Product Images from "Electron Transfer Flavoprotein Subunit Beta Is a Candidate Endothelial Cell Autoantigen in Behçet’s Disease"

Article Title: Electron Transfer Flavoprotein Subunit Beta Is a Candidate Endothelial Cell Autoantigen in Behçet’s Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124760

ETFB was confirmed as an autoantigen in BD. (A) The expression and purification of the ETFB protein. M: Marker; 1: E . coli BL21 containing a recombinant plasmid after the induction of expression with IPTG; 2: Purified recombinant ETFB protein. (B) Verification of the ETFB protein by mass spectrometry. The Mascot score was 474 (Matrix Sciences, London, UK; www.matrixscience.com ). (C) Western blotting results for ETFB probed with serum from patient BD1 (1:500 dilution). (D) Western blotting results for ETFB probed with serum from patient BD3. (E) Healthy control.
Figure Legend Snippet: ETFB was confirmed as an autoantigen in BD. (A) The expression and purification of the ETFB protein. M: Marker; 1: E . coli BL21 containing a recombinant plasmid after the induction of expression with IPTG; 2: Purified recombinant ETFB protein. (B) Verification of the ETFB protein by mass spectrometry. The Mascot score was 474 (Matrix Sciences, London, UK; www.matrixscience.com ). (C) Western blotting results for ETFB probed with serum from patient BD1 (1:500 dilution). (D) Western blotting results for ETFB probed with serum from patient BD3. (E) Healthy control.

Techniques Used: Expressing, Purification, Marker, Recombinant, Plasmid Preparation, Mass Spectrometry, Western Blot

27) Product Images from "Setting up a platform for plant-based influenza virus vaccine production in South Africa"

Article Title: Setting up a platform for plant-based influenza virus vaccine production in South Africa

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-12-14

A) Coomassie-stained SDS-PAGE gel of transiently expressed H5 and H5tr. Crude, clarified extracts were prepared from N. benthamiana leaves that were expressing the H5 and H5tr proteins transiently. The extracts were separated by SDS-PAGE and compared to known BSA concentrations after staining with Coomassie Blue. Lane 1, prestained protein ladder (Fermentas); lane 2, 6.25 μg BSA; lane 3, 3.12 μg BSA; lane 4, 1.56 μg BSA; lane 5, 0.78 μg BSA; lane 6, concentrated H5, lane 7 concentrated H5tr, lane 8, crude H5 extract, lane 9 crude H5tr extract. The arrow indicates the position of the H5 protein. B) Coomassie-stained SDS-PAGE gel of H5 expressed in stable transgenic plants. Apoplast-targeted H5 from transgenic plants was separated lanes 4 and 5. Lane 1, protein ladder (Fermentas); lane 2, 5 μg BSA; lane 3, 2.5 μg BSA; lane 4, crude H5 extract; and lane 5, concentrated H5.
Figure Legend Snippet: A) Coomassie-stained SDS-PAGE gel of transiently expressed H5 and H5tr. Crude, clarified extracts were prepared from N. benthamiana leaves that were expressing the H5 and H5tr proteins transiently. The extracts were separated by SDS-PAGE and compared to known BSA concentrations after staining with Coomassie Blue. Lane 1, prestained protein ladder (Fermentas); lane 2, 6.25 μg BSA; lane 3, 3.12 μg BSA; lane 4, 1.56 μg BSA; lane 5, 0.78 μg BSA; lane 6, concentrated H5, lane 7 concentrated H5tr, lane 8, crude H5 extract, lane 9 crude H5tr extract. The arrow indicates the position of the H5 protein. B) Coomassie-stained SDS-PAGE gel of H5 expressed in stable transgenic plants. Apoplast-targeted H5 from transgenic plants was separated lanes 4 and 5. Lane 1, protein ladder (Fermentas); lane 2, 5 μg BSA; lane 3, 2.5 μg BSA; lane 4, crude H5 extract; and lane 5, concentrated H5.

Techniques Used: Staining, SDS Page, Expressing, Transgenic Assay

Western blot indicating the presence of H5-specific antibodies in sera from mice immunised with plant-produced H5 or H5tr. Purified H5N1 HA protein (Sino Biological) was detected using sera from H5-immunised mice (lanes 3–5), or H5tr-immunised mice (lanes 6–8). Lane 1, protein ladder (Fermentas); lane 2, HA detection using anti-H5N1 HA mouse monoclonal antibody (1:5000, Abcam) and lane 9 is the negative control where sera from animals vaccinated with PBS was used (dilution 1:4000). The arrow indicates the presence of the correct band size in all the H5 and H5tr candidate vaccine sera samples.
Figure Legend Snippet: Western blot indicating the presence of H5-specific antibodies in sera from mice immunised with plant-produced H5 or H5tr. Purified H5N1 HA protein (Sino Biological) was detected using sera from H5-immunised mice (lanes 3–5), or H5tr-immunised mice (lanes 6–8). Lane 1, protein ladder (Fermentas); lane 2, HA detection using anti-H5N1 HA mouse monoclonal antibody (1:5000, Abcam) and lane 9 is the negative control where sera from animals vaccinated with PBS was used (dilution 1:4000). The arrow indicates the presence of the correct band size in all the H5 and H5tr candidate vaccine sera samples.

Techniques Used: Western Blot, Mouse Assay, Produced, Purification, Negative Control

Western blot analysis of H5 and H5tr transient expression in N. benthamiana , showing accumulation in various subcellular compartments 7 days post infiltration. HA protein was detected using a primary rabbit anti-H5N1 polyclonal antibody. Crude plant extracts were analysed from plant tissue infiltrated with Agrobacterium strains carrying the following expression vectors: Lane 1, pTRAERH-H5; lane 2, pTRAERH-H5tr; lane 3, pTRAc-H5; lane 4, pTRAc-H5tr; lane 5, pTRACTP-H5; lane 6, pTRACTP-H5tr; lane 7, pTRAa-H5; lane 8, pTRAa-H5tr; lane 9, protein ladder (Fermentas). The arrow indicates the position of the H5 and H5tr proteins.
Figure Legend Snippet: Western blot analysis of H5 and H5tr transient expression in N. benthamiana , showing accumulation in various subcellular compartments 7 days post infiltration. HA protein was detected using a primary rabbit anti-H5N1 polyclonal antibody. Crude plant extracts were analysed from plant tissue infiltrated with Agrobacterium strains carrying the following expression vectors: Lane 1, pTRAERH-H5; lane 2, pTRAERH-H5tr; lane 3, pTRAc-H5; lane 4, pTRAc-H5tr; lane 5, pTRACTP-H5; lane 6, pTRACTP-H5tr; lane 7, pTRAa-H5; lane 8, pTRAa-H5tr; lane 9, protein ladder (Fermentas). The arrow indicates the position of the H5 and H5tr proteins.

Techniques Used: Western Blot, Expressing

Western blot analysis showing HA expression in a representative sample of transgenic plant lines. Samples were harvested from T 3 plant lines that were transformed with pTRAa-H5 (lanes 2–4) or pTRAERH-H5tr (lanes 5–7). Lane 1, protein ladder (Fermentas).
Figure Legend Snippet: Western blot analysis showing HA expression in a representative sample of transgenic plant lines. Samples were harvested from T 3 plant lines that were transformed with pTRAa-H5 (lanes 2–4) or pTRAERH-H5tr (lanes 5–7). Lane 1, protein ladder (Fermentas).

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

28) Product Images from "Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage"

Article Title: Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage

Journal: bioRxiv

doi: 10.1101/2020.04.25.061606

GABPB2 upregulation can compensate for loss of GABPB1L A , Similarity between GABP proteins. Partial protein alignment (MUSCLE) of GABPB1L, GABPB1S, and GABPB2. GABPB1L is the long isoform, and GABPB1S the short isoform, of the GABPB1 gene. GABPB2 is a distinct gene. Amino acids matching across all three proteins are highlighted in blue, those matching across two proteins are highlighted in green. GABPA (alpha) subunits of the GABP complex bind to both the native ETS site (ETS) and the mutation-derived ETS sites (G228A or G250A) at the TERT promoter locus. The GABPB1 short (beta-1S) or long (beta-1L) isoform subunits bind to the alpha subunits to form either heterodimers (beta-1S) or heterotetramers (beta-1L). Heterotetramer formation is presumably mediated through the leucine zipper-like domain of GABPB1L, which is also present in GABPB2. B , GABPB2 mRNA expression measured via qRT-PCR in GABPB1L knockout clones, plotted relative to control (wild-type) cells, in TERT p mutant cell lines. Data represent mean +/- SEM. C , Competitive proliferation assay in U-251 cells using pairs of sgRNAs targeting a positive control locus (sgRPA1.2-3) and total GABPB1 (targeting exon 3, sgGABPV.33-34), in the presence of a lentiviral vector expressing either GABPB2 or mTagBFP2 (control). Data represent mean +/- standard deviation of triplicates. D , TERT mRNA expression measured via qRT-PCR following siRNA-mediated knockdown of GABPA or GABPB2 in LN-229 wild-type, FKO, or heterozygous KO cells. p, p-value (unpaired, two-tailed student’s t-test). n.s., not significant (alpha level = 0.01). Data represent mean +/- SEM.
Figure Legend Snippet: GABPB2 upregulation can compensate for loss of GABPB1L A , Similarity between GABP proteins. Partial protein alignment (MUSCLE) of GABPB1L, GABPB1S, and GABPB2. GABPB1L is the long isoform, and GABPB1S the short isoform, of the GABPB1 gene. GABPB2 is a distinct gene. Amino acids matching across all three proteins are highlighted in blue, those matching across two proteins are highlighted in green. GABPA (alpha) subunits of the GABP complex bind to both the native ETS site (ETS) and the mutation-derived ETS sites (G228A or G250A) at the TERT promoter locus. The GABPB1 short (beta-1S) or long (beta-1L) isoform subunits bind to the alpha subunits to form either heterodimers (beta-1S) or heterotetramers (beta-1L). Heterotetramer formation is presumably mediated through the leucine zipper-like domain of GABPB1L, which is also present in GABPB2. B , GABPB2 mRNA expression measured via qRT-PCR in GABPB1L knockout clones, plotted relative to control (wild-type) cells, in TERT p mutant cell lines. Data represent mean +/- SEM. C , Competitive proliferation assay in U-251 cells using pairs of sgRNAs targeting a positive control locus (sgRPA1.2-3) and total GABPB1 (targeting exon 3, sgGABPV.33-34), in the presence of a lentiviral vector expressing either GABPB2 or mTagBFP2 (control). Data represent mean +/- standard deviation of triplicates. D , TERT mRNA expression measured via qRT-PCR following siRNA-mediated knockdown of GABPA or GABPB2 in LN-229 wild-type, FKO, or heterozygous KO cells. p, p-value (unpaired, two-tailed student’s t-test). n.s., not significant (alpha level = 0.01). Data represent mean +/- SEM.

Techniques Used: Mutagenesis, Derivative Assay, Expressing, Quantitative RT-PCR, Knock-Out, Clone Assay, Proliferation Assay, Positive Control, Plasmid Preparation, Standard Deviation, Two Tailed Test

A GABP transcription factor tetramer binds directly to the mutant but not WT TERT promoter A , Schematic of the wild-type versus mutant TERT promoter. The mutant promoter contains two adjacent ETS family transcription factor binding sites that are hypothesized to recruit a GABP heterotetramer complex. B , GABP – TERT p binding curve analysis. The plot shows change in a fraction of bound wild type and mutant TERT p dependent on the concentration of GABPBA-B1L heterodimers with their respective Kd values. C , Representative immunoblot of GABPB1 in U-251 cells with doxycycline-induced shRNAs targeting GABPB1S (shGB1S.82, shGB1S.77, shGB1S.32, shGB1S.110) and GABPB1L (shGB1L.377, shGB1L.699, shGB1L.968, shGB1L.1202) compared to negative control (olfactory receptor OR2B6, shOR2B6.910) and non-targeting (renilla luciferase, shRen.713) shRNAs. Cells were incubated with doxycycline for 6 days prior to harvest. The lower band represents GABPB1S, the upper band represents GABPB1L. D , TERT mRNA expression measured via qRT-PCR in lines from ( C ).
Figure Legend Snippet: A GABP transcription factor tetramer binds directly to the mutant but not WT TERT promoter A , Schematic of the wild-type versus mutant TERT promoter. The mutant promoter contains two adjacent ETS family transcription factor binding sites that are hypothesized to recruit a GABP heterotetramer complex. B , GABP – TERT p binding curve analysis. The plot shows change in a fraction of bound wild type and mutant TERT p dependent on the concentration of GABPBA-B1L heterodimers with their respective Kd values. C , Representative immunoblot of GABPB1 in U-251 cells with doxycycline-induced shRNAs targeting GABPB1S (shGB1S.82, shGB1S.77, shGB1S.32, shGB1S.110) and GABPB1L (shGB1L.377, shGB1L.699, shGB1L.968, shGB1L.1202) compared to negative control (olfactory receptor OR2B6, shOR2B6.910) and non-targeting (renilla luciferase, shRen.713) shRNAs. Cells were incubated with doxycycline for 6 days prior to harvest. The lower band represents GABPB1S, the upper band represents GABPB1L. D , TERT mRNA expression measured via qRT-PCR in lines from ( C ).

Techniques Used: Mutagenesis, Binding Assay, Concentration Assay, Negative Control, Luciferase, Incubation, Expressing, Quantitative RT-PCR

29) Product Images from "Immunologic perturbations in severe COVID-19/SARS-CoV-2 infection"

Article Title: Immunologic perturbations in severe COVID-19/SARS-CoV-2 infection

Journal: bioRxiv

doi: 10.1101/2020.05.18.101717

Elevated frequency of plasmablasts, changes in B cell subsets and SARS-CoV-2-specific antibody production in COVID-19 individuals. Multiparametric flow cytometry analyses on fresh whole blood after red blood cell lysis characterizing plasmablast and B cell subset frequencies from HD (n= 12), and moderate (n=7), severe (n=27), and recovered (n=6) COVID-19 individuals. A), B) Distribution and representative plots of B cell plasmablasts (defined as CD27+ CD38+ B cells) and non-plasmablast subsets defined by CD21 and CD27 expression in HD (n= 12), and moderate (n=7), severe (n=27), and recovered (n=6) COVID-19 individuals. Numbers inside the plots indicate the subset proportion of the corresponding parent population (within total B cells for plasmablasts, within non-plasmablasts for CD21/CD27 subsets). C) Frequencies of CD11c and Ki-67 in non-plasmablast B cell subsets defined in a). Analyses of CD11c are shown for half of the individuals with moderate COVID-19. Plots from a representative HD and severe COVID-19 individual shown. Numbers in each plot indicate the frequency within the parent gate. D) Levels of SARS-CoV-2 spike RBD-specific IgM and IgG antibodies in serum or plasma of HD (n= 12), moderate (n=7), severe (n=27), and recovered (n=6) COVID-19 individuals. Antibody measurements were performed by ELISA using plates coated with the receptor binding domain (RBD) from the SARS-CoV-2 spike protein. Sera and plasma samples were heat-inactivated at 56°C for 1 hour prior to testing in ELISA to inactivate virus. Antibody levels were reported as µg/ml amounts relative to the CR3022 monoclonal antibody (recombinant human anti-SARS-CoV-2, specifically binds to spike protein RBD). E) Spearman correlations of plasma/serum levels of SARS-CoV-2 RBD-specific IgM (top) and IgG (bottom) and days since onset of symptoms on moderate and severe COVID-19 individuals. Specific color coding was assigned per individual for cross comparison across graphs and Figs. Lines on the graphs indicate the median of the group. Differences between groups were calculated using Kruskal-Wallis test with Dunn’s multiple comparison post-test. **** p
Figure Legend Snippet: Elevated frequency of plasmablasts, changes in B cell subsets and SARS-CoV-2-specific antibody production in COVID-19 individuals. Multiparametric flow cytometry analyses on fresh whole blood after red blood cell lysis characterizing plasmablast and B cell subset frequencies from HD (n= 12), and moderate (n=7), severe (n=27), and recovered (n=6) COVID-19 individuals. A), B) Distribution and representative plots of B cell plasmablasts (defined as CD27+ CD38+ B cells) and non-plasmablast subsets defined by CD21 and CD27 expression in HD (n= 12), and moderate (n=7), severe (n=27), and recovered (n=6) COVID-19 individuals. Numbers inside the plots indicate the subset proportion of the corresponding parent population (within total B cells for plasmablasts, within non-plasmablasts for CD21/CD27 subsets). C) Frequencies of CD11c and Ki-67 in non-plasmablast B cell subsets defined in a). Analyses of CD11c are shown for half of the individuals with moderate COVID-19. Plots from a representative HD and severe COVID-19 individual shown. Numbers in each plot indicate the frequency within the parent gate. D) Levels of SARS-CoV-2 spike RBD-specific IgM and IgG antibodies in serum or plasma of HD (n= 12), moderate (n=7), severe (n=27), and recovered (n=6) COVID-19 individuals. Antibody measurements were performed by ELISA using plates coated with the receptor binding domain (RBD) from the SARS-CoV-2 spike protein. Sera and plasma samples were heat-inactivated at 56°C for 1 hour prior to testing in ELISA to inactivate virus. Antibody levels were reported as µg/ml amounts relative to the CR3022 monoclonal antibody (recombinant human anti-SARS-CoV-2, specifically binds to spike protein RBD). E) Spearman correlations of plasma/serum levels of SARS-CoV-2 RBD-specific IgM (top) and IgG (bottom) and days since onset of symptoms on moderate and severe COVID-19 individuals. Specific color coding was assigned per individual for cross comparison across graphs and Figs. Lines on the graphs indicate the median of the group. Differences between groups were calculated using Kruskal-Wallis test with Dunn’s multiple comparison post-test. **** p

Techniques Used: Flow Cytometry, Lysis, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant

30) Product Images from "The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila TelomeresAKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance"

Article Title: The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila TelomeresAKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1005260

Peo directly interacts with HOAP and is also likely to bind Moi and Ver. (A) Schematic of HOAP truncations used in GST pulldown experiments; R indicates the repeated segments found in the C-proximal half of the protein. (B) Bacterially purified GST-HOAP segments spanning the N proximal half of the protein precipitate bacterially expressed His-Peo, which is not pulled down by GST alone or the C-proximal half of HOAP. His-Peo was detected using our anti-Peo antibody; the two Peo bands are likely to correspond to different translation products generated in E . coli starting from the two closely spaced ATG codons present on peo cDNA (see Fig 2 ) (C) GST-HOAP, GST-Moi and GST-Ver precipitate Peo-FLAG from HeLa cell extracts. Peo-FLAG was detected with anti-FLAG antibody.
Figure Legend Snippet: Peo directly interacts with HOAP and is also likely to bind Moi and Ver. (A) Schematic of HOAP truncations used in GST pulldown experiments; R indicates the repeated segments found in the C-proximal half of the protein. (B) Bacterially purified GST-HOAP segments spanning the N proximal half of the protein precipitate bacterially expressed His-Peo, which is not pulled down by GST alone or the C-proximal half of HOAP. His-Peo was detected using our anti-Peo antibody; the two Peo bands are likely to correspond to different translation products generated in E . coli starting from the two closely spaced ATG codons present on peo cDNA (see Fig 2 ) (C) GST-HOAP, GST-Moi and GST-Ver precipitate Peo-FLAG from HeLa cell extracts. Peo-FLAG was detected with anti-FLAG antibody.

Techniques Used: Purification, Generated

Mapping the Peo regions that interact with terminin. (A) A tridimensional molecular model for Peo. The arrow pointing to “N-ter” indicates the N-terminus of the protein; the arrow pointing to “C-ter” indicates the starting site of the disordered C-terminal region of Peo (not depicted); the variant Asp residue and His-Pro-His motif are represented as sticks and indicated by red and purple arrows, respectively (see Materials and Methods and S2 Fig for construction of the Peo 3D model). (B) Schematic organization of the Peo protein and Peo truncations used for GST pulldown. (C-E) GST-pulldown from S2 cells extracts expressing HOAP-FLAG (C), Ver-FLAG (D) or Moi-HA (E). HOAP-FLAG, Ver-FLAG and Moi-HA were detected with anti-FLAG and anti-HA antibodies. The C-terminal disordered region (included in the Peo 3 fragment) does not interact with any of the terminin components. HOAP specifically interacts with N-terminal region of Peo; in contrast, Moi and Ver interact with both the N terminal and the UEV-containing central regions of the protein.
Figure Legend Snippet: Mapping the Peo regions that interact with terminin. (A) A tridimensional molecular model for Peo. The arrow pointing to “N-ter” indicates the N-terminus of the protein; the arrow pointing to “C-ter” indicates the starting site of the disordered C-terminal region of Peo (not depicted); the variant Asp residue and His-Pro-His motif are represented as sticks and indicated by red and purple arrows, respectively (see Materials and Methods and S2 Fig for construction of the Peo 3D model). (B) Schematic organization of the Peo protein and Peo truncations used for GST pulldown. (C-E) GST-pulldown from S2 cells extracts expressing HOAP-FLAG (C), Ver-FLAG (D) or Moi-HA (E). HOAP-FLAG, Ver-FLAG and Moi-HA were detected with anti-FLAG and anti-HA antibodies. The C-terminal disordered region (included in the Peo 3 fragment) does not interact with any of the terminin components. HOAP specifically interacts with N-terminal region of Peo; in contrast, Moi and Ver interact with both the N terminal and the UEV-containing central regions of the protein.

Techniques Used: Variant Assay, Expressing

31) Product Images from "The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila TelomeresAKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance"

Article Title: The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila TelomeresAKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1005260

Peo directly interacts with HOAP and is also likely to bind Moi and Ver. (A) Schematic of HOAP truncations used in GST pulldown experiments; R indicates the repeated segments found in the C-proximal half of the protein. (B) Bacterially purified GST-HOAP segments spanning the N proximal half of the protein precipitate bacterially expressed His-Peo, which is not pulled down by GST alone or the C-proximal half of HOAP. His-Peo was detected using our anti-Peo antibody; the two Peo bands are likely to correspond to different translation products generated in E . coli starting from the two closely spaced ATG codons present on peo cDNA (see Fig 2 ) (C) GST-HOAP, GST-Moi and GST-Ver precipitate Peo-FLAG from HeLa cell extracts. Peo-FLAG was detected with anti-FLAG antibody.
Figure Legend Snippet: Peo directly interacts with HOAP and is also likely to bind Moi and Ver. (A) Schematic of HOAP truncations used in GST pulldown experiments; R indicates the repeated segments found in the C-proximal half of the protein. (B) Bacterially purified GST-HOAP segments spanning the N proximal half of the protein precipitate bacterially expressed His-Peo, which is not pulled down by GST alone or the C-proximal half of HOAP. His-Peo was detected using our anti-Peo antibody; the two Peo bands are likely to correspond to different translation products generated in E . coli starting from the two closely spaced ATG codons present on peo cDNA (see Fig 2 ) (C) GST-HOAP, GST-Moi and GST-Ver precipitate Peo-FLAG from HeLa cell extracts. Peo-FLAG was detected with anti-FLAG antibody.

Techniques Used: Purification, Generated

Mapping the Peo regions that interact with terminin. (A) A tridimensional molecular model for Peo. The arrow pointing to “N-ter” indicates the N-terminus of the protein; the arrow pointing to “C-ter” indicates the starting site of the disordered C-terminal region of Peo (not depicted); the variant Asp residue and His-Pro-His motif are represented as sticks and indicated by red and purple arrows, respectively (see Materials and Methods and S2 Fig for construction of the Peo 3D model). (B) Schematic organization of the Peo protein and Peo truncations used for GST pulldown. (C-E) GST-pulldown from S2 cells extracts expressing HOAP-FLAG (C), Ver-FLAG (D) or Moi-HA (E). HOAP-FLAG, Ver-FLAG and Moi-HA were detected with anti-FLAG and anti-HA antibodies. The C-terminal disordered region (included in the Peo 3 fragment) does not interact with any of the terminin components. HOAP specifically interacts with N-terminal region of Peo; in contrast, Moi and Ver interact with both the N terminal and the UEV-containing central regions of the protein.
Figure Legend Snippet: Mapping the Peo regions that interact with terminin. (A) A tridimensional molecular model for Peo. The arrow pointing to “N-ter” indicates the N-terminus of the protein; the arrow pointing to “C-ter” indicates the starting site of the disordered C-terminal region of Peo (not depicted); the variant Asp residue and His-Pro-His motif are represented as sticks and indicated by red and purple arrows, respectively (see Materials and Methods and S2 Fig for construction of the Peo 3D model). (B) Schematic organization of the Peo protein and Peo truncations used for GST pulldown. (C-E) GST-pulldown from S2 cells extracts expressing HOAP-FLAG (C), Ver-FLAG (D) or Moi-HA (E). HOAP-FLAG, Ver-FLAG and Moi-HA were detected with anti-FLAG and anti-HA antibodies. The C-terminal disordered region (included in the Peo 3 fragment) does not interact with any of the terminin components. HOAP specifically interacts with N-terminal region of Peo; in contrast, Moi and Ver interact with both the N terminal and the UEV-containing central regions of the protein.

Techniques Used: Variant Assay, Expressing

32) Product Images from "Establishment of platform for screening insulin-like growth factor-1 receptor inhibitors and evaluation of novel inhibitors"

Article Title: Establishment of platform for screening insulin-like growth factor-1 receptor inhibitors and evaluation of novel inhibitors

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2011.23

Hematoxylin inhibits IGF1R activity in vitro and binds directly to IGF1R-CD. (A) Inhibitory activity of hematoxylin on IGF1R detected by ELISA assay. Tests were performed three times independently. (B) Hematoxylin binds to IGF1R-CD. SPR assay was performed as described in Materials and methods. (C) The binding mode of hematoxylin to the kinase domain of IGF1R predicted by molecular docking. The ligand (green carbon) and residues (cyan carbon) are represented as sticks and lines, respectively. The yellow dashed lines denote the hydrogen bonds and the oxygen atoms are colored in red. The molecular surface of the binding site was shown as green dots. The structure figure was prepared using PyMol (www.pymol.org). Data shown were mean±SD from three independent experiments.
Figure Legend Snippet: Hematoxylin inhibits IGF1R activity in vitro and binds directly to IGF1R-CD. (A) Inhibitory activity of hematoxylin on IGF1R detected by ELISA assay. Tests were performed three times independently. (B) Hematoxylin binds to IGF1R-CD. SPR assay was performed as described in Materials and methods. (C) The binding mode of hematoxylin to the kinase domain of IGF1R predicted by molecular docking. The ligand (green carbon) and residues (cyan carbon) are represented as sticks and lines, respectively. The yellow dashed lines denote the hydrogen bonds and the oxygen atoms are colored in red. The molecular surface of the binding site was shown as green dots. The structure figure was prepared using PyMol (www.pymol.org). Data shown were mean±SD from three independent experiments.

Techniques Used: Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

Effect of hematoxylin on the proliferation and the phosphorylation of IGF1R and downstream signaling pathways in HL-60 cells. (A) Growth inhibition of HL-60 cells by hematoxylin. Cells were treated with the indicated concentrations of hematoxylin for 72 h. Cell viability was determined by sulforhodamine B assay. The tests were repeated three times independently. (B) Hematoxlyin inhibits the phosphorylation of IGF1R and the activation of downstream signaling pathways in HL-60 cells. Cells were cultured in the presence of different doses of hematoxylin for 24 h and harvested. Whole cell lysates were assayed for different proteins by immunoblotting. A representative anti-GAPDH immunoblot is shown as a loading control. Data shown were mean±SD from three independent experiments.
Figure Legend Snippet: Effect of hematoxylin on the proliferation and the phosphorylation of IGF1R and downstream signaling pathways in HL-60 cells. (A) Growth inhibition of HL-60 cells by hematoxylin. Cells were treated with the indicated concentrations of hematoxylin for 72 h. Cell viability was determined by sulforhodamine B assay. The tests were repeated three times independently. (B) Hematoxlyin inhibits the phosphorylation of IGF1R and the activation of downstream signaling pathways in HL-60 cells. Cells were cultured in the presence of different doses of hematoxylin for 24 h and harvested. Whole cell lysates were assayed for different proteins by immunoblotting. A representative anti-GAPDH immunoblot is shown as a loading control. Data shown were mean±SD from three independent experiments.

Techniques Used: Inhibition, Sulforhodamine B Assay, Activation Assay, Cell Culture

ELISA assays determining the phosphorylation of a synthetic peptide. (A) Plot of A 490 vs IGF1R quantity illustrating the relationship between substrate phosphorylation status and IGF1R level. (B) Plot of A 490 vs ATP concentration, and (C) Plot of A 490 vs concentration of synthetic substrate poly(Glu,Tyr) 4:1 , used to determine the optimal concentration of substrate in the screening model. Data shown were mean±SD from three independent experiments.
Figure Legend Snippet: ELISA assays determining the phosphorylation of a synthetic peptide. (A) Plot of A 490 vs IGF1R quantity illustrating the relationship between substrate phosphorylation status and IGF1R level. (B) Plot of A 490 vs ATP concentration, and (C) Plot of A 490 vs concentration of synthetic substrate poly(Glu,Tyr) 4:1 , used to determine the optimal concentration of substrate in the screening model. Data shown were mean±SD from three independent experiments.

Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay

Titration of the divalent cations and determination of IGF1R inhibitory activity of I-OMe-AG 538 in the established screening model. (A-B) Plot of A 490 vs concentrations of divalent cations, used to determine the metal requirement for catalysis of substrate phosphorylation. (C) Effect of positive IGF1R inhibitor, I-OMe-AG 538, on IGF1R tyrosine kinase activity. Data shown were mean±SD from three independent experiments.
Figure Legend Snippet: Titration of the divalent cations and determination of IGF1R inhibitory activity of I-OMe-AG 538 in the established screening model. (A-B) Plot of A 490 vs concentrations of divalent cations, used to determine the metal requirement for catalysis of substrate phosphorylation. (C) Effect of positive IGF1R inhibitor, I-OMe-AG 538, on IGF1R tyrosine kinase activity. Data shown were mean±SD from three independent experiments.

Techniques Used: Titration, Activity Assay

SDS-polyacrylamide gel analysis of purified IGF1R-CD. (A) IGF1R-CD was Ni-NTA column purified from T ni insect cells, and the purity of the fusion protein was examined in aliquots from different steps of the purification. Lane 1, whole infected cell lysate; Lane 2, supernatant sample; Lane 3, cell debris after lysis; Lane 4, wash step fraction; Lane 5, elution step fraction 1; Lane 6, elution step fraction 2; M, molecular weight marker. Proteins were separated by 15% SDS-PAGE, and stained with Coomassie blue dye. (B) Parallel samples were immunoprecipitated using a polyclonal antibody against His-Tag and th en Western blotted with an anti-IGF1R antibody.
Figure Legend Snippet: SDS-polyacrylamide gel analysis of purified IGF1R-CD. (A) IGF1R-CD was Ni-NTA column purified from T ni insect cells, and the purity of the fusion protein was examined in aliquots from different steps of the purification. Lane 1, whole infected cell lysate; Lane 2, supernatant sample; Lane 3, cell debris after lysis; Lane 4, wash step fraction; Lane 5, elution step fraction 1; Lane 6, elution step fraction 2; M, molecular weight marker. Proteins were separated by 15% SDS-PAGE, and stained with Coomassie blue dye. (B) Parallel samples were immunoprecipitated using a polyclonal antibody against His-Tag and th en Western blotted with an anti-IGF1R antibody.

Techniques Used: Purification, Infection, Lysis, Molecular Weight, Marker, SDS Page, Staining, Immunoprecipitation, Western Blot

33) Product Images from "A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome"

Article Title: A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome

Journal: Nature

doi: 10.1038/nature13234

Pol α contains a Ctf4-interacting motif that binds to the helical domain of Ctf4 a , Identification of the Ctf4-binding motif of Pol1, the yeast orthologue of Pol α. GST-tagged constructs spanning progressively smaller N-terminal regions of Pol1 were tested for interaction with Ctf4 CTD in pull-down experiments on glutathione sepharose beads. The top panel shows the boundaries of the GST-Pol1 constructs; the bottom panel shows the result of the pull-down experiments, analysed by SDS-PAGE. The last lane on the right-hand side of the gel contains only Ctf4 CTD . The position of the Ctf4 CTD band in the pull-down experiments is highlighted by a box. The asterisk marks the position of GST-Pol1 121-348 , which overlaps partially with Ctf4 CTD . b , Multiple sequence alignment of the Ctf4-binding motif of yeast Pol1 (Sc; S. cerevisiae ) with Pol α sequences from S. pombe (Sp), D. rerio (Dr), D. melanogaster (Dm) and H. sapiens (Hs). Invariant residues are highlighted in green, identical residues in yellow and similar residues in cyan. The asterisk marks amino acids that are essential for interaction with Ctf4 CTD (see panel c). c , Alanine-scanning mutagenesis of the Ctf4-binding motif. Pol1 residues 137 to 149 were fused to GST and each amino acid between 140 and 149 (except G145) was mutated to alanine. The effect of each single-point mutation on the interaction with Ctf4 CTD was tested by GST pull-down and analysed by SDS-PAGE. d , The budding yeast strains POL1-9MYC ( Control ) and pol1-A-9MYC ( pol1-A , containing the D141A, D142A, L144A and F147A mutations in the endogenous POL1 locus) were grown at 24°C, arrested in G1-phase and released into S-phase for 30 minutes. The MYC-tagged proteins were isolated from cell extracts by immunoprecipitation on anti-MYC beads and the indicated proteins were detected by immunoblotting with the corresponding antibodies 23 . e , Co-crystal structure of Ctf4 CTD bound to a peptide corresponding to the Ctf4-binding motif of Pol α. Ctf4 is drawn as in Fig. 1a , the Ctf4-binding motif of Pol α is drawn as green ribbon. f , Detailed view of the interaction between the Ctf4-binding motif of Pol α (green tube) and the helical domain of Ctf4 (yellow ribbon). The side chains of Pol1 residues F140, D142, I143, L144, F147 and Ctf4 residue R904 are shown as sticks.
Figure Legend Snippet: Pol α contains a Ctf4-interacting motif that binds to the helical domain of Ctf4 a , Identification of the Ctf4-binding motif of Pol1, the yeast orthologue of Pol α. GST-tagged constructs spanning progressively smaller N-terminal regions of Pol1 were tested for interaction with Ctf4 CTD in pull-down experiments on glutathione sepharose beads. The top panel shows the boundaries of the GST-Pol1 constructs; the bottom panel shows the result of the pull-down experiments, analysed by SDS-PAGE. The last lane on the right-hand side of the gel contains only Ctf4 CTD . The position of the Ctf4 CTD band in the pull-down experiments is highlighted by a box. The asterisk marks the position of GST-Pol1 121-348 , which overlaps partially with Ctf4 CTD . b , Multiple sequence alignment of the Ctf4-binding motif of yeast Pol1 (Sc; S. cerevisiae ) with Pol α sequences from S. pombe (Sp), D. rerio (Dr), D. melanogaster (Dm) and H. sapiens (Hs). Invariant residues are highlighted in green, identical residues in yellow and similar residues in cyan. The asterisk marks amino acids that are essential for interaction with Ctf4 CTD (see panel c). c , Alanine-scanning mutagenesis of the Ctf4-binding motif. Pol1 residues 137 to 149 were fused to GST and each amino acid between 140 and 149 (except G145) was mutated to alanine. The effect of each single-point mutation on the interaction with Ctf4 CTD was tested by GST pull-down and analysed by SDS-PAGE. d , The budding yeast strains POL1-9MYC ( Control ) and pol1-A-9MYC ( pol1-A , containing the D141A, D142A, L144A and F147A mutations in the endogenous POL1 locus) were grown at 24°C, arrested in G1-phase and released into S-phase for 30 minutes. The MYC-tagged proteins were isolated from cell extracts by immunoprecipitation on anti-MYC beads and the indicated proteins were detected by immunoblotting with the corresponding antibodies 23 . e , Co-crystal structure of Ctf4 CTD bound to a peptide corresponding to the Ctf4-binding motif of Pol α. Ctf4 is drawn as in Fig. 1a , the Ctf4-binding motif of Pol α is drawn as green ribbon. f , Detailed view of the interaction between the Ctf4-binding motif of Pol α (green tube) and the helical domain of Ctf4 (yellow ribbon). The side chains of Pol1 residues F140, D142, I143, L144, F147 and Ctf4 residue R904 are shown as sticks.

Techniques Used: Binding Assay, Construct, SDS Page, Sequencing, Mutagenesis, Isolation, Immunoprecipitation

Architecture of yeast Ctf4 a , Ctf4 self-associates in a trimer of novel design. The panel shows top and side views of the crystal structure of the C-terminal region of yeast Ctf4 (Ctf4 CTD ; amino acids 471 to 927). The protein is drawn as ribbon, coloured according to its domain structure: the β-propeller domain is in light blue and the helical domain in yellow. Above the drawing, a bar diagram shows the domain structure of full-length yeast Ctf4 and the extent of the region crystallized in our study. b , Analysis of full-length Ctf4 by single-particle electron microscopy. Multivariate statistical symmetry analysis detects a threefold symmetry component for the full-length Ctf4 particle. Reference-free class averages of full-length Ctf4 reveal a core structure flexibly linked to up to three satellite domains. c , Analysis of Ctf4 CTD by single-particle electron microscopy. The C-terminal domain of Ctf4 maintains a trimeric structure, as shown by multivariate statistical symmetry analysis and reference-free class averages. d , Size exclusion chromatography - multi-angle laser scattering analysis of yeast Ctf4 CTD . The light scattering is plotted alongside the fitted molecular weights. The protein eluted in a single peak, corresponding to a measured molecular weight of 161.1 kDa. The predicted molecular weight for the trimeric species is 163.1 kDa. e , Native mass-spectrometry analysis of yeast Ctf4 CTD . The measured molecular weight of 163195 Da matches closely the predicted molecular weight of 163148 Da for a trimeric species.
Figure Legend Snippet: Architecture of yeast Ctf4 a , Ctf4 self-associates in a trimer of novel design. The panel shows top and side views of the crystal structure of the C-terminal region of yeast Ctf4 (Ctf4 CTD ; amino acids 471 to 927). The protein is drawn as ribbon, coloured according to its domain structure: the β-propeller domain is in light blue and the helical domain in yellow. Above the drawing, a bar diagram shows the domain structure of full-length yeast Ctf4 and the extent of the region crystallized in our study. b , Analysis of full-length Ctf4 by single-particle electron microscopy. Multivariate statistical symmetry analysis detects a threefold symmetry component for the full-length Ctf4 particle. Reference-free class averages of full-length Ctf4 reveal a core structure flexibly linked to up to three satellite domains. c , Analysis of Ctf4 CTD by single-particle electron microscopy. The C-terminal domain of Ctf4 maintains a trimeric structure, as shown by multivariate statistical symmetry analysis and reference-free class averages. d , Size exclusion chromatography - multi-angle laser scattering analysis of yeast Ctf4 CTD . The light scattering is plotted alongside the fitted molecular weights. The protein eluted in a single peak, corresponding to a measured molecular weight of 161.1 kDa. The predicted molecular weight for the trimeric species is 163.1 kDa. e , Native mass-spectrometry analysis of yeast Ctf4 CTD . The measured molecular weight of 163195 Da matches closely the predicted molecular weight of 163148 Da for a trimeric species.

Techniques Used: Electron Microscopy, Size-exclusion Chromatography, Molecular Weight, Mass Spectrometry

The Sld5 subunit of yeast GINS shares a common mechanism of Ctf4 binding with Pol α a , Analysis of the Ctf4 - GINS interaction by gel filtration chromatography, using Ctf4 CTD and versions of GINS that contain either full-length (top panel) or N-terminally truncated Sld5 (Sld5ΔN; bottom panel). b , Multiple sequence alignment of the N-terminus of fungal Sld5 sequences (Sc, Saccharomyces cerevisiae ; Ag, Ashbya gossypii ; An, Aspergillus niger ; Gz, Gibberella zeae ; Ca, Candida albicans ). Invariant residues are highlighted in green, identical residues in yellow and similar residues in cyan. The Ctf4-binding motif of yeast Pol1 is reported below the alignment. c , Mapping of the Ctf4-binding sequence in the N-terminus of Sld5 by GST-pull down analysis. The top panel shows the boundaries of the GSTSld5 constructs tested for interaction with Ctf4 CTD ; the bottom panel shows the results of the pull-down experiments, analysed by SDS-PAGE. The band marked with an asterisk corresponds to free GST. d, Alanine-scanning mutagenesis of the Ctf4-binding motif. Residues 1 to 20 of yeast Sld5 were fused to GST and each position between 3 and 13 (except A10) was mutated to alanine. The effect of each single-point mutation on the interaction with Ctf4 CTD was tested by GST pull-down and analysed by SDS-PAGE. e , Co-crystal structure of Ctf4 CTD bound to a peptide corresponding to the Ctf4-binding motif of Sld5. Ctf4 is drawn as in Fig. 1a , the Ctf4-binding motif of Sld5 is drawn as red ribbon. f , Detailed view of the interaction between the Ctf4-binding motif of Sld5 (red tube) and the helical domain of Ctf4 (yellow ribbon). The side chains of Sld5 residues I3, I5, D7, I8, L9, L12 and Ctf4 residue R904 are shown as sticks.
Figure Legend Snippet: The Sld5 subunit of yeast GINS shares a common mechanism of Ctf4 binding with Pol α a , Analysis of the Ctf4 - GINS interaction by gel filtration chromatography, using Ctf4 CTD and versions of GINS that contain either full-length (top panel) or N-terminally truncated Sld5 (Sld5ΔN; bottom panel). b , Multiple sequence alignment of the N-terminus of fungal Sld5 sequences (Sc, Saccharomyces cerevisiae ; Ag, Ashbya gossypii ; An, Aspergillus niger ; Gz, Gibberella zeae ; Ca, Candida albicans ). Invariant residues are highlighted in green, identical residues in yellow and similar residues in cyan. The Ctf4-binding motif of yeast Pol1 is reported below the alignment. c , Mapping of the Ctf4-binding sequence in the N-terminus of Sld5 by GST-pull down analysis. The top panel shows the boundaries of the GSTSld5 constructs tested for interaction with Ctf4 CTD ; the bottom panel shows the results of the pull-down experiments, analysed by SDS-PAGE. The band marked with an asterisk corresponds to free GST. d, Alanine-scanning mutagenesis of the Ctf4-binding motif. Residues 1 to 20 of yeast Sld5 were fused to GST and each position between 3 and 13 (except A10) was mutated to alanine. The effect of each single-point mutation on the interaction with Ctf4 CTD was tested by GST pull-down and analysed by SDS-PAGE. e , Co-crystal structure of Ctf4 CTD bound to a peptide corresponding to the Ctf4-binding motif of Sld5. Ctf4 is drawn as in Fig. 1a , the Ctf4-binding motif of Sld5 is drawn as red ribbon. f , Detailed view of the interaction between the Ctf4-binding motif of Sld5 (red tube) and the helical domain of Ctf4 (yellow ribbon). The side chains of Sld5 residues I3, I5, D7, I8, L9, L12 and Ctf4 residue R904 are shown as sticks.

Techniques Used: Binding Assay, Filtration, Chromatography, Sequencing, Construct, SDS Page, Mutagenesis

The Ctf4 trimer coordinates the recruitment of replication factors to the fork a , Superposition of the structures of Ctf4 CTD bound to the Ctf4-binding motif of Pol α (green tube) and Sld5 (red tube). Ctf4 CTD is displayed as molecular surface, in light brown. b , Ctf4 CTD can associate in principle with up to three partner proteins. To illustrate this point, the Ctf4-binding motif of Pol α was modelled in each of the three binding sites of Ctf4 CTD . The helical Ctf4-binding motif is shown as a white cylinder, and Ctf4 CTD is drawn as a molecular surface, in light blue. c , Native mass spectrometry analysis of the Ctf4 CTD trimer in the presence of peptides corresponding to the Ctf4-binding motifs of Pol α (top) and Sld5 (bottom). d , Single-particle electron microscopy analysis of the interaction of GINS with the Ctf4 CTD trimer. Reference-free class averages of Ctf4 CTD bound to one (top row), two (middle row) or three copies (bottom row) of GINS are shown. e , Reference-free class averages of the Ctf4 CTD - Pol1 NTD (top row), Ctf4 CTD - Pol1 NTD - GINS (middle row) and Ctf4 CTD - Pol1 NTD - (GINS) 2 (bottom row) heteroassemblies. f , The panel shows the crystal structure of human GINS ( ref. 25 ) docked into the electron microscopy reconstruction of the CMG helicase ( ref. 24 ). The Sld5 subunit of GINS is coloured orange and the rest of GINS is shown in white. The density for the MCM and Cdc45 subunits of the CMG helicase is shown as a semi-transparent grey surface, whereas the density of the GINS tetramer is shown as an outline. The position of MCM2, MCM3, MCM5 and Cdc45, which surround GINS in the helicase complex, is indicated. An arrow marks the amino-terminal residue in the Sld5 structure. g , A model of Ctf4 function at the replication fork, as the physical bridge between the CMG helicase and the DNA polymerase α/primase complex. The additional contacts between Ctf4 and GINS suggested by the EM analysis (panel d) are indicated by dashed lines.
Figure Legend Snippet: The Ctf4 trimer coordinates the recruitment of replication factors to the fork a , Superposition of the structures of Ctf4 CTD bound to the Ctf4-binding motif of Pol α (green tube) and Sld5 (red tube). Ctf4 CTD is displayed as molecular surface, in light brown. b , Ctf4 CTD can associate in principle with up to three partner proteins. To illustrate this point, the Ctf4-binding motif of Pol α was modelled in each of the three binding sites of Ctf4 CTD . The helical Ctf4-binding motif is shown as a white cylinder, and Ctf4 CTD is drawn as a molecular surface, in light blue. c , Native mass spectrometry analysis of the Ctf4 CTD trimer in the presence of peptides corresponding to the Ctf4-binding motifs of Pol α (top) and Sld5 (bottom). d , Single-particle electron microscopy analysis of the interaction of GINS with the Ctf4 CTD trimer. Reference-free class averages of Ctf4 CTD bound to one (top row), two (middle row) or three copies (bottom row) of GINS are shown. e , Reference-free class averages of the Ctf4 CTD - Pol1 NTD (top row), Ctf4 CTD - Pol1 NTD - GINS (middle row) and Ctf4 CTD - Pol1 NTD - (GINS) 2 (bottom row) heteroassemblies. f , The panel shows the crystal structure of human GINS ( ref. 25 ) docked into the electron microscopy reconstruction of the CMG helicase ( ref. 24 ). The Sld5 subunit of GINS is coloured orange and the rest of GINS is shown in white. The density for the MCM and Cdc45 subunits of the CMG helicase is shown as a semi-transparent grey surface, whereas the density of the GINS tetramer is shown as an outline. The position of MCM2, MCM3, MCM5 and Cdc45, which surround GINS in the helicase complex, is indicated. An arrow marks the amino-terminal residue in the Sld5 structure. g , A model of Ctf4 function at the replication fork, as the physical bridge between the CMG helicase and the DNA polymerase α/primase complex. The additional contacts between Ctf4 and GINS suggested by the EM analysis (panel d) are indicated by dashed lines.

Techniques Used: Binding Assay, Mass Spectrometry, Electron Microscopy

34) Product Images from "A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome"

Article Title: A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome

Journal: Nature

doi: 10.1038/nature13234

Pol α contains a Ctf4-interacting motif that binds to the helical domain of Ctf4 a , Identification of the Ctf4-binding motif of Pol1, the yeast orthologue of Pol α. GST-tagged constructs spanning progressively smaller N-terminal regions of Pol1 were tested for interaction with Ctf4 CTD in pull-down experiments on glutathione sepharose beads. The top panel shows the boundaries of the GST-Pol1 constructs; the bottom panel shows the result of the pull-down experiments, analysed by SDS-PAGE. The last lane on the right-hand side of the gel contains only Ctf4 CTD . The position of the Ctf4 CTD band in the pull-down experiments is highlighted by a box. The asterisk marks the position of GST-Pol1 121-348 , which overlaps partially with Ctf4 CTD . b , Multiple sequence alignment of the Ctf4-binding motif of yeast Pol1 (Sc; S. cerevisiae ) with Pol α sequences from S. pombe (Sp), D. rerio (Dr), D. melanogaster (Dm) and H. sapiens (Hs). Invariant residues are highlighted in green, identical residues in yellow and similar residues in cyan. The asterisk marks amino acids that are essential for interaction with Ctf4 CTD (see panel c). c , Alanine-scanning mutagenesis of the Ctf4-binding motif. Pol1 residues 137 to 149 were fused to GST and each amino acid between 140 and 149 (except G145) was mutated to alanine. The effect of each single-point mutation on the interaction with Ctf4 CTD was tested by GST pull-down and analysed by SDS-PAGE. d , The budding yeast strains POL1-9MYC ( Control ) and pol1-A-9MYC ( pol1-A , containing the D141A, D142A, L144A and F147A mutations in the endogenous POL1 locus) were grown at 24°C, arrested in G1-phase and released into S-phase for 30 minutes. The MYC-tagged proteins were isolated from cell extracts by immunoprecipitation on anti-MYC beads and the indicated proteins were detected by immunoblotting with the corresponding antibodies 23 . e , Co-crystal structure of Ctf4 CTD bound to a peptide corresponding to the Ctf4-binding motif of Pol α. Ctf4 is drawn as in Fig. 1a , the Ctf4-binding motif of Pol α is drawn as green ribbon. f , Detailed view of the interaction between the Ctf4-binding motif of Pol α (green tube) and the helical domain of Ctf4 (yellow ribbon). The side chains of Pol1 residues F140, D142, I143, L144, F147 and Ctf4 residue R904 are shown as sticks.
Figure Legend Snippet: Pol α contains a Ctf4-interacting motif that binds to the helical domain of Ctf4 a , Identification of the Ctf4-binding motif of Pol1, the yeast orthologue of Pol α. GST-tagged constructs spanning progressively smaller N-terminal regions of Pol1 were tested for interaction with Ctf4 CTD in pull-down experiments on glutathione sepharose beads. The top panel shows the boundaries of the GST-Pol1 constructs; the bottom panel shows the result of the pull-down experiments, analysed by SDS-PAGE. The last lane on the right-hand side of the gel contains only Ctf4 CTD . The position of the Ctf4 CTD band in the pull-down experiments is highlighted by a box. The asterisk marks the position of GST-Pol1 121-348 , which overlaps partially with Ctf4 CTD . b , Multiple sequence alignment of the Ctf4-binding motif of yeast Pol1 (Sc; S. cerevisiae ) with Pol α sequences from S. pombe (Sp), D. rerio (Dr), D. melanogaster (Dm) and H. sapiens (Hs). Invariant residues are highlighted in green, identical residues in yellow and similar residues in cyan. The asterisk marks amino acids that are essential for interaction with Ctf4 CTD (see panel c). c , Alanine-scanning mutagenesis of the Ctf4-binding motif. Pol1 residues 137 to 149 were fused to GST and each amino acid between 140 and 149 (except G145) was mutated to alanine. The effect of each single-point mutation on the interaction with Ctf4 CTD was tested by GST pull-down and analysed by SDS-PAGE. d , The budding yeast strains POL1-9MYC ( Control ) and pol1-A-9MYC ( pol1-A , containing the D141A, D142A, L144A and F147A mutations in the endogenous POL1 locus) were grown at 24°C, arrested in G1-phase and released into S-phase for 30 minutes. The MYC-tagged proteins were isolated from cell extracts by immunoprecipitation on anti-MYC beads and the indicated proteins were detected by immunoblotting with the corresponding antibodies 23 . e , Co-crystal structure of Ctf4 CTD bound to a peptide corresponding to the Ctf4-binding motif of Pol α. Ctf4 is drawn as in Fig. 1a , the Ctf4-binding motif of Pol α is drawn as green ribbon. f , Detailed view of the interaction between the Ctf4-binding motif of Pol α (green tube) and the helical domain of Ctf4 (yellow ribbon). The side chains of Pol1 residues F140, D142, I143, L144, F147 and Ctf4 residue R904 are shown as sticks.

Techniques Used: Binding Assay, Construct, SDS Page, Sequencing, Mutagenesis, Isolation, Immunoprecipitation

Architecture of yeast Ctf4 a , Ctf4 self-associates in a trimer of novel design. The panel shows top and side views of the crystal structure of the C-terminal region of yeast Ctf4 (Ctf4 CTD ; amino acids 471 to 927). The protein is drawn as ribbon, coloured according to its domain structure: the β-propeller domain is in light blue and the helical domain in yellow. Above the drawing, a bar diagram shows the domain structure of full-length yeast Ctf4 and the extent of the region crystallized in our study. b , Analysis of full-length Ctf4 by single-particle electron microscopy. Multivariate statistical symmetry analysis detects a threefold symmetry component for the full-length Ctf4 particle. Reference-free class averages of full-length Ctf4 reveal a core structure flexibly linked to up to three satellite domains. c , Analysis of Ctf4 CTD by single-particle electron microscopy. The C-terminal domain of Ctf4 maintains a trimeric structure, as shown by multivariate statistical symmetry analysis and reference-free class averages. d , Size exclusion chromatography - multi-angle laser scattering analysis of yeast Ctf4 CTD . The light scattering is plotted alongside the fitted molecular weights. The protein eluted in a single peak, corresponding to a measured molecular weight of 161.1 kDa. The predicted molecular weight for the trimeric species is 163.1 kDa. e , Native mass-spectrometry analysis of yeast Ctf4 CTD . The measured molecular weight of 163195 Da matches closely the predicted molecular weight of 163148 Da for a trimeric species.
Figure Legend Snippet: Architecture of yeast Ctf4 a , Ctf4 self-associates in a trimer of novel design. The panel shows top and side views of the crystal structure of the C-terminal region of yeast Ctf4 (Ctf4 CTD ; amino acids 471 to 927). The protein is drawn as ribbon, coloured according to its domain structure: the β-propeller domain is in light blue and the helical domain in yellow. Above the drawing, a bar diagram shows the domain structure of full-length yeast Ctf4 and the extent of the region crystallized in our study. b , Analysis of full-length Ctf4 by single-particle electron microscopy. Multivariate statistical symmetry analysis detects a threefold symmetry component for the full-length Ctf4 particle. Reference-free class averages of full-length Ctf4 reveal a core structure flexibly linked to up to three satellite domains. c , Analysis of Ctf4 CTD by single-particle electron microscopy. The C-terminal domain of Ctf4 maintains a trimeric structure, as shown by multivariate statistical symmetry analysis and reference-free class averages. d , Size exclusion chromatography - multi-angle laser scattering analysis of yeast Ctf4 CTD . The light scattering is plotted alongside the fitted molecular weights. The protein eluted in a single peak, corresponding to a measured molecular weight of 161.1 kDa. The predicted molecular weight for the trimeric species is 163.1 kDa. e , Native mass-spectrometry analysis of yeast Ctf4 CTD . The measured molecular weight of 163195 Da matches closely the predicted molecular weight of 163148 Da for a trimeric species.

Techniques Used: Electron Microscopy, Size-exclusion Chromatography, Molecular Weight, Mass Spectrometry

The Sld5 subunit of yeast GINS shares a common mechanism of Ctf4 binding with Pol α a , Analysis of the Ctf4 - GINS interaction by gel filtration chromatography, using Ctf4 CTD and versions of GINS that contain either full-length (top panel) or N-terminally truncated Sld5 (Sld5ΔN; bottom panel). b , Multiple sequence alignment of the N-terminus of fungal Sld5 sequences (Sc, Saccharomyces cerevisiae ; Ag, Ashbya gossypii ; An, Aspergillus niger ; Gz, Gibberella zeae ; Ca, Candida albicans ). Invariant residues are highlighted in green, identical residues in yellow and similar residues in cyan. The Ctf4-binding motif of yeast Pol1 is reported below the alignment. c , Mapping of the Ctf4-binding sequence in the N-terminus of Sld5 by GST-pull down analysis. The top panel shows the boundaries of the GSTSld5 constructs tested for interaction with Ctf4 CTD ; the bottom panel shows the results of the pull-down experiments, analysed by SDS-PAGE. The band marked with an asterisk corresponds to free GST. d, Alanine-scanning mutagenesis of the Ctf4-binding motif. Residues 1 to 20 of yeast Sld5 were fused to GST and each position between 3 and 13 (except A10) was mutated to alanine. The effect of each single-point mutation on the interaction with Ctf4 CTD was tested by GST pull-down and analysed by SDS-PAGE. e , Co-crystal structure of Ctf4 CTD bound to a peptide corresponding to the Ctf4-binding motif of Sld5. Ctf4 is drawn as in Fig. 1a , the Ctf4-binding motif of Sld5 is drawn as red ribbon. f , Detailed view of the interaction between the Ctf4-binding motif of Sld5 (red tube) and the helical domain of Ctf4 (yellow ribbon). The side chains of Sld5 residues I3, I5, D7, I8, L9, L12 and Ctf4 residue R904 are shown as sticks.
Figure Legend Snippet: The Sld5 subunit of yeast GINS shares a common mechanism of Ctf4 binding with Pol α a , Analysis of the Ctf4 - GINS interaction by gel filtration chromatography, using Ctf4 CTD and versions of GINS that contain either full-length (top panel) or N-terminally truncated Sld5 (Sld5ΔN; bottom panel). b , Multiple sequence alignment of the N-terminus of fungal Sld5 sequences (Sc, Saccharomyces cerevisiae ; Ag, Ashbya gossypii ; An, Aspergillus niger ; Gz, Gibberella zeae ; Ca, Candida albicans ). Invariant residues are highlighted in green, identical residues in yellow and similar residues in cyan. The Ctf4-binding motif of yeast Pol1 is reported below the alignment. c , Mapping of the Ctf4-binding sequence in the N-terminus of Sld5 by GST-pull down analysis. The top panel shows the boundaries of the GSTSld5 constructs tested for interaction with Ctf4 CTD ; the bottom panel shows the results of the pull-down experiments, analysed by SDS-PAGE. The band marked with an asterisk corresponds to free GST. d, Alanine-scanning mutagenesis of the Ctf4-binding motif. Residues 1 to 20 of yeast Sld5 were fused to GST and each position between 3 and 13 (except A10) was mutated to alanine. The effect of each single-point mutation on the interaction with Ctf4 CTD was tested by GST pull-down and analysed by SDS-PAGE. e , Co-crystal structure of Ctf4 CTD bound to a peptide corresponding to the Ctf4-binding motif of Sld5. Ctf4 is drawn as in Fig. 1a , the Ctf4-binding motif of Sld5 is drawn as red ribbon. f , Detailed view of the interaction between the Ctf4-binding motif of Sld5 (red tube) and the helical domain of Ctf4 (yellow ribbon). The side chains of Sld5 residues I3, I5, D7, I8, L9, L12 and Ctf4 residue R904 are shown as sticks.

Techniques Used: Binding Assay, Filtration, Chromatography, Sequencing, Construct, SDS Page, Mutagenesis

The Ctf4 trimer coordinates the recruitment of replication factors to the fork a , Superposition of the structures of Ctf4 CTD bound to the Ctf4-binding motif of Pol α (green tube) and Sld5 (red tube). Ctf4 CTD is displayed as molecular surface, in light brown. b , Ctf4 CTD can associate in principle with up to three partner proteins. To illustrate this point, the Ctf4-binding motif of Pol α was modelled in each of the three binding sites of Ctf4 CTD . The helical Ctf4-binding motif is shown as a white cylinder, and Ctf4 CTD is drawn as a molecular surface, in light blue. c , Native mass spectrometry analysis of the Ctf4 CTD trimer in the presence of peptides corresponding to the Ctf4-binding motifs of Pol α (top) and Sld5 (bottom). d , Single-particle electron microscopy analysis of the interaction of GINS with the Ctf4 CTD trimer. Reference-free class averages of Ctf4 CTD bound to one (top row), two (middle row) or three copies (bottom row) of GINS are shown. e , Reference-free class averages of the Ctf4 CTD - Pol1 NTD (top row), Ctf4 CTD - Pol1 NTD - GINS (middle row) and Ctf4 CTD - Pol1 NTD - (GINS) 2 (bottom row) heteroassemblies. f , The panel shows the crystal structure of human GINS ( ref. 25 ) docked into the electron microscopy reconstruction of the CMG helicase ( ref. 24 ). The Sld5 subunit of GINS is coloured orange and the rest of GINS is shown in white. The density for the MCM and Cdc45 subunits of the CMG helicase is shown as a semi-transparent grey surface, whereas the density of the GINS tetramer is shown as an outline. The position of MCM2, MCM3, MCM5 and Cdc45, which surround GINS in the helicase complex, is indicated. An arrow marks the amino-terminal residue in the Sld5 structure. g , A model of Ctf4 function at the replication fork, as the physical bridge between the CMG helicase and the DNA polymerase α/primase complex. The additional contacts between Ctf4 and GINS suggested by the EM analysis (panel d) are indicated by dashed lines.
Figure Legend Snippet: The Ctf4 trimer coordinates the recruitment of replication factors to the fork a , Superposition of the structures of Ctf4 CTD bound to the Ctf4-binding motif of Pol α (green tube) and Sld5 (red tube). Ctf4 CTD is displayed as molecular surface, in light brown. b , Ctf4 CTD can associate in principle with up to three partner proteins. To illustrate this point, the Ctf4-binding motif of Pol α was modelled in each of the three binding sites of Ctf4 CTD . The helical Ctf4-binding motif is shown as a white cylinder, and Ctf4 CTD is drawn as a molecular surface, in light blue. c , Native mass spectrometry analysis of the Ctf4 CTD trimer in the presence of peptides corresponding to the Ctf4-binding motifs of Pol α (top) and Sld5 (bottom). d , Single-particle electron microscopy analysis of the interaction of GINS with the Ctf4 CTD trimer. Reference-free class averages of Ctf4 CTD bound to one (top row), two (middle row) or three copies (bottom row) of GINS are shown. e , Reference-free class averages of the Ctf4 CTD - Pol1 NTD (top row), Ctf4 CTD - Pol1 NTD - GINS (middle row) and Ctf4 CTD - Pol1 NTD - (GINS) 2 (bottom row) heteroassemblies. f , The panel shows the crystal structure of human GINS ( ref. 25 ) docked into the electron microscopy reconstruction of the CMG helicase ( ref. 24 ). The Sld5 subunit of GINS is coloured orange and the rest of GINS is shown in white. The density for the MCM and Cdc45 subunits of the CMG helicase is shown as a semi-transparent grey surface, whereas the density of the GINS tetramer is shown as an outline. The position of MCM2, MCM3, MCM5 and Cdc45, which surround GINS in the helicase complex, is indicated. An arrow marks the amino-terminal residue in the Sld5 structure. g , A model of Ctf4 function at the replication fork, as the physical bridge between the CMG helicase and the DNA polymerase α/primase complex. The additional contacts between Ctf4 and GINS suggested by the EM analysis (panel d) are indicated by dashed lines.

Techniques Used: Binding Assay, Mass Spectrometry, Electron Microscopy

35) Product Images from "Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo"

Article Title: Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo

Journal: BioMed Research International

doi: 10.1155/2015/863845

Inhibition of HUVEC sprouting by recombinant PEDF. (a) Exemplary fluorescence micrographs of DiO labeled HUVEC spheroids cultured in the presence of 2% serum, VEGF, VEGF plus rPEDF, and VEGF plus bevacizumab (scale bars: 50 μ m). (b) Effect of rPEDF and bevacizumab on VEGF-stimulated sprouting of human umbilical vein endothelial cells (HUVEC) spheroids. VEGF increased the cumulative sprouting length by more than 60% above control. The addition of rPEDF at a concentration of 200 ng/mL reduced sprouting below the control level. To achieve the same level of inhibition as 200 ng/mL of rPEDF, it required 250 μ g/mL of bevacizumab. The differences between VEGF-induced HUVEC sprouting and all concentrations of rPEDF and bevacizumab were statistically significant ( ∗∗∗∗ p
Figure Legend Snippet: Inhibition of HUVEC sprouting by recombinant PEDF. (a) Exemplary fluorescence micrographs of DiO labeled HUVEC spheroids cultured in the presence of 2% serum, VEGF, VEGF plus rPEDF, and VEGF plus bevacizumab (scale bars: 50 μ m). (b) Effect of rPEDF and bevacizumab on VEGF-stimulated sprouting of human umbilical vein endothelial cells (HUVEC) spheroids. VEGF increased the cumulative sprouting length by more than 60% above control. The addition of rPEDF at a concentration of 200 ng/mL reduced sprouting below the control level. To achieve the same level of inhibition as 200 ng/mL of rPEDF, it required 250 μ g/mL of bevacizumab. The differences between VEGF-induced HUVEC sprouting and all concentrations of rPEDF and bevacizumab were statistically significant ( ∗∗∗∗ p

Techniques Used: Inhibition, Recombinant, Fluorescence, Labeling, Cell Culture, Concentration Assay

Effect of recombinant PEDF on apoptosis of HUVEC. The addition of VEGF protected HUVEC from apoptosis induced by low serum. Apoptosis of HUVEC was significantly increased by the addition of rPEDF or bevacizumab ( ∗∗∗∗ p
Figure Legend Snippet: Effect of recombinant PEDF on apoptosis of HUVEC. The addition of VEGF protected HUVEC from apoptosis induced by low serum. Apoptosis of HUVEC was significantly increased by the addition of rPEDF or bevacizumab ( ∗∗∗∗ p

Techniques Used: Recombinant

PEDF secretion by immortalized RPE cells in vitro and in vivo . (a) ELISA-based quantification and Western blot analysis of total PEDF secretion by immortalized rat RPE cells in vitro at passages 1, 2, 5, and 10 (grey bars) after SB100X -mediated transfection with the pT2-CMV-PEDF/EGFP transposon plasmid. Note that nontransfected control cells secreted only very small amounts of endogenous PEDF (white bar). Data represent the mean ± SD from 2 independent measurements ( ∗∗∗ p = 0.0001). (b) Western blot analysis of total PEDF extracted from rat control eyes and eyes at 1 day, 1, 2, and 3 weeks after subretinal transplantation of PEDF-transfected rat RPE cells. Double bands indicated the slightly different molecular weights of endogenous (46.5 kDa) and recombinant human PEDF (47.5 kDa), which included a His-tag. Loading of equal protein amounts was confirmed by similar densities of GAPDH protein bands (36 kDa). Note that for these experiments the number of cells transplanted was 1 × 10 5 , since the low sensitivity of the anti-PEDF antibodies required a larger amount of protein. Data represent the mean ± SD from 2 independent measurements. (c) ELISA-based quantification of recombinant PEDF secretion in vivo in lysates of rat eyes at 1 day, 1, 2, 3, and 4 weeks after subretinal transplantation of 1 × 10 4 PEDF-transfected RPE cells, showed a constant level of recombinant human PEDF. For nontransfected control cells recombinant PEDF was not detectable (left bar), because the analysis was carried out with Ni-NTA HisSorb plates. Each bar represents the average data of three injected eyes ( ∗∗∗∗ p
Figure Legend Snippet: PEDF secretion by immortalized RPE cells in vitro and in vivo . (a) ELISA-based quantification and Western blot analysis of total PEDF secretion by immortalized rat RPE cells in vitro at passages 1, 2, 5, and 10 (grey bars) after SB100X -mediated transfection with the pT2-CMV-PEDF/EGFP transposon plasmid. Note that nontransfected control cells secreted only very small amounts of endogenous PEDF (white bar). Data represent the mean ± SD from 2 independent measurements ( ∗∗∗ p = 0.0001). (b) Western blot analysis of total PEDF extracted from rat control eyes and eyes at 1 day, 1, 2, and 3 weeks after subretinal transplantation of PEDF-transfected rat RPE cells. Double bands indicated the slightly different molecular weights of endogenous (46.5 kDa) and recombinant human PEDF (47.5 kDa), which included a His-tag. Loading of equal protein amounts was confirmed by similar densities of GAPDH protein bands (36 kDa). Note that for these experiments the number of cells transplanted was 1 × 10 5 , since the low sensitivity of the anti-PEDF antibodies required a larger amount of protein. Data represent the mean ± SD from 2 independent measurements. (c) ELISA-based quantification of recombinant PEDF secretion in vivo in lysates of rat eyes at 1 day, 1, 2, 3, and 4 weeks after subretinal transplantation of 1 × 10 4 PEDF-transfected RPE cells, showed a constant level of recombinant human PEDF. For nontransfected control cells recombinant PEDF was not detectable (left bar), because the analysis was carried out with Ni-NTA HisSorb plates. Each bar represents the average data of three injected eyes ( ∗∗∗∗ p

Techniques Used: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Plasmid Preparation, Transplantation Assay, Recombinant, Injection

Effects of subretinal transplantation of RPE cells transfected with the PEDF gene on laser-induced CNV in a rat model. (a) Fluorescein angiograms of rat eyes 1 and 2 weeks after laser injury and cell transplantation showed the decrease in leakage in eyes transplanted with transfected RPE cells secreting 2 ng rPEDF/day, but not in eyes transplanted with Venus -expressing cells or sham-operated eyes. (b) Distribution of total CNV lesions, classified by a blinded observer into grade 0 (no leakage), grade 1 (very mild leakage), and grade 2 (clear leakage), showed that the eyes transplanted with PEDF-transfected cells had fewer grade 2 lesions than the other groups. Each group comprised at least 6 animals. (c) Quantification of clinically relevant grade 2 CNV lesions in rat eyes after 1 and 2 weeks showed that eyes transplanted with rPEDF-expressing cells had significantly fewer grade 2 lesions (1 week: ∗ p = 0.042; 2 weeks: ∗∗ p = 0.0023). (d) Exemplary fluorescein angiograms of grades 0, 1, and 2 lesions.
Figure Legend Snippet: Effects of subretinal transplantation of RPE cells transfected with the PEDF gene on laser-induced CNV in a rat model. (a) Fluorescein angiograms of rat eyes 1 and 2 weeks after laser injury and cell transplantation showed the decrease in leakage in eyes transplanted with transfected RPE cells secreting 2 ng rPEDF/day, but not in eyes transplanted with Venus -expressing cells or sham-operated eyes. (b) Distribution of total CNV lesions, classified by a blinded observer into grade 0 (no leakage), grade 1 (very mild leakage), and grade 2 (clear leakage), showed that the eyes transplanted with PEDF-transfected cells had fewer grade 2 lesions than the other groups. Each group comprised at least 6 animals. (c) Quantification of clinically relevant grade 2 CNV lesions in rat eyes after 1 and 2 weeks showed that eyes transplanted with rPEDF-expressing cells had significantly fewer grade 2 lesions (1 week: ∗ p = 0.042; 2 weeks: ∗∗ p = 0.0023). (d) Exemplary fluorescein angiograms of grades 0, 1, and 2 lesions.

Techniques Used: Transplantation Assay, Transfection, Expressing

Choroidal flat-mounts of retinas from a rat model of laser-induced CNV. (a) Exemplary fluorescence micrographs of isolectin B4 (vascular leakage) stained choroidal flat-mounts 2 weeks after laser treatment. Note the decrease in B4 staining in the retinas transplanted with RPE cells transfected with the PEDF gene secreting 2 ng rPEDF/day. (b) Quantitative analysis of area of vascular leakage 2 weeks after laser injury showed that transplantation of PEDF-transfected RPE cells reduced the area of leakage by over 50% compared to control, sham-operated eyes, and eyes transplanted with Venus -expressing cells ( ∗∗ p = 0.0012, rPEDF versus no injection, sham injection, and Venus -expressing cells). (c) Choroidal flat-mounts of rat eyes 8 weeks after laser injury showed that the reduction in leakage observed at 2 weeks in eyes transplanted with PEDF-transfected RPE cells was still approximately 50% of the leakage in eyes transplanted with Venus -expressing cells ( ∗∗ p = 0.0022).
Figure Legend Snippet: Choroidal flat-mounts of retinas from a rat model of laser-induced CNV. (a) Exemplary fluorescence micrographs of isolectin B4 (vascular leakage) stained choroidal flat-mounts 2 weeks after laser treatment. Note the decrease in B4 staining in the retinas transplanted with RPE cells transfected with the PEDF gene secreting 2 ng rPEDF/day. (b) Quantitative analysis of area of vascular leakage 2 weeks after laser injury showed that transplantation of PEDF-transfected RPE cells reduced the area of leakage by over 50% compared to control, sham-operated eyes, and eyes transplanted with Venus -expressing cells ( ∗∗ p = 0.0012, rPEDF versus no injection, sham injection, and Venus -expressing cells). (c) Choroidal flat-mounts of rat eyes 8 weeks after laser injury showed that the reduction in leakage observed at 2 weeks in eyes transplanted with PEDF-transfected RPE cells was still approximately 50% of the leakage in eyes transplanted with Venus -expressing cells ( ∗∗ p = 0.0022).

Techniques Used: Fluorescence, Staining, Transfection, Transplantation Assay, Expressing, Injection

Neurogenic activity of purified recombinant PEDF. (a) 5 days after the addition of 20 and 100 ng/mL of rPEDF, it was evident that rPEDF stimulated neurite outgrowth (scale bar: 50 μ m). (b) Quantification showed a 3-fold increase of neurite outgrowth in the presence of 20 ng/mL rPEDF and a 6-fold increase in the presence of 100 ng/mL rPEDF. Data represents the mean ± SD of measurements from 6 microscopic fields from 2 independent experiments ( ∗∗∗∗ p
Figure Legend Snippet: Neurogenic activity of purified recombinant PEDF. (a) 5 days after the addition of 20 and 100 ng/mL of rPEDF, it was evident that rPEDF stimulated neurite outgrowth (scale bar: 50 μ m). (b) Quantification showed a 3-fold increase of neurite outgrowth in the presence of 20 ng/mL rPEDF and a 6-fold increase in the presence of 100 ng/mL rPEDF. Data represents the mean ± SD of measurements from 6 microscopic fields from 2 independent experiments ( ∗∗∗∗ p

Techniques Used: Activity Assay, Purification, Recombinant

Effect of recombinant PEDF on migration of HUVEC. Quantitative analysis showed that migration of HUVEC was stimulated by VEGF and that VEGF-stimulated migration was significantly inhibited by rPEDF and bevacizumab ( ∗∗∗∗ p
Figure Legend Snippet: Effect of recombinant PEDF on migration of HUVEC. Quantitative analysis showed that migration of HUVEC was stimulated by VEGF and that VEGF-stimulated migration was significantly inhibited by rPEDF and bevacizumab ( ∗∗∗∗ p

Techniques Used: Recombinant, Migration

36) Product Images from "Arabidopsis DNA polymerase ϵ recruits components of Polycomb repressor complex to mediate epigenetic gene silencing"

Article Title: Arabidopsis DNA polymerase ϵ recruits components of Polycomb repressor complex to mediate epigenetic gene silencing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw156

Interaction of ESD7 with PRC2 components. ( A ) Diagrams showing ESD7 protein and the fragments used in these analyses, POLA5 and POLA3. The N-terminal catalytic domain is indicated by the black box and the C-terminal domain (white box) includes the POLA5 fragment carrying a MIR region and the POLA3 fragment carrying a zinc finger domain. ( B ) Yeast two-hybrid experiment showing interaction of ESD7 fragments with EMF2 and CLF. One out of three independent co-transformants is shown in the picture growing onto -LWH, -LWHA and -LWHA media containing different 3AT concentrations. ( C ) In vitro binding assay of a fragment of CLF containing the SET domain fused to GST with POLA3 and POLA5 ESD7 radiolabelled fragments. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins. ( D ) In vitro binding assays of the fusion proteins GST-POLA5 and His 6 -EMF2-VEFS containing different fragments of the VEFS domain (4AD and 5AD). The EMF2-derived proteins were revealed by western blot with an αHis antibody. Protein molecular weights are indicated. The lower bands in the input lanes correspond to His 6 -EMF2 VEFS degradation products. Arrows indicate the expected size for the corresponding fusion proteins. ( E ) In vivo co-IP of POLA5 and CLF proteins. Nuclei proteins were extracted from plants overexpressing functional GFP-CLF protein in clf-16 mutant background, POLA5-HA (L4) and from 35S::GFP-CLF 35S::POLA5-HA lines in c lf-16 mutant background. An αHA antibody was used to pull-down the protein complexes and CLF protein was detected using an αGFP antibody. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins. ( F ) In vivo co-IP of POLA5 and EMF2 proteins. Nuclei proteins were extracted from plants overexpressing functional GFP-EMF2 protein in emf2-2 mutant background, POLA5-HA and from 35S::GFP-EMF2 35S::POLA5-HA lines in emf2-2 mutant background. An αHA antibody was used to pull-down the protein complexes and EMF2 protein was detected using an αGFP antibody. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins.
Figure Legend Snippet: Interaction of ESD7 with PRC2 components. ( A ) Diagrams showing ESD7 protein and the fragments used in these analyses, POLA5 and POLA3. The N-terminal catalytic domain is indicated by the black box and the C-terminal domain (white box) includes the POLA5 fragment carrying a MIR region and the POLA3 fragment carrying a zinc finger domain. ( B ) Yeast two-hybrid experiment showing interaction of ESD7 fragments with EMF2 and CLF. One out of three independent co-transformants is shown in the picture growing onto -LWH, -LWHA and -LWHA media containing different 3AT concentrations. ( C ) In vitro binding assay of a fragment of CLF containing the SET domain fused to GST with POLA3 and POLA5 ESD7 radiolabelled fragments. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins. ( D ) In vitro binding assays of the fusion proteins GST-POLA5 and His 6 -EMF2-VEFS containing different fragments of the VEFS domain (4AD and 5AD). The EMF2-derived proteins were revealed by western blot with an αHis antibody. Protein molecular weights are indicated. The lower bands in the input lanes correspond to His 6 -EMF2 VEFS degradation products. Arrows indicate the expected size for the corresponding fusion proteins. ( E ) In vivo co-IP of POLA5 and CLF proteins. Nuclei proteins were extracted from plants overexpressing functional GFP-CLF protein in clf-16 mutant background, POLA5-HA (L4) and from 35S::GFP-CLF 35S::POLA5-HA lines in c lf-16 mutant background. An αHA antibody was used to pull-down the protein complexes and CLF protein was detected using an αGFP antibody. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins. ( F ) In vivo co-IP of POLA5 and EMF2 proteins. Nuclei proteins were extracted from plants overexpressing functional GFP-EMF2 protein in emf2-2 mutant background, POLA5-HA and from 35S::GFP-EMF2 35S::POLA5-HA lines in emf2-2 mutant background. An αHA antibody was used to pull-down the protein complexes and EMF2 protein was detected using an αGFP antibody. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins.

Techniques Used: In Vitro, Binding Assay, Derivative Assay, Western Blot, In Vivo, Co-Immunoprecipitation Assay, Functional Assay, Mutagenesis

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

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr170

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

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

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

Techniques Used: Incubation, Labeling, Autoradiography

38) Product Images from "Non-canonical reader modules of BAZ1A promote recovery from DNA damage"

Article Title: Non-canonical reader modules of BAZ1A promote recovery from DNA damage

Journal: Nature Communications

doi: 10.1038/s41467-017-00866-0

The PHD module of BAZ1A binds free and nucleosomal DNA. a Sequence alignment of the PHD modules of BAZ1A, BAZ1B, and the most N-terminal PHD module of dAcf1 amino acid boundaries are indicated, and identical residues are highlighted in gray . Filled and open circles indicate the Zn-binding Cys 4 HisCys 3 motif characteristic of the PHD fold. Green arrowheads indicate K1181 and K1183 that are required for BAZ1A-PHD to bind DNA. b Electrostatic surface representation of BAZ1A-PHD (model) and BAZ1B-PHD (PDB: 1F62). Red and blue indicate negatively and positively charged areas, respectively. The positively charged surface formed by K1181 and K1183 in the BAZ1A-PHD model and the corresponding region in BAZ1B-PHD are outlined in dotted green . c Steady-state biolayer interferometry measurement of DNA binding to wild-type BAZ1A-PHD, BAZ1B-PHD, or mutant versions of BAZ1A-PHD bearing the single K1181A or the double K1181A/K1183Y substitutions. d Average relative deuterium uptake difference (ARDD) for BAZ1A-PHD bound to DNA compared to uncomplexed BAZ1A-PHD, measured by HDX-MS. The values for six matching peptides (recovered from both DNA-bound and uncomplexed samples) are represented by histograms spanning the corresponding amino acid sequence of the BAZ1A-PHD construct. No matching peptides were obtained for residues 32–42 and histograms of overlapping peptides are shown in transparency. Residue numbering refers to the tag-free expression construct used for this experiment; residues colored in red do not belong to the endogenous BAZ1A sequence. The two lysine residues colored in blue and highlighted with blue arrowheads correspond to K1181 and K1183. Peptide sequences with the highest ARDD values ( > 10%) are highlighted in green ; see Supplementary Fig. 7a , and the “Methods” section for more details. e Peptides with the highest ARDD values ( > 10%) were mapped on the BAZ1A-PHD model surface and shown in green . K1181 and K1183 (residues 47 and 49 in d ) are colored in blue , and white areas correspond to residues 32–42, for which no ARDD data was obtained. f Same as c but using mononucleosomes. Each data point is the mean value ± s.e.m from four independent experiments. Fitted K D s were determined from the averaged data and reported ± standard error
Figure Legend Snippet: The PHD module of BAZ1A binds free and nucleosomal DNA. a Sequence alignment of the PHD modules of BAZ1A, BAZ1B, and the most N-terminal PHD module of dAcf1 amino acid boundaries are indicated, and identical residues are highlighted in gray . Filled and open circles indicate the Zn-binding Cys 4 HisCys 3 motif characteristic of the PHD fold. Green arrowheads indicate K1181 and K1183 that are required for BAZ1A-PHD to bind DNA. b Electrostatic surface representation of BAZ1A-PHD (model) and BAZ1B-PHD (PDB: 1F62). Red and blue indicate negatively and positively charged areas, respectively. The positively charged surface formed by K1181 and K1183 in the BAZ1A-PHD model and the corresponding region in BAZ1B-PHD are outlined in dotted green . c Steady-state biolayer interferometry measurement of DNA binding to wild-type BAZ1A-PHD, BAZ1B-PHD, or mutant versions of BAZ1A-PHD bearing the single K1181A or the double K1181A/K1183Y substitutions. d Average relative deuterium uptake difference (ARDD) for BAZ1A-PHD bound to DNA compared to uncomplexed BAZ1A-PHD, measured by HDX-MS. The values for six matching peptides (recovered from both DNA-bound and uncomplexed samples) are represented by histograms spanning the corresponding amino acid sequence of the BAZ1A-PHD construct. No matching peptides were obtained for residues 32–42 and histograms of overlapping peptides are shown in transparency. Residue numbering refers to the tag-free expression construct used for this experiment; residues colored in red do not belong to the endogenous BAZ1A sequence. The two lysine residues colored in blue and highlighted with blue arrowheads correspond to K1181 and K1183. Peptide sequences with the highest ARDD values ( > 10%) are highlighted in green ; see Supplementary Fig. 7a , and the “Methods” section for more details. e Peptides with the highest ARDD values ( > 10%) were mapped on the BAZ1A-PHD model surface and shown in green . K1181 and K1183 (residues 47 and 49 in d ) are colored in blue , and white areas correspond to residues 32–42, for which no ARDD data was obtained. f Same as c but using mononucleosomes. Each data point is the mean value ± s.e.m from four independent experiments. Fitted K D s were determined from the averaged data and reported ± standard error

Techniques Used: Sequencing, Binding Assay, Mutagenesis, Mass Spectrometry, Construct, Expressing

A non-canonical glutamic acid “gatekeeper” residue reduces the affinity of BAZ1A-BD for acetylated histone peptides. a Schematic representation of the BAZ1A and BAZ1B proteins. b Sequence of BAZ1A-BD and BAZ1B-BD surrounding the “anchor” and the “gatekeeper” residues; amino-acid boundaries are indicated. c Cartoon representation of BAZ1A-BD from the 1.7 Å resolution crystal structure (Table 1 ). d Top view of the binding pockets of BAZ1A-BD and BAZ1B-BD (model) in surface representation. The side chains of the gatekeeper residues are shown in stick representation with carbon atoms colored green and the oxygen atoms of the negatively charged BAZ1A-BD E1515 shown in red . e Representative examples of histone peptide array binding images for BAZ1A-BD, BAZ1A-BD E1515V , BAZ1B-BD, and BAZ1B-BD V1425E protein modules. Refer to Supplementary Fig. 3c, d for peptide array information. f Biolayer interferometry binding study of wild-type BAZ1A-BD, BAZ1A-BD E1515V (BD EV ), BAZ1A-BD N1509Y (BD NY ), or the double mutant BAZ1A-BD E1515V/N1509Y (BD EVNY ). Steady-state binding was determined using biotinylated histone H4 1–19, either with or without acetylation of the four lysines present (see “Methods”). Each data point is the mean value ± s.e.m from four independent experiments. The fitted K D for BAZ1A-BD E1515V was determined from the averaged data and reported ± standard error
Figure Legend Snippet: A non-canonical glutamic acid “gatekeeper” residue reduces the affinity of BAZ1A-BD for acetylated histone peptides. a Schematic representation of the BAZ1A and BAZ1B proteins. b Sequence of BAZ1A-BD and BAZ1B-BD surrounding the “anchor” and the “gatekeeper” residues; amino-acid boundaries are indicated. c Cartoon representation of BAZ1A-BD from the 1.7 Å resolution crystal structure (Table 1 ). d Top view of the binding pockets of BAZ1A-BD and BAZ1B-BD (model) in surface representation. The side chains of the gatekeeper residues are shown in stick representation with carbon atoms colored green and the oxygen atoms of the negatively charged BAZ1A-BD E1515 shown in red . e Representative examples of histone peptide array binding images for BAZ1A-BD, BAZ1A-BD E1515V , BAZ1B-BD, and BAZ1B-BD V1425E protein modules. Refer to Supplementary Fig. 3c, d for peptide array information. f Biolayer interferometry binding study of wild-type BAZ1A-BD, BAZ1A-BD E1515V (BD EV ), BAZ1A-BD N1509Y (BD NY ), or the double mutant BAZ1A-BD E1515V/N1509Y (BD EVNY ). Steady-state binding was determined using biotinylated histone H4 1–19, either with or without acetylation of the four lysines present (see “Methods”). Each data point is the mean value ± s.e.m from four independent experiments. The fitted K D for BAZ1A-BD E1515V was determined from the averaged data and reported ± standard error

Techniques Used: Sequencing, Binding Assay, Peptide Microarray, Mutagenesis

39) Product Images from "Llama immunization with full-length VAR2CSA generates cross-reactive and inhibitory single-domain antibodies against the DBL1X domain"

Article Title: Llama immunization with full-length VAR2CSA generates cross-reactive and inhibitory single-domain antibodies against the DBL1X domain

Journal: Scientific Reports

doi: 10.1038/srep07373

Mapping of the VAR2CSA domains targeted by the nanobodies. ELISA plates were coated at 1 µg/ml with (a) recombinant VAR2CSA multi-domains 3D7-DBL1X-6ε, 3D7-DBL1X-3X, 3D7-DBL4ε-6ε and FCR3-DBL3X-4ε, (b) recombinant VAR2CSA single-domains 3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X and (c) recombinant VAR2CSA single-domains 3D7-DBL5ε, 3D7-DBL6ε and FCR3-DBL6ε, and incubated with the 29 nanobodies (0.3 µg/ml). Nanobody binding was detected with an HRP-conjugated goat anti-llama IgG and revealed with TMB substrate. Optical density was measured at 655nm. (d) Representation of the different nanobody specificities on VAR2CSA.
Figure Legend Snippet: Mapping of the VAR2CSA domains targeted by the nanobodies. ELISA plates were coated at 1 µg/ml with (a) recombinant VAR2CSA multi-domains 3D7-DBL1X-6ε, 3D7-DBL1X-3X, 3D7-DBL4ε-6ε and FCR3-DBL3X-4ε, (b) recombinant VAR2CSA single-domains 3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X and (c) recombinant VAR2CSA single-domains 3D7-DBL5ε, 3D7-DBL6ε and FCR3-DBL6ε, and incubated with the 29 nanobodies (0.3 µg/ml). Nanobody binding was detected with an HRP-conjugated goat anti-llama IgG and revealed with TMB substrate. Optical density was measured at 655nm. (d) Representation of the different nanobody specificities on VAR2CSA.

Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Binding Assay

RaPID plot of the nanobodies. k on and k off values for each VHH to the VAR2CSA antigen are indicated as dots on the 2-dimensional plot next to labels denoting the VHH clone.
Figure Legend Snippet: RaPID plot of the nanobodies. k on and k off values for each VHH to the VAR2CSA antigen are indicated as dots on the 2-dimensional plot next to labels denoting the VHH clone.

Techniques Used:

Nanobodies recognize mostly conformational epitopes. Reduced and non-reduced recombinant protein VAR2CSA (3D7-DBL1X-6ε) was transferred onto membranes by Western blotting and probed with the nanobodies (0.5 µg/ml). Nanobody binding was detected with an HRP-conjugated goat anti-llama IgG. (a) Western blot with anti-DBL1X nanobodies. (b) Western blot with anti-DBL4ε nanobody. (c) Western blot with anti-DBL5ε nanobodies. (d) Western blot with anti-DBL6ε nanobodies.
Figure Legend Snippet: Nanobodies recognize mostly conformational epitopes. Reduced and non-reduced recombinant protein VAR2CSA (3D7-DBL1X-6ε) was transferred onto membranes by Western blotting and probed with the nanobodies (0.5 µg/ml). Nanobody binding was detected with an HRP-conjugated goat anti-llama IgG. (a) Western blot with anti-DBL1X nanobodies. (b) Western blot with anti-DBL4ε nanobody. (c) Western blot with anti-DBL5ε nanobodies. (d) Western blot with anti-DBL6ε nanobodies.

Techniques Used: Recombinant, Western Blot, Binding Assay

Nanobodies raised against VAR2CSA recognize placental-type infected erythrocytes. Nanobody recognition by flow cytometry of native VAR2CSA expressed at the surface of infected erythrocytes with parasite strains NF54 (homologous parasite strain), FCR3 and 7G8 selected for CSA-binding and FCR3 selected for CD36-binding. Nanobody binding was detected with a mouse anti-His IgG and a PE-conjugated goat anti-mouse IgG. The Geometric Mean PE ratio was calculated using the negative control (without nanobody). The different DBLs targeted by the nanobodies are depicted in the figure. Error bars represent the standard deviation of data obtained from three independent experiments (NF54-CSA) and two independent experiments (7G8-CSA).
Figure Legend Snippet: Nanobodies raised against VAR2CSA recognize placental-type infected erythrocytes. Nanobody recognition by flow cytometry of native VAR2CSA expressed at the surface of infected erythrocytes with parasite strains NF54 (homologous parasite strain), FCR3 and 7G8 selected for CSA-binding and FCR3 selected for CD36-binding. Nanobody binding was detected with a mouse anti-His IgG and a PE-conjugated goat anti-mouse IgG. The Geometric Mean PE ratio was calculated using the negative control (without nanobody). The different DBLs targeted by the nanobodies are depicted in the figure. Error bars represent the standard deviation of data obtained from three independent experiments (NF54-CSA) and two independent experiments (7G8-CSA).

Techniques Used: Infection, Flow Cytometry, Cytometry, Binding Assay, Negative Control, Standard Deviation

Recognition of full-length VAR2CSA recombinant protein by the different nanobodies. ELISA plates were coated with 1 µg/ml of full-length recombinant VAR2CSA protein (3D7-DBL1X-6ε) and incubated with the 29 nanobodies (0.3 µg/ml). Nanobody binding was detected with an HRP-conjugated goat anti-llama IgG and revealed with TMB substrate. Optical density was measured at 655nm.
Figure Legend Snippet: Recognition of full-length VAR2CSA recombinant protein by the different nanobodies. ELISA plates were coated with 1 µg/ml of full-length recombinant VAR2CSA protein (3D7-DBL1X-6ε) and incubated with the 29 nanobodies (0.3 µg/ml). Nanobody binding was detected with an HRP-conjugated goat anti-llama IgG and revealed with TMB substrate. Optical density was measured at 655nm.

Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Four nanobodies against DBL1X reproducibly inhibit CSA adhesion of the homologous NF54-CSA line. Adhesion assays on CSA-coated plastic spots were performed using purified erythrocytes infected with the NF54 parasite line and selected for the CSA-binding phenotype. Nineteen different nanobodies were tested for their ability to inhibit the IEs binding to CSA. The percentage of binding for a given condition was calculated using the number of adherent cells obtained with IEs pre-incubated with PBS alone as a reference (100% binding). The different DBLs targeted by the nanobodies are depicted in the figure. Error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Four nanobodies against DBL1X reproducibly inhibit CSA adhesion of the homologous NF54-CSA line. Adhesion assays on CSA-coated plastic spots were performed using purified erythrocytes infected with the NF54 parasite line and selected for the CSA-binding phenotype. Nineteen different nanobodies were tested for their ability to inhibit the IEs binding to CSA. The percentage of binding for a given condition was calculated using the number of adherent cells obtained with IEs pre-incubated with PBS alone as a reference (100% binding). The different DBLs targeted by the nanobodies are depicted in the figure. Error bars represent the standard deviation of three independent experiments.

Techniques Used: Purification, Infection, Binding Assay, Incubation, Standard Deviation

40) Product Images from "Evaluating the effect of enzymatic pretreatment on the anaerobic digestibility of pulp and paper biosludge"

Article Title: Evaluating the effect of enzymatic pretreatment on the anaerobic digestibility of pulp and paper biosludge

Journal: Biotechnology Reports

doi: 10.1016/j.btre.2017.12.009

Specific biogas production, SBP, of biosludge pretreated with enzymes over the 62 days of anaerobic digestion. a) protease from A. oryzae ; b) lysozyme; c) protease from B. licheniformis ; d) glycosidase SCO6604; e) protease BCE_2078 and f) CTec 2. Untreated (control) for all samples had phosphate buffer added to biosludge instead of enzyme solution. Range differences between BMP 1 (a, c, e) and BMP 2 (b, d, f) are mainly due to differences in biosludge and granules used in each BMP, inoculum to substrate ratios and, soluble chemical oxygen demand (sCOD) variations.
Figure Legend Snippet: Specific biogas production, SBP, of biosludge pretreated with enzymes over the 62 days of anaerobic digestion. a) protease from A. oryzae ; b) lysozyme; c) protease from B. licheniformis ; d) glycosidase SCO6604; e) protease BCE_2078 and f) CTec 2. Untreated (control) for all samples had phosphate buffer added to biosludge instead of enzyme solution. Range differences between BMP 1 (a, c, e) and BMP 2 (b, d, f) are mainly due to differences in biosludge and granules used in each BMP, inoculum to substrate ratios and, soluble chemical oxygen demand (sCOD) variations.

Techniques Used:

Biogas production from enzyme solutions. Total biogas productions (TBP) are presented for BMP 3, samples that contained enzyme solutions and inoculum. a) protease from A. oryzae ; b) lysozyme; c) protease from B. licheniformis ; d) glycosidase SCO6604. Inoculum only is the control, i.e. no enzyme added. Error bars show standard deviation of triplicates.
Figure Legend Snippet: Biogas production from enzyme solutions. Total biogas productions (TBP) are presented for BMP 3, samples that contained enzyme solutions and inoculum. a) protease from A. oryzae ; b) lysozyme; c) protease from B. licheniformis ; d) glycosidase SCO6604. Inoculum only is the control, i.e. no enzyme added. Error bars show standard deviation of triplicates.

Techniques Used: Standard Deviation

Related Articles

Affinity Chromatography:

Article Title: The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier
Article Snippet: .. Nanobodies were purified from the periplasmic extract by Ni-NTA (QIAGEN) affinity chromatography. .. Purification of BKA-inhibited TtAac-Nb complexes Mitochondria (500-650 mg total protein) were inhibited with 2 nmol bongkrekic acid (BKA, Sigma Aldrich) per mg mitochondrial protein, supplemented with 20 μM ADP (final volume of 25 mL), at room temperature for 2 h. All subsequent steps were performed at 4°C.

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: Transmission Characteristics of Barley Yellow Striate Mosaic Virus in Its Planthopper Vector Laodelphax striatellus
Article Snippet: .. BYSMV N and Actin proteins were purified from the final suspension of transformed cell treated using Ni-NTA resin (Qiagen, Hilden, Germany) as previous report ( ). .. The purified proteins immunized rabbits, and the specific polyclonal antisera was used to purify Immunoglobulin G (IgG) using A-Sepharose affinity column (Sigma–Aldrich).

Article Title: The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier
Article Snippet: .. Nanobodies were purified from the periplasmic extract by Ni-NTA (QIAGEN) affinity chromatography. .. Purification of BKA-inhibited TtAac-Nb complexes Mitochondria (500-650 mg total protein) were inhibited with 2 nmol bongkrekic acid (BKA, Sigma Aldrich) per mg mitochondrial protein, supplemented with 20 μM ADP (final volume of 25 mL), at room temperature for 2 h. All subsequent steps were performed at 4°C.

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: Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition
Article Snippet: .. Recombinant polypeptides composing the N-terminal region of Kb CesA1p (amino acids 1 to 107), produced in E. coli (with His-tag), and purified by Ni-NTA resin (under denaturing conditions, QIAexpressionist; Qiagen), was used as immunogens for the generation of anti-CesA1p (anti-KbCesA1p) polyclonal antibody. .. Rabbit polyclonal antibodies were generated in the Animal and Plant Care Facility at the Hong Kong University of Science and Technology following institutional and National Institutes of Health guidelines.

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).

Produced:

Article Title: Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition
Article Snippet: .. Recombinant polypeptides composing the N-terminal region of Kb CesA1p (amino acids 1 to 107), produced in E. coli (with His-tag), and purified by Ni-NTA resin (under denaturing conditions, QIAexpressionist; Qiagen), was used as immunogens for the generation of anti-CesA1p (anti-KbCesA1p) polyclonal antibody. .. Rabbit polyclonal antibodies were generated in the Animal and Plant Care Facility at the Hong Kong University of Science and Technology following institutional and National Institutes of Health guidelines.

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).

Transformation Assay:

Article Title: Transmission Characteristics of Barley Yellow Striate Mosaic Virus in Its Planthopper Vector Laodelphax striatellus
Article Snippet: .. BYSMV N and Actin proteins were purified from the final suspension of transformed cell treated using Ni-NTA resin (Qiagen, Hilden, Germany) as previous report ( ). .. The purified proteins immunized rabbits, and the specific polyclonal antisera was used to purify Immunoglobulin G (IgG) using A-Sepharose affinity column (Sigma–Aldrich).

Recombinant:

Article Title: Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition
Article Snippet: .. Recombinant polypeptides composing the N-terminal region of Kb CesA1p (amino acids 1 to 107), produced in E. coli (with His-tag), and purified by Ni-NTA resin (under denaturing conditions, QIAexpressionist; Qiagen), was used as immunogens for the generation of anti-CesA1p (anti-KbCesA1p) polyclonal antibody. .. Rabbit polyclonal antibodies were generated in the Animal and Plant Care Facility at the Hong Kong University of Science and Technology following institutional and National Institutes of Health guidelines.

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).

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    Qiagen ni nta resin
    The human Coq9 polypeptide associates with yeast Coq6. Mitochondria were isolated from <t>CNAP6</t> and CNAP6: mcHCOQ9 . Purified mitochondria (13 mg) were solubilized and co-precipitation was then performed on the solubilized mitochondria with <t>Ni-NTA</t> resin. Flow-through (FT), wash (W1 and W2), eluate (E1 and E2), and beads from co-precipitation were collected. 0.17% of the FT, 0.25% of W1, 0.25% of W2, 1% of E1, 0.5% of E2, and 1.25% of Ni-NTA resin were analyzed by SDS-PAGE followed by immunoblotting with antibodies against yeast Coq9, Coq6, human COQ9, and Atp2. Purified mitochondria (15 μg) from CNAP6: mcHCOQ9 were included as control.
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    The human Coq9 polypeptide associates with yeast Coq6. Mitochondria were isolated from CNAP6 and CNAP6: mcHCOQ9 . Purified mitochondria (13 mg) were solubilized and co-precipitation was then performed on the solubilized mitochondria with Ni-NTA resin. Flow-through (FT), wash (W1 and W2), eluate (E1 and E2), and beads from co-precipitation were collected. 0.17% of the FT, 0.25% of W1, 0.25% of W2, 1% of E1, 0.5% of E2, and 1.25% of Ni-NTA resin were analyzed by SDS-PAGE followed by immunoblotting with antibodies against yeast Coq9, Coq6, human COQ9, and Atp2. Purified mitochondria (15 μg) from CNAP6: mcHCOQ9 were included as control.

    Journal: Frontiers in Physiology

    Article Title: Human COQ9 Rescues a coq9 Yeast Mutant by Enhancing Coenzyme Q Biosynthesis from 4-Hydroxybenzoic Acid and Stabilizing the CoQ-Synthome

    doi: 10.3389/fphys.2017.00463

    Figure Lengend Snippet: The human Coq9 polypeptide associates with yeast Coq6. Mitochondria were isolated from CNAP6 and CNAP6: mcHCOQ9 . Purified mitochondria (13 mg) were solubilized and co-precipitation was then performed on the solubilized mitochondria with Ni-NTA resin. Flow-through (FT), wash (W1 and W2), eluate (E1 and E2), and beads from co-precipitation were collected. 0.17% of the FT, 0.25% of W1, 0.25% of W2, 1% of E1, 0.5% of E2, and 1.25% of Ni-NTA resin were analyzed by SDS-PAGE followed by immunoblotting with antibodies against yeast Coq9, Coq6, human COQ9, and Atp2. Purified mitochondria (15 μg) from CNAP6: mcHCOQ9 were included as control.

    Article Snippet: Mitochondria purified from CNAP6:mcHCOQ9 were solubilized with digitonin and subjected to co-precipitation over Ni-NTA resin.

    Techniques: Isolation, Purification, Flow Cytometry, SDS Page

    Purified PduP-E and non-tagged E from whole cells and purified BMCs. a SDS-PAGE analysis of PduP-E purified from whole cells and non-tagged E after Factor Xa proteolysis. Lane 1 molecular weight markers with sizes of standards in kDa on the left. Lane 2 PduP-E purified from Ec3087 induced with 0.1 mM rhamnose only. Lane 3 PduP-E purified from Ec3087 co-induced with 0.1 mM rhamnose and 0.5 mM IPTG. Lane 4 non-tagged E after Factor Xa proteolysis. Lanes 2 – 4 were loaded with 1 µg of protein. b SDS-PAGE and anti-His 6 Western blot analysis during purification of PduP-E from purified BMCs. Lane 1 molecular weight markers. Lane 2 purified BMCs from Ec3087 co-induced with rhamnose and IPTG (starting material). Lane 3 flow through after binding to the Ni–NTA column. Lanes 4 – 6 sequential wash fractions containing 20 mM imidazole. Lanes 7 – 15 sequential elution fractions containing 200 mM imidazole

    Journal: Microbial Cell Factories

    Article Title: Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ϕX174 in Escherichia coli

    doi: 10.1186/s12934-017-0685-x

    Figure Lengend Snippet: Purified PduP-E and non-tagged E from whole cells and purified BMCs. a SDS-PAGE analysis of PduP-E purified from whole cells and non-tagged E after Factor Xa proteolysis. Lane 1 molecular weight markers with sizes of standards in kDa on the left. Lane 2 PduP-E purified from Ec3087 induced with 0.1 mM rhamnose only. Lane 3 PduP-E purified from Ec3087 co-induced with 0.1 mM rhamnose and 0.5 mM IPTG. Lane 4 non-tagged E after Factor Xa proteolysis. Lanes 2 – 4 were loaded with 1 µg of protein. b SDS-PAGE and anti-His 6 Western blot analysis during purification of PduP-E from purified BMCs. Lane 1 molecular weight markers. Lane 2 purified BMCs from Ec3087 co-induced with rhamnose and IPTG (starting material). Lane 3 flow through after binding to the Ni–NTA column. Lanes 4 – 6 sequential wash fractions containing 20 mM imidazole. Lanes 7 – 15 sequential elution fractions containing 200 mM imidazole

    Article Snippet: The non-tagged E was found to no longer bind to Ni–NTA resin, allowing for partial removal of the major contaminant at ~25 kDa by incubation with Ni–NTA resin.

    Techniques: Purification, SDS Page, Molecular Weight, Western Blot, Flow Cytometry, Binding Assay

    Heteromultimerization by PCVRs Lysates from B. anthracis atxA- null pXO1+ pXO2- strains (UT423) containing plasmids that encode IPTG-inducible AtxA-His (pUTE991), AcpA-FLAG (pUTE1079), or GFP-FLAG (pUTE1013); were co-incubated as indicated, then co-affinity purified with Ni 2+ -NTA resin. Proteins present in the mixed lysates prior to (Load, lanes 1–3) and after purification (Eluate, lanes 4–6) were subjected to SDS-PAGE and Western blot with α-His and α-FLAG antibodies as indicated. Arrows indicate the predicted sizes of AtxA, AcpA, and GFP.

    Journal: Molecular microbiology

    Article Title: Regulons and Protein-Protein Interactions of PRD-containing Bacillus anthracis Virulence Regulators Reveal Overlapping but Distinct Functions

    doi: 10.1111/mmi.13961

    Figure Lengend Snippet: Heteromultimerization by PCVRs Lysates from B. anthracis atxA- null pXO1+ pXO2- strains (UT423) containing plasmids that encode IPTG-inducible AtxA-His (pUTE991), AcpA-FLAG (pUTE1079), or GFP-FLAG (pUTE1013); were co-incubated as indicated, then co-affinity purified with Ni 2+ -NTA resin. Proteins present in the mixed lysates prior to (Load, lanes 1–3) and after purification (Eluate, lanes 4–6) were subjected to SDS-PAGE and Western blot with α-His and α-FLAG antibodies as indicated. Arrows indicate the predicted sizes of AtxA, AcpA, and GFP.

    Article Snippet: Preparations of affinity-purified AcpA-His and AcpB-His used for BMH crosslinking experiments were purified using NTA-Ni resin and eluted using imidazole.

    Techniques: Incubation, Affinity Purification, Purification, SDS Page, Western Blot

    Homomultimerization AcpA and AcpB Lysates from B. anthracis atxA- null pXO1+ pXO2- strains (UT423) containing plasmids that encode IPTG-inducible (A) AcpA-His (pUTE1090), AcpA-FLAG (pUTE1079), or GFP-FLAG (pUTE1013); (B) AcpB-His (pUTE1091), AcpB-FLAG (pUTE1093), or GFP-FLAG (pUTE1013) were co-incubated as indicated, then co-affinity purified with Ni 2+ -NTA resin. Proteins present in the mixed lysates prior to (Load, lanes 1–3) and after purification (Eluate, lanes 4–6) were subjected to SDS-PAGE and Western blot with α-His and α-FLAG antibodies as indicated. Arrows indicate the predicted sizes of AcpA-His, AcpA-FLAG, AcpB-His, AcpB-FLAG, and GFP-FLAG; (C) FLAG-tagged AcpA (pUTE1079) and AcpB (pUTE1093) were induced by IPTG in a B. anthracis atxA- null pXO1+ pXO2- strain. Lysates were incubated with or without the crosslinking agent BMH and subjected to SDS-PAGE and Western blot. Proteins were detected with α-FLAG antibody. (D) Affinity purified AcpA-His from B. anthracis ANR-1 (pUTE1090) incubated with or without BMH and subjected to SDS-PAGE and Western blot. Proteins were detected with α-His antibody.

    Journal: Molecular microbiology

    Article Title: Regulons and Protein-Protein Interactions of PRD-containing Bacillus anthracis Virulence Regulators Reveal Overlapping but Distinct Functions

    doi: 10.1111/mmi.13961

    Figure Lengend Snippet: Homomultimerization AcpA and AcpB Lysates from B. anthracis atxA- null pXO1+ pXO2- strains (UT423) containing plasmids that encode IPTG-inducible (A) AcpA-His (pUTE1090), AcpA-FLAG (pUTE1079), or GFP-FLAG (pUTE1013); (B) AcpB-His (pUTE1091), AcpB-FLAG (pUTE1093), or GFP-FLAG (pUTE1013) were co-incubated as indicated, then co-affinity purified with Ni 2+ -NTA resin. Proteins present in the mixed lysates prior to (Load, lanes 1–3) and after purification (Eluate, lanes 4–6) were subjected to SDS-PAGE and Western blot with α-His and α-FLAG antibodies as indicated. Arrows indicate the predicted sizes of AcpA-His, AcpA-FLAG, AcpB-His, AcpB-FLAG, and GFP-FLAG; (C) FLAG-tagged AcpA (pUTE1079) and AcpB (pUTE1093) were induced by IPTG in a B. anthracis atxA- null pXO1+ pXO2- strain. Lysates were incubated with or without the crosslinking agent BMH and subjected to SDS-PAGE and Western blot. Proteins were detected with α-FLAG antibody. (D) Affinity purified AcpA-His from B. anthracis ANR-1 (pUTE1090) incubated with or without BMH and subjected to SDS-PAGE and Western blot. Proteins were detected with α-His antibody.

    Article Snippet: Preparations of affinity-purified AcpA-His and AcpB-His used for BMH crosslinking experiments were purified using NTA-Ni resin and eluted using imidazole.

    Techniques: Incubation, Affinity Purification, Purification, SDS Page, Western Blot

    Interaction of hRPA with RMI1. A , mixtures of BLM, Topo IIIα, and RMI1 with hRPA were incubated with Ni-NTA resin, which was washed and then treated with SDS to elute bound proteins. hRPA alone was also incubated with the resin. The supernatant

    Journal: The Journal of Biological Chemistry

    Article Title: Role of Replication Protein A in Double Holliday Junction Dissolution Mediated by the BLM-Topo III?-RMI1-RMI2 Protein Complex *

    doi: 10.1074/jbc.M113.465609

    Figure Lengend Snippet: Interaction of hRPA with RMI1. A , mixtures of BLM, Topo IIIα, and RMI1 with hRPA were incubated with Ni-NTA resin, which was washed and then treated with SDS to elute bound proteins. hRPA alone was also incubated with the resin. The supernatant

    Article Snippet: When Ni-NTA resin was used to pull down RMI1 through its (His)6 tag, hRPA was also retained on the affinity matrix ( A , lane 9 ).

    Techniques: Incubation