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    Name:
    e coli protein expression
    Description:
    BL21 DE3 T1R are competent E coli that are suitable for high level induction and expression genes regulated by expression vectors with T7 promoter The cells have transformation efficiency of 1x107 cfu mug when transformed with non saturating amounts of pUC19 plasmid DNA
    Catalog Number:
    b2935
    Price:
    None
    Applications:
    Suitable for induction and expression of genes directed by the expression systems with T7 promoter
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    Structured Review

    Millipore protein expression
    BL21 DE3 T1R are competent E coli that are suitable for high level induction and expression genes regulated by expression vectors with T7 promoter The cells have transformation efficiency of 1x107 cfu mug when transformed with non saturating amounts of pUC19 plasmid DNA
    https://www.bioz.com/result/protein expression/product/Millipore
    Average 99 stars, based on 328 article reviews
    Price from $9.99 to $1999.99
    protein expression - by Bioz Stars, 2021-01
    99/100 stars

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    Related Articles

    Electron Microscopy:

    Article Title: The Potent and Broadly Neutralizing Human Dengue Virus-Specific Monoclonal Antibody 1C19 Reveals a Unique Cross-Reactive Epitope on the bc Loop of Domain II of the Envelope Protein
    Article Snippet: .. Each E protein mutant was individually transfected into human HEK-293T cells and allowed to express for 22 h. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences), and permeabilized with 0.1% (wt/vol) saponin (Sigma-Aldrich) in PBS plus calcium and magnesium (PBS++). .. Cells were stained with purified MAbs (0.1 to 0.6 µg/ml) diluted in 10% normal goat serum (NGS) (Sigma)-0.1% w/v saponin, pH 9.

    Transfection:

    Article Title: The Potent and Broadly Neutralizing Human Dengue Virus-Specific Monoclonal Antibody 1C19 Reveals a Unique Cross-Reactive Epitope on the bc Loop of Domain II of the Envelope Protein
    Article Snippet: .. Each E protein mutant was individually transfected into human HEK-293T cells and allowed to express for 22 h. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences), and permeabilized with 0.1% (wt/vol) saponin (Sigma-Aldrich) in PBS plus calcium and magnesium (PBS++). .. Cells were stained with purified MAbs (0.1 to 0.6 µg/ml) diluted in 10% normal goat serum (NGS) (Sigma)-0.1% w/v saponin, pH 9.

    Clone Assay:

    Article Title: β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry
    Article Snippet: .. Escherichia coli NEB5α (New England Biolabs, Ipswich, MA, USA) and E. coli MB2159 (D-alanine auxotrophe) [ ] were used as cloning hosts in the transformation of DNA fragments; whereas E. coli BL21 Star DE3 (Invitrogen, Carlsbad, CA, USA) and E. coli T7 Express (Novagen, Darmstadt, Germany) were used as the expression hosts. ..

    Mutagenesis:

    Article Title: The Potent and Broadly Neutralizing Human Dengue Virus-Specific Monoclonal Antibody 1C19 Reveals a Unique Cross-Reactive Epitope on the bc Loop of Domain II of the Envelope Protein
    Article Snippet: .. Each E protein mutant was individually transfected into human HEK-293T cells and allowed to express for 22 h. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences), and permeabilized with 0.1% (wt/vol) saponin (Sigma-Aldrich) in PBS plus calcium and magnesium (PBS++). .. Cells were stained with purified MAbs (0.1 to 0.6 µg/ml) diluted in 10% normal goat serum (NGS) (Sigma)-0.1% w/v saponin, pH 9.

    Protease Inhibitor:

    Article Title: Isolation of Neutralizing Monoclonal Antibodies to Human Astrovirus and Characterization of Virus Variants That Escape Neutralization
    Article Snippet: .. Plasmids were transformed into E. coli strain BL21(DE3), and protein production was induced with 1 mM IPTG at 18°C for 16 h. E. coli cells were lysed by ultrasonication in 20 mM Tris-HCl (pH 8.0)–500 mM NaCl–20 mM imidazole containing 2 μM MgCl2 , 1,250 U Benzonase (Millipore), and 1× protease inhibitor cocktail (set V, EDTA free) (Millipore). .. Proteins were purified from soluble lysates by HisTrap metal affinity chromatography.

    Purification:

    Article Title: A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation
    Article Snippet: .. Protein expression and purification All proteins were expressed in BL21(DE3) (for expression or co-expression of pCDF-1b vectors) or BL21 (for pGEX-6p vectors) by overnight induction with 0.2 mM isopropyl β-d -1-thiogalactopyranoside at 18°C. .. In general, affinity purifications followed the HiTrap manufacturer's protocols and included cOmplete™ EDTA-free protease inhibitors (Millipore-Sigma), 6 mM β-mercapto-ethanol, and 0.2 tris(2-carboxyethyl) phosphine (TCEP) reducing agents in the buffers.

    Incubation:

    Article Title: Surfactant protein A is expressed in the central nervous system of rats with experimental autoimmune encephalomyelitis, and suppresses inflammation in human astrocytes and microglia
    Article Snippet: .. Evaluation of LPS stimulation of SP-A protein expression in human astrocytes and microglia Human astrocytes and microglia were incubated with a range of LPS concentrations from Escherichia coli 0111: B4 (0, 1, 5 and 10 µg/ml; Sigma; St. Louis, MO, USA) for 24 h, or 10 µg/ml LPS for different durations (2, 4, 8, 16 and 24 h). .. Cells were collected for analysis of SP-A protein expression by western blotting.

    Expressing:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: .. The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. Competent BL21 cells were transformed with the plasmid to express the protein at high levels.

    Article Title: Site-specific receptor methylation of FrzCD in Myxococcus xanthus is controlled by a tetra-trico peptide repeat (TPR) containing regulatory domain of the FrzF methyltransferase
    Article Snippet: .. The plasmid was sequenced and analysed using the Sequencher program (Gene Codes) and transformed into the E. coli Tuner protein expression strain (Novagen). .. Cells were grown to mid-log phase at 37°C and expression was induced by adding 1 mM of IPTG and transferring cells to 18°C for 16 h. Cells were isolated by centrifugation at 9000 g .

    Article Title: Surfactant protein A is expressed in the central nervous system of rats with experimental autoimmune encephalomyelitis, and suppresses inflammation in human astrocytes and microglia
    Article Snippet: .. Evaluation of LPS stimulation of SP-A protein expression in human astrocytes and microglia Human astrocytes and microglia were incubated with a range of LPS concentrations from Escherichia coli 0111: B4 (0, 1, 5 and 10 µg/ml; Sigma; St. Louis, MO, USA) for 24 h, or 10 µg/ml LPS for different durations (2, 4, 8, 16 and 24 h). .. Cells were collected for analysis of SP-A protein expression by western blotting.

    Article Title: A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation
    Article Snippet: .. Protein expression and purification All proteins were expressed in BL21(DE3) (for expression or co-expression of pCDF-1b vectors) or BL21 (for pGEX-6p vectors) by overnight induction with 0.2 mM isopropyl β-d -1-thiogalactopyranoside at 18°C. .. In general, affinity purifications followed the HiTrap manufacturer's protocols and included cOmplete™ EDTA-free protease inhibitors (Millipore-Sigma), 6 mM β-mercapto-ethanol, and 0.2 tris(2-carboxyethyl) phosphine (TCEP) reducing agents in the buffers.

    Article Title: β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry
    Article Snippet: .. Escherichia coli NEB5α (New England Biolabs, Ipswich, MA, USA) and E. coli MB2159 (D-alanine auxotrophe) [ ] were used as cloning hosts in the transformation of DNA fragments; whereas E. coli BL21 Star DE3 (Invitrogen, Carlsbad, CA, USA) and E. coli T7 Express (Novagen, Darmstadt, Germany) were used as the expression hosts. ..

    Transformation Assay:

    Article Title: Site-specific receptor methylation of FrzCD in Myxococcus xanthus is controlled by a tetra-trico peptide repeat (TPR) containing regulatory domain of the FrzF methyltransferase
    Article Snippet: .. The plasmid was sequenced and analysed using the Sequencher program (Gene Codes) and transformed into the E. coli Tuner protein expression strain (Novagen). .. Cells were grown to mid-log phase at 37°C and expression was induced by adding 1 mM of IPTG and transferring cells to 18°C for 16 h. Cells were isolated by centrifugation at 9000 g .

    Article Title: β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry
    Article Snippet: .. Escherichia coli NEB5α (New England Biolabs, Ipswich, MA, USA) and E. coli MB2159 (D-alanine auxotrophe) [ ] were used as cloning hosts in the transformation of DNA fragments; whereas E. coli BL21 Star DE3 (Invitrogen, Carlsbad, CA, USA) and E. coli T7 Express (Novagen, Darmstadt, Germany) were used as the expression hosts. ..

    Article Title: Isolation of Neutralizing Monoclonal Antibodies to Human Astrovirus and Characterization of Virus Variants That Escape Neutralization
    Article Snippet: .. Plasmids were transformed into E. coli strain BL21(DE3), and protein production was induced with 1 mM IPTG at 18°C for 16 h. E. coli cells were lysed by ultrasonication in 20 mM Tris-HCl (pH 8.0)–500 mM NaCl–20 mM imidazole containing 2 μM MgCl2 , 1,250 U Benzonase (Millipore), and 1× protease inhibitor cocktail (set V, EDTA free) (Millipore). .. Proteins were purified from soluble lysates by HisTrap metal affinity chromatography.

    Recombinant:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: .. The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. Competent BL21 cells were transformed with the plasmid to express the protein at high levels.

    Plasmid Preparation:

    Article Title: Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys
    Article Snippet: .. The H5 HA gene was ligated to the E. coli protein expression vector pET21b (Novagen), from which the recombinant protein was expressed as a fusion protein with a histidine tag. .. Competent BL21 cells were transformed with the plasmid to express the protein at high levels.

    Article Title: Site-specific receptor methylation of FrzCD in Myxococcus xanthus is controlled by a tetra-trico peptide repeat (TPR) containing regulatory domain of the FrzF methyltransferase
    Article Snippet: .. The plasmid was sequenced and analysed using the Sequencher program (Gene Codes) and transformed into the E. coli Tuner protein expression strain (Novagen). .. Cells were grown to mid-log phase at 37°C and expression was induced by adding 1 mM of IPTG and transferring cells to 18°C for 16 h. Cells were isolated by centrifugation at 9000 g .

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    Millipore bat3 mammalian expression construct
    Modulation of macrophage functions by soluble <t>BAT3.</t> A. J774A.1 cells were first stimulated with IFN-γ (20 ng/ml) for 2 hours and then purified recombinant BAT3 was added into culture at different concentrations. Total nitrite levels in cell culture supernatants were determined after 18 hours using a colorimetric assay kit. **P
    Bat3 Mammalian Expression Construct, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bat3 mammalian expression construct/product/Millipore
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    bat3 mammalian expression construct - by Bioz Stars, 2021-01
    85/100 stars
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    85
    Millipore plasmids expressing cyclin d1
    FBXO31-mediated <t>cyclin</t> D1 degradation occurs through the proteasomal pathway. a , Cyclin D1 levels in SK-MEL-28 cells transduced with a retrovirus expressing FBXO31 or empty vector and treated in the presence or absence of lactacystin. b , Quantitative real-time RT-PCR monitoring cyclin D1 mRNA levels (error bars, s.d., n=3). c , Cyclin D1 levels in SK-MEL-28 cells stably transfected with a plasmid expressing either HA-tagged cyclin D1 or cyclin D1(T286A) and transduced with an FBXO31 retrovirus. d , FACS analysis in cells described in ( d ). e , DNA replication assay (error bars, s.d., n=3).
    Plasmids Expressing Cyclin D1, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmids expressing cyclin d1/product/Millipore
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    91
    Millipore integrin expression
    A conserved <t>integrin-binding</t> motif exists in the aMPV/B and hMPV F proteins. A , F protein sequences from 12 clinical isolates of aMPV/B and 13 clinical isolates of hMPV were aligned. The RDD motif is highlighted in the boxes. B , the three-dimensional
    Integrin Expression, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/integrin expression/product/Millipore
    Average 91 stars, based on 3 article reviews
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    88
    Millipore recombinant protein expression codon optimized genes
    iRBCs <t>expressing</t> a different PfEMP1 variant show different level of reduction in cytoadherence upon conditional depletion of PFE1605w. A. Preselected iRBCs binding to either <t>recombinant</t> CD36, ICAM‐1 or CSA immobilized on tissue‐treated glass slides at 50 µg ml −1 (CD36, ICAM‐1) or 20 µg ml −1 (CSA) concentrations. Parasites expressing PFE1605w as a C‐terminally tagged DD fusion <t>protein</t> were grown for 96 h in the presence (PFE1605w ON ) or absence (PFE1605w OFF ) of 625 nM Shield‐1. The graphs display overall mean values across triplicate experiments using linear regression with a random effect for experiment. The error bars represent the SDs of the triplicate experiments. An arbitrary threshold (dashed line) for unspecific binding was calculated as the mean level of iRBC binding to 1% w/v BSA plus two SDs. P values were calculated by using a two‐tailed Student's t ‐test. The asterisks indicate P ≤ 0.0001. ‘ns’ indicates P ≥ 0.05. B. Pie charts show the var transcript distribution in the selected lines. qPCR was performed with specific primers for each var <t>gene</t> as previously reported.
    Recombinant Protein Expression Codon Optimized Genes, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Modulation of macrophage functions by soluble BAT3. A. J774A.1 cells were first stimulated with IFN-γ (20 ng/ml) for 2 hours and then purified recombinant BAT3 was added into culture at different concentrations. Total nitrite levels in cell culture supernatants were determined after 18 hours using a colorimetric assay kit. **P

    Journal: PLoS ONE

    Article Title: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages

    doi: 10.1371/journal.pone.0040836

    Figure Lengend Snippet: Modulation of macrophage functions by soluble BAT3. A. J774A.1 cells were first stimulated with IFN-γ (20 ng/ml) for 2 hours and then purified recombinant BAT3 was added into culture at different concentrations. Total nitrite levels in cell culture supernatants were determined after 18 hours using a colorimetric assay kit. **P

    Article Snippet: Expression and Purification of Recombinant BAT3 293T cells were transfected with BAT3 mammalian expression construct using Nanojuice transfection kit (EMD Biosciences).

    Techniques: Purification, Recombinant, Cell Culture, Colorimetric Assay

    Regulation of ESAT-6 induced apoptosis by BAT3. A. BMDMs and J774A.1 cells were incubated with different concentrations of ESAT-6 for 24 hours. Cell culture supernatants were collected and subjected to colorimetric caspase-3 assay. Fold change in comparison to unstimulated controls was calculated and plotted in bar graph. *P

    Journal: PLoS ONE

    Article Title: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages

    doi: 10.1371/journal.pone.0040836

    Figure Lengend Snippet: Regulation of ESAT-6 induced apoptosis by BAT3. A. BMDMs and J774A.1 cells were incubated with different concentrations of ESAT-6 for 24 hours. Cell culture supernatants were collected and subjected to colorimetric caspase-3 assay. Fold change in comparison to unstimulated controls was calculated and plotted in bar graph. *P

    Article Snippet: Expression and Purification of Recombinant BAT3 293T cells were transfected with BAT3 mammalian expression construct using Nanojuice transfection kit (EMD Biosciences).

    Techniques: Incubation, Cell Culture, Caspase-3 Assay

    ESAT-6 induces the expression of BAT3 in macrophages. A. Bone marrow-derived macrophages (BMDM) were incubated with different concentrations of ESAT-6 in Opti-MEM medium for 6 hours. Cell culture supernatants were collected and concentrated. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blot was developed using a rabbit polyclonal antibody against BAT3 protein. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. B. BMDM were incubated with 5 µg/ml of ESAT-6. Nuclear and cytoplasmic extracts were prepared and western blots were developed as mentioned above in figure 3A . Histogram plot shows the densitometric quantification of the change in BAT3 protein levels in nuclear and cytoplasmic extracts. C. Total RNA was isolated from BMDM that were stimulated with ESAT-6 (5 µg/ml) for 6 hours; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. D. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and western blots of nuclear and cytoplasmic fractions were developed as mentioned above in figure 3A . Histogram plot shows the densitometric quantification of the change in BAT3 protein levels in nuclear and cytoplasmic extracts. E. J774A.1 cells were stimulated with ESAT-6 (5 µg/ml) for 6 hours. Total RNA was isolated; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. F. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and culture supernatants were collected at different time points. Heat shock-treated cell supernatants were used as positive control. Western blots were developed as mentioned above in figure 3A . Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. G. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and cytoplasmic extracts were collected at different time points. The western blots were developed as mentioned above in figure 3A . Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. H. J774A.1 cells were incubated with 5 µg/ml of Ag85B for 9 hours and different cellular fractions (Nuclear extract, Cytoplasmic extract and Supernatants) were collected at different time points. The western blots were developed as mentioned above in figure 3A . The cytoplasmic marker GAPDH and nuclear marker histone H1 served as positive controls for the cytoplasmic extracts and nuclear extracts, respectively, in western blotting experiments.

    Journal: PLoS ONE

    Article Title: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages

    doi: 10.1371/journal.pone.0040836

    Figure Lengend Snippet: ESAT-6 induces the expression of BAT3 in macrophages. A. Bone marrow-derived macrophages (BMDM) were incubated with different concentrations of ESAT-6 in Opti-MEM medium for 6 hours. Cell culture supernatants were collected and concentrated. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blot was developed using a rabbit polyclonal antibody against BAT3 protein. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. B. BMDM were incubated with 5 µg/ml of ESAT-6. Nuclear and cytoplasmic extracts were prepared and western blots were developed as mentioned above in figure 3A . Histogram plot shows the densitometric quantification of the change in BAT3 protein levels in nuclear and cytoplasmic extracts. C. Total RNA was isolated from BMDM that were stimulated with ESAT-6 (5 µg/ml) for 6 hours; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. D. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and western blots of nuclear and cytoplasmic fractions were developed as mentioned above in figure 3A . Histogram plot shows the densitometric quantification of the change in BAT3 protein levels in nuclear and cytoplasmic extracts. E. J774A.1 cells were stimulated with ESAT-6 (5 µg/ml) for 6 hours. Total RNA was isolated; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. F. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and culture supernatants were collected at different time points. Heat shock-treated cell supernatants were used as positive control. Western blots were developed as mentioned above in figure 3A . Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. G. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and cytoplasmic extracts were collected at different time points. The western blots were developed as mentioned above in figure 3A . Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. H. J774A.1 cells were incubated with 5 µg/ml of Ag85B for 9 hours and different cellular fractions (Nuclear extract, Cytoplasmic extract and Supernatants) were collected at different time points. The western blots were developed as mentioned above in figure 3A . The cytoplasmic marker GAPDH and nuclear marker histone H1 served as positive controls for the cytoplasmic extracts and nuclear extracts, respectively, in western blotting experiments.

    Article Snippet: Expression and Purification of Recombinant BAT3 293T cells were transfected with BAT3 mammalian expression construct using Nanojuice transfection kit (EMD Biosciences).

    Techniques: Expressing, Derivative Assay, Incubation, Cell Culture, Protein Concentration, SDS Page, Electrophoresis, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Amplification, Positive Control, Marker

    Expression of BAT3 in J774A.1 cells and bone marrow-derived macrophages (BMDM) in response to non-lethal heat shock. A. J774A.1 cells and BMDM were subjected to non-lethal heat shock at 42°C for 10 minutes and rested for 1 hour. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on SDS-PAGE gels. Following electrophoresis and western blotting, the blots were developed using a rabbit polyclonal antibody against BAT3 protein. The molecular weight of BAT3 was ∼122 kDa. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blots. B. Total RNA was isolated from cells; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. C. The exosomal pellet (Ex) and soluble fractions (SF), of both J774A.1 cells and bone marrow-derived macrophages (BMDM) culture supernatants were collected. Total 25 µg of protein from each sample was run on a 12% SDS-PAGE gel followed by western blotting to detect BAT3 (upper panel) and HSP70 (lower panel). The cytoplasmic marker GAPDH and nuclear marker histone H1 served as positive controls for the cytoplasmic extracts and nuclear extracts, respectively, in western blotting experiments.

    Journal: PLoS ONE

    Article Title: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages

    doi: 10.1371/journal.pone.0040836

    Figure Lengend Snippet: Expression of BAT3 in J774A.1 cells and bone marrow-derived macrophages (BMDM) in response to non-lethal heat shock. A. J774A.1 cells and BMDM were subjected to non-lethal heat shock at 42°C for 10 minutes and rested for 1 hour. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on SDS-PAGE gels. Following electrophoresis and western blotting, the blots were developed using a rabbit polyclonal antibody against BAT3 protein. The molecular weight of BAT3 was ∼122 kDa. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blots. B. Total RNA was isolated from cells; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. C. The exosomal pellet (Ex) and soluble fractions (SF), of both J774A.1 cells and bone marrow-derived macrophages (BMDM) culture supernatants were collected. Total 25 µg of protein from each sample was run on a 12% SDS-PAGE gel followed by western blotting to detect BAT3 (upper panel) and HSP70 (lower panel). The cytoplasmic marker GAPDH and nuclear marker histone H1 served as positive controls for the cytoplasmic extracts and nuclear extracts, respectively, in western blotting experiments.

    Article Snippet: Expression and Purification of Recombinant BAT3 293T cells were transfected with BAT3 mammalian expression construct using Nanojuice transfection kit (EMD Biosciences).

    Techniques: Expressing, Derivative Assay, Protein Concentration, SDS Page, Electrophoresis, Western Blot, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Amplification, Marker

    Inhibition of caspase-3 and proteasome provides protection of BAT3 and BCL-2. J774A.1 cells were pre-incubated with 85 µM zVAD-FMK or 10 µM proteasome inhibitor MG-132 or both for 4 hours and then stimulated with 5 µg/ml of ESAT-6 protein. Cytoplasmic extracts were collected at different time points. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blots were developed using a rabbit polyclonal antibody against BAT3 protein ( Figure 6A ) and a mouse monoclonal antibody against BCL-2 protein ( Figure 6B ). All experiments were done with 0.05% DMSO vehicle control and ESAT-6 alone as positive control (data not shown). The cytoplasmic marker GAPDH served as positive control for the western blots of cytoplasmic extracts.

    Journal: PLoS ONE

    Article Title: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages

    doi: 10.1371/journal.pone.0040836

    Figure Lengend Snippet: Inhibition of caspase-3 and proteasome provides protection of BAT3 and BCL-2. J774A.1 cells were pre-incubated with 85 µM zVAD-FMK or 10 µM proteasome inhibitor MG-132 or both for 4 hours and then stimulated with 5 µg/ml of ESAT-6 protein. Cytoplasmic extracts were collected at different time points. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blots were developed using a rabbit polyclonal antibody against BAT3 protein ( Figure 6A ) and a mouse monoclonal antibody against BCL-2 protein ( Figure 6B ). All experiments were done with 0.05% DMSO vehicle control and ESAT-6 alone as positive control (data not shown). The cytoplasmic marker GAPDH served as positive control for the western blots of cytoplasmic extracts.

    Article Snippet: Expression and Purification of Recombinant BAT3 293T cells were transfected with BAT3 mammalian expression construct using Nanojuice transfection kit (EMD Biosciences).

    Techniques: Inhibition, Incubation, Protein Concentration, SDS Page, Electrophoresis, Western Blot, Positive Control, Marker

    Interaction of BAT3 with BCL-2 and expression of BCL-2 in ESAT-6 stimulated J774A.1 cells. A. J774A.1 cells were co-transfected with FLAG-tagged BAT3 plasmid and HIS-tagged BCL-2 plasmid or control vectors and incubated with ESAT-6. Cytoplasmic extracts of the cells were subjected to immunoprecipitation (IP) with antibodies against FLAG peptide or HIS tag, followed by western blotting with respective antibodies as indicated. Total 25 µg of each protein sample was loaded in 12% SDS-PAGE gel for the development of western blot. Upper panel: IP with anti-FLAG antibody and western blot with anti-HIS antibody. The molecular weight of BCL-2 was ∼25 kDa. Lower panel: IP with anti-HIS antibody and western blot with anti-FLAG antibody. The molecular weight of BAT3 was ∼122 kDa. Whole cell lysates of the J774A.1 cells expressing recombinant BAT3 and BCL-2 proteins served as positive control for the western blotting. B. Upper panel: J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and cytoplasmic extracts were collected at different time points. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blot was developed using a mouse monoclonal antibody against BCL-2 protein. J774A.1 cells were either transfected with BAT3 plasmid (lower panel) or control vector (middle panel) for 72 hours, incubated with ESAT-6 and cytoplasmic extracts were collected at different time points. Western blots were developed for the BCL-2 protein as described earlier.

    Journal: PLoS ONE

    Article Title: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages

    doi: 10.1371/journal.pone.0040836

    Figure Lengend Snippet: Interaction of BAT3 with BCL-2 and expression of BCL-2 in ESAT-6 stimulated J774A.1 cells. A. J774A.1 cells were co-transfected with FLAG-tagged BAT3 plasmid and HIS-tagged BCL-2 plasmid or control vectors and incubated with ESAT-6. Cytoplasmic extracts of the cells were subjected to immunoprecipitation (IP) with antibodies against FLAG peptide or HIS tag, followed by western blotting with respective antibodies as indicated. Total 25 µg of each protein sample was loaded in 12% SDS-PAGE gel for the development of western blot. Upper panel: IP with anti-FLAG antibody and western blot with anti-HIS antibody. The molecular weight of BCL-2 was ∼25 kDa. Lower panel: IP with anti-HIS antibody and western blot with anti-FLAG antibody. The molecular weight of BAT3 was ∼122 kDa. Whole cell lysates of the J774A.1 cells expressing recombinant BAT3 and BCL-2 proteins served as positive control for the western blotting. B. Upper panel: J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and cytoplasmic extracts were collected at different time points. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blot was developed using a mouse monoclonal antibody against BCL-2 protein. J774A.1 cells were either transfected with BAT3 plasmid (lower panel) or control vector (middle panel) for 72 hours, incubated with ESAT-6 and cytoplasmic extracts were collected at different time points. Western blots were developed for the BCL-2 protein as described earlier.

    Article Snippet: Expression and Purification of Recombinant BAT3 293T cells were transfected with BAT3 mammalian expression construct using Nanojuice transfection kit (EMD Biosciences).

    Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Western Blot, SDS Page, Molecular Weight, Recombinant, Positive Control, Protein Concentration, Electrophoresis

    FBXO31-mediated cyclin D1 degradation occurs through the proteasomal pathway. a , Cyclin D1 levels in SK-MEL-28 cells transduced with a retrovirus expressing FBXO31 or empty vector and treated in the presence or absence of lactacystin. b , Quantitative real-time RT-PCR monitoring cyclin D1 mRNA levels (error bars, s.d., n=3). c , Cyclin D1 levels in SK-MEL-28 cells stably transfected with a plasmid expressing either HA-tagged cyclin D1 or cyclin D1(T286A) and transduced with an FBXO31 retrovirus. d , FACS analysis in cells described in ( d ). e , DNA replication assay (error bars, s.d., n=3).

    Journal: Nature

    Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage

    doi: 10.1038/nature08011

    Figure Lengend Snippet: FBXO31-mediated cyclin D1 degradation occurs through the proteasomal pathway. a , Cyclin D1 levels in SK-MEL-28 cells transduced with a retrovirus expressing FBXO31 or empty vector and treated in the presence or absence of lactacystin. b , Quantitative real-time RT-PCR monitoring cyclin D1 mRNA levels (error bars, s.d., n=3). c , Cyclin D1 levels in SK-MEL-28 cells stably transfected with a plasmid expressing either HA-tagged cyclin D1 or cyclin D1(T286A) and transduced with an FBXO31 retrovirus. d , FACS analysis in cells described in ( d ). e , DNA replication assay (error bars, s.d., n=3).

    Article Snippet: Cell cycle analysis by two-colour FACS Cells transduced with a retrovirus expressing FBXO31 or empty vector in the presence or absence of plasmids expressing cyclin D1 or cyclin D1(T286A), or cells stably expressing luciferase or cyclin D1 siRNAs, were incubated with BrdU (20 µM) for 4 h, fixed in 70% ethanol and stained sequentially with an α-BrdU mouse antibody (Calbiochem), Alexa 488 conjugated goat α-mouse antibody (Invitrogen) and propidium iodide (Sigma).

    Techniques: Transduction, Expressing, Plasmid Preparation, Quantitative RT-PCR, Stable Transfection, Transfection, FACS

    Ectopic expression of FBXO31 induces G1 arrest and selective degradation of cyclin D1. a , FACS analysis in SK-MEL-28 cells transduced with a retrovirus expressing empty vector or FBXO31 in the absence or presence of nocodazole. b , DNA replication assay, monitored by bromodeoxyuridine (BrdU) incorporation (error bars, s.d., n=3). c–e , Immunoblot analysis showing levels of cyclins ( c ), CDKs ( d ) and CDK inhibitors ( e ) at various time points following transduction with an FBXO31 retrovirus. Tubulin was monitored as a loading control. Ectopic expression of FBXO31 did not induce a DDR ( Supplementary Figure 17 ).

    Journal: Nature

    Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage

    doi: 10.1038/nature08011

    Figure Lengend Snippet: Ectopic expression of FBXO31 induces G1 arrest and selective degradation of cyclin D1. a , FACS analysis in SK-MEL-28 cells transduced with a retrovirus expressing empty vector or FBXO31 in the absence or presence of nocodazole. b , DNA replication assay, monitored by bromodeoxyuridine (BrdU) incorporation (error bars, s.d., n=3). c–e , Immunoblot analysis showing levels of cyclins ( c ), CDKs ( d ) and CDK inhibitors ( e ) at various time points following transduction with an FBXO31 retrovirus. Tubulin was monitored as a loading control. Ectopic expression of FBXO31 did not induce a DDR ( Supplementary Figure 17 ).

    Article Snippet: Cell cycle analysis by two-colour FACS Cells transduced with a retrovirus expressing FBXO31 or empty vector in the presence or absence of plasmids expressing cyclin D1 or cyclin D1(T286A), or cells stably expressing luciferase or cyclin D1 siRNAs, were incubated with BrdU (20 µM) for 4 h, fixed in 70% ethanol and stained sequentially with an α-BrdU mouse antibody (Calbiochem), Alexa 488 conjugated goat α-mouse antibody (Invitrogen) and propidium iodide (Sigma).

    Techniques: Expressing, FACS, Transduction, Plasmid Preparation, BrdU Incorporation Assay

    FBXO31 interacts with and directs ubiquitination of cyclin D1. a , Co-immunoprecipitation of FBXO31 with cyclin D1 and SCF complex components. The FBXO31-cyclin D1 interaction was specific ( Supplementary Fig. 18 ) and predominantly cytoplasmic ( Supplementary Fig. 19 ). b , Co-immunoprecipitation of endogenous FBXO31 and cyclin D1. c , Cyclin D1 levels in SK-MEL-28 cells ectopically expressing vector, FBXO31 or FBXO31ΔF. d , In vivo ubiquitination assay. Polyubiquitinated cyclin D1 was detected by immunoprecipitation of Flag-tagged ubiquitin followed by immunoblotting for HA-cyclin D1. e , In vitro ubiquitination assay. Immunopurified SCF complexes were incubated with GST-cyclin D1, Erk2, E1, E2, ATP and ubiquitin.

    Journal: Nature

    Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage

    doi: 10.1038/nature08011

    Figure Lengend Snippet: FBXO31 interacts with and directs ubiquitination of cyclin D1. a , Co-immunoprecipitation of FBXO31 with cyclin D1 and SCF complex components. The FBXO31-cyclin D1 interaction was specific ( Supplementary Fig. 18 ) and predominantly cytoplasmic ( Supplementary Fig. 19 ). b , Co-immunoprecipitation of endogenous FBXO31 and cyclin D1. c , Cyclin D1 levels in SK-MEL-28 cells ectopically expressing vector, FBXO31 or FBXO31ΔF. d , In vivo ubiquitination assay. Polyubiquitinated cyclin D1 was detected by immunoprecipitation of Flag-tagged ubiquitin followed by immunoblotting for HA-cyclin D1. e , In vitro ubiquitination assay. Immunopurified SCF complexes were incubated with GST-cyclin D1, Erk2, E1, E2, ATP and ubiquitin.

    Article Snippet: Cell cycle analysis by two-colour FACS Cells transduced with a retrovirus expressing FBXO31 or empty vector in the presence or absence of plasmids expressing cyclin D1 or cyclin D1(T286A), or cells stably expressing luciferase or cyclin D1 siRNAs, were incubated with BrdU (20 µM) for 4 h, fixed in 70% ethanol and stained sequentially with an α-BrdU mouse antibody (Calbiochem), Alexa 488 conjugated goat α-mouse antibody (Invitrogen) and propidium iodide (Sigma).

    Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, In Vivo, Ubiquitin Assay, In Vitro, Incubation

    Cell cycle arrest following DNA damage requires ATM-mediated induction of FBXO31. a , Immunoblot analysis of FBXO31 following γ-irradiation. For comparison to ectopically expressed FBXO31 levels see Supplementary Fig. 20 . b , Cyclin D1 levels in cells expressing a NS or FBXO31 shRNA following γ-irradiation. c , FACS analysis. d , (Top) Putative ATM sites in FBXO31. (Bottom) In vitro phosphorylation of GST-FBXO31 fusion proteins harbouring wild type (WT) or mutated (SDM) SQ sites. ATM-WT, wild type ATM; ATM-KD, kinase dead ATM mutant. e–f , Levels of ectopically expressed WT or mutant FBXO31 following γ-irradiation (e) or ectopic ATM expression (f). g , FBXO31, cyclin D1, ATM and phosphorylated ATM in γ-irradiated cells expressing a NS or ATM shRNA. h , Colony formation assay in untreated or γ-irradiated cells. i , FBXO31 and cyclin D1 levels following treatment with DNA damaging agents. j , Model.

    Journal: Nature

    Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage

    doi: 10.1038/nature08011

    Figure Lengend Snippet: Cell cycle arrest following DNA damage requires ATM-mediated induction of FBXO31. a , Immunoblot analysis of FBXO31 following γ-irradiation. For comparison to ectopically expressed FBXO31 levels see Supplementary Fig. 20 . b , Cyclin D1 levels in cells expressing a NS or FBXO31 shRNA following γ-irradiation. c , FACS analysis. d , (Top) Putative ATM sites in FBXO31. (Bottom) In vitro phosphorylation of GST-FBXO31 fusion proteins harbouring wild type (WT) or mutated (SDM) SQ sites. ATM-WT, wild type ATM; ATM-KD, kinase dead ATM mutant. e–f , Levels of ectopically expressed WT or mutant FBXO31 following γ-irradiation (e) or ectopic ATM expression (f). g , FBXO31, cyclin D1, ATM and phosphorylated ATM in γ-irradiated cells expressing a NS or ATM shRNA. h , Colony formation assay in untreated or γ-irradiated cells. i , FBXO31 and cyclin D1 levels following treatment with DNA damaging agents. j , Model.

    Article Snippet: Cell cycle analysis by two-colour FACS Cells transduced with a retrovirus expressing FBXO31 or empty vector in the presence or absence of plasmids expressing cyclin D1 or cyclin D1(T286A), or cells stably expressing luciferase or cyclin D1 siRNAs, were incubated with BrdU (20 µM) for 4 h, fixed in 70% ethanol and stained sequentially with an α-BrdU mouse antibody (Calbiochem), Alexa 488 conjugated goat α-mouse antibody (Invitrogen) and propidium iodide (Sigma).

    Techniques: Irradiation, Expressing, shRNA, FACS, In Vitro, Mutagenesis, Colony Assay

    A conserved integrin-binding motif exists in the aMPV/B and hMPV F proteins. A , F protein sequences from 12 clinical isolates of aMPV/B and 13 clinical isolates of hMPV were aligned. The RDD motif is highlighted in the boxes. B , the three-dimensional

    Journal: The Journal of Biological Chemistry

    Article Title: Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection *

    doi: 10.1074/jbc.M115.711382

    Figure Lengend Snippet: A conserved integrin-binding motif exists in the aMPV/B and hMPV F proteins. A , F protein sequences from 12 clinical isolates of aMPV/B and 13 clinical isolates of hMPV were aligned. The RDD motif is highlighted in the boxes. B , the three-dimensional

    Article Snippet: As a reference control for the analysis of F protein and integrin expression on the cell membrane, Na+ /K+ -ATPase expression on the cell membrane was analyzed using an anti-Na+ /K+ -ATPase β-1 antibody (monoclonal antibody, catalogue number: 05-382, lot number: 2685213, Millipore).

    Techniques: Binding Assay

    Knockdown of integrin αv- and β1-specific siRNAs inhibits the fusogenic activity of the aMPV/B F protein and the replication of aMPV/B. A, siRNAs targeting monkey integrin αv or β1, mouse integrin αv or β1,

    Journal: The Journal of Biological Chemistry

    Article Title: Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection *

    doi: 10.1074/jbc.M115.711382

    Figure Lengend Snippet: Knockdown of integrin αv- and β1-specific siRNAs inhibits the fusogenic activity of the aMPV/B F protein and the replication of aMPV/B. A, siRNAs targeting monkey integrin αv or β1, mouse integrin αv or β1,

    Article Snippet: As a reference control for the analysis of F protein and integrin expression on the cell membrane, Na+ /K+ -ATPase expression on the cell membrane was analyzed using an anti-Na+ /K+ -ATPase β-1 antibody (monoclonal antibody, catalogue number: 05-382, lot number: 2685213, Millipore).

    Techniques: Activity Assay

    Overexpression of integrin αv and β1 enhances the fusogenic activity of the aMPV/B F protein and the replication of aMPV/B. A–D , cells were transfected with plasmid DNAs expressing integrin αv and β1 from different

    Journal: The Journal of Biological Chemistry

    Article Title: Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection *

    doi: 10.1074/jbc.M115.711382

    Figure Lengend Snippet: Overexpression of integrin αv and β1 enhances the fusogenic activity of the aMPV/B F protein and the replication of aMPV/B. A–D , cells were transfected with plasmid DNAs expressing integrin αv and β1 from different

    Article Snippet: As a reference control for the analysis of F protein and integrin expression on the cell membrane, Na+ /K+ -ATPase expression on the cell membrane was analyzed using an anti-Na+ /K+ -ATPase β-1 antibody (monoclonal antibody, catalogue number: 05-382, lot number: 2685213, Millipore).

    Techniques: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Expressing

    Overexpression of integrin αv and β1 enhances the binding activity of the aMPV/B F protein and infection of aMPV/B. A , the binding activity of the aMPV/B F protein and infection with aMPV/B in CHO cells overexpressing the integrins αv

    Journal: The Journal of Biological Chemistry

    Article Title: Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection *

    doi: 10.1074/jbc.M115.711382

    Figure Lengend Snippet: Overexpression of integrin αv and β1 enhances the binding activity of the aMPV/B F protein and infection of aMPV/B. A , the binding activity of the aMPV/B F protein and infection with aMPV/B in CHO cells overexpressing the integrins αv

    Article Snippet: As a reference control for the analysis of F protein and integrin expression on the cell membrane, Na+ /K+ -ATPase expression on the cell membrane was analyzed using an anti-Na+ /K+ -ATPase β-1 antibody (monoclonal antibody, catalogue number: 05-382, lot number: 2685213, Millipore).

    Techniques: Over Expression, Binding Assay, Activity Assay, Infection

    Antibodies specific for αv and β1 integrin block the fusogenic activity of the aMPV/B F protein and aMPV/B replication. A and B , in the presence of integrin-specific antibodies, aMPV/B F protein fusogenic activities were measured by syncytium

    Journal: The Journal of Biological Chemistry

    Article Title: Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection *

    doi: 10.1074/jbc.M115.711382

    Figure Lengend Snippet: Antibodies specific for αv and β1 integrin block the fusogenic activity of the aMPV/B F protein and aMPV/B replication. A and B , in the presence of integrin-specific antibodies, aMPV/B F protein fusogenic activities were measured by syncytium

    Article Snippet: As a reference control for the analysis of F protein and integrin expression on the cell membrane, Na+ /K+ -ATPase expression on the cell membrane was analyzed using an anti-Na+ /K+ -ATPase β-1 antibody (monoclonal antibody, catalogue number: 05-382, lot number: 2685213, Millipore).

    Techniques: Blocking Assay, Activity Assay

    iRBCs expressing a different PfEMP1 variant show different level of reduction in cytoadherence upon conditional depletion of PFE1605w. A. Preselected iRBCs binding to either recombinant CD36, ICAM‐1 or CSA immobilized on tissue‐treated glass slides at 50 µg ml −1 (CD36, ICAM‐1) or 20 µg ml −1 (CSA) concentrations. Parasites expressing PFE1605w as a C‐terminally tagged DD fusion protein were grown for 96 h in the presence (PFE1605w ON ) or absence (PFE1605w OFF ) of 625 nM Shield‐1. The graphs display overall mean values across triplicate experiments using linear regression with a random effect for experiment. The error bars represent the SDs of the triplicate experiments. An arbitrary threshold (dashed line) for unspecific binding was calculated as the mean level of iRBC binding to 1% w/v BSA plus two SDs. P values were calculated by using a two‐tailed Student's t ‐test. The asterisks indicate P ≤ 0.0001. ‘ns’ indicates P ≥ 0.05. B. Pie charts show the var transcript distribution in the selected lines. qPCR was performed with specific primers for each var gene as previously reported.

    Journal: Cellular Microbiology

    Article Title: Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton) Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton

    doi: 10.1111/cmi.12583

    Figure Lengend Snippet: iRBCs expressing a different PfEMP1 variant show different level of reduction in cytoadherence upon conditional depletion of PFE1605w. A. Preselected iRBCs binding to either recombinant CD36, ICAM‐1 or CSA immobilized on tissue‐treated glass slides at 50 µg ml −1 (CD36, ICAM‐1) or 20 µg ml −1 (CSA) concentrations. Parasites expressing PFE1605w as a C‐terminally tagged DD fusion protein were grown for 96 h in the presence (PFE1605w ON ) or absence (PFE1605w OFF ) of 625 nM Shield‐1. The graphs display overall mean values across triplicate experiments using linear regression with a random effect for experiment. The error bars represent the SDs of the triplicate experiments. An arbitrary threshold (dashed line) for unspecific binding was calculated as the mean level of iRBC binding to 1% w/v BSA plus two SDs. P values were calculated by using a two‐tailed Student's t ‐test. The asterisks indicate P ≤ 0.0001. ‘ns’ indicates P ≥ 0.05. B. Pie charts show the var transcript distribution in the selected lines. qPCR was performed with specific primers for each var gene as previously reported.

    Article Snippet: Recombinant protein expression Codon‐optimized genes encoding the ATS‐C fragments of PfEMP1 variants ( ) were cloned in a modified pET‐16 vector (Merc Millipore).

    Techniques: Expressing, Variant Assay, Binding Assay, Recombinant, Two Tailed Test, Real-time Polymerase Chain Reaction