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 "SPIN1 promotes tumorigenesis by blocking the uL18 (universal large ribosomal subunit protein 18)-MDM2-p53 pathway in human cancer"

Article Title: SPIN1 promotes tumorigenesis by blocking the uL18 (universal large ribosomal subunit protein 18)-MDM2-p53 pathway in human cancer

Journal: eLife

doi: 10.7554/eLife.31275

SPIN1-dT2 fails to inhibit p53.
Figure Legend Snippet: SPIN1-dT2 fails to inhibit p53.

Techniques Used:

Western blotting analyses of human colon cancer tissues (n = 22) and normal colon tissue (n = 20) and quantification of SPIN1 expression (Mean ± SEM, p
Figure Legend Snippet: Western blotting analyses of human colon cancer tissues (n = 22) and normal colon tissue (n = 20) and quantification of SPIN1 expression (Mean ± SEM, p

Techniques Used: Western Blot, Expressing

SPIN1 interacts with uL18 in HEK293 cells. HEK293 cells were transfected with plasmids encoding Myc-SPIN1 and Flag-uL18, and 48 hr later cell lysates were collected for IP-WB analysis using anti-Flag antibody.
Figure Legend Snippet: SPIN1 interacts with uL18 in HEK293 cells. HEK293 cells were transfected with plasmids encoding Myc-SPIN1 and Flag-uL18, and 48 hr later cell lysates were collected for IP-WB analysis using anti-Flag antibody.

Techniques Used: Transfection, Western Blot

SPIN1 reduces p53 stability by enhancing MDM2-mediated ubiquitination. ( A ) and ( B ) p53-half-life is increased by SPIN1 knockdown. ( A ) HCT116 p53+/+ cells transfected with scramble or SPIN1 siRNA for 48 hr, were treated with 100 μg/ml of cycloheximide (CHX), and harvested at different time points as indicated. The p53 protein was detected by WB analysis, quantified by densitometry and plotted against time to determine p53-half-lives ( B ). ( C ) and ( D ) SPIN1 overexpression shortens the half-life of p53. HCT116 p53+/+ cells transfected with pcDNA or Flag-SPIN1 for 48 hr were treated with 100 μg/ml of cycloheximide and harvested at indicated time points for WB analysis with indicated antibodies ( C ). The intensity of each band was quantified, and normalized with β-actin and plotted ( D ). ( E ) SPIN1 promotes MDM2-induced p53 ubiquitination. HCT116 p53-/- cells were transfected with combinations of plasmids encoding HA-MDM2, p53, His-Ub or Myc-SPIN1, and treated with MG132 for 6 hr before being harvested for in vivo ubiquitination assay. Bound and input proteins were detected by WB analysis with indicated antibodies. ( F ) SPIN1 enhances MDM2-mediated p53 proteasomal degradation. HCT116 p53+/+ cells were transfected with plasmids encoding HA-MDM2 and Flag-SPIN1, and treated with MG132 for 6 hr before harvested, followed by WB analysis with antibodies as indicated. ( G ) Ectopic SPIN1 does not change p53 protein level without MDM2. MEF p53-/-; Mdm2-/- cells were transfected with combinations of plasmids encoding p53 with or without Flag-SPIN1, followed by WB analysis using antibodies as indicated.
Figure Legend Snippet: SPIN1 reduces p53 stability by enhancing MDM2-mediated ubiquitination. ( A ) and ( B ) p53-half-life is increased by SPIN1 knockdown. ( A ) HCT116 p53+/+ cells transfected with scramble or SPIN1 siRNA for 48 hr, were treated with 100 μg/ml of cycloheximide (CHX), and harvested at different time points as indicated. The p53 protein was detected by WB analysis, quantified by densitometry and plotted against time to determine p53-half-lives ( B ). ( C ) and ( D ) SPIN1 overexpression shortens the half-life of p53. HCT116 p53+/+ cells transfected with pcDNA or Flag-SPIN1 for 48 hr were treated with 100 μg/ml of cycloheximide and harvested at indicated time points for WB analysis with indicated antibodies ( C ). The intensity of each band was quantified, and normalized with β-actin and plotted ( D ). ( E ) SPIN1 promotes MDM2-induced p53 ubiquitination. HCT116 p53-/- cells were transfected with combinations of plasmids encoding HA-MDM2, p53, His-Ub or Myc-SPIN1, and treated with MG132 for 6 hr before being harvested for in vivo ubiquitination assay. Bound and input proteins were detected by WB analysis with indicated antibodies. ( F ) SPIN1 enhances MDM2-mediated p53 proteasomal degradation. HCT116 p53+/+ cells were transfected with plasmids encoding HA-MDM2 and Flag-SPIN1, and treated with MG132 for 6 hr before harvested, followed by WB analysis with antibodies as indicated. ( G ) Ectopic SPIN1 does not change p53 protein level without MDM2. MEF p53-/-; Mdm2-/- cells were transfected with combinations of plasmids encoding p53 with or without Flag-SPIN1, followed by WB analysis using antibodies as indicated.

Techniques Used: Transfection, Western Blot, Over Expression, In Vivo, Ubiquitin Assay

SPIN1 knockdown increase p53 and its targets protein levels in a dose-dependent manner. HCT116 p53+/+ ( A ) and U2OS ( B ) cells were transfected with a titrated concentration of SPIN1 siRNA (20 nM, 40 nM and 60 nM). And cells were harvested 48 hr after transfection and followed by WB analysis with indicated antibodies. ( C ) U2OS cells were co-transfected with scramble siRNA (40 nM), SPIN1-siRNA (40 nM), with or without FLAG-SPIN1 as indicated. Cells were harvested 48 hr after transfection for WB analysis.
Figure Legend Snippet: SPIN1 knockdown increase p53 and its targets protein levels in a dose-dependent manner. HCT116 p53+/+ ( A ) and U2OS ( B ) cells were transfected with a titrated concentration of SPIN1 siRNA (20 nM, 40 nM and 60 nM). And cells were harvested 48 hr after transfection and followed by WB analysis with indicated antibodies. ( C ) U2OS cells were co-transfected with scramble siRNA (40 nM), SPIN1-siRNA (40 nM), with or without FLAG-SPIN1 as indicated. Cells were harvested 48 hr after transfection for WB analysis.

Techniques Used: Transfection, Concentration Assay, Western Blot

Expression of genes involved in p53 pathway is correlated with SPIN1 expression. mRNA expression levels of 664 colorectal tumors were retrieved from Genomic Data Commons ( https://portal.gdc.cancer.gov/ ). In the data set, gene expression levels were measured with FPKM (Fragments Per Kilobase of transcript per Million mapped reads) and normalized using the Upper Quantile method. All 644 tumor samples were sorted based on the expression level of SPIN1 from low to high.
Figure Legend Snippet: Expression of genes involved in p53 pathway is correlated with SPIN1 expression. mRNA expression levels of 664 colorectal tumors were retrieved from Genomic Data Commons ( https://portal.gdc.cancer.gov/ ). In the data set, gene expression levels were measured with FPKM (Fragments Per Kilobase of transcript per Million mapped reads) and normalized using the Upper Quantile method. All 644 tumor samples were sorted based on the expression level of SPIN1 from low to high.

Techniques Used: Expressing

A model for SPIN1 regulation of the uL18-MDM2-p53 pathway in cancer.(see text in the Discussion for details).
Figure Legend Snippet: A model for SPIN1 regulation of the uL18-MDM2-p53 pathway in cancer.(see text in the Discussion for details).

Techniques Used:

SPIN1 knockdown reduces rRNA expression and SPIN1-Y170A mutant retains activity to repress p53. ( A ) Scramble siRNA or SPIN1 siRNA was introduced into U2OS cells. RNA levels were analyzed using Q-PCR (*p value
Figure Legend Snippet: SPIN1 knockdown reduces rRNA expression and SPIN1-Y170A mutant retains activity to repress p53. ( A ) Scramble siRNA or SPIN1 siRNA was introduced into U2OS cells. RNA levels were analyzed using Q-PCR (*p value

Techniques Used: Expressing, Mutagenesis, Activity Assay, Polymerase Chain Reaction

High expression of SPIN1 is detected in multiple cancers and associated with poor prognosis in cancer patients. ( A ) TCGA database was utilized, and the data were modified from the cBioPortal for Cancer Genomics ( http://www.cbioportal.org/ ). ( B ) The expression profile of SPIN1 in cancers and normal tissues was searched in Oncomine Gene Browser ( http://www.oncomine.org/ ). The results were from Talantov Melanoma database. Seven cases of normal skin and 45 cases of melanoma were analyzed in this figure. Correlation between SPIN1 upregulation and tumor stage, poorer prognosis or treatment resistance is not clear. ( C and D ) Overexpression of SPIN1 is correlated with overall survival and disease-free survival in breast cancer ( http://www.cbioportal.org/ ), although the sample number of high SPIN1 patients is small and more samples are desired. ( E ) SPIN1 overexpression was associated with poor prognosis in colorectal cancer patients in an expression profile study from GSE17537 ( http://www.PrognoScan.org/ ). ( F ) High expression of SPIN1 was correlated with poor overall survival in gastric cancer patients ( http://www.kmplot.com ).
Figure Legend Snippet: High expression of SPIN1 is detected in multiple cancers and associated with poor prognosis in cancer patients. ( A ) TCGA database was utilized, and the data were modified from the cBioPortal for Cancer Genomics ( http://www.cbioportal.org/ ). ( B ) The expression profile of SPIN1 in cancers and normal tissues was searched in Oncomine Gene Browser ( http://www.oncomine.org/ ). The results were from Talantov Melanoma database. Seven cases of normal skin and 45 cases of melanoma were analyzed in this figure. Correlation between SPIN1 upregulation and tumor stage, poorer prognosis or treatment resistance is not clear. ( C and D ) Overexpression of SPIN1 is correlated with overall survival and disease-free survival in breast cancer ( http://www.cbioportal.org/ ), although the sample number of high SPIN1 patients is small and more samples are desired. ( E ) SPIN1 overexpression was associated with poor prognosis in colorectal cancer patients in an expression profile study from GSE17537 ( http://www.PrognoScan.org/ ). ( F ) High expression of SPIN1 was correlated with poor overall survival in gastric cancer patients ( http://www.kmplot.com ).

Techniques Used: Expressing, Modification, Over Expression

Mapping of domains responsible for uL18-SPIN1 and uL18-MDM2 binding. ( A ) uL18 interacts with the second Tudor like domain of SPIN1. Purified GST-tagged SPIN1 fragments, including aa 1-262(FL), aa 50–120, aa 121–262, aa 121–193, aa 194–262 and GST protein alone were incubated with purified His-uL18 protein for 3 hr at 4°C. Bound proteins were detected by WB analysis using anti-uL18 or coomassie staining. ( B ) A schematic diagram of uL18-binding regions on SPIN1 based on the result from ( A ). ( C ) SPIN1 interacts with both the N- and C-termini of uL18. Purified GST-tagged uL18 fragments, including aa1-297(FL), aa 1–50, aa 112–297, aa 39–253 or GST protein alone were rotated with purified His-SPIN1 protein for 1 hr at 4°C. Bound proteins were detected by WB analysis using anti-SPIN1 or coomassie staining. ( D ) A schematic diagram of SPIN1-binding regions on uL18 derived from the result from ( C ). ( E ) MDM2 interacts with both the N- and C-termini of uL18. Purified GST-tagged uL18 fragments, including aa1-297(FL), aa 1–50, aa 112–297, aa 39–253, aa 1–251 or GST protein alone were rotated with purified His-MDM2 protein for 4 hr at 4°C. Bound proteins were detected by WB analysis using anti-MDM2 (2A10) or coomassie staining. ( F ) A schematic diagram of uL18 binding regions on MDM2 based on the result from ( E ).
Figure Legend Snippet: Mapping of domains responsible for uL18-SPIN1 and uL18-MDM2 binding. ( A ) uL18 interacts with the second Tudor like domain of SPIN1. Purified GST-tagged SPIN1 fragments, including aa 1-262(FL), aa 50–120, aa 121–262, aa 121–193, aa 194–262 and GST protein alone were incubated with purified His-uL18 protein for 3 hr at 4°C. Bound proteins were detected by WB analysis using anti-uL18 or coomassie staining. ( B ) A schematic diagram of uL18-binding regions on SPIN1 based on the result from ( A ). ( C ) SPIN1 interacts with both the N- and C-termini of uL18. Purified GST-tagged uL18 fragments, including aa1-297(FL), aa 1–50, aa 112–297, aa 39–253 or GST protein alone were rotated with purified His-SPIN1 protein for 1 hr at 4°C. Bound proteins were detected by WB analysis using anti-SPIN1 or coomassie staining. ( D ) A schematic diagram of SPIN1-binding regions on uL18 derived from the result from ( C ). ( E ) MDM2 interacts with both the N- and C-termini of uL18. Purified GST-tagged uL18 fragments, including aa1-297(FL), aa 1–50, aa 112–297, aa 39–253, aa 1–251 or GST protein alone were rotated with purified His-MDM2 protein for 4 hr at 4°C. Bound proteins were detected by WB analysis using anti-MDM2 (2A10) or coomassie staining. ( F ) A schematic diagram of uL18 binding regions on MDM2 based on the result from ( E ).

Techniques Used: Binding Assay, Purification, Incubation, Western Blot, Staining, Derivative Assay

SPIN1 does not bind to MDM2, and SPIN1 and uL18 co-localize in the nucleolus. ( A ) SPIN1 does not bind to MDM2. HCT116 p53-/- cells were transfected with combination of plasmids encoding Myc-SPIN1 and HA-MDM2, followed by IP-WB analysis with indicated antibodies. ( B ) SPIN1 and uL18 co-localize in the nucleolus. HEK293 and H1299 SPIN1 stable cells were transfected with Flag-uL18 for 36 hr and then immunostained with anti-Myc (red) and anti-Flag antibody (green), and counterstained with DAPI.
Figure Legend Snippet: SPIN1 does not bind to MDM2, and SPIN1 and uL18 co-localize in the nucleolus. ( A ) SPIN1 does not bind to MDM2. HCT116 p53-/- cells were transfected with combination of plasmids encoding Myc-SPIN1 and HA-MDM2, followed by IP-WB analysis with indicated antibodies. ( B ) SPIN1 and uL18 co-localize in the nucleolus. HEK293 and H1299 SPIN1 stable cells were transfected with Flag-uL18 for 36 hr and then immunostained with anti-Myc (red) and anti-Flag antibody (green), and counterstained with DAPI.

Techniques Used: Transfection, Western Blot

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-27869-1

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

Techniques Used: Fluorescence, Microscopy, Staining

4) 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

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-019-38982-0

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

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

6) Product Images from "Substrate channeling in oxylipin biosynthesis through a protein complex in the plastid envelope of Arabidopsis thaliana"

Article Title: Substrate channeling in oxylipin biosynthesis through a protein complex in the plastid envelope of Arabidopsis thaliana

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erz015

In vitro import and differential membrane binding of AOS and HPL in chloroplasts. (A) Levels of 35 S-Met-labelled 35 S-AOS (a) and 35 S-HPL (b) before and after import into isolated Arabidopsis chloroplasts. Thl, thermolysin; TP, translation product. The positions of precursor proteins (pAOS and pHPL) and mature proteins (AOS, HPL) are indicated. (B) Detection by SDS-PAGE and autoradiography of 35 S-AOS (a) and 35 S-HPL (b) in trypsin (Try)-treated (+Try) and untreated (-Try) mixed outer and inner plastid envelopes (ME), inner plastid envelope (IM), outer plastid envelope (OM), thylakoids (Th), and stroma (St). CP, Chloroplast reference fraction prior to import and protease treatment. The western blot in panel c shows the levels of the inner chloroplast envelope translocon protein TIC110 and the outer chloroplast envelope protein TOC75 in non-trypsin-treated chloroplasts (CP) versus trypsin-treated chloroplasts containing imported 35 S-AOS/HPL and respective subfractions. Signal detection was made with an enhanced chemiluminescence (ECL) system. (C) Western blot analysis of the endogenous AOS (AOS endo ) and HPL (HPL endo ) (predicted molecular masses 54.5 kDa and 51.3 kDa, respectively; panel a) compared with TOC75, the translocon at the inner chloroplast envelope membrane protein TIC55, the chlorophyll a/b binding protein LHCII (CAB), and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU) (panel b) in the indicated fractions of non-trypsin-treated chloroplasts. Signal detection was made with either ECL or alkaline phosphatase-5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (NBT)-based systems, as indicated. (D) Membrane binding of imported 35 S-AOS (a) and 35 S-HPL (b), as assessed by their extractability by 1 M NaCl and 0.1 M Na 2 CO 3 , pH 11. Both pellet (P) and supernatant (S) fractions, obtained after sedimentation of the membranes, were tested by SDS-PAGE and autoradiography for the two labeled proteins.
Figure Legend Snippet: In vitro import and differential membrane binding of AOS and HPL in chloroplasts. (A) Levels of 35 S-Met-labelled 35 S-AOS (a) and 35 S-HPL (b) before and after import into isolated Arabidopsis chloroplasts. Thl, thermolysin; TP, translation product. The positions of precursor proteins (pAOS and pHPL) and mature proteins (AOS, HPL) are indicated. (B) Detection by SDS-PAGE and autoradiography of 35 S-AOS (a) and 35 S-HPL (b) in trypsin (Try)-treated (+Try) and untreated (-Try) mixed outer and inner plastid envelopes (ME), inner plastid envelope (IM), outer plastid envelope (OM), thylakoids (Th), and stroma (St). CP, Chloroplast reference fraction prior to import and protease treatment. The western blot in panel c shows the levels of the inner chloroplast envelope translocon protein TIC110 and the outer chloroplast envelope protein TOC75 in non-trypsin-treated chloroplasts (CP) versus trypsin-treated chloroplasts containing imported 35 S-AOS/HPL and respective subfractions. Signal detection was made with an enhanced chemiluminescence (ECL) system. (C) Western blot analysis of the endogenous AOS (AOS endo ) and HPL (HPL endo ) (predicted molecular masses 54.5 kDa and 51.3 kDa, respectively; panel a) compared with TOC75, the translocon at the inner chloroplast envelope membrane protein TIC55, the chlorophyll a/b binding protein LHCII (CAB), and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU) (panel b) in the indicated fractions of non-trypsin-treated chloroplasts. Signal detection was made with either ECL or alkaline phosphatase-5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (NBT)-based systems, as indicated. (D) Membrane binding of imported 35 S-AOS (a) and 35 S-HPL (b), as assessed by their extractability by 1 M NaCl and 0.1 M Na 2 CO 3 , pH 11. Both pellet (P) and supernatant (S) fractions, obtained after sedimentation of the membranes, were tested by SDS-PAGE and autoradiography for the two labeled proteins.

Techniques Used: In Vitro, Binding Assay, Isolation, SDS Page, Autoradiography, Western Blot, Sedimentation, Labeling

The Vick and Zimmerman pathway leading to the synthesis of jasmonic acid, and the concurrent AOS and HPL reactions. Pathway intermediates are indicated as follows: 13-HPOT, (9 Z 11 E 15 Z 13 S )-13-hydroperoxy-9,11,15-octadecatrienoic acid; EOT, 12,13( S )-epoxy-9( Z ),11,15( Z )-octadecatrienoic acid; ODA, 12-oxo- cis -9-dodecenoic acid; OPDA, cis -(+)-12-oxophytodienoic acid. Enzymes are indicated as follows: AOC, allene oxide cyclase; AOS, allene oxide synthase; HPL, hydroperoxy lyase; LOX, 13-lipoxygenase. Note that EOT is short-lived and spontaneously disintegrates into volatile α-ketols and γ-ketols as well as racemic OPDA.
Figure Legend Snippet: The Vick and Zimmerman pathway leading to the synthesis of jasmonic acid, and the concurrent AOS and HPL reactions. Pathway intermediates are indicated as follows: 13-HPOT, (9 Z 11 E 15 Z 13 S )-13-hydroperoxy-9,11,15-octadecatrienoic acid; EOT, 12,13( S )-epoxy-9( Z ),11,15( Z )-octadecatrienoic acid; ODA, 12-oxo- cis -9-dodecenoic acid; OPDA, cis -(+)-12-oxophytodienoic acid. Enzymes are indicated as follows: AOC, allene oxide cyclase; AOS, allene oxide synthase; HPL, hydroperoxy lyase; LOX, 13-lipoxygenase. Note that EOT is short-lived and spontaneously disintegrates into volatile α-ketols and γ-ketols as well as racemic OPDA.

Techniques Used:

Detection of AOS complexes in vitro and in planta . (A) Gel filtration elution profile of AOS-FLAG in protein extracts of transgenic plants expressing FLAG-tagged AOS (solid line) and in isolated chloroplasts after in vitro import of AOS-(His) 6 (dashed line). AOS-FLAG was quantified by western blotting using FLAG-specific antibodies and an enhanced chemiluminescence (ECL) system. Similarly, AOS-(His) 6 signals were quantified by either western blotting using FLAG-specific antibodies and ECL detection, or radioactivity measurements in the case of 35 S-AOS-(His) 6 . For easier comparison, the different curves were normalized and values are expressed as relative signal intensities (int.). The positions of apo-ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), and carbonic anhydrase (37 kDa), used as molecular size standards, are indicated (squares and dotted line). (B) Western blot analysis of AOS-FLAG. Positions of purified 14 Leu-labeled tomato heat shock proteins used as molecular size markers are indicated ( M r ). (C) Panel a, SDS-PAGE pattern of plastid envelope proteins co-purifying with AOS-(His) 6 (lane 1) and HPL-(His) 6 (lane 2). The indicated bands were identified by protein sequencing. Panel b, western blotting to identify LOX2 among the proteins detected in panel a.
Figure Legend Snippet: Detection of AOS complexes in vitro and in planta . (A) Gel filtration elution profile of AOS-FLAG in protein extracts of transgenic plants expressing FLAG-tagged AOS (solid line) and in isolated chloroplasts after in vitro import of AOS-(His) 6 (dashed line). AOS-FLAG was quantified by western blotting using FLAG-specific antibodies and an enhanced chemiluminescence (ECL) system. Similarly, AOS-(His) 6 signals were quantified by either western blotting using FLAG-specific antibodies and ECL detection, or radioactivity measurements in the case of 35 S-AOS-(His) 6 . For easier comparison, the different curves were normalized and values are expressed as relative signal intensities (int.). The positions of apo-ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), and carbonic anhydrase (37 kDa), used as molecular size standards, are indicated (squares and dotted line). (B) Western blot analysis of AOS-FLAG. Positions of purified 14 Leu-labeled tomato heat shock proteins used as molecular size markers are indicated ( M r ). (C) Panel a, SDS-PAGE pattern of plastid envelope proteins co-purifying with AOS-(His) 6 (lane 1) and HPL-(His) 6 (lane 2). The indicated bands were identified by protein sequencing. Panel b, western blotting to identify LOX2 among the proteins detected in panel a.

Techniques Used: In Vitro, Filtration, Transgenic Assay, Expressing, Isolation, Western Blot, Radioactivity, Purification, Labeling, SDS Page, Sequencing

7) 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

8) Product Images from "The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier"

Article Title: The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier

Journal: Cell

doi: 10.1016/j.cell.2018.11.025

Interactions between Nanobody and ADP/ATP Carrier, Related to Figure 1 (A) Crystal packing shows alternating layers of carrier proteins (Aac, gray cartoon, with one molecule highlighted as a rainbow cartoon) and nanobodies (Nb, wheat cartoon), with the nanobodies playing key roles in forming crystal contacts. (B) In the crystal, each ADP/ATP carrier molecule (Aac, gray cartoon) interacts with one nanobody (wheat cartoon, interactions highlighted in black box) via the nanobody CDR2 (blue) and CDR3 (red) loops, and with a symmetry-related nanobody (violet cartoon, interactions highlighted in blue box). (C) An enlarged view of the interactions involving the nanobody CDR2 and 3 loops. The CDR3 loop interacts with the matrix end of H1, the matrix helix h12, and the loop connecting them, via hydrogen bond and electrostatic interactions (between D56 of the carrier and R99 of the nanobody). (D) An enlarged view of the interactions with a symmetry-related nanobody (violet cartoon), with A155 and R161 of the carrier forming hydrogen bonds with Q119. These interactions are likely to be solely due to crystal packing.
Figure Legend Snippet: Interactions between Nanobody and ADP/ATP Carrier, Related to Figure 1 (A) Crystal packing shows alternating layers of carrier proteins (Aac, gray cartoon, with one molecule highlighted as a rainbow cartoon) and nanobodies (Nb, wheat cartoon), with the nanobodies playing key roles in forming crystal contacts. (B) In the crystal, each ADP/ATP carrier molecule (Aac, gray cartoon) interacts with one nanobody (wheat cartoon, interactions highlighted in black box) via the nanobody CDR2 (blue) and CDR3 (red) loops, and with a symmetry-related nanobody (violet cartoon, interactions highlighted in blue box). (C) An enlarged view of the interactions involving the nanobody CDR2 and 3 loops. The CDR3 loop interacts with the matrix end of H1, the matrix helix h12, and the loop connecting them, via hydrogen bond and electrostatic interactions (between D56 of the carrier and R99 of the nanobody). (D) An enlarged view of the interactions with a symmetry-related nanobody (violet cartoon), with A155 and R161 of the carrier forming hydrogen bonds with Q119. These interactions are likely to be solely due to crystal packing.

Techniques Used:

9) 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

10) Product Images from "Peptidyl prolyl cis/trans isomerase activity on the cell surface correlates with extracellular matrix development"

Article Title: Peptidyl prolyl cis/trans isomerase activity on the cell surface correlates with extracellular matrix development

Journal: Communications Biology

doi: 10.1038/s42003-019-0315-8

Parallel activity and inhibition measurements. a Inhibition of FKBP12 by rapamycin (black square) and inhibition of CypA (red cycle), CypB (blue upward pointing triangle), and Cyp40 (dark yellow downward-pointing triangle) by CsA. PPIase activities are shown as the mean of triplicates ± SD and expressed as percentages of enzyme activity relative to an inhibitor-free control. The K i values were 1.5 ± 1.2 nM for FKBP/rapamycin, 8.6 ± 1.2 nM for CypA/CsA, 12.9 ± 1.3 nM for CypB/CsA, and 176.8 ± 27.6 nM for Cyp40/CsA. The resulting IC 50 values are in good agreement with the previously reported values using standard PPIase assays. b Concentration-dependent Pin1 activity. The addition of PO 4 3- resulted in Pin1 inhibition. CsA, cyclosporin A; CypA, cyclophilin A; CypB, cyclophilin B; Cyp40, cyclophilin 40; PPIase, peptidyl prolyl cis / trans isomerase
Figure Legend Snippet: Parallel activity and inhibition measurements. a Inhibition of FKBP12 by rapamycin (black square) and inhibition of CypA (red cycle), CypB (blue upward pointing triangle), and Cyp40 (dark yellow downward-pointing triangle) by CsA. PPIase activities are shown as the mean of triplicates ± SD and expressed as percentages of enzyme activity relative to an inhibitor-free control. The K i values were 1.5 ± 1.2 nM for FKBP/rapamycin, 8.6 ± 1.2 nM for CypA/CsA, 12.9 ± 1.3 nM for CypB/CsA, and 176.8 ± 27.6 nM for Cyp40/CsA. The resulting IC 50 values are in good agreement with the previously reported values using standard PPIase assays. b Concentration-dependent Pin1 activity. The addition of PO 4 3- resulted in Pin1 inhibition. CsA, cyclosporin A; CypA, cyclophilin A; CypB, cyclophilin B; Cyp40, cyclophilin 40; PPIase, peptidyl prolyl cis / trans isomerase

Techniques Used: Activity Assay, Inhibition, Concentration Assay

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

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

Journal: Current HIV Research

doi: 10.2174/1570162X17666181206111859

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

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

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

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

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

Techniques Used: Purification, Positive Control, MTT Assay

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

Techniques Used:

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

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

12) 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

13) 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

14) 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

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

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2018.157

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

Techniques Used: Construct

16) 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

17) 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

18) 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

19) Product Images from "A noncognate aminoacyl-tRNA synthetase that may resolve a missing link in protein evolution"

Article Title: A noncognate aminoacyl-tRNA synthetase that may resolve a missing link in protein evolution

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

doi: 10.1073/pnas.1932482100

Overexpression of GluRS2 induces a toxic growth phenotype in E. coli , caused by misacylation of E. coli tRNA Gln1 . ( A ) E. coli DH5α was transformed with pSS001 (GluRS1, □), pSS002 (GluRS2, •), or unmodified pQE-80 (○). Cultures were grown in LB, supplemented with 100 μg/ml ampicillin. Growth was monitored at 600 nm. After each growth, pSS001 and pSS002 were purified and each insert was resequenced to verify that random mutations did not occur. Similar results were obtained by using E. coli K-12 (data not shown). ( B ) Acid gel and Northern blot analysis of E. coli tRNA Gln1 misacylation by H. pylori GluRS2. Hybridization was conducted with an E. coli tRNA Gln1 -specific oligonucleotide (Table 2); the oligonucleotide was kinased with [γ- 32 P]ATP before hybridization.
Figure Legend Snippet: Overexpression of GluRS2 induces a toxic growth phenotype in E. coli , caused by misacylation of E. coli tRNA Gln1 . ( A ) E. coli DH5α was transformed with pSS001 (GluRS1, □), pSS002 (GluRS2, •), or unmodified pQE-80 (○). Cultures were grown in LB, supplemented with 100 μg/ml ampicillin. Growth was monitored at 600 nm. After each growth, pSS001 and pSS002 were purified and each insert was resequenced to verify that random mutations did not occur. Similar results were obtained by using E. coli K-12 (data not shown). ( B ) Acid gel and Northern blot analysis of E. coli tRNA Gln1 misacylation by H. pylori GluRS2. Hybridization was conducted with an E. coli tRNA Gln1 -specific oligonucleotide (Table 2); the oligonucleotide was kinased with [γ- 32 P]ATP before hybridization.

Techniques Used: Over Expression, Transformation Assay, Purification, Northern Blot, Hybridization

Transfer RNA specificities of GluRS1 and GluRS2. ( A ) Glu-tRNA AA biosynthesis by GluRS1 (black) versus GluRS2 (red). ( Top ) tRNA Glu1 .( Middle ) tRNA Glu2 . ( Bottom ) tRNA Gln . Assays shown are the average of triplicate experiments (see Materials and Methods ). ( B ) Acid gel and Northern blot analysis of H. pylori tRNA Glu1 ( Top ), tRNA Glu2 ( Middle ), and tRNA Gln ( Bottom ). The black line in the middle of the bottom two gels represents empty gel lanes that were removed for clarity. Each tRNA/GluRS combination was evaluated individually (lanes 2 and 3) and as a mixture of tRNAs (lanes 4 and 5). For comparison, lane 1 shows each tRNA preparation in the deacylated form, before incubation with either GluRS1 or GluRS2. ( C ) Glutamine is not a substrate for GluRS2. A standard aminoacylation assay was performed (see Materials and Methods ) with GluRS2, 2 μM tRNA Gln , and 100 μM glutamate (□) or 100 μM glutamine (▪). The graph represents averaged data from experiments run in triplicate.
Figure Legend Snippet: Transfer RNA specificities of GluRS1 and GluRS2. ( A ) Glu-tRNA AA biosynthesis by GluRS1 (black) versus GluRS2 (red). ( Top ) tRNA Glu1 .( Middle ) tRNA Glu2 . ( Bottom ) tRNA Gln . Assays shown are the average of triplicate experiments (see Materials and Methods ). ( B ) Acid gel and Northern blot analysis of H. pylori tRNA Glu1 ( Top ), tRNA Glu2 ( Middle ), and tRNA Gln ( Bottom ). The black line in the middle of the bottom two gels represents empty gel lanes that were removed for clarity. Each tRNA/GluRS combination was evaluated individually (lanes 2 and 3) and as a mixture of tRNAs (lanes 4 and 5). For comparison, lane 1 shows each tRNA preparation in the deacylated form, before incubation with either GluRS1 or GluRS2. ( C ) Glutamine is not a substrate for GluRS2. A standard aminoacylation assay was performed (see Materials and Methods ) with GluRS2, 2 μM tRNA Gln , and 100 μM glutamate (□) or 100 μM glutamine (▪). The graph represents averaged data from experiments run in triplicate.

Techniques Used: Northern Blot, Incubation, Aminoacylation Assay

Evolution of bacterial GluRSs. ( A ) Distance tree of bacterial GluRS, GluRS1, and GluRS2 sequences. An ancient gene duplication event ( * ) separates GluRS1 (dark gray) and GluRS2 (light gray) sequences in the proteobacteria. The genomes of organisms labeled with bullets (•) contain a glnS ORF. ( B ) Model for the evolution of GluRS in the proteobacteria. Dashed arrows indicate possible future events in evolution of tRNA specificities. See Materials and Methods for tree derivations.
Figure Legend Snippet: Evolution of bacterial GluRSs. ( A ) Distance tree of bacterial GluRS, GluRS1, and GluRS2 sequences. An ancient gene duplication event ( * ) separates GluRS1 (dark gray) and GluRS2 (light gray) sequences in the proteobacteria. The genomes of organisms labeled with bullets (•) contain a glnS ORF. ( B ) Model for the evolution of GluRS in the proteobacteria. Dashed arrows indicate possible future events in evolution of tRNA specificities. See Materials and Methods for tree derivations.

Techniques Used: Labeling

20) 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

21) 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

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

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

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

doi: 10.1073/pnas.192206699

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

Techniques Used:

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

Techniques Used: Binding Assay

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

Techniques Used: Binding Assay

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

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

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

doi: 10.1073/pnas.192206699

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

Techniques Used:

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

Techniques Used: Binding Assay

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

Techniques Used: Binding Assay

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

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

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

doi: 10.1073/pnas.1619473114

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

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

25) 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

26) 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

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

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

Journal: Nucleic Acids Research

doi:

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

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

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

Techniques Used: Transfection, Construct, Luciferase, Activity Assay

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

Techniques Used: Transfection, Staining, Fluorescence, Microscopy

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

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

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

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

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

Techniques Used: Sequencing, Binding Assay, Incubation, Labeling

28) 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

29) Product Images from "SPLUNC1 Deficiency Enhances Airway Eosinophilic Inflammation in Mice"

Article Title: SPLUNC1 Deficiency Enhances Airway Eosinophilic Inflammation in Mice

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2012-0064OC

SPLUNC1 reduces eotaxin-2 in murine macrophages. ( A ) Full-length human SPLUNC1 cDNA with histidine (His) tag at the C-terminus (C-ter) was cloned into a pRK-7–neo-vector and expressed in human embryonic kidney–293 (HEK293) cells by transient transfection. ( B ) His-tagged SPLUNC1 protein was purified by affinity chromatography. Western blotting was used to verify recombinant SPLUNC1 (rSPLUNC1) protein secretion and purity. ( C and D ) Raw 264.7 cells were seeded (5 × 10 5 cells/well) onto a 48-well tissue culture plate. The next day, cells were pretreated with BSA (control) or murine IL-4/IL-13 (10 ng/ml each) for 1 hour. Thereafter, cells were treated with rSPLUNC1 at different concentrations or with the control solution purified from HEK293 cells transfected with an empty plasmid vector. One hour later, cells were stimulated with LPS (10 ng/ml) or medium alone for 24 hours. Murine eotaxin-2 protein ( C ) and mRNA ( D ) were measured using ELISA and real-time PCR, respectively. Data are expressed as means ± SEM ( n = 3–4 replicates). * P
Figure Legend Snippet: SPLUNC1 reduces eotaxin-2 in murine macrophages. ( A ) Full-length human SPLUNC1 cDNA with histidine (His) tag at the C-terminus (C-ter) was cloned into a pRK-7–neo-vector and expressed in human embryonic kidney–293 (HEK293) cells by transient transfection. ( B ) His-tagged SPLUNC1 protein was purified by affinity chromatography. Western blotting was used to verify recombinant SPLUNC1 (rSPLUNC1) protein secretion and purity. ( C and D ) Raw 264.7 cells were seeded (5 × 10 5 cells/well) onto a 48-well tissue culture plate. The next day, cells were pretreated with BSA (control) or murine IL-4/IL-13 (10 ng/ml each) for 1 hour. Thereafter, cells were treated with rSPLUNC1 at different concentrations or with the control solution purified from HEK293 cells transfected with an empty plasmid vector. One hour later, cells were stimulated with LPS (10 ng/ml) or medium alone for 24 hours. Murine eotaxin-2 protein ( C ) and mRNA ( D ) were measured using ELISA and real-time PCR, respectively. Data are expressed as means ± SEM ( n = 3–4 replicates). * P

Techniques Used: Clone Assay, Plasmid Preparation, Transfection, Purification, Affinity Chromatography, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

30) 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

31) Product Images from "Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies"

Article Title: Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies

Journal: Cell Death & Disease

doi: 10.1038/cddis.2012.117

Cell type-specific expression of Bcl-G revealed by immunohistochemical staining. The Bcl-G-specific mAb 2E11 was used for immunohistochemical staining of tissue sections from the ( a) male reproductive tract, ( b ) GI tract, ( c ) respiratory tract and ( d ) other organs of WT and Bcl-G −/− (negative control) mice. Biotinylated goat anti-rat IgG antibodies were used as the secondary reagent followed by peroxidase conjugation and detection with the DAB reagent (Vector Laboratories). Tissue sections were counterstained with hematoxylin. Representative photomicrographs are shown at × 100 (bar represents 100 μ m) and × 400 magnification (bar represents 40 μ m). The asterisks indicate cases where tissues from Bcl-G −/− mice were unavailable and WT tissues stained only with secondary antibodies were used instead as negative controls
Figure Legend Snippet: Cell type-specific expression of Bcl-G revealed by immunohistochemical staining. The Bcl-G-specific mAb 2E11 was used for immunohistochemical staining of tissue sections from the ( a) male reproductive tract, ( b ) GI tract, ( c ) respiratory tract and ( d ) other organs of WT and Bcl-G −/− (negative control) mice. Biotinylated goat anti-rat IgG antibodies were used as the secondary reagent followed by peroxidase conjugation and detection with the DAB reagent (Vector Laboratories). Tissue sections were counterstained with hematoxylin. Representative photomicrographs are shown at × 100 (bar represents 100 μ m) and × 400 magnification (bar represents 40 μ m). The asterisks indicate cases where tissues from Bcl-G −/− mice were unavailable and WT tissues stained only with secondary antibodies were used instead as negative controls

Techniques Used: Expressing, Immunohistochemistry, Staining, Negative Control, Mouse Assay, Conjugation Assay, Plasmid Preparation

Detection of Bcl-G subcellular localisation using immunofluorescence staining. The Bcl-G-specific mAb clone 2E11 was used in immunofluorescence staining to determine the subcellular localisation of Bcl-G in ( a ) 293T cells transiently overexpressing HA-tagged Bcl-G or empty vector, ( b ) FACS-purified splenic CD8 + conventional DCs, ( c ) paraffin-embedded lymph node and ( d ) testis sections from WT mice. For b – d , Bcl-G −/− DCs and control tissue sections that showed no staining (negative controls) are not shown. Bcl-G immunostaining was detected using Alexa488-conjugated goat anti-rat IgG antibodies (Molecular Probes). DAPI and Mitotracker Red (for a ) were used to stain the nuclei and mitochondria, respectively
Figure Legend Snippet: Detection of Bcl-G subcellular localisation using immunofluorescence staining. The Bcl-G-specific mAb clone 2E11 was used in immunofluorescence staining to determine the subcellular localisation of Bcl-G in ( a ) 293T cells transiently overexpressing HA-tagged Bcl-G or empty vector, ( b ) FACS-purified splenic CD8 + conventional DCs, ( c ) paraffin-embedded lymph node and ( d ) testis sections from WT mice. For b – d , Bcl-G −/− DCs and control tissue sections that showed no staining (negative controls) are not shown. Bcl-G immunostaining was detected using Alexa488-conjugated goat anti-rat IgG antibodies (Molecular Probes). DAPI and Mitotracker Red (for a ) were used to stain the nuclei and mitochondria, respectively

Techniques Used: Immunofluorescence, Staining, Plasmid Preparation, FACS, Purification, Mouse Assay, Immunostaining

Bcl-G-specific mAbs bind to different regions on mouse Bcl-G. ( A ) A panel of ten truncated Bcl-G deletion mutants (M1–M10) was generated and these proteins overexpressed in 293T cells. The region on Bcl-G recognised by each mAb was determined by testing its ability to detect each Bcl-G truncation mutant in western blotting. Probing with HA-specific antibodies served as a loading control. ‘FL' indicates full-length Bcl-G. ( B ) The epitope region recognised by mAb 2E11 was further narrowed using five mutants (a–e) within M4 (amino acids 102–135). ‘−' Indicates extracts from cells transfected with empty vector. ( C ) Intracellular FACS staining of transiently transfected 293T cells was used to map the epitope of mAb 10C9. Cells stained with HA antibody (solid line) or FITC-conjugated anti-rat secondary antibodies (dotted line) were used as positive and negative controls, respectively. ( D ) Schematic diagram of Bcl-G and the epitope regions recognised by each mAb
Figure Legend Snippet: Bcl-G-specific mAbs bind to different regions on mouse Bcl-G. ( A ) A panel of ten truncated Bcl-G deletion mutants (M1–M10) was generated and these proteins overexpressed in 293T cells. The region on Bcl-G recognised by each mAb was determined by testing its ability to detect each Bcl-G truncation mutant in western blotting. Probing with HA-specific antibodies served as a loading control. ‘FL' indicates full-length Bcl-G. ( B ) The epitope region recognised by mAb 2E11 was further narrowed using five mutants (a–e) within M4 (amino acids 102–135). ‘−' Indicates extracts from cells transfected with empty vector. ( C ) Intracellular FACS staining of transiently transfected 293T cells was used to map the epitope of mAb 10C9. Cells stained with HA antibody (solid line) or FITC-conjugated anti-rat secondary antibodies (dotted line) were used as positive and negative controls, respectively. ( D ) Schematic diagram of Bcl-G and the epitope regions recognised by each mAb

Techniques Used: Generated, Mutagenesis, Western Blot, Transfection, Plasmid Preparation, FACS, Staining

Detection of endogenous Bcl-G by western blotting and immunoprecipitation using Bcl-G-specific monoclonal antibodies. ( a ) Western blotting using the Bcl-G-specific mAbs 2E11, 2F7 and 4B10 showed that they are capable of specifcially detecting endogenous mBcl-G in protein lysates from WT mouse testes. Extracts from Bcl-G −/− testes were used as a negative control. Reprobing the membranes for HSP70 served as loading controls. ( b ) Bcl-G from WT mouse testis lysate was immunoprecipitated using each of the four mAbs and detected in western blots by probing with 2E11. Bcl-G −/− testis lysates were used as a negative control. The mAbs 2E11 and 10C9 specifically pulled down endogenous mouse Bcl-G while clones 2F7 and 4B10 did not. Probing for Bcl-G and HSP70 in the flowthrough (unbound) served as loading controls. Asterisks indicate the Ig heavy and light chains
Figure Legend Snippet: Detection of endogenous Bcl-G by western blotting and immunoprecipitation using Bcl-G-specific monoclonal antibodies. ( a ) Western blotting using the Bcl-G-specific mAbs 2E11, 2F7 and 4B10 showed that they are capable of specifcially detecting endogenous mBcl-G in protein lysates from WT mouse testes. Extracts from Bcl-G −/− testes were used as a negative control. Reprobing the membranes for HSP70 served as loading controls. ( b ) Bcl-G from WT mouse testis lysate was immunoprecipitated using each of the four mAbs and detected in western blots by probing with 2E11. Bcl-G −/− testis lysates were used as a negative control. The mAbs 2E11 and 10C9 specifically pulled down endogenous mouse Bcl-G while clones 2F7 and 4B10 did not. Probing for Bcl-G and HSP70 in the flowthrough (unbound) served as loading controls. Asterisks indicate the Ig heavy and light chains

Techniques Used: Western Blot, Immunoprecipitation, Negative Control, Clone Assay

Screening of hybridoma clones for Bcl-G-specific monoclonal antibodies. Hybridoma supernatants were screened for antibodies that detect HA-tagged Bcl-G overexpressed by 293T cells using intracellular immunofluorescent staining and FACS analysis. FITC-conjugated goat anti-rat Ig secondary antibodies (dotted line in a – c ) were used as the secondary reagent after a first incubation with hybridoma culture supernatant (solid line in a – c ), sera from immunised rats ( b ) or supernatant from hybridoma clone 2E11 ( c ). Antibodies against HA (dotted line represents FITC-conjugated anti-mouse secondary antibody only, used as a negative control) were used as a positive control ( d )
Figure Legend Snippet: Screening of hybridoma clones for Bcl-G-specific monoclonal antibodies. Hybridoma supernatants were screened for antibodies that detect HA-tagged Bcl-G overexpressed by 293T cells using intracellular immunofluorescent staining and FACS analysis. FITC-conjugated goat anti-rat Ig secondary antibodies (dotted line in a – c ) were used as the secondary reagent after a first incubation with hybridoma culture supernatant (solid line in a – c ), sera from immunised rats ( b ) or supernatant from hybridoma clone 2E11 ( c ). Antibodies against HA (dotted line represents FITC-conjugated anti-mouse secondary antibody only, used as a negative control) were used as a positive control ( d )

Techniques Used: Clone Assay, Staining, FACS, Incubation, Negative Control, Positive Control

Intracellular staining and FACS analysis using Bcl-G-specific mAbs reveals higher Bcl-G expression in CD8 + splenic cDCs compared with other DC subsets. To determine the levels of Bcl-G expression in the CD8 + and CD8 − DC subsets, DC-enriched splenocyte cell suspensions were surface stained with CD11c-FITC and CD8-APC monoclonal antibodies followed by intracellular staining with biotinylated Bcl-G-specific mAbs and incubation with PE-streptavidin (solid line). Cells stained with PE-streptavidin alone were used as negative controls (tinted grey). Samples were analysed using a flow cytometer and representative FACS plots are shown
Figure Legend Snippet: Intracellular staining and FACS analysis using Bcl-G-specific mAbs reveals higher Bcl-G expression in CD8 + splenic cDCs compared with other DC subsets. To determine the levels of Bcl-G expression in the CD8 + and CD8 − DC subsets, DC-enriched splenocyte cell suspensions were surface stained with CD11c-FITC and CD8-APC monoclonal antibodies followed by intracellular staining with biotinylated Bcl-G-specific mAbs and incubation with PE-streptavidin (solid line). Cells stained with PE-streptavidin alone were used as negative controls (tinted grey). Samples were analysed using a flow cytometer and representative FACS plots are shown

Techniques Used: Staining, FACS, Expressing, Incubation, Flow Cytometry, Cytometry

32) 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

33) Product Images from "Alterations of the Transcriptome of Sulfolobus acidocaldarius by Exoribonuclease aCPSF2"

Article Title: Alterations of the Transcriptome of Sulfolobus acidocaldarius by Exoribonuclease aCPSF2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076569

Saci-aCPSF2 displays 5´ to 3´ exonuclease activity. Lane 1, [α- 32 P]ATP was loaded on the gel. 5´-PPP-40A1 labeled RNA was incubated for 0´ to 60´ at 65°C in the presence of 500 ng recombinant Saci-aCPSF2 (lanes 2-6) or in the absence of the enzyme (lanes 7-11). The PPP-40A1 substrate contains a single labeled adenosine residue at position +3 (top; Table S2 ).
Figure Legend Snippet: Saci-aCPSF2 displays 5´ to 3´ exonuclease activity. Lane 1, [α- 32 P]ATP was loaded on the gel. 5´-PPP-40A1 labeled RNA was incubated for 0´ to 60´ at 65°C in the presence of 500 ng recombinant Saci-aCPSF2 (lanes 2-6) or in the absence of the enzyme (lanes 7-11). The PPP-40A1 substrate contains a single labeled adenosine residue at position +3 (top; Table S2 ).

Techniques Used: Activity Assay, Labeling, Incubation, Recombinant

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

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

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003275

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

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

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066947

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

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

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

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

36) 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

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

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

Journal: Scientific Reports

doi: 10.1038/srep03199

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

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

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

Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot

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

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot

38) Product Images from "Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System"

Article Title: Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System

Journal: PLoS ONE

doi: 10.1371/journal.pone.0079562

Functionality and disulfide-bonding patterns of the co-expressed cysteine variants of OutL and OutM. ( A ), secretion activity of OutL/M variants. ( B and C ), disulfide-bonding analysis of OutL/M variants. D. dadantii A4229 wt cells, carrying a pTdB-oLoM plasmid co-expressing mutant outL and outM alleles (indicated on top), were grown, treated and analyzed with either PelD and PelI antibodies ( A ), or with GST-OutL antibodies ( B ), or with OutM antibodies ( C ), as in Figure 4 . The positions of OutL and OutM monomers (1-m), dimers (2-m) and OutL-M heterodimers (L-M) are indicated by arrowheads. Non-specific specie interacting with OutM-antibodies are shown by asterisks and OutL-degradation products, by dots. The amounts of formed dimers reflect the proximity of the respective residues from adjacent protomers.
Figure Legend Snippet: Functionality and disulfide-bonding patterns of the co-expressed cysteine variants of OutL and OutM. ( A ), secretion activity of OutL/M variants. ( B and C ), disulfide-bonding analysis of OutL/M variants. D. dadantii A4229 wt cells, carrying a pTdB-oLoM plasmid co-expressing mutant outL and outM alleles (indicated on top), were grown, treated and analyzed with either PelD and PelI antibodies ( A ), or with GST-OutL antibodies ( B ), or with OutM antibodies ( C ), as in Figure 4 . The positions of OutL and OutM monomers (1-m), dimers (2-m) and OutL-M heterodimers (L-M) are indicated by arrowheads. Non-specific specie interacting with OutM-antibodies are shown by asterisks and OutL-degradation products, by dots. The amounts of formed dimers reflect the proximity of the respective residues from adjacent protomers.

Techniques Used: Activity Assay, Plasmid Preparation, Expressing, Mutagenesis

Dissection of the interacting regions of OutC, OutL and OutM in pull-down assays. The GST-fused derivatives of OutM ( A ), OutL ( B ) or OutC ( C ) (indicated at the top) were immobilized on Glutathione Sepharose beads, to constitute the affinity matrices (upper panels). Next, the indicated proteins of interest were incubated with these matrices for 1 h and unbound proteins were washed away. Bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE and either stained (upper panels), or (lower panels) probed with the indicated antibodies, or revealed by autoradiography ( 35 S). GST-fused degradation products are indicated by asterisks. Schematic representation of the used derivatives is shown in Figure S1 .
Figure Legend Snippet: Dissection of the interacting regions of OutC, OutL and OutM in pull-down assays. The GST-fused derivatives of OutM ( A ), OutL ( B ) or OutC ( C ) (indicated at the top) were immobilized on Glutathione Sepharose beads, to constitute the affinity matrices (upper panels). Next, the indicated proteins of interest were incubated with these matrices for 1 h and unbound proteins were washed away. Bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE and either stained (upper panels), or (lower panels) probed with the indicated antibodies, or revealed by autoradiography ( 35 S). GST-fused degradation products are indicated by asterisks. Schematic representation of the used derivatives is shown in Figure S1 .

Techniques Used: Dissection, Incubation, SDS Page, Staining, Autoradiography

39) 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

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056715

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

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

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Article Title: Substrate channeling in oxylipin biosynthesis through a protein complex in the plastid envelope of Arabidopsis thaliana
Article Snippet: .. Isolation of AOS-containing and HPL-containing higher molecular mass complexes AOS-(His)6 and HPL-(His)6 were imported into isolated Arabidopsis chloroplasts, and proteins interacting with AOS-(His)6 - or HPL-(His)6 were purified from detergent-solubilized envelopes by Ni-NTA affinity chromatography (Qiagen). .. For comparison, AOS- or HPL-containing plastid envelope protein complexes were purified from plants expressing FLAG-tagged AOS (AOS-FLAG) or HPL (HPL-FLAG) (see ).

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Article Snippet: .. HrpE-Trx-6 His (HrpE), NHrpE-Trx-6 His (N-HrpE), CHrpE-Trx-6 His (C-HrpE) and Trx-6 His (Trx) were purified with a Ni2+ -nitrilotriacetate (Ni-NTA) agarose column (Qiagen) and CsGRP-GST (CsGRP) and GST with a Glutathione Sepharose column (GE Healthcare). ..

Article Title: 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: 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: SPIN1 promotes tumorigenesis by blocking the uL18 (universal large ribosomal subunit protein 18)-MDM2-p53 pathway in human cancer
Article Snippet: .. His-tagged SPIN1 and His-tagged uL18 were purified using a Ni-NTA (QIAGEN, Valencia, CA, USA) column, and eluted with 0.5 M imidazole. ..

Immunoprecipitation:

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

Incubation:

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

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

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).

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).

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    Qiagen ni nta chromatography
    G58T-mediated loading of siRNA on EVs: SDS-PAGE of the G58T-based proteins purified by <t>Ni-NTA</t> chromatography. Gel is stained with Coomassie brilliant blue dye. (b) Gel shift assay of G58T EVs (upper) and G58TF EVs (lower), reflecting binding of siRNA to EVs. 20pmoles of siRNA were incubated with the given number of EVs. Bands trapped near the wells represent bound siRNA. siRNA alone was used as negative control. Based on the gel shift assay, around 550 siRNAs bind to each G58T EV and 714 siRNAs to each G58TF EV. Higher binding of siRNA to G58TF is due to the arginine-rich FHV peptide. (c) RNase A protection assay of G58TF EVs bound to 50 pmoles of siRNA. Bands in the agarose gel represent siRNA isolated from G58TF EVs after treatment with RNase A (0.2 mg/ml). G58TF EVs bound to siRNA were incubated with RNase A for 6 h at 37°C. (d) Representative confocal microscopy images of N2a cells after 4h of treatment with G58TF EVs carrying FAM-labelled siRNA (green). Cells were treated with lysotracker red dye (red dots) to label late-endosomes. Inset in the merged figure represents magnified image of N2a cells showing colocalization of siRNA with lysotracker dye (yellow spots). Images were captured using 60X objective lens of Olympus confocal microscope FV1000. (e) <t>GAPDH</t> gene silencing by G58T EVs at mRNA level in N2a cells. Different amounts of GAPDH siRNA bound to G58T EVs were added to cells. Level of mRNA was quantified after 48h of treatment by probe-based real time PCR. Data were analysed using linear regression analysis and ΔΔCt method. Level of mRNA in control was assigned as 1 and used to determine the relative level of GAPDH mRNA in the treated cells. Results are shown as mean ± s.d, n= 3, ***p
    Ni Nta Chromatography, supplied by Qiagen, used in various techniques. Bioz Stars score: 91/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Qiagen ni nta beads
    <t>rCsHscB</t> is a novel agonist for TLR2 to induce immune responses of macrophage. A Molecular docking analysis of binding TLR2 with CsHscB. B Pull-down assay analysis of interaction of TLR2 and rCsHscB. The cells stimulated by supernatant of lysate from E. Coli transfecting with Vector controls (pET-28, His-tagged control, Lane 1), pET-28a-CsHscB vectors (pET-28-CsHscB, unpurified, Lane 2), purified rCsHscB-stimulated cells (Lane 3), binding buffer (Lane 4) and medium (Lane 6) for 24 h, the cells were lysed and incubated with rCsHscB immobilized on <t>Ni-NTA</t> beads, and bead-bound proteins were loaded onto a gel for immunoblotting for TLR2 and rCsHscB, respectively. Lane 5 represents negative control for pull-down assay; C∼E The production of IL-6 (C), IL-10 (D) and TNF-α (E) were hindered in rCsHscB-stimulated cells when TLR2 was blocked by neutralizing antibody. PM 3 CSK 4 were used as TLR2 ligand for positive controls. Quantitative data are representative of mean ± SEM of at least three independent experiments. * P
    Ni Nta Beads, supplied by Qiagen, used in various techniques. Bioz Stars score: 94/100, based on 653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Qiagen ni nta beads pull down assay purified recombinant δn290mia40sps
    The noncovalent ITS-dependent binding of substrates to Mia40 is mediated by hydrophobic interactions. (A) Purified His-tagged <t>ΔN290Mia40SPS</t> was bound to Ni 2+ <t>-NTA</t> beads. 35 S-labeled proteins were incubated with Ni-NTA beads with or without ΔN290Mia40SPS at 15°C for 2 h. (B) 35 S-labeled Tim10 was incubated with Ni-NTA bead–immobilized ΔN290Mia40SPS for 2 h at 30°C in the presence of 50, 150, or 500 mM NaCl. (C) As in B, but binding was performed in the presence of 0.01, 0.05, 0.1, or 0.5% Triton X-100. (D) ITC of 0.25 mM ΔN290Mia40SPS alone (bottom right) or with 0.025 mM of wild-type (WT) Tim10 (top left), Tim10 ΔN30 (top right), or Tim10 ΔN39 (bottom left).
    Ni Nta Beads Pull Down Assay Purified Recombinant δn290mia40sps, supplied by Qiagen, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    G58T-mediated loading of siRNA on EVs: SDS-PAGE of the G58T-based proteins purified by Ni-NTA chromatography. Gel is stained with Coomassie brilliant blue dye. (b) Gel shift assay of G58T EVs (upper) and G58TF EVs (lower), reflecting binding of siRNA to EVs. 20pmoles of siRNA were incubated with the given number of EVs. Bands trapped near the wells represent bound siRNA. siRNA alone was used as negative control. Based on the gel shift assay, around 550 siRNAs bind to each G58T EV and 714 siRNAs to each G58TF EV. Higher binding of siRNA to G58TF is due to the arginine-rich FHV peptide. (c) RNase A protection assay of G58TF EVs bound to 50 pmoles of siRNA. Bands in the agarose gel represent siRNA isolated from G58TF EVs after treatment with RNase A (0.2 mg/ml). G58TF EVs bound to siRNA were incubated with RNase A for 6 h at 37°C. (d) Representative confocal microscopy images of N2a cells after 4h of treatment with G58TF EVs carrying FAM-labelled siRNA (green). Cells were treated with lysotracker red dye (red dots) to label late-endosomes. Inset in the merged figure represents magnified image of N2a cells showing colocalization of siRNA with lysotracker dye (yellow spots). Images were captured using 60X objective lens of Olympus confocal microscope FV1000. (e) GAPDH gene silencing by G58T EVs at mRNA level in N2a cells. Different amounts of GAPDH siRNA bound to G58T EVs were added to cells. Level of mRNA was quantified after 48h of treatment by probe-based real time PCR. Data were analysed using linear regression analysis and ΔΔCt method. Level of mRNA in control was assigned as 1 and used to determine the relative level of GAPDH mRNA in the treated cells. Results are shown as mean ± s.d, n= 3, ***p

    Journal: bioRxiv

    Article Title: GAPDH controls extracellular vesicle biogenesis and enhances therapeutic potential of EVs in silencing the Huntingtin gene in mice via siRNA delivery

    doi: 10.1101/2020.01.09.899880

    Figure Lengend Snippet: G58T-mediated loading of siRNA on EVs: SDS-PAGE of the G58T-based proteins purified by Ni-NTA chromatography. Gel is stained with Coomassie brilliant blue dye. (b) Gel shift assay of G58T EVs (upper) and G58TF EVs (lower), reflecting binding of siRNA to EVs. 20pmoles of siRNA were incubated with the given number of EVs. Bands trapped near the wells represent bound siRNA. siRNA alone was used as negative control. Based on the gel shift assay, around 550 siRNAs bind to each G58T EV and 714 siRNAs to each G58TF EV. Higher binding of siRNA to G58TF is due to the arginine-rich FHV peptide. (c) RNase A protection assay of G58TF EVs bound to 50 pmoles of siRNA. Bands in the agarose gel represent siRNA isolated from G58TF EVs after treatment with RNase A (0.2 mg/ml). G58TF EVs bound to siRNA were incubated with RNase A for 6 h at 37°C. (d) Representative confocal microscopy images of N2a cells after 4h of treatment with G58TF EVs carrying FAM-labelled siRNA (green). Cells were treated with lysotracker red dye (red dots) to label late-endosomes. Inset in the merged figure represents magnified image of N2a cells showing colocalization of siRNA with lysotracker dye (yellow spots). Images were captured using 60X objective lens of Olympus confocal microscope FV1000. (e) GAPDH gene silencing by G58T EVs at mRNA level in N2a cells. Different amounts of GAPDH siRNA bound to G58T EVs were added to cells. Level of mRNA was quantified after 48h of treatment by probe-based real time PCR. Data were analysed using linear regression analysis and ΔΔCt method. Level of mRNA in control was assigned as 1 and used to determine the relative level of GAPDH mRNA in the treated cells. Results are shown as mean ± s.d, n= 3, ***p

    Article Snippet: GAPDH protein was purified by Ni-NTA chromatography (Qiagen) under native conditions, using standard protocols recommended by the manufacturer.

    Techniques: SDS Page, Purification, Chromatography, Staining, Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Negative Control, Agarose Gel Electrophoresis, Isolation, Confocal Microscopy, Microscopy, Real-time Polymerase Chain Reaction

    Surface binding of GAPDH leads to aggregation of EVs: (a) Western blot showing exogenous binding of GAPDH to HEK293T EVs. Increasing concentrations of histidine-(His6) and Flag-tagged GAPDH (lane 3,4 and 5) were incubated with a fixed number of EVs (see details in methods). Endogenous GAPDH (Endo. GAPDH) present naturally on the EV surface is shown in the top blot along with the exogenous GAPDH (Exo. GAPDH). Alix and CD81 are EV protein markers used as a positive control. Calnexin (bottom blot) was used to demonstrate the purity of EV samples. In this blot, lane 2, 3 and 4 represent EVs, and lane 5 represents cell lysate. Representative blots (n > 3). (b) UV-absorbance spectrum of EVs after passing through gel-filtration column. Increase in the absorbance of EVs+GAPDH peak indicates binding of GAPDH protein. Purified EVs were used for incubation with either GAPDH (upper chromatogram) or BSA protein (lower chromatogram). The first peak (at ∼10 ml elution) represents EVs and the second peak (around 20 ml elution) represents unbound proteins. Representative graphs (n > 3). (c) NTA profile showing the size distribution of purified HEK293T EVs after incubation with either GAPDH or BSA proteins respectively. Binding of GAPDH to the EVs, shifts their size. Inset is the scatter plot representing size (mean) of EVs (red; EVs+GAPDH, black; EVs+BSA). Data shown as mean ± s.d, n=9, ***p

    Journal: bioRxiv

    Article Title: GAPDH controls extracellular vesicle biogenesis and enhances therapeutic potential of EVs in silencing the Huntingtin gene in mice via siRNA delivery

    doi: 10.1101/2020.01.09.899880

    Figure Lengend Snippet: Surface binding of GAPDH leads to aggregation of EVs: (a) Western blot showing exogenous binding of GAPDH to HEK293T EVs. Increasing concentrations of histidine-(His6) and Flag-tagged GAPDH (lane 3,4 and 5) were incubated with a fixed number of EVs (see details in methods). Endogenous GAPDH (Endo. GAPDH) present naturally on the EV surface is shown in the top blot along with the exogenous GAPDH (Exo. GAPDH). Alix and CD81 are EV protein markers used as a positive control. Calnexin (bottom blot) was used to demonstrate the purity of EV samples. In this blot, lane 2, 3 and 4 represent EVs, and lane 5 represents cell lysate. Representative blots (n > 3). (b) UV-absorbance spectrum of EVs after passing through gel-filtration column. Increase in the absorbance of EVs+GAPDH peak indicates binding of GAPDH protein. Purified EVs were used for incubation with either GAPDH (upper chromatogram) or BSA protein (lower chromatogram). The first peak (at ∼10 ml elution) represents EVs and the second peak (around 20 ml elution) represents unbound proteins. Representative graphs (n > 3). (c) NTA profile showing the size distribution of purified HEK293T EVs after incubation with either GAPDH or BSA proteins respectively. Binding of GAPDH to the EVs, shifts their size. Inset is the scatter plot representing size (mean) of EVs (red; EVs+GAPDH, black; EVs+BSA). Data shown as mean ± s.d, n=9, ***p

    Article Snippet: GAPDH protein was purified by Ni-NTA chromatography (Qiagen) under native conditions, using standard protocols recommended by the manufacturer.

    Techniques: Binding Assay, Western Blot, Incubation, Positive Control, Filtration, Purification

    rCsHscB is a novel agonist for TLR2 to induce immune responses of macrophage. A Molecular docking analysis of binding TLR2 with CsHscB. B Pull-down assay analysis of interaction of TLR2 and rCsHscB. The cells stimulated by supernatant of lysate from E. Coli transfecting with Vector controls (pET-28, His-tagged control, Lane 1), pET-28a-CsHscB vectors (pET-28-CsHscB, unpurified, Lane 2), purified rCsHscB-stimulated cells (Lane 3), binding buffer (Lane 4) and medium (Lane 6) for 24 h, the cells were lysed and incubated with rCsHscB immobilized on Ni-NTA beads, and bead-bound proteins were loaded onto a gel for immunoblotting for TLR2 and rCsHscB, respectively. Lane 5 represents negative control for pull-down assay; C∼E The production of IL-6 (C), IL-10 (D) and TNF-α (E) were hindered in rCsHscB-stimulated cells when TLR2 was blocked by neutralizing antibody. PM 3 CSK 4 were used as TLR2 ligand for positive controls. Quantitative data are representative of mean ± SEM of at least three independent experiments. * P

    Journal: bioRxiv

    Article Title: CsHscB as a novel TLR2 agonist from carcinogenic liver fluke Clonorchis sinensis modulates host immune response

    doi: 10.1101/858670

    Figure Lengend Snippet: rCsHscB is a novel agonist for TLR2 to induce immune responses of macrophage. A Molecular docking analysis of binding TLR2 with CsHscB. B Pull-down assay analysis of interaction of TLR2 and rCsHscB. The cells stimulated by supernatant of lysate from E. Coli transfecting with Vector controls (pET-28, His-tagged control, Lane 1), pET-28a-CsHscB vectors (pET-28-CsHscB, unpurified, Lane 2), purified rCsHscB-stimulated cells (Lane 3), binding buffer (Lane 4) and medium (Lane 6) for 24 h, the cells were lysed and incubated with rCsHscB immobilized on Ni-NTA beads, and bead-bound proteins were loaded onto a gel for immunoblotting for TLR2 and rCsHscB, respectively. Lane 5 represents negative control for pull-down assay; C∼E The production of IL-6 (C), IL-10 (D) and TNF-α (E) were hindered in rCsHscB-stimulated cells when TLR2 was blocked by neutralizing antibody. PM 3 CSK 4 were used as TLR2 ligand for positive controls. Quantitative data are representative of mean ± SEM of at least three independent experiments. * P

    Article Snippet: The rCsHscB were incubated with Ni-NTA beads (QIAGEN, GER) for 12h at 4°C after the agaroses were balanced with binding buffer at 4 times in 4°C. rCsHscB immobilized on bead were incubated with total cell lysates (RAW264.7) for 12h at 4°C.

    Techniques: Binding Assay, Pull Down Assay, Plasmid Preparation, Positron Emission Tomography, Purification, Incubation, Negative Control

    The noncovalent ITS-dependent binding of substrates to Mia40 is mediated by hydrophobic interactions. (A) Purified His-tagged ΔN290Mia40SPS was bound to Ni 2+ -NTA beads. 35 S-labeled proteins were incubated with Ni-NTA beads with or without ΔN290Mia40SPS at 15°C for 2 h. (B) 35 S-labeled Tim10 was incubated with Ni-NTA bead–immobilized ΔN290Mia40SPS for 2 h at 30°C in the presence of 50, 150, or 500 mM NaCl. (C) As in B, but binding was performed in the presence of 0.01, 0.05, 0.1, or 0.5% Triton X-100. (D) ITC of 0.25 mM ΔN290Mia40SPS alone (bottom right) or with 0.025 mM of wild-type (WT) Tim10 (top left), Tim10 ΔN30 (top right), or Tim10 ΔN39 (bottom left).

    Journal: The Journal of Cell Biology

    Article Title: A novel intermembrane space-targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

    doi: 10.1083/jcb.200905134

    Figure Lengend Snippet: The noncovalent ITS-dependent binding of substrates to Mia40 is mediated by hydrophobic interactions. (A) Purified His-tagged ΔN290Mia40SPS was bound to Ni 2+ -NTA beads. 35 S-labeled proteins were incubated with Ni-NTA beads with or without ΔN290Mia40SPS at 15°C for 2 h. (B) 35 S-labeled Tim10 was incubated with Ni-NTA bead–immobilized ΔN290Mia40SPS for 2 h at 30°C in the presence of 50, 150, or 500 mM NaCl. (C) As in B, but binding was performed in the presence of 0.01, 0.05, 0.1, or 0.5% Triton X-100. (D) ITC of 0.25 mM ΔN290Mia40SPS alone (bottom right) or with 0.025 mM of wild-type (WT) Tim10 (top left), Tim10 ΔN30 (top right), or Tim10 ΔN39 (bottom left).

    Article Snippet: Ni-NTA beads pull-down assay Purified recombinant ΔN290Mia40SPS was incubated with Ni-NTA beads (QIAGEN) for 20 min at 4°C.

    Techniques: Binding Assay, Purification, Labeling, Incubation