Structured Review

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Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the <t>EDTA/DTT</t> based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
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1) Product Images from "DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile"

Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055636

Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
Figure Legend Snippet: Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

Techniques Used: FACS, Selection, Staining, Isolation, Expressing, Whisker Assay

Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
Figure Legend Snippet: Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

Techniques Used: Isolation, Expressing, Whisker Assay

2) Product Images from "Maintaining mRNA Integrity during Decalcification of Mineralized Tissues"

Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

Journal: PLoS ONE

doi: 10.1371/journal.pone.0058154

Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.
Figure Legend Snippet: Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.
Figure Legend Snippet: Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.

Techniques Used: Immunohistochemistry, Staining

In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).
Figure Legend Snippet: In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).

Techniques Used: In Situ Hybridization, Labeling, Expressing

Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.
Figure Legend Snippet: Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.

Techniques Used: Staining

Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).
Figure Legend Snippet: Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).

Techniques Used: Electrophoresis, Isolation

3) Product Images from "A role for caveolin-1 in desmoglein binding and desmosome dynamics"

Article Title: A role for caveolin-1 in desmoglein binding and desmosome dynamics

Journal: Oncogene

doi: 10.1038/onc.2011.346

Localization of Dsg2 and Cav-1 to membrane lipid rafts. ( A ) A431 cells were treated with MβCD (10 mM) for 1 hr and extracted in a Tris-NaCl-EDTA buffer containing Tx. Proteins were subjected to a discontinous (5-35%) sucrose-gradient separation, resolved over SDS-PAGE and immunoblotted for Cav-1, Cav-2, Flo1, Flo2, β-Cat, γ-Cat, actin, E-Cad and Dsg2. Immunoblotting revealed that Cav-1 localized predominantly to low-density fractions 4 and 5 (top left panel), corresponding to lipid rafts. Dsg2 was distributed through all fractions from 4-12. Treatment with MβCD (10 mM) for 1 hr, disrupted lipid rafts, and shifted both Cav-1 and Dsg2 to the more dense fractions. In addition to the 160 kDa Dsg2 full-length protein, we observed a 65 kDa band in the lipid raft fraction # 4 (vertical arrow). Accumulation of this fragment was enhanced and shifted to the denser fractions in the presence of MβCD (arrow head). We note that β-Cat, γ-Cat, E-Cad and actin fractioned to the lower, denser fractions, and remained relatively unchanged in the presence of MβCD. ( B ) Proteins from fractions 4 (lipid raft fraction) and 12 (high molecular weight, non-raft fraction) from above were resolved over SDS-PAGE and immunoblotted for Dsg2 using two different antibodies, 10D2 and DG3.10. Treatment with MβCD increased the level of the 65-kDa Dsg2 fragment as detected by DG3.10, but not 10D2.
Figure Legend Snippet: Localization of Dsg2 and Cav-1 to membrane lipid rafts. ( A ) A431 cells were treated with MβCD (10 mM) for 1 hr and extracted in a Tris-NaCl-EDTA buffer containing Tx. Proteins were subjected to a discontinous (5-35%) sucrose-gradient separation, resolved over SDS-PAGE and immunoblotted for Cav-1, Cav-2, Flo1, Flo2, β-Cat, γ-Cat, actin, E-Cad and Dsg2. Immunoblotting revealed that Cav-1 localized predominantly to low-density fractions 4 and 5 (top left panel), corresponding to lipid rafts. Dsg2 was distributed through all fractions from 4-12. Treatment with MβCD (10 mM) for 1 hr, disrupted lipid rafts, and shifted both Cav-1 and Dsg2 to the more dense fractions. In addition to the 160 kDa Dsg2 full-length protein, we observed a 65 kDa band in the lipid raft fraction # 4 (vertical arrow). Accumulation of this fragment was enhanced and shifted to the denser fractions in the presence of MβCD (arrow head). We note that β-Cat, γ-Cat, E-Cad and actin fractioned to the lower, denser fractions, and remained relatively unchanged in the presence of MβCD. ( B ) Proteins from fractions 4 (lipid raft fraction) and 12 (high molecular weight, non-raft fraction) from above were resolved over SDS-PAGE and immunoblotted for Dsg2 using two different antibodies, 10D2 and DG3.10. Treatment with MβCD increased the level of the 65-kDa Dsg2 fragment as detected by DG3.10, but not 10D2.

Techniques Used: SDS Page, Molecular Weight

4) Product Images from "Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression"

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression

Journal: American journal of physiology. Heart and circulatory physiology

doi: 10.1152/ajpheart.00350.2003

Electroporation-mediated delivery of oligonucleotides to the intact vasculature. FITC-labeled wtDNAzyme [100 μM in 10 mM Tris (pH 8), 1 mM EDTA, and 140 mM NaCl] was transferred to rat mesenteric arteries using electroporation (8 pulses at 10 ms each, 200 V/cm). One minute to 24 h postelectroporation, vessels were excised, rinsed extensively with PBS, fixed in formalin, paraffin embedded, and sectioned to 10 μm. Immunohistochemistry was performed with antibodies against fluorescein and visualized using Vector Blue (Vector Labs). Control vessel receiving no oligonucleotide ( A ) or vessels electroporated with oligonucleotide and removed at 1 min postelectroporation ( B and C ) are shown. The oligonucleotide is stained in blue, and the tissue was counterstained with eosin. Bars = 50 μm.
Figure Legend Snippet: Electroporation-mediated delivery of oligonucleotides to the intact vasculature. FITC-labeled wtDNAzyme [100 μM in 10 mM Tris (pH 8), 1 mM EDTA, and 140 mM NaCl] was transferred to rat mesenteric arteries using electroporation (8 pulses at 10 ms each, 200 V/cm). One minute to 24 h postelectroporation, vessels were excised, rinsed extensively with PBS, fixed in formalin, paraffin embedded, and sectioned to 10 μm. Immunohistochemistry was performed with antibodies against fluorescein and visualized using Vector Blue (Vector Labs). Control vessel receiving no oligonucleotide ( A ) or vessels electroporated with oligonucleotide and removed at 1 min postelectroporation ( B and C ) are shown. The oligonucleotide is stained in blue, and the tissue was counterstained with eosin. Bars = 50 μm.

Techniques Used: Electroporation, Labeling, Mass Spectrometry, Immunohistochemistry, Plasmid Preparation, Staining

5) Product Images from "The lipid phosphatase Synaptojanin 1 undergoes a significant alteration in expression and solubility and is associated with brain lesions in Alzheimer’s disease"

Article Title: The lipid phosphatase Synaptojanin 1 undergoes a significant alteration in expression and solubility and is associated with brain lesions in Alzheimer’s disease

Journal: Acta Neuropathologica Communications

doi: 10.1186/s40478-020-00954-1

In vitro cleavage assay for SYNJ1 by calpain. a HEK 293 cells were transiently transfected with Flag-SYNJ1 145 and cultured for 24 h. The cell lysate was incubated at 37 °C for 1 h in the presence or absence of calcium. In the presence of calcium in the lysate, SYNJ1 was significantly decreased by 50% and a 140-kDa band appeared (open circle). The proteolysis was inhibited by adding calcium chelators (EDTA and EGTA) or calpain inhibitor I. b The graph shows the OD of SYNJ1 normalised to actin in each condition of three independent experiments. * p
Figure Legend Snippet: In vitro cleavage assay for SYNJ1 by calpain. a HEK 293 cells were transiently transfected with Flag-SYNJ1 145 and cultured for 24 h. The cell lysate was incubated at 37 °C for 1 h in the presence or absence of calcium. In the presence of calcium in the lysate, SYNJ1 was significantly decreased by 50% and a 140-kDa band appeared (open circle). The proteolysis was inhibited by adding calcium chelators (EDTA and EGTA) or calpain inhibitor I. b The graph shows the OD of SYNJ1 normalised to actin in each condition of three independent experiments. * p

Techniques Used: In Vitro, Cleavage Assay, Transfection, Cell Culture, Incubation

6) Product Images from "Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells"

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells

Journal: Bioscience Reports

doi: 10.1042/BSR20130042

High molecular mass 46 kDa NDRG1 isoform identified in DU145 cells is not due to phosphorylation Analysis of ( A ) NDRG1 dephosphorylation assay using phosphatases and ( B, C ) Mn 2+ -Phos-tag SDS/PAGE assays. ( A ) Western blotting of DU145 whole cell lysates treated with or without λ-PPase or CIAP. Loss of immunoreactive pNDRG1 Ser330 protein was used as a positive control to show active phosphatases. PhosStop and/or EDTA treatment was used to inhibit both endogenous (control lysates) and exogenous (λ-PPase or CIAP treated lysates) phosphatases. Blots were probed for β-actin which was used as a loading control. ( B ) A comparison of the NDRG1 Phos-tag SDS/PAGE immunoblot relative to the control NDRG1 SDS/PAGE immunoblot using DU145 cell lysates (duplicate samples are shown). β-actin was used as a loading control. ( C ) CBB staining of α-casein and dephosphorylated α-casein after performing Phos-tag SDS/PAGE or standard SDS/PAGE to confirm active phosphatases and Phos-tag efficacy. The figure is representative of three individual experiments.
Figure Legend Snippet: High molecular mass 46 kDa NDRG1 isoform identified in DU145 cells is not due to phosphorylation Analysis of ( A ) NDRG1 dephosphorylation assay using phosphatases and ( B, C ) Mn 2+ -Phos-tag SDS/PAGE assays. ( A ) Western blotting of DU145 whole cell lysates treated with or without λ-PPase or CIAP. Loss of immunoreactive pNDRG1 Ser330 protein was used as a positive control to show active phosphatases. PhosStop and/or EDTA treatment was used to inhibit both endogenous (control lysates) and exogenous (λ-PPase or CIAP treated lysates) phosphatases. Blots were probed for β-actin which was used as a loading control. ( B ) A comparison of the NDRG1 Phos-tag SDS/PAGE immunoblot relative to the control NDRG1 SDS/PAGE immunoblot using DU145 cell lysates (duplicate samples are shown). β-actin was used as a loading control. ( C ) CBB staining of α-casein and dephosphorylated α-casein after performing Phos-tag SDS/PAGE or standard SDS/PAGE to confirm active phosphatases and Phos-tag efficacy. The figure is representative of three individual experiments.

Techniques Used: De-Phosphorylation Assay, SDS Page, Western Blot, Positive Control, Staining

7) Product Images from "Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †"

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.01366-10

) with hybrid E2 proteins expressed in COS-7 cells. Hybrid E2 proteins were mixed with relevant DNA targets, and MAb 1E4 was added simultaneously. Oligo, oligonucleotide; mock, cells which were transfected with carrier DNA only. Protein-DNA complexes were resolved by 6% PAGE in 0.25× Tris-borate-EDTA. (C) Measurement of cooperative DNA binding of E2, E2C, and LANA:E2C at two neighboring binding sites. Graphs represent quantified data from cooperative DNA binding assays performed with different amounts of E2, E2C, and LANA:E2C COS-7 lysates (0.0625 to 2 μl) and with two E2-binding-site-radiolabeled probes. Open triangles, free probe; filled triangles, one E2 binding site occupied; filled squares, two E2 binding sites occupied. The maximal occupancy of the single E2 binding site and the respective k ab ).
Figure Legend Snippet: ) with hybrid E2 proteins expressed in COS-7 cells. Hybrid E2 proteins were mixed with relevant DNA targets, and MAb 1E4 was added simultaneously. Oligo, oligonucleotide; mock, cells which were transfected with carrier DNA only. Protein-DNA complexes were resolved by 6% PAGE in 0.25× Tris-borate-EDTA. (C) Measurement of cooperative DNA binding of E2, E2C, and LANA:E2C at two neighboring binding sites. Graphs represent quantified data from cooperative DNA binding assays performed with different amounts of E2, E2C, and LANA:E2C COS-7 lysates (0.0625 to 2 μl) and with two E2-binding-site-radiolabeled probes. Open triangles, free probe; filled triangles, one E2 binding site occupied; filled squares, two E2 binding sites occupied. The maximal occupancy of the single E2 binding site and the respective k ab ).

Techniques Used: Transfection, Polyacrylamide Gel Electrophoresis, Binding Assay

8) Product Images from "Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞"

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M800189200

IFN γ induces cell surface-specific reduction in annexin 2 expression. A, biotin-labeled surface proteins detected by avidin staining following two-dimensional gel electrophoresis ( 2DE ). Biotinylation was performed prior to and after 72 h of IFNγ stimulation. Proteins down-regulated by IFNγ are indicated by upward arrows . The protein indicated by a white rectangle undergoes a series of acidic post-translational modifications after IFNγ treatment. B, Coomassie-stained proteins in whole cell extracts and avidin-purified biotinylated membrane fractions from 1542CP3TX cells grown in the presence or absence of IFNγ. The protein band indicated by oblique arrows was analyzed by mass spectrometry. C, 36-kDa protein band was excised, trypsinized, and identified as human annexin 2 ( anx2 ) (NCBI gi number 18645167) by matrix-assisted laser desorption/ionization-mass spectrometry analysis. 43% of its amino acid sequence was covered by the 15 matched peptides indicated by circles . The 54-kDa protein band indicated by a horizontal arrow in B was identified as interferon-inducible protein 53 (IFP53) in a similar fashion. Immunoblot ( IB ) of avidin purified membrane fractions confirming that IFNγ suppresses the surface density of annexin 2 in the prostate cancer cells. A monoclonal antibody against annexin 2 was employed in a 1:2000 dilution (catalog number 03-4400, Zymed Laboratories Inc.). D, immunoblot of EDTA-released surface proteins demonstrating temporal reduction in the membrane density of annexin 2 in response to sustained IFNγ treatment. E, abundance of annexin 2 in whole cell OGP extracts of 1542CP3TX cells was determined by immunostaining ( top ). Treatment with interferon for 24 h had no effect on the global annexin 2 levels. β-Actin in the separated detergent samples serves as loading control (1:1000 dilution of A-S441, Sigma).
Figure Legend Snippet: IFN γ induces cell surface-specific reduction in annexin 2 expression. A, biotin-labeled surface proteins detected by avidin staining following two-dimensional gel electrophoresis ( 2DE ). Biotinylation was performed prior to and after 72 h of IFNγ stimulation. Proteins down-regulated by IFNγ are indicated by upward arrows . The protein indicated by a white rectangle undergoes a series of acidic post-translational modifications after IFNγ treatment. B, Coomassie-stained proteins in whole cell extracts and avidin-purified biotinylated membrane fractions from 1542CP3TX cells grown in the presence or absence of IFNγ. The protein band indicated by oblique arrows was analyzed by mass spectrometry. C, 36-kDa protein band was excised, trypsinized, and identified as human annexin 2 ( anx2 ) (NCBI gi number 18645167) by matrix-assisted laser desorption/ionization-mass spectrometry analysis. 43% of its amino acid sequence was covered by the 15 matched peptides indicated by circles . The 54-kDa protein band indicated by a horizontal arrow in B was identified as interferon-inducible protein 53 (IFP53) in a similar fashion. Immunoblot ( IB ) of avidin purified membrane fractions confirming that IFNγ suppresses the surface density of annexin 2 in the prostate cancer cells. A monoclonal antibody against annexin 2 was employed in a 1:2000 dilution (catalog number 03-4400, Zymed Laboratories Inc.). D, immunoblot of EDTA-released surface proteins demonstrating temporal reduction in the membrane density of annexin 2 in response to sustained IFNγ treatment. E, abundance of annexin 2 in whole cell OGP extracts of 1542CP3TX cells was determined by immunostaining ( top ). Treatment with interferon for 24 h had no effect on the global annexin 2 levels. β-Actin in the separated detergent samples serves as loading control (1:1000 dilution of A-S441, Sigma).

Techniques Used: Expressing, Labeling, Avidin-Biotin Assay, Staining, Two-Dimensional Gel Electrophoresis, Electrophoresis, Purification, Mass Spectrometry, Sequencing, Immunostaining

9) Product Images from "Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist"

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist

Journal: Scientific Reports

doi: 10.1038/srep24880

IL-36Ra cleavage by activated PMN supernatant is prevented by serine protease inhibitors. SUMO tagged IL-36Ra was incubated with supernatant from PMNs stimulated with PMA for 1 hour at 37 °C. Incubation was performed in the presence or absence of a range of protease inhibitors. PI = complete protease inhibitor cocktail (Roche), EDTA = Ethylenediaminetetraacetic acid, inhibitor of proteases where a metal ion is required for cleavage, PI Roche = complete ultra-protease inhibitor cocktail which includes aspartic proteases (Roche), IAA = iodoacetic acid a pan cysteine protease inhibitor, PMSF = phenylmethylsulfonyl fluoride a pan serine protease inhibitor, α1 anti-trypsin is another pan serine protease inhibitor. Samples were then analysed using WB.
Figure Legend Snippet: IL-36Ra cleavage by activated PMN supernatant is prevented by serine protease inhibitors. SUMO tagged IL-36Ra was incubated with supernatant from PMNs stimulated with PMA for 1 hour at 37 °C. Incubation was performed in the presence or absence of a range of protease inhibitors. PI = complete protease inhibitor cocktail (Roche), EDTA = Ethylenediaminetetraacetic acid, inhibitor of proteases where a metal ion is required for cleavage, PI Roche = complete ultra-protease inhibitor cocktail which includes aspartic proteases (Roche), IAA = iodoacetic acid a pan cysteine protease inhibitor, PMSF = phenylmethylsulfonyl fluoride a pan serine protease inhibitor, α1 anti-trypsin is another pan serine protease inhibitor. Samples were then analysed using WB.

Techniques Used: Incubation, Protease Inhibitor, Western Blot

10) Product Images from "Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)"

Article Title: Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0171464

Cif expression regulates the CDC25-H and proline-rich domains in SOS1. (A,B) HEK293T cells were transfected with the indicated expression plasmids for two days, followed by FLAG immunoprecipitation of the cell lysates using FLAG-agarose and Western blotting of the immunoprecipitates and aliquots of the cell lysates using the indicated antibodies. (C) All cells were transfected with FLAG-SOS1 as well as V5-Cif or empty vector, as indicated, for two days. Cells were then lysed in hypotonic lysis buffer (25 mM Tris-HCL, 2 mM EDTA, 2 mM EGTA, 0.1% β-Mercaptoethanol, Roche protease inhibitor cocktail, pH 7.5) and cell lysates subjected to freeze-thawing at -80°C. The cytosolic fraction was separated from the remaining cellular compartments (membranes and nuclear fraction) through centrifugation. The proteins in the cytosolic supernatant and the pellet were denatured using SDS loading buffer and analysed using Western blotting with the indicated antibodies. Equal corresponding amounts of cytosol and pellet were loaded. GAPDH served as a control for the cytosolic fraction and calnexin as a control for the membrane fraction. Glut1 (55 kda) served as an additional control for the membrane fraction, as the higher molecular weight, glycosylated Glut1 species can only be detected in the membrane fraction. (D) Cells were transfected with the indicated FLAG-mSOS1 truncation constructs for two days, followed by Western blotting using the indicated antibodies. The bottom FLAG blot in the Western blot panel was obtained from running a duplicate set of lysates through another SDS-PAGE gel of a lower resolving gel percentage for a longer time to increase the resolving power.
Figure Legend Snippet: Cif expression regulates the CDC25-H and proline-rich domains in SOS1. (A,B) HEK293T cells were transfected with the indicated expression plasmids for two days, followed by FLAG immunoprecipitation of the cell lysates using FLAG-agarose and Western blotting of the immunoprecipitates and aliquots of the cell lysates using the indicated antibodies. (C) All cells were transfected with FLAG-SOS1 as well as V5-Cif or empty vector, as indicated, for two days. Cells were then lysed in hypotonic lysis buffer (25 mM Tris-HCL, 2 mM EDTA, 2 mM EGTA, 0.1% β-Mercaptoethanol, Roche protease inhibitor cocktail, pH 7.5) and cell lysates subjected to freeze-thawing at -80°C. The cytosolic fraction was separated from the remaining cellular compartments (membranes and nuclear fraction) through centrifugation. The proteins in the cytosolic supernatant and the pellet were denatured using SDS loading buffer and analysed using Western blotting with the indicated antibodies. Equal corresponding amounts of cytosol and pellet were loaded. GAPDH served as a control for the cytosolic fraction and calnexin as a control for the membrane fraction. Glut1 (55 kda) served as an additional control for the membrane fraction, as the higher molecular weight, glycosylated Glut1 species can only be detected in the membrane fraction. (D) Cells were transfected with the indicated FLAG-mSOS1 truncation constructs for two days, followed by Western blotting using the indicated antibodies. The bottom FLAG blot in the Western blot panel was obtained from running a duplicate set of lysates through another SDS-PAGE gel of a lower resolving gel percentage for a longer time to increase the resolving power.

Techniques Used: Expressing, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Lysis, Protease Inhibitor, Centrifugation, Molecular Weight, Construct, SDS Page

11) Product Images from "Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy"

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy

Journal: Current Biology

doi: 10.1016/j.cub.2017.06.021

Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .
Figure Legend Snippet: Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

Techniques Used: Expressing, Plasmid Preparation, Construct, Incubation, Recombinant, Immunoprecipitation, Lysis, Mass Spectrometry, Mutagenesis

12) Product Images from "Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli"

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli

Journal: PLoS ONE

doi: 10.1371/journal.pone.0098042

PBP3 oligomerization. (a) Chromatogram of PBP3 88–165 gel filtration on a Superdex 75 10/300 GL. The first peak elutes at 12.16 ml and the second at 13.52 ml. Carbonic anhydrase (31 kDa) elutes at 11.05 ml and lysozyme (14 kDa) at 15.25 ml (data not shown). The buffer was 0.15 M NaCl and 0.1 M Tris, pH 8 1 mM EDTA. (b) Chromatogram of PBP3 57–577 gel filtration on a Superdex 200 10/300 GL. The first small peak elutes at 13.3 ml, the second at 14.77 ml. Bovine serum albumin used as a standard elutes at 14.12 ml (molecular mass 67 kDa, data not shown). The masses calculated on the basis of the mass standards are 108.5 kDa for the first peak (PBP3 57–577 dimer) and 58.5 kDa for the second peak (PBP3 57–577 monomer). The buffer was 20 mM Tris HCl pH 8, 0.5 M NaCl.
Figure Legend Snippet: PBP3 oligomerization. (a) Chromatogram of PBP3 88–165 gel filtration on a Superdex 75 10/300 GL. The first peak elutes at 12.16 ml and the second at 13.52 ml. Carbonic anhydrase (31 kDa) elutes at 11.05 ml and lysozyme (14 kDa) at 15.25 ml (data not shown). The buffer was 0.15 M NaCl and 0.1 M Tris, pH 8 1 mM EDTA. (b) Chromatogram of PBP3 57–577 gel filtration on a Superdex 200 10/300 GL. The first small peak elutes at 13.3 ml, the second at 14.77 ml. Bovine serum albumin used as a standard elutes at 14.12 ml (molecular mass 67 kDa, data not shown). The masses calculated on the basis of the mass standards are 108.5 kDa for the first peak (PBP3 57–577 dimer) and 58.5 kDa for the second peak (PBP3 57–577 monomer). The buffer was 20 mM Tris HCl pH 8, 0.5 M NaCl.

Techniques Used: Filtration

13) Product Images from "The Structure and Regulation of Human Muscle α-Actinin"

Article Title: The Structure and Regulation of Human Muscle α-Actinin

Journal: Cell

doi: 10.1016/j.cell.2014.10.056

Neck Peptide of Wild-Type α-Actinin-2 Is Unstructured when Not Bound to the CaM-like Domain, Related to Figure 4 Fingerprint area of the 2D NOESY spectrum of the WT neck-peptide in 90:10 H 2 O:D 2 O with neck-peptide in 25mM phosphate pH 6.5, 25 mM NaCl, 3 mM DTT and 0.5 mM EDTA recorded at 298K. Hα i -HN i and Hα i -HN i+1 cross peaks are shown in red and the sequential walk of the peptide is indicated with horizontal and vertical lines. No additional NOE cross peaks were found that indicated an existence of any kind of stable α-helical structural in the fingerprint area.
Figure Legend Snippet: Neck Peptide of Wild-Type α-Actinin-2 Is Unstructured when Not Bound to the CaM-like Domain, Related to Figure 4 Fingerprint area of the 2D NOESY spectrum of the WT neck-peptide in 90:10 H 2 O:D 2 O with neck-peptide in 25mM phosphate pH 6.5, 25 mM NaCl, 3 mM DTT and 0.5 mM EDTA recorded at 298K. Hα i -HN i and Hα i -HN i+1 cross peaks are shown in red and the sequential walk of the peptide is indicated with horizontal and vertical lines. No additional NOE cross peaks were found that indicated an existence of any kind of stable α-helical structural in the fingerprint area.

Techniques Used: Chick Chorioallantoic Membrane Assay

14) Product Images from "Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70genomic locus✩"

Article Title: Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70genomic locus✩

Journal: International journal for parasitology

doi: 10.1016/j.ijpara.2003.11.020

Gel shift assay. (A) electrophoretic mobility shift assay (EMSA) (using biotin labeled oligonucleotides) demonstrating binding of a putative regulatory protein (arrowhead) to Tg-hsp70-REG. ‘*’ indicates the location of unbound (free) probe. Toxoplasma gondii ME49 strain protein lysate was prepared by sonication of purified parasites in 20 mM Hepes (pH 8.0), 0.1 KCL, 0.5 mM DTT 0.2 mM EDTA 20% glycerol containing a protease inhibitor cocktail tablet (Roche) followed by centrifugation at 3000 × g to remove insoluble materials. Lane 1: Biotin labeled Tg-hsp70-REG, Lane 2: 10 × unlabeled Tg-hsp70-REG added to reaction, Lane 3: 20 × unlabeled Tg-hsp70-REG added to reaction, Lane 4: 50 × unlabeled Tg-hsp70-REG added to reaction, Lane 5: no oligomers added to the reaction (negative control). As can be seen cold unlabeled Tg-hsp70-REG competes with the binding of this protein to the biotin labeled Tg-hsp70-REG. (B) EMSA (using 32 P labeled oligonucleotides). Lane 1: 32 P labeled Tg-hsp70-REG demonstrating binding to putative regulatory protein (arrowhead), Lane 2: 50 × unlabeled Tg-hsp70-REG added to the reaction, Lane 3: 32 P labeled Tg-hsp70-REG, Lane 4: 50 × unlabeled Tg-hsp70-CTL added to 32 P labeled Tg-hsp70-REG, Lane 5: 50 × unlabeled BAG1 added to 32 P labeled Tg-hsp70-REG. ‘*’ indicates the location of unbound (free) probe.
Figure Legend Snippet: Gel shift assay. (A) electrophoretic mobility shift assay (EMSA) (using biotin labeled oligonucleotides) demonstrating binding of a putative regulatory protein (arrowhead) to Tg-hsp70-REG. ‘*’ indicates the location of unbound (free) probe. Toxoplasma gondii ME49 strain protein lysate was prepared by sonication of purified parasites in 20 mM Hepes (pH 8.0), 0.1 KCL, 0.5 mM DTT 0.2 mM EDTA 20% glycerol containing a protease inhibitor cocktail tablet (Roche) followed by centrifugation at 3000 × g to remove insoluble materials. Lane 1: Biotin labeled Tg-hsp70-REG, Lane 2: 10 × unlabeled Tg-hsp70-REG added to reaction, Lane 3: 20 × unlabeled Tg-hsp70-REG added to reaction, Lane 4: 50 × unlabeled Tg-hsp70-REG added to reaction, Lane 5: no oligomers added to the reaction (negative control). As can be seen cold unlabeled Tg-hsp70-REG competes with the binding of this protein to the biotin labeled Tg-hsp70-REG. (B) EMSA (using 32 P labeled oligonucleotides). Lane 1: 32 P labeled Tg-hsp70-REG demonstrating binding to putative regulatory protein (arrowhead), Lane 2: 50 × unlabeled Tg-hsp70-REG added to the reaction, Lane 3: 32 P labeled Tg-hsp70-REG, Lane 4: 50 × unlabeled Tg-hsp70-CTL added to 32 P labeled Tg-hsp70-REG, Lane 5: 50 × unlabeled BAG1 added to 32 P labeled Tg-hsp70-REG. ‘*’ indicates the location of unbound (free) probe.

Techniques Used: Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Sonication, Purification, Protease Inhibitor, Centrifugation, Negative Control, CTL Assay

15) Product Images from "pH Dependence of the Stress Regulator DksA"

Article Title: pH Dependence of the Stress Regulator DksA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0120746

DksA is sensitive to changes in pH. (A) DksA activity increases at low pH. Increasing concentrations of DksA were added to holo RNAP (30 nM), ApC dinucleotide (0.2 mM), UTP (0.2 mM), GTP (4 μM) and [α- 32 P]-GTP (10 μCi of 3000 Ci mmol −1 ) followed by incubation for 15 minutes in Transcription buffer (20 mM Tris-HCl pH 7.9, 20 mM NaCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, 0.1 mM EDTA). A linear DNA fragment containing the rrnB P1 promoter was added to initiate transcription and the formation of a 4 nucleotide RNA product was monitored on a denaturing 8% acrylamide gel. A dotted line marks the inhibition of 50% of transcription and is denoted as IC 50 . The IC 50 values (calculated using a single-site binding equation from three independent repeats combined in a best-fit curve, in μM) were: pH 7.6 − 0.7 ± 0.28, pH 6.7 − 0.11 ± 0.016. (B) DksA affinity to core increases at lower pH. DksA binding to core RNAP was performed using the localized Fe 2+ mediated cleavage assay at different pH. DksA concentrations were: 0, 25, 50, 100, 200 and 400 nM. FL—Full length protein, Cl—cleaved protein, Kd app—apparent Kd.
Figure Legend Snippet: DksA is sensitive to changes in pH. (A) DksA activity increases at low pH. Increasing concentrations of DksA were added to holo RNAP (30 nM), ApC dinucleotide (0.2 mM), UTP (0.2 mM), GTP (4 μM) and [α- 32 P]-GTP (10 μCi of 3000 Ci mmol −1 ) followed by incubation for 15 minutes in Transcription buffer (20 mM Tris-HCl pH 7.9, 20 mM NaCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, 0.1 mM EDTA). A linear DNA fragment containing the rrnB P1 promoter was added to initiate transcription and the formation of a 4 nucleotide RNA product was monitored on a denaturing 8% acrylamide gel. A dotted line marks the inhibition of 50% of transcription and is denoted as IC 50 . The IC 50 values (calculated using a single-site binding equation from three independent repeats combined in a best-fit curve, in μM) were: pH 7.6 − 0.7 ± 0.28, pH 6.7 − 0.11 ± 0.016. (B) DksA affinity to core increases at lower pH. DksA binding to core RNAP was performed using the localized Fe 2+ mediated cleavage assay at different pH. DksA concentrations were: 0, 25, 50, 100, 200 and 400 nM. FL—Full length protein, Cl—cleaved protein, Kd app—apparent Kd.

Techniques Used: Activity Assay, Incubation, Acrylamide Gel Assay, Inhibition, Binding Assay, Cleavage Assay

16) Product Images from "The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin"

Article Title: The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin

Journal: Matrix biology : journal of the International Society for Matrix Biology

doi: 10.1016/j.matbio.2006.02.002

Cell-bound FITC–70K does not form detergent-insoluble multimers. FN –/– cells were plated on laminin-coated tissue culture plastic for 2h. 40nM FITC–70K or 20nM FITC–FN were added for 10min, 1h or 24h in 400nM LPA, DMEM and 0.2% BSA. Monolayers were washed and extracted with 1% deoxycholate in TBS, 2mM EDTA, 2mM iodoacetamide and proteinase inhibitor complex. Samples were centrifuged to separate insoluble from soluble fractions. Samples were run unreduced on 8% acrylamide gels and immunoblotted with rabbit anti-FITC antibodies, followed by peroxidase anti-rabbit conjugates and chemiluminescence development. Lanes: (1) FITC–70K, (2) FITC–FN. Migration of molecular weight standards (kDa) is depicted to the left. The arrow indicates the well level at the top of the 3.5% stacking gel.
Figure Legend Snippet: Cell-bound FITC–70K does not form detergent-insoluble multimers. FN –/– cells were plated on laminin-coated tissue culture plastic for 2h. 40nM FITC–70K or 20nM FITC–FN were added for 10min, 1h or 24h in 400nM LPA, DMEM and 0.2% BSA. Monolayers were washed and extracted with 1% deoxycholate in TBS, 2mM EDTA, 2mM iodoacetamide and proteinase inhibitor complex. Samples were centrifuged to separate insoluble from soluble fractions. Samples were run unreduced on 8% acrylamide gels and immunoblotted with rabbit anti-FITC antibodies, followed by peroxidase anti-rabbit conjugates and chemiluminescence development. Lanes: (1) FITC–70K, (2) FITC–FN. Migration of molecular weight standards (kDa) is depicted to the left. The arrow indicates the well level at the top of the 3.5% stacking gel.

Techniques Used: Migration, Molecular Weight

17) Product Images from "In Vitro Assembly of Catalase *"

Article Title: In Vitro Assembly of Catalase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.596148

Apocatalase polypeptide proteolysis is induced in late exponential growth phase E. faecalis cells. A , lysates from strain OG1RF cells grown in heme-free medium until early exponential growth phase ( sample A ) or late exponential growth phase ( sample B ) and lysates from cells grown in heme-supplemented medium until late exponential growth phase ( sample C ) were analyzed individually or as mixtures as indicated. The samples were incubated at 37 °C, and the presence of KatA polypeptide in aliquots removed at the indicated time points was determined by immuno-blot. B , proteolysis of KatA in sample A mixed with sample B in the presence of 1 m m PMSF and/or EDTA or Complete EDTA-free protease inhibitor cocktail. The reference, sample A + B without any additions, is indicated by Ctrl .
Figure Legend Snippet: Apocatalase polypeptide proteolysis is induced in late exponential growth phase E. faecalis cells. A , lysates from strain OG1RF cells grown in heme-free medium until early exponential growth phase ( sample A ) or late exponential growth phase ( sample B ) and lysates from cells grown in heme-supplemented medium until late exponential growth phase ( sample C ) were analyzed individually or as mixtures as indicated. The samples were incubated at 37 °C, and the presence of KatA polypeptide in aliquots removed at the indicated time points was determined by immuno-blot. B , proteolysis of KatA in sample A mixed with sample B in the presence of 1 m m PMSF and/or EDTA or Complete EDTA-free protease inhibitor cocktail. The reference, sample A + B without any additions, is indicated by Ctrl .

Techniques Used: Incubation, Protease Inhibitor

18) Product Images from "Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles"

Article Title: Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

Journal: Virology

doi: 10.1016/j.virol.2016.04.012

Deletion mapping of HBZ, APH-2 and NF110a interaction domains. (A) Schematic of HBZ and deletion mutants used in co-immunoprecipitations. Functional domains are: AD: activation domain; CD: central domain; bZIP: basic leucine zipper. Black boxes represent the LXXLL motifs. (B) NFARs interact with HBZ central domain. 293T cells were seeded into 60 mm cell cultures dishes and transfected with 8 μg of pFLAG-HBZ or pFLAG-HBZΔAD and 10 μg of pFLAG-HBZΔCD or pFLAG-HBZΔbZIP as indicated. 24 hours post-transfection cells were lysed in RIPA buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1% Triton ® X-100, 0.1% SDS, 0.5% sodium deoxycholate. An anti-FLAG M2 resin was used to precipitate protein complexes from lysates overnight. Interactions were analysed by western blot using anti-FLAG and anti-ILF3 antibodies. (C) Schematic of APH-2 and mutants used in co-immunoprecipitations. Functional domains are: IXXLL: LXXLL-like motif; ncbZIP: non-canonical basic leucine zipper domain; LXXLL: LXXLL motif. (D) 293T cells were seeded into 60 mm cell cultures dishes and transfected with 8 mg of full length APH-2 or the indicated mutants. Lysates from transfected cells were prepared using a buffer containing 1X TBS, 0.005M EDTA, 1% Triton-X 100 24 h post-transfection and subjected to immunoprecipitation using FLAG M2 resin. Precipitates were analysed by western blot using anti-FLAG and anti-ILF-3 antibodies. (E) Schematic representation of NF110a and mutants used in co-immunoprecipitations. Mutants are: NF110a-167-394 incorporating the EIF2α-homology region and NLS; 398–603 incorporating two double-stranded RNA-binding domains (DRBD-1/-2). Y2H represents the minimum domain in NF110a required for interaction with APH-2 obtained from yeast two-hybrid screen. (F and G) HBZ and APH-2 interact with NF110a 167-394. 293T cells were seeded into 60 mm cell cultures dishes and co-transfected with 2 μg pCAGGS-NF110a-167-394-His or 8 μg pCAGGS-NF110a-398-603-His and 4 μg pFLAG-HBZ (F) or pFLAG-APH-2 (G) as indicated. Cells were lysed in 1X TBS, 0.005M EDTA, 1% Triton-X 100 buffer as above. Interactions were analysed by immunoblotting using anti-FLAG and anti-His antibodies. Arrows indicate specific protein bands.
Figure Legend Snippet: Deletion mapping of HBZ, APH-2 and NF110a interaction domains. (A) Schematic of HBZ and deletion mutants used in co-immunoprecipitations. Functional domains are: AD: activation domain; CD: central domain; bZIP: basic leucine zipper. Black boxes represent the LXXLL motifs. (B) NFARs interact with HBZ central domain. 293T cells were seeded into 60 mm cell cultures dishes and transfected with 8 μg of pFLAG-HBZ or pFLAG-HBZΔAD and 10 μg of pFLAG-HBZΔCD or pFLAG-HBZΔbZIP as indicated. 24 hours post-transfection cells were lysed in RIPA buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1% Triton ® X-100, 0.1% SDS, 0.5% sodium deoxycholate. An anti-FLAG M2 resin was used to precipitate protein complexes from lysates overnight. Interactions were analysed by western blot using anti-FLAG and anti-ILF3 antibodies. (C) Schematic of APH-2 and mutants used in co-immunoprecipitations. Functional domains are: IXXLL: LXXLL-like motif; ncbZIP: non-canonical basic leucine zipper domain; LXXLL: LXXLL motif. (D) 293T cells were seeded into 60 mm cell cultures dishes and transfected with 8 mg of full length APH-2 or the indicated mutants. Lysates from transfected cells were prepared using a buffer containing 1X TBS, 0.005M EDTA, 1% Triton-X 100 24 h post-transfection and subjected to immunoprecipitation using FLAG M2 resin. Precipitates were analysed by western blot using anti-FLAG and anti-ILF-3 antibodies. (E) Schematic representation of NF110a and mutants used in co-immunoprecipitations. Mutants are: NF110a-167-394 incorporating the EIF2α-homology region and NLS; 398–603 incorporating two double-stranded RNA-binding domains (DRBD-1/-2). Y2H represents the minimum domain in NF110a required for interaction with APH-2 obtained from yeast two-hybrid screen. (F and G) HBZ and APH-2 interact with NF110a 167-394. 293T cells were seeded into 60 mm cell cultures dishes and co-transfected with 2 μg pCAGGS-NF110a-167-394-His or 8 μg pCAGGS-NF110a-398-603-His and 4 μg pFLAG-HBZ (F) or pFLAG-APH-2 (G) as indicated. Cells were lysed in 1X TBS, 0.005M EDTA, 1% Triton-X 100 buffer as above. Interactions were analysed by immunoblotting using anti-FLAG and anti-His antibodies. Arrows indicate specific protein bands.

Techniques Used: Functional Assay, Activation Assay, Transfection, Western Blot, Immunoprecipitation, RNA Binding Assay, Two Hybrid Screening

19) Product Images from "Positive feedback regulation of p53 transactivity by DNA damage-induced ISG15 modification"

Article Title: Positive feedback regulation of p53 transactivity by DNA damage-induced ISG15 modification

Journal: Nature Communications

doi: 10.1038/ncomms12513

ATM/ATR kinases induce the expression of ISG15-conjugating system. p53 +/+ HCT116 cells were treated with 1 μM doxorubicin (DOX) ( a ), 0.25 μM camptothecin (CPT) ( b ) and 30 J m −2 ultraviolet (UV) ( c ). Immediately after the treatment, the cells were incubated with 5 mM caffeine for increasing periods. The cells were washed with iced PBS, and lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM NEM, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 0.2% (v/v) Triton X-100 and 1 X protease inhibitor cocktail. The samples were centrifuged for 30 min at 16,000 g and the supernatant fractions (cell lysates) were subjected to immunoblot with anti-ISG15, anti-UBE1L anti-UBCH8, anti-EFP, anti-phospho-p53 (p-p53), anti-p53, anti-phospho-Chk1 (p-Chk1) and anti-β-actin antibodies (from the top to the bottom, respectively).
Figure Legend Snippet: ATM/ATR kinases induce the expression of ISG15-conjugating system. p53 +/+ HCT116 cells were treated with 1 μM doxorubicin (DOX) ( a ), 0.25 μM camptothecin (CPT) ( b ) and 30 J m −2 ultraviolet (UV) ( c ). Immediately after the treatment, the cells were incubated with 5 mM caffeine for increasing periods. The cells were washed with iced PBS, and lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM NEM, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 0.2% (v/v) Triton X-100 and 1 X protease inhibitor cocktail. The samples were centrifuged for 30 min at 16,000 g and the supernatant fractions (cell lysates) were subjected to immunoblot with anti-ISG15, anti-UBE1L anti-UBCH8, anti-EFP, anti-phospho-p53 (p-p53), anti-p53, anti-phospho-Chk1 (p-Chk1) and anti-β-actin antibodies (from the top to the bottom, respectively).

Techniques Used: Expressing, Cycling Probe Technology, Incubation, Protease Inhibitor

20) Product Images from "Comparison of Blood Collected in Acid-Citrate-Dextrose and EDTA for Use in Human Immunodeficiency Virus Peripheral Blood Mononuclear Cell Cultures"

Article Title: Comparison of Blood Collected in Acid-Citrate-Dextrose and EDTA for Use in Human Immunodeficiency Virus Peripheral Blood Mononuclear Cell Cultures

Journal: Journal of Clinical Microbiology

doi:

Sensitivity of HIV PBMC cultures according to log 10 HIV RNA plasma viral loads with ACD or EDTA as the anticoagulant.
Figure Legend Snippet: Sensitivity of HIV PBMC cultures according to log 10 HIV RNA plasma viral loads with ACD or EDTA as the anticoagulant.

Techniques Used:

21) Product Images from "Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins"

Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins

Journal: Cell

doi: 10.1016/j.cell.2013.01.037

IFNγ Treatment Does Not Cause a Significant Accumulation of Polyubiquitin Conjugates during the Induction of Immunoproteasomes, Related to Figure 1 (A and B) Murine embryonic (A) and B8 (B) fibroblasts were stimulated with 100U/ml IFNγ for the indicated time periods. As a control for ubiquitin accumulation, cells were treated with the proteasome inhibitor lactacystin (10μM, 4hr) or vehicle (DMSO). The cells were then lysed by sonication in 25mM HEPES pH 7.4, 1mM ATP, 1mM DTT, 5mM MgCl 2 and 10% glycerol. Lysates were subsequently centrifuged for one hour at 100,000 x g. Protein concentrations of the lysates were determined using the DC Protein Assay (Biorad) and equal amounts of protein were separated by SDS-PAGE and analyzed by western blotting. α-tubulin served as a loading control. One representative experiment out of three independent experiments with similar outcomes is shown. The induction of LMP7 by IFNγ stimulation was confirmed by immunoblot analysis; GAPDH served as a loading control. The ubiquitin levels were determined by densitometric analyses (ImageJ) of five different western blots; shown are the mean values ± SD obtained after normalization to the loading control and relative to the value for unstimulated cells which was set to unity. (C and D) HeLa cells were treated with 100U/ml IFNγ as described (C) or with 10 μM lactacystin for 4 hr (D). The cells were lysed with nonionic detergent according to the methods described by Seifert et al. (20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM and Complete Protease Inhibitor Cocktail (Roche)) and immunblotted for ubiquitin (C, left). The ubiquitin levels, (relative to the β-actin control) were determined by densitometric analyses (ImageJ) as described (C, right). The mean values ± SEM for three replicates are shown (C).
Figure Legend Snippet: IFNγ Treatment Does Not Cause a Significant Accumulation of Polyubiquitin Conjugates during the Induction of Immunoproteasomes, Related to Figure 1 (A and B) Murine embryonic (A) and B8 (B) fibroblasts were stimulated with 100U/ml IFNγ for the indicated time periods. As a control for ubiquitin accumulation, cells were treated with the proteasome inhibitor lactacystin (10μM, 4hr) or vehicle (DMSO). The cells were then lysed by sonication in 25mM HEPES pH 7.4, 1mM ATP, 1mM DTT, 5mM MgCl 2 and 10% glycerol. Lysates were subsequently centrifuged for one hour at 100,000 x g. Protein concentrations of the lysates were determined using the DC Protein Assay (Biorad) and equal amounts of protein were separated by SDS-PAGE and analyzed by western blotting. α-tubulin served as a loading control. One representative experiment out of three independent experiments with similar outcomes is shown. The induction of LMP7 by IFNγ stimulation was confirmed by immunoblot analysis; GAPDH served as a loading control. The ubiquitin levels were determined by densitometric analyses (ImageJ) of five different western blots; shown are the mean values ± SD obtained after normalization to the loading control and relative to the value for unstimulated cells which was set to unity. (C and D) HeLa cells were treated with 100U/ml IFNγ as described (C) or with 10 μM lactacystin for 4 hr (D). The cells were lysed with nonionic detergent according to the methods described by Seifert et al. (20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM and Complete Protease Inhibitor Cocktail (Roche)) and immunblotted for ubiquitin (C, left). The ubiquitin levels, (relative to the β-actin control) were determined by densitometric analyses (ImageJ) as described (C, right). The mean values ± SEM for three replicates are shown (C).

Techniques Used: Sonication, DC Protein Assay, SDS Page, Western Blot, Protease Inhibitor

22) Product Images from "Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients"

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

Journal: Journal of Virology

doi: 10.1128/JVI.00798-18

CsCl density gradient separation and analysis of viral particles from HepAD38 cell culture supernatant. (A) Native agarose gel analysis of viral particles. Culture supernatant of HepAD38 cells was concentrated (via ultrafiltration) and fractionated by CsCl density gradient centrifugation (3 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 1.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral particles in each fraction were resolved by native agarose gel electrophoresis, followed by detection of viral antigens with anti-HBsAg and anti-HBcAg antibodies and viral DNA by hybridization with minus-strand-specific riboprobe. (B to F) Southern and Northern blot detection of viral nucleic acids. Viral DNAs were separated by electrophoresis through Tris-acetate-EDTA (TAE) or alkaline (ALK) agarose gel for Southern blotting with minus- or plus-strand-specific riboprobes. Viral RNA was obtained by treatment with total nucleic acids with DNase I and separated by formaldehyde-MOPS agarose gel, followed by Northern blotting. (G) Quantification of viral DNA and RNA in naked capsids or virions. Fractions containing naked capsids (fractions 3 to 7) or virions (fractions 10 to 21) were pooled, and viral DNA and RNA were quantified by PCR. (H) DNA and RNA ratios in naked capsids and virions calculated based on quantitative results. Asterisks indicate unknown high-density viral particles detected by anti-HBcAg or anti-HBsAg antibodies but devoid of any HBV-specific nucleic acids. M, markers ( E. coli -derived HBV capsids or DNA and RNA ladders as described in the legend to Fig. 1 ).
Figure Legend Snippet: CsCl density gradient separation and analysis of viral particles from HepAD38 cell culture supernatant. (A) Native agarose gel analysis of viral particles. Culture supernatant of HepAD38 cells was concentrated (via ultrafiltration) and fractionated by CsCl density gradient centrifugation (3 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 1.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral particles in each fraction were resolved by native agarose gel electrophoresis, followed by detection of viral antigens with anti-HBsAg and anti-HBcAg antibodies and viral DNA by hybridization with minus-strand-specific riboprobe. (B to F) Southern and Northern blot detection of viral nucleic acids. Viral DNAs were separated by electrophoresis through Tris-acetate-EDTA (TAE) or alkaline (ALK) agarose gel for Southern blotting with minus- or plus-strand-specific riboprobes. Viral RNA was obtained by treatment with total nucleic acids with DNase I and separated by formaldehyde-MOPS agarose gel, followed by Northern blotting. (G) Quantification of viral DNA and RNA in naked capsids or virions. Fractions containing naked capsids (fractions 3 to 7) or virions (fractions 10 to 21) were pooled, and viral DNA and RNA were quantified by PCR. (H) DNA and RNA ratios in naked capsids and virions calculated based on quantitative results. Asterisks indicate unknown high-density viral particles detected by anti-HBcAg or anti-HBsAg antibodies but devoid of any HBV-specific nucleic acids. M, markers ( E. coli -derived HBV capsids or DNA and RNA ladders as described in the legend to Fig. 1 ).

Techniques Used: Cell Culture, Agarose Gel Electrophoresis, Gradient Centrifugation, DNA Hybridization, Northern Blot, Electrophoresis, Southern Blot, Polymerase Chain Reaction, Derivative Assay

23) Product Images from "L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation"

Article Title: L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation

Journal: Journal of Virology

doi: 10.1128/JVI.75.9.4332-4342.2001

In vitro binding of L2 to L1 pentamers, but not to intact VLPs. BPV1 L1 VLPs disassemble into component capsomers after dialysis in disassembly buffer. BPV1 L1 VLPs purified from recombinant baculovirus-infected insect cells was left intact or was disassembled via dialysis against disassembly buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 3 mM DTT) at 4°C overnight. Samples of intact BPV1 L1 VLPs (A) and disassembled BPV1 L1 VLPs (B) were examined via electron microscopy at 36,000× magnification (bar, 100 nm). Intact VLPs and pentamer are indicated with large and small arrows, respectively. (C) In vitro-transcribed L2 mRNA was translated in rabbit reticulocyte lysate supplemented with [ 35 S]cysteine. The in vitro translation product was incubated with purified L1 VLPs or capsomer and then immunoprecipitated and subjected to SDS-PAGE and autoradiography. Lane 1, in vitro-translated L2 immunoprecipitated with anti-L1 serum; lane 2, in vitro-translated L2 immunoprecipitated with anti-L2 serum; lane 3, in vitro-translated L2 incubated with intact L1 VLPs and then immunoprecipitated with anti-L1 serum; lane 4, in vitro-translated L2 incubated with disassembled L1 VLPs and then immunoprecipitated with anti-L1 serum; lane 5, in vitro-translated L2 incubated with disassembled L1 VLPs and nonradiolabeled L2 with a C-terminal six-histidine tag and then immunoprecipitated with anti-L1 serum; lane 6, in vitro-translated L2 incubated with disassembled L1 VLPs and nonradiolabeled overlapping L2 peptides (A through F) with C-terminal six-histidine tags; lane 7, in vitro-translated luciferase incubated with disassembled L1 VLPs and then immunoprecipitated with anti-L1 serum. Molecular masses (in kilodaltons) are shown.
Figure Legend Snippet: In vitro binding of L2 to L1 pentamers, but not to intact VLPs. BPV1 L1 VLPs disassemble into component capsomers after dialysis in disassembly buffer. BPV1 L1 VLPs purified from recombinant baculovirus-infected insect cells was left intact or was disassembled via dialysis against disassembly buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 3 mM DTT) at 4°C overnight. Samples of intact BPV1 L1 VLPs (A) and disassembled BPV1 L1 VLPs (B) were examined via electron microscopy at 36,000× magnification (bar, 100 nm). Intact VLPs and pentamer are indicated with large and small arrows, respectively. (C) In vitro-transcribed L2 mRNA was translated in rabbit reticulocyte lysate supplemented with [ 35 S]cysteine. The in vitro translation product was incubated with purified L1 VLPs or capsomer and then immunoprecipitated and subjected to SDS-PAGE and autoradiography. Lane 1, in vitro-translated L2 immunoprecipitated with anti-L1 serum; lane 2, in vitro-translated L2 immunoprecipitated with anti-L2 serum; lane 3, in vitro-translated L2 incubated with intact L1 VLPs and then immunoprecipitated with anti-L1 serum; lane 4, in vitro-translated L2 incubated with disassembled L1 VLPs and then immunoprecipitated with anti-L1 serum; lane 5, in vitro-translated L2 incubated with disassembled L1 VLPs and nonradiolabeled L2 with a C-terminal six-histidine tag and then immunoprecipitated with anti-L1 serum; lane 6, in vitro-translated L2 incubated with disassembled L1 VLPs and nonradiolabeled overlapping L2 peptides (A through F) with C-terminal six-histidine tags; lane 7, in vitro-translated luciferase incubated with disassembled L1 VLPs and then immunoprecipitated with anti-L1 serum. Molecular masses (in kilodaltons) are shown.

Techniques Used: In Vitro, Binding Assay, Purification, Recombinant, Infection, Electron Microscopy, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Luciferase

24) Product Images from "Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A"

Article Title: Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp157

Human mtTFA is an asymmetric monomer in the absence of DNA. ( A ) Deletion constructs of h-mtTFA. The upper panel shows a schematic diagram and the lower panel shows SDS–PAGE of h-mtTFA deletion constructs on a 15% polyacrylamide gel. ( B ) Size-exclusion chromatography (Superdex 200; GE Healthcare) elution profiles of h-mtTFA and h-mtTFA deletion constructs, mtTFA 1–179 , mtTFA 1–109 , mtTFA 1–79 , mtTFA 80–204 , mtTFA 110–204 and mtTFA 110–179 (top panel), and the single HMG domains, HMGB1 box A and HMGD (lower panel) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The position of each size standard is indicated by arrows above the top panel for amylase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and RNase A (14 kDa). The void volume was at 45 ml and is not shown. ( C ) Sedimentation velocity profiles for the raw data acquired at different time points and the residuals after fittings had been performed using SEDFIT in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. ( D ) Calculated sedimentation coefficient distributions for the full-length h-mtTFA.
Figure Legend Snippet: Human mtTFA is an asymmetric monomer in the absence of DNA. ( A ) Deletion constructs of h-mtTFA. The upper panel shows a schematic diagram and the lower panel shows SDS–PAGE of h-mtTFA deletion constructs on a 15% polyacrylamide gel. ( B ) Size-exclusion chromatography (Superdex 200; GE Healthcare) elution profiles of h-mtTFA and h-mtTFA deletion constructs, mtTFA 1–179 , mtTFA 1–109 , mtTFA 1–79 , mtTFA 80–204 , mtTFA 110–204 and mtTFA 110–179 (top panel), and the single HMG domains, HMGB1 box A and HMGD (lower panel) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The position of each size standard is indicated by arrows above the top panel for amylase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and RNase A (14 kDa). The void volume was at 45 ml and is not shown. ( C ) Sedimentation velocity profiles for the raw data acquired at different time points and the residuals after fittings had been performed using SEDFIT in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. ( D ) Calculated sedimentation coefficient distributions for the full-length h-mtTFA.

Techniques Used: Construct, SDS Page, Size-exclusion Chromatography, Sedimentation

Human mtTFA box B interacts with other regions of mtTFA. An N-terminal GST fusion with box B (mtTFA 110–179 ) was tested for its ability to interact with the various deletion constructs of h-mtTFA (mtTFA 1-109 , mtTFA 1–79 , mtTFA 1–80–204 , mtTFA 110–204 , mtTFA 110–179 , mtTFA 80–179 , mtTFA 1–95 and mtTFA 96–179 ) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The reactions were electrophoresed on a 15% SDS–PAGE gel and transferred to nitrocellulose membrane (Millipore) and probed with anti-His and anti-GST antibodies, respectively.
Figure Legend Snippet: Human mtTFA box B interacts with other regions of mtTFA. An N-terminal GST fusion with box B (mtTFA 110–179 ) was tested for its ability to interact with the various deletion constructs of h-mtTFA (mtTFA 1-109 , mtTFA 1–79 , mtTFA 1–80–204 , mtTFA 110–204 , mtTFA 110–179 , mtTFA 80–179 , mtTFA 1–95 and mtTFA 96–179 ) in 50 mM HEPES–Na pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. The reactions were electrophoresed on a 15% SDS–PAGE gel and transferred to nitrocellulose membrane (Millipore) and probed with anti-His and anti-GST antibodies, respectively.

Techniques Used: Construct, SDS Page

25) Product Images from "Platelets and Erythrocyte-Bound Platelets Bind Infectious HIV-1 in Plasma of Chronically Infected Patients"

Article Title: Platelets and Erythrocyte-Bound Platelets Bind Infectious HIV-1 in Plasma of Chronically Infected Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081002

HIV-1 cell-cell infection of PBMC is eliminated by EDTA. PLT-RBC enriched RBC native were prepared by pre-incubation of normal RBC native (10 9 cells) with the indicated numbers of PLT, washed 3 times, and pre-incubated with HIV-1 Bal , washed 3 times, and then examined for HIV-1 infection of PBMC in the presence or absence of 5 mM EDTA. Cell-cell infection was eliminated by EDTA (p
Figure Legend Snippet: HIV-1 cell-cell infection of PBMC is eliminated by EDTA. PLT-RBC enriched RBC native were prepared by pre-incubation of normal RBC native (10 9 cells) with the indicated numbers of PLT, washed 3 times, and pre-incubated with HIV-1 Bal , washed 3 times, and then examined for HIV-1 infection of PBMC in the presence or absence of 5 mM EDTA. Cell-cell infection was eliminated by EDTA (p

Techniques Used: Infection, Incubation

PLT-RBC is a subpopulation of cells in whole blood and in preparations of RBC native from HIV-1-infected patients. A–B. Flow cytometry was performed with EDTA-anticoagulated whole blood obtained from an HIV-positive individual. A. Representative dot plot shows scatter properties of the RBC, PLT-RBC, and PLT. CD41a+/CD235a+ cells (red) represent PLT-RBC; CD41a+/CD235a- cells (green) represent free PLT; CD41a-/CD235a+ cells (blue) represent RBC; and CD41a-/CD235a- particles (gray) represent small vesicles or debris. B. Quantification of cells shown in frame A that express CD41a and/or CD235a. C–D. Flow cytometry was performed with an EDTA-free RBC native preparation (see fraction D in Figure 1 ) obtained from an HIV-positive individual. RBC native were analyzed as in frames A–B.
Figure Legend Snippet: PLT-RBC is a subpopulation of cells in whole blood and in preparations of RBC native from HIV-1-infected patients. A–B. Flow cytometry was performed with EDTA-anticoagulated whole blood obtained from an HIV-positive individual. A. Representative dot plot shows scatter properties of the RBC, PLT-RBC, and PLT. CD41a+/CD235a+ cells (red) represent PLT-RBC; CD41a+/CD235a- cells (green) represent free PLT; CD41a-/CD235a+ cells (blue) represent RBC; and CD41a-/CD235a- particles (gray) represent small vesicles or debris. B. Quantification of cells shown in frame A that express CD41a and/or CD235a. C–D. Flow cytometry was performed with an EDTA-free RBC native preparation (see fraction D in Figure 1 ) obtained from an HIV-positive individual. RBC native were analyzed as in frames A–B.

Techniques Used: Infection, Flow Cytometry, Cytometry

Fractionation of blood from HIV-1-infected patients for obtaining plasma and RBC native in the presence or absence of EDTA. Whole blood collected from a patient in the presence (A,B) or absence (C,D) of EDTA, and separated into four plasma and erythrocyte fractions, A, B, C, and D, as shown. Fraction D was treated with 5 mM EDTA, resulting in Fraction E. All of the samples were used immediately for co-culture with PBMC to detect capability for causing HIV-1 infection of the PBMC. Aliquots were stored at −20°C for viral RNA measurements. See Materials and Methods for further details.
Figure Legend Snippet: Fractionation of blood from HIV-1-infected patients for obtaining plasma and RBC native in the presence or absence of EDTA. Whole blood collected from a patient in the presence (A,B) or absence (C,D) of EDTA, and separated into four plasma and erythrocyte fractions, A, B, C, and D, as shown. Fraction D was treated with 5 mM EDTA, resulting in Fraction E. All of the samples were used immediately for co-culture with PBMC to detect capability for causing HIV-1 infection of the PBMC. Aliquots were stored at −20°C for viral RNA measurements. See Materials and Methods for further details.

Techniques Used: Fractionation, Infection, Co-Culture Assay

Cell-cell infection of PBMC by RBC native from HIV-positive patients. A . EDTA-free RBC native from 11 chronically-infected patients were obtained as in Fig. 1D and co-incubated with PBMC to examine infection of the PBMC. 25 µl of EDTA-free RBC native were diluted in 75 µl of IL-2 medium and added to 50 µl of 1.5×10 5 PHA-stimulated PBMC/well in IL-2 medium. B . 100 µl of EDTA-free plasmas from the patients, obtained as in Fig. 1C and supplemented with 20 U/ml of recombinant IL-2, did not infect co-incubated PBMC. C . The RBC native shown in frame A were treated with 5 mM EDTA followed by washing 3 times in IL-2 medium before infecting PBMCs (see Fig. 1E ) showed no HIV-1 infection of co-incubated PBMC. The infection exhibited by the group of EDTA-free RBC native ( A ) was significantly higher using a paired t-test, than the infection exhibited both by the group of EDTA-free plasma ( B ) and by the group of EDTA-stripped RBC native ( C ) at 8 days post-infection (p = 0.0404) and 10 days post-infection (p = 0.038).
Figure Legend Snippet: Cell-cell infection of PBMC by RBC native from HIV-positive patients. A . EDTA-free RBC native from 11 chronically-infected patients were obtained as in Fig. 1D and co-incubated with PBMC to examine infection of the PBMC. 25 µl of EDTA-free RBC native were diluted in 75 µl of IL-2 medium and added to 50 µl of 1.5×10 5 PHA-stimulated PBMC/well in IL-2 medium. B . 100 µl of EDTA-free plasmas from the patients, obtained as in Fig. 1C and supplemented with 20 U/ml of recombinant IL-2, did not infect co-incubated PBMC. C . The RBC native shown in frame A were treated with 5 mM EDTA followed by washing 3 times in IL-2 medium before infecting PBMCs (see Fig. 1E ) showed no HIV-1 infection of co-incubated PBMC. The infection exhibited by the group of EDTA-free RBC native ( A ) was significantly higher using a paired t-test, than the infection exhibited both by the group of EDTA-free plasma ( B ) and by the group of EDTA-stripped RBC native ( C ) at 8 days post-infection (p = 0.0404) and 10 days post-infection (p = 0.038).

Techniques Used: Infection, Incubation, Recombinant

26) Product Images from "Hepatitis B Virus e Antigen Activates the Suppressor of Cytokine Signaling 2 to Repress Interferon Action"

Article Title: Hepatitis B Virus e Antigen Activates the Suppressor of Cytokine Signaling 2 to Repress Interferon Action

Journal: Scientific Reports

doi: 10.1038/s41598-017-01773-6

The impacts of HBeAg on ISG expression and antiviral activity induced by IFNs. ( A and B ) HepG2 cells were transfected with pCMV-Tag2B, pCMV-HBeAg, or pCMV-HBeAg-1896mut for 24 h and treated with rhIFN-α ( A ) or rhIFN-λ1 ( B ) for another 24 h. Total RNA was extracted from the treated cells, and mRNA levels of IFN-stimulated genes, OAS (upper panels) and PKR (lower panels), in the RNA extracts were detected by real-time PCR. ( C and D ) HepG2 cells were incubated with PBS, rHBeAg at 50 ng/ml, or heat-inactivated rHBeAg for 12 h and treated with rhIFN-α ( C ) or rhIFN-λ1 ( D ) for another 24 h. Total RNA was extracted from the treated cells, and mRNA levels of OAS (upper panels) and PKR (lower panels) in the RNA extracts were detected by real-time PCR. ( E and F ) HepG2 cells were co-transfected with pHBV1.3 and pCMV-Tag2B or pCMV-HBeAg for 24 h and treated with rhIFN-α (upper panels) or rhIFN-λ1 (lower panels) for another 24 h. Cells were harvested and lysed, and the levels of hepatitis B s antigen (HBsAg) in culture supernatants were measured by ELISA using an HBsAg diagnostic kit ( E ). Cells were homogenized in lyses buffer and treated with DNase. The viral cores were precipitated by adding EDTA and polyethylene glycol and concentrated by centrifugation. HBV capsid-associated DNA was quantified by real-time PCR ( F ). ( G and H ) Huh7 cells were transfected with pHBV1.3 or pHBV1.3-1896mut for 24 h, and treated with rhIFN-α (upper panels) or rhIFN-λ1 (lower panels) for another 24 h. Cells were harvested and lysed, and the levels of hepatitis B s antigen (HBsAg) in culture supernatants were measured by ELISA ( G ). Cells were homogenized in lyses buffer and treated with DNase. The viral cores were precipitated by adding EDTA and polyethylene glycol and concentrated by centrifugation. HBV capsid-associated DNA was quantified by real-time PCR ( H ). Data shown were means ± SE; n = 3. *p
Figure Legend Snippet: The impacts of HBeAg on ISG expression and antiviral activity induced by IFNs. ( A and B ) HepG2 cells were transfected with pCMV-Tag2B, pCMV-HBeAg, or pCMV-HBeAg-1896mut for 24 h and treated with rhIFN-α ( A ) or rhIFN-λ1 ( B ) for another 24 h. Total RNA was extracted from the treated cells, and mRNA levels of IFN-stimulated genes, OAS (upper panels) and PKR (lower panels), in the RNA extracts were detected by real-time PCR. ( C and D ) HepG2 cells were incubated with PBS, rHBeAg at 50 ng/ml, or heat-inactivated rHBeAg for 12 h and treated with rhIFN-α ( C ) or rhIFN-λ1 ( D ) for another 24 h. Total RNA was extracted from the treated cells, and mRNA levels of OAS (upper panels) and PKR (lower panels) in the RNA extracts were detected by real-time PCR. ( E and F ) HepG2 cells were co-transfected with pHBV1.3 and pCMV-Tag2B or pCMV-HBeAg for 24 h and treated with rhIFN-α (upper panels) or rhIFN-λ1 (lower panels) for another 24 h. Cells were harvested and lysed, and the levels of hepatitis B s antigen (HBsAg) in culture supernatants were measured by ELISA using an HBsAg diagnostic kit ( E ). Cells were homogenized in lyses buffer and treated with DNase. The viral cores were precipitated by adding EDTA and polyethylene glycol and concentrated by centrifugation. HBV capsid-associated DNA was quantified by real-time PCR ( F ). ( G and H ) Huh7 cells were transfected with pHBV1.3 or pHBV1.3-1896mut for 24 h, and treated with rhIFN-α (upper panels) or rhIFN-λ1 (lower panels) for another 24 h. Cells were harvested and lysed, and the levels of hepatitis B s antigen (HBsAg) in culture supernatants were measured by ELISA ( G ). Cells were homogenized in lyses buffer and treated with DNase. The viral cores were precipitated by adding EDTA and polyethylene glycol and concentrated by centrifugation. HBV capsid-associated DNA was quantified by real-time PCR ( H ). Data shown were means ± SE; n = 3. *p

Techniques Used: Expressing, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Centrifugation

27) Product Images from "7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases"

Article Title: 7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1149

DNA repair activities towards 8oxoA containing duplex oligonucleotides in extracts from E. coli and mouse cells. 2.5 nM 5′-[ 32 P]-labelled 40 mer 8oxoA•N oligonucleotide duplexes was incubated with either 30 µg of MEFs extract or 20 µg of E. coli cell extract. The repair assay (volume 100 µl) was performed in BER + EDTA buffer containing 50 mM KCl, 20 mM HEPES–KOH (pH 7.6), 0.1 mg/ml BSA, 1 mM DTT and 1 mM EDTA, for 1 h at 37°C. The reactions were stopped by adding SDS and proteinase K. ( A ) Denaturing PAGE analysis of the cleavage products after incubation of 8oxoA•N duplexes with mouse cell extracts. ( B ) Graphic representation of the mean values of cleavage activities in mouse cell extracts. ( C ) Denaturing PAGE analysis of the cleavage products after incubation of 8oxoA•N duplexes with E. coli cell extracts. ( D ) Graphic representation of the mean values of cleavage activities in E. coli cell extracts. For details see ‘Materials and Methods’ section.
Figure Legend Snippet: DNA repair activities towards 8oxoA containing duplex oligonucleotides in extracts from E. coli and mouse cells. 2.5 nM 5′-[ 32 P]-labelled 40 mer 8oxoA•N oligonucleotide duplexes was incubated with either 30 µg of MEFs extract or 20 µg of E. coli cell extract. The repair assay (volume 100 µl) was performed in BER + EDTA buffer containing 50 mM KCl, 20 mM HEPES–KOH (pH 7.6), 0.1 mg/ml BSA, 1 mM DTT and 1 mM EDTA, for 1 h at 37°C. The reactions were stopped by adding SDS and proteinase K. ( A ) Denaturing PAGE analysis of the cleavage products after incubation of 8oxoA•N duplexes with mouse cell extracts. ( B ) Graphic representation of the mean values of cleavage activities in mouse cell extracts. ( C ) Denaturing PAGE analysis of the cleavage products after incubation of 8oxoA•N duplexes with E. coli cell extracts. ( D ) Graphic representation of the mean values of cleavage activities in E. coli cell extracts. For details see ‘Materials and Methods’ section.

Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

28) Product Images from "EWI‐2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry"

Article Title: EWI‐2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry

Journal: Cellular Microbiology

doi: 10.1111/cmi.12112

MT 81w recognizes clusters of mCD 81. A. Schematic representation of mTM 3 and mTM 4 chimeras. These chimeras were generated by replacing the third ( mTM 3) or the fourth ( mTM 4) transmembrane domain of mCD 81 by the corresponding domain of CD 82. B. Huh ‐7w7 cells were transiently transfected with the empty vector ( pcDNA 3.1), mCD 81, mTM 3 or mTM 4, lysed in PBS / BrijO 10/ EDTA and analysed by W estern blot with MT 81 mAb . C. Huh ‐7w7 cells transiently expressing pcDNA 3.1, mCD 81, mTM 3 or mTM 4 were labelled with MT 81 and MT 81w mAbs followed by PE ‐conjugated secondary antibody and analysed by flow cytometry. MT 81w/ MT 81 ratios are presented for each cell line and reported as the mean ± SD of three independent experiments. ** corresponds to a P value below 0.001, in comparison with cells expressing mCD 81, as determined by the M ann– W hitney U ‐test. D. Huh ‐7 clones expressing EWI ‐2 or EWI ‐2wint were used in cross‐link experiments. Huh ‐7 and Huh ‐7w7 cells were used as controls. Cells were treated with 2‐ BP to expose membrane proximal cysteines and cross‐linked with the DTME reagent. After lysis in PBS / BrijO 10/ C ysteine, 5 A 6 anti‐ CD 81 mAb was used to immunoprecipitate CD 81 complexes. Proteins were revealed using biotinylated 1.3.3.22 anti‐ CD 81 mAb followed by HRP ‐ S treptavidin. The molecular weights of the prestained molecular ladder are indicated in kDa. Black arrows indicate a ≈ 90 kDa band probably corresponding to CD 81 tetramers. In (B) and (D) asterisk indicates a longer exposure of mTM 4 and EWI ‐2wint 3 respectively.
Figure Legend Snippet: MT 81w recognizes clusters of mCD 81. A. Schematic representation of mTM 3 and mTM 4 chimeras. These chimeras were generated by replacing the third ( mTM 3) or the fourth ( mTM 4) transmembrane domain of mCD 81 by the corresponding domain of CD 82. B. Huh ‐7w7 cells were transiently transfected with the empty vector ( pcDNA 3.1), mCD 81, mTM 3 or mTM 4, lysed in PBS / BrijO 10/ EDTA and analysed by W estern blot with MT 81 mAb . C. Huh ‐7w7 cells transiently expressing pcDNA 3.1, mCD 81, mTM 3 or mTM 4 were labelled with MT 81 and MT 81w mAbs followed by PE ‐conjugated secondary antibody and analysed by flow cytometry. MT 81w/ MT 81 ratios are presented for each cell line and reported as the mean ± SD of three independent experiments. ** corresponds to a P value below 0.001, in comparison with cells expressing mCD 81, as determined by the M ann– W hitney U ‐test. D. Huh ‐7 clones expressing EWI ‐2 or EWI ‐2wint were used in cross‐link experiments. Huh ‐7 and Huh ‐7w7 cells were used as controls. Cells were treated with 2‐ BP to expose membrane proximal cysteines and cross‐linked with the DTME reagent. After lysis in PBS / BrijO 10/ C ysteine, 5 A 6 anti‐ CD 81 mAb was used to immunoprecipitate CD 81 complexes. Proteins were revealed using biotinylated 1.3.3.22 anti‐ CD 81 mAb followed by HRP ‐ S treptavidin. The molecular weights of the prestained molecular ladder are indicated in kDa. Black arrows indicate a ≈ 90 kDa band probably corresponding to CD 81 tetramers. In (B) and (D) asterisk indicates a longer exposure of mTM 4 and EWI ‐2wint 3 respectively.

Techniques Used: Generated, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Clone Assay, Lysis

EWI ‐2wint needs to interact with CD 81 to inhibit HCV infection. A. Schematic representation of EWI ‐2 and mutated EWI ‐2 Fur proteins. EWI ‐2wint corresponds to EWI ‐2 without its first Ig domain. In the LAL mutant, mutation of a glycine zipper motif in the transmembrane domain of EWI ‐2 Fur abolishes the interaction of EWI ‐2/ EWI ‐2wint with CD 81. In the Qcc mutant, the cytosolic tail of EWI ‐2 Fur was replaced by that of MHC II but which still contains the two juxtamembranous palmitoylatable cysteines. B. Huh ‐7 clones expressing the different FLAG and HA ‐tagged EWI constructs were cell surface biotinylated and lysed in PBS / BrijO 10/ EDTA . Lysates were used to carry out immunoprecipation assays with the TS 81 anti‐ CD 81 mAb , the M 2 anti‐ FLAG mAb or the HA 11 anti‐ HA mAb . The control lines ( CTL ) correspond to lysates incubated with no mAb . Proteins are detected using HRP ‐ S treptavidin. C. Each Huh ‐7 clone was infected with HCVcc and stained using anti‐ NS 5 mAb (2 F 6/ G 11) and a secondary antibody conjugated with PE . Non‐infected cells served as negative controls. A representative histogram of every clone is shown. Percentages corresponding to infected cells were calculated using W easel software.
Figure Legend Snippet: EWI ‐2wint needs to interact with CD 81 to inhibit HCV infection. A. Schematic representation of EWI ‐2 and mutated EWI ‐2 Fur proteins. EWI ‐2wint corresponds to EWI ‐2 without its first Ig domain. In the LAL mutant, mutation of a glycine zipper motif in the transmembrane domain of EWI ‐2 Fur abolishes the interaction of EWI ‐2/ EWI ‐2wint with CD 81. In the Qcc mutant, the cytosolic tail of EWI ‐2 Fur was replaced by that of MHC II but which still contains the two juxtamembranous palmitoylatable cysteines. B. Huh ‐7 clones expressing the different FLAG and HA ‐tagged EWI constructs were cell surface biotinylated and lysed in PBS / BrijO 10/ EDTA . Lysates were used to carry out immunoprecipation assays with the TS 81 anti‐ CD 81 mAb , the M 2 anti‐ FLAG mAb or the HA 11 anti‐ HA mAb . The control lines ( CTL ) correspond to lysates incubated with no mAb . Proteins are detected using HRP ‐ S treptavidin. C. Each Huh ‐7 clone was infected with HCVcc and stained using anti‐ NS 5 mAb (2 F 6/ G 11) and a secondary antibody conjugated with PE . Non‐infected cells served as negative controls. A representative histogram of every clone is shown. Percentages corresponding to infected cells were calculated using W easel software.

Techniques Used: Infection, Mutagenesis, Clone Assay, Expressing, Construct, Incubation, Staining, Software

29) Product Images from "Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease"

Article Title: Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease

Journal: Protein Expression and Purification

doi: 10.1016/j.pep.2007.10.008

Impacts of temperature, ionic strength, and protease inhibitors on the enzymatic activity of recombinant PPV NIa protease. (a) Effect of temperature on the enzymatic activity of recombinant PPV NIa protease. Fifty micrograms of substrate sG and 1 μg of protease were incubated at different temperatures (4, 20, 30, and 40 °C) with a final substrate concentration of 1 μg/μl in the reaction system. The corresponding aliquots were collected and analyzed at different time points (5, 20, 30, 40, 60, 120, 240, and 600 min). (b) Effect of salt on the enzymatic activity of recombinant PPV NIa protease. Fifty micrograms of substrate sG and 1 μg of protease were incubated at 30 °C in the reaction buffer containing additional NaCl at increasing concentration (0, 50, 100, 200, 300, and 500 mM). The aliquots were collected and analyzed at each time point (5, 30, 60, 120, 240, and 360 min). (c) Effect of protease inhibitors on the enzymatic activity of recombinant PPV NIa protease. Fifty micrograms of substrate sG and 1 μg of protease were included in a 50 μl reaction system at 30 °C for 2 h in the presence of the indicated protease inhibitors. PMSF, phenylmethanesulfonyl fluoride; Benz, benzamidine; Pep. A, pepstatin A; Apro, aprotinin; Leu, leupeptin; Anti, antipain-dihydrochloride; EDTA, ethylene diamine tetraacetic acid. The reaction mixture in the absence of protease inhibitors was used as the positive control. (d) Effect of detergents and denaturing reagents on the enzymatic activity of recombinant PPV NIa protease. Eighty micrograms of substrate sG and 1 μg of protease were included in a 50 μl reaction system at 30 °C for 3 h in the presence of the indicated reagent. The concentration units of urea, Triton X-100, and SDS were mol/L, % (v/v), % (w/v), respectively. The reaction mixture without additional reagents was used as the positive control and uncleaved substrate sG was used as negative control.
Figure Legend Snippet: Impacts of temperature, ionic strength, and protease inhibitors on the enzymatic activity of recombinant PPV NIa protease. (a) Effect of temperature on the enzymatic activity of recombinant PPV NIa protease. Fifty micrograms of substrate sG and 1 μg of protease were incubated at different temperatures (4, 20, 30, and 40 °C) with a final substrate concentration of 1 μg/μl in the reaction system. The corresponding aliquots were collected and analyzed at different time points (5, 20, 30, 40, 60, 120, 240, and 600 min). (b) Effect of salt on the enzymatic activity of recombinant PPV NIa protease. Fifty micrograms of substrate sG and 1 μg of protease were incubated at 30 °C in the reaction buffer containing additional NaCl at increasing concentration (0, 50, 100, 200, 300, and 500 mM). The aliquots were collected and analyzed at each time point (5, 30, 60, 120, 240, and 360 min). (c) Effect of protease inhibitors on the enzymatic activity of recombinant PPV NIa protease. Fifty micrograms of substrate sG and 1 μg of protease were included in a 50 μl reaction system at 30 °C for 2 h in the presence of the indicated protease inhibitors. PMSF, phenylmethanesulfonyl fluoride; Benz, benzamidine; Pep. A, pepstatin A; Apro, aprotinin; Leu, leupeptin; Anti, antipain-dihydrochloride; EDTA, ethylene diamine tetraacetic acid. The reaction mixture in the absence of protease inhibitors was used as the positive control. (d) Effect of detergents and denaturing reagents on the enzymatic activity of recombinant PPV NIa protease. Eighty micrograms of substrate sG and 1 μg of protease were included in a 50 μl reaction system at 30 °C for 3 h in the presence of the indicated reagent. The concentration units of urea, Triton X-100, and SDS were mol/L, % (v/v), % (w/v), respectively. The reaction mixture without additional reagents was used as the positive control and uncleaved substrate sG was used as negative control.

Techniques Used: Activity Assay, Recombinant, Incubation, Concentration Assay, Positive Control, Negative Control

30) Product Images from "Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions"

Article Title: Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00299-16

(A) Expression analysis of troC and yciC using qRT-PCR. (A) RNA samples were isolated from log-phase cells of the wild type (WT/pBBR), the zur mutant ( zur ::Gm/pBBR), and the complemented strain ( zur ::Gm/pZUR) grown in LB medium. pBBR is the plasmid vector. pZUR is the plasmid containing a functional zur gene. The expression of the target genes was normalized to that of 16S rRNA, and the fold changes in gene expression in zur ::Gm/pBBR and zur ::Gm/pZUR are relative to those in WT/pBBR (regarded as 1). The experiment was performed in biological triplicate, and the error bars indicate the standard deviations. (B) Induction of troC and yciC is specific to zinc limitation. Shown is qRT-PCR analysis of troC and yciC expression. Wild-type NTL4 cells were grown in LB medium and under metal-limiting conditions (LB plus 1 mM EDTA). Metals (CdCl 2 , CoCl 2 , CuSO 4 , FeCl 3 , MnCl 2 , NiCl 2 , and ZnCl 2 ) were supplemented at a final concentration of 0.45 mM. The expression levels are presented as percentages and are relative to those in cells grown in LB plus 1 mM EDTA (100%).
Figure Legend Snippet: (A) Expression analysis of troC and yciC using qRT-PCR. (A) RNA samples were isolated from log-phase cells of the wild type (WT/pBBR), the zur mutant ( zur ::Gm/pBBR), and the complemented strain ( zur ::Gm/pZUR) grown in LB medium. pBBR is the plasmid vector. pZUR is the plasmid containing a functional zur gene. The expression of the target genes was normalized to that of 16S rRNA, and the fold changes in gene expression in zur ::Gm/pBBR and zur ::Gm/pZUR are relative to those in WT/pBBR (regarded as 1). The experiment was performed in biological triplicate, and the error bars indicate the standard deviations. (B) Induction of troC and yciC is specific to zinc limitation. Shown is qRT-PCR analysis of troC and yciC expression. Wild-type NTL4 cells were grown in LB medium and under metal-limiting conditions (LB plus 1 mM EDTA). Metals (CdCl 2 , CoCl 2 , CuSO 4 , FeCl 3 , MnCl 2 , NiCl 2 , and ZnCl 2 ) were supplemented at a final concentration of 0.45 mM. The expression levels are presented as percentages and are relative to those in cells grown in LB plus 1 mM EDTA (100%).

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Mutagenesis, Plasmid Preparation, Functional Assay, Concentration Assay

Effects of a single mutation (A) and double mutations (B) in the troC and yciC genes on the sensitivity of A. tumefaciens to EDTA. (A) The strains are wild-type NTL4 (WT), PS132 ( znuA ::Gm), PC135 ( zinT ::Gm), TC142 ( troC ::Gm), and YC154 ( yciC ::Km). (B) The WT, TC142 ( troC ::Gm), and TCYC15 ( troC ::Gm yciC ::Km) strains carry the plasmid vector (pBBR). The mutant strains were complemented with functional troCBA or yciC from the multicopy plasmids pTROCBA and pYciC, respectively. Cells were adjusted, serially diluted, and spotted onto plates containing AB and AB plus EDTA (0.6, 0.9, and 1.2 mM) with or without 50 μM ZnCl 2 . Tenfold serial dilutions are indicated. The plates were incubated at 28°C for 48 h.
Figure Legend Snippet: Effects of a single mutation (A) and double mutations (B) in the troC and yciC genes on the sensitivity of A. tumefaciens to EDTA. (A) The strains are wild-type NTL4 (WT), PS132 ( znuA ::Gm), PC135 ( zinT ::Gm), TC142 ( troC ::Gm), and YC154 ( yciC ::Km). (B) The WT, TC142 ( troC ::Gm), and TCYC15 ( troC ::Gm yciC ::Km) strains carry the plasmid vector (pBBR). The mutant strains were complemented with functional troCBA or yciC from the multicopy plasmids pTROCBA and pYciC, respectively. Cells were adjusted, serially diluted, and spotted onto plates containing AB and AB plus EDTA (0.6, 0.9, and 1.2 mM) with or without 50 μM ZnCl 2 . Tenfold serial dilutions are indicated. The plates were incubated at 28°C for 48 h.

Techniques Used: Mutagenesis, Plasmid Preparation, Functional Assay, Incubation

(A) Genomic context of the A. tumefaciens troCBA operon and yciC . The rnhA (Atu3177) gene encoding RNase H is located downstream of the troCBA operon. The Atu3182 gene encoding a hypothetical protein with unknown function is located downstream of the yciC gene. The primer sets used to amplify the junctions between troC and troB (BT4265 and BT4266) and between troB and troA (BT4267 and BT4268) with RT-PCR analysis are indicated. (B) troC and yciC promoters. The ATG start codons for troC and yciC are shown in boldface with bent arrows. The transcriptional +1 start site of troC and yciC was determined using 5′ RACE and is indicated by asterisks. The predicted −10 and −35 sequences are underlined. The Zur-binding sites are shaded. The putative ribosome-binding sites (RBS) are underlined. (C) Conserved Zur-binding site (Zur box) for A. tumefaciens ). The sequences of Zur boxes found in the promoter regions of A. tumefaciens znuA , zinT , troC , and yciC (P znuA , P zinT , P troC , and P yciC , respectively) are shown. The conserved residues in all the Zur boxes are marked with dots. (D) Schematic representation of the promoter- lacZ fusions (not drawn to scale) from plasmids p027troC-lacZ and p027yciC-lacZ. (E) β-Galactosidase activity assay. Wild-type NTL4 (WT/pBBR), the zur mutant strain SPP12 ( zur ::Gm/pBBR), and the complemented strain ( zur ::Gm/pZUR) contain either p027troC-lacZ or p027yciC-lacZ. pBBR is a plasmid vector, while pZur contains a functional zur gene. Cells were grown in LB for 4 h and then were left untreated (UN) or treated with 1 mM EDTA for 1 h. The results are the means and SD of triplicate samples.
Figure Legend Snippet: (A) Genomic context of the A. tumefaciens troCBA operon and yciC . The rnhA (Atu3177) gene encoding RNase H is located downstream of the troCBA operon. The Atu3182 gene encoding a hypothetical protein with unknown function is located downstream of the yciC gene. The primer sets used to amplify the junctions between troC and troB (BT4265 and BT4266) and between troB and troA (BT4267 and BT4268) with RT-PCR analysis are indicated. (B) troC and yciC promoters. The ATG start codons for troC and yciC are shown in boldface with bent arrows. The transcriptional +1 start site of troC and yciC was determined using 5′ RACE and is indicated by asterisks. The predicted −10 and −35 sequences are underlined. The Zur-binding sites are shaded. The putative ribosome-binding sites (RBS) are underlined. (C) Conserved Zur-binding site (Zur box) for A. tumefaciens ). The sequences of Zur boxes found in the promoter regions of A. tumefaciens znuA , zinT , troC , and yciC (P znuA , P zinT , P troC , and P yciC , respectively) are shown. The conserved residues in all the Zur boxes are marked with dots. (D) Schematic representation of the promoter- lacZ fusions (not drawn to scale) from plasmids p027troC-lacZ and p027yciC-lacZ. (E) β-Galactosidase activity assay. Wild-type NTL4 (WT/pBBR), the zur mutant strain SPP12 ( zur ::Gm/pBBR), and the complemented strain ( zur ::Gm/pZUR) contain either p027troC-lacZ or p027yciC-lacZ. pBBR is a plasmid vector, while pZur contains a functional zur gene. Cells were grown in LB for 4 h and then were left untreated (UN) or treated with 1 mM EDTA for 1 h. The results are the means and SD of triplicate samples.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay, Mutagenesis, Plasmid Preparation, Functional Assay

31) Product Images from "Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue"

Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

Journal: Journal of neuroscience methods

doi: 10.1016/j.jneumeth.2017.02.002

Simultaneous optimization of EDTA, NaCl, and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein
Figure Legend Snippet: Simultaneous optimization of EDTA, NaCl, and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein

Techniques Used:

3.1 EDTA, NaCl, and SDS concentrations modulate protein yield during solubilization of TRIzol-precipitated protein
Figure Legend Snippet: 3.1 EDTA, NaCl, and SDS concentrations modulate protein yield during solubilization of TRIzol-precipitated protein

Techniques Used:

EDTA, NaCl, and SDS concentrations significantly alter protein yield during solubilization of TRIzol-precipitated protein
Figure Legend Snippet: EDTA, NaCl, and SDS concentrations significantly alter protein yield during solubilization of TRIzol-precipitated protein

Techniques Used:

Related Articles

Transfection:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Affinity Chromatography:

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Article Snippet: .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). ..

Isolation:

Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues
Article Snippet: .. Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany). .. Quantitative PCR (qPCR) reactions containing 2.5 ng cDNA were prepared to 5µL in 384 well PCR plates (Roche) using the LightCycler® 480 SYBR Green I Master (Roche) as per the manufacturer’s instructions and run on a LC480 II thermo cycler (Roche).

Avidin-Biotin Assay:

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). ..

Electrophoretic Mobility Shift Assay:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Real-time Polymerase Chain Reaction:

Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues
Article Snippet: .. Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany). .. Quantitative PCR (qPCR) reactions containing 2.5 ng cDNA were prepared to 5µL in 384 well PCR plates (Roche) using the LightCycler® 480 SYBR Green I Master (Roche) as per the manufacturer’s instructions and run on a LC480 II thermo cycler (Roche).

Incubation:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: A role for caveolin-1 in desmoglein binding and desmosome dynamics
Article Snippet: .. For Tx-soluble proteins, cells were incubated on ice for 20 min in a 1% Tx-containing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA), complete with protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma). ..

Article Title: The lipid phosphatase Synaptojanin 1 undergoes a significant alteration in expression and solubility and is associated with brain lesions in Alzheimer’s disease
Article Snippet: .. Preparation of brain homogenates for biochemical analysisAbout 200 mg of frozen T1 isocortex was homogenised as reported [ , ] in 10 volumes of ice-cold RIPA buffer containing 50 mM Tris pH 7.4 containing 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 5 mM EDTA, 1 mM EGTA, Roche complete protease inhibitors, 1 mM PMSF, and phosphatase inhibitor cocktail 2, (Sigma, P-5726) and incubated for 60 min at 4 °C on a rotator. .. One hundred microliter of the total homogenate was supplemented with Laemmli buffer, sonicated on ice and analysed as the total fraction.

Cell Culture:

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression
Article Snippet: .. Cultured cells (100-mm dishes) were washed twice with PBS and lysed with 1 ml of 50 mM Tris (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and complete protease inhibitor (Roche). ..

Expressing:

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Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Protease Inhibitor:

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice. .. Cell lysates were incubated on a rotator for 3 h at room temperature with 1.5 mg of Dynabeads protein G (Invitrogen) that had been pre-incubated with anti-NDRG1 (1:12.5, C-terminal) antibody diluted in PBS with 0.02% (v/v) Tween-20 for 3 h at room temperature.

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression
Article Snippet: .. Cultured cells (100-mm dishes) were washed twice with PBS and lysed with 1 ml of 50 mM Tris (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and complete protease inhibitor (Roche). ..

Article Title: Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells *Interferon-? Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells * S⃞
Article Snippet: .. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β- d -glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). ..

Sonication:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice. .. Cell lysates were incubated on a rotator for 3 h at room temperature with 1.5 mg of Dynabeads protein G (Invitrogen) that had been pre-incubated with anti-NDRG1 (1:12.5, C-terminal) antibody diluted in PBS with 0.02% (v/v) Tween-20 for 3 h at room temperature.

Lysis:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Article Title: Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells
Article Snippet: .. Cells were then lysed using RIPA lysis buffer [65 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 10% (v/v) glycerol, protease inhibitor cocktail [Complete™ EDTA-free; Roche Applied Science] and PhosStop and sonicated on ice. .. Cell lysates were incubated on a rotator for 3 h at room temperature with 1.5 mg of Dynabeads protein G (Invitrogen) that had been pre-incubated with anti-NDRG1 (1:12.5, C-terminal) antibody diluted in PBS with 0.02% (v/v) Tween-20 for 3 h at room temperature.

Binding Assay:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

Plasmid Preparation:

Article Title: Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †
Article Snippet: .. Briefly, COS-7 cell extracts were transfected with 1 μg expression plasmid pCG and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 0.35% Nonidet P-40, 10 mM dithiothreitol [DTT], and protease inhibitors [Roche]) on ice for 30 min. For gel shift assays, 0.2 μl of cell extract was incubated in 10 μl binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 15% glycerol, 5 mg of bovine serum albumin [BSA] per ml, protease inhibitors [Roche]) at room temperature for 20 min in the presence of 1 μg of sonicated salmon sperm DNA and 0.5 ng of a 32 P-labeled, double-stranded, high-affinity probe for BPV1 E2 binding site 9 (BS9; 5′-ACAAAGT ACCGTTGCCGGT CGAA-3′ [binding site is shown in bold]). ..

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  • edta  (Roche)
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    Roche edta
    Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the <t>EDTA/DTT</t> based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p
    Edta, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche bj edta
    The interaction between HCV glycoproteins and CD81 is blocked when CD81 interacts with partner protein(s). A, HCV E1E2 heterodimers immobilized onto anti-E2 coated beads interacted with CD81 in all cells lysed in Triton X-100 (TX). However, the interaction of HCV-E1E2 with CD81 was blocked in Daudi and Ramos cells lysed in Brij (Bj). Maintenance of CD81 with other tetraspanins (T) and partners (P) is disrupted by the indicated lysis conditions, as diagrammed. Anti-CD81 and irrelevant (Cont) mAbs were used in immunoprecipitations as controls. Precipitation of CD81 was revealed by western blotting with the anti-CD81 5A6 mAb. CD81-LEL corresponds to the large extracellular loop of CD81 fused to the glutathione-S transferase. B, After cell surface biotinylation, the indicated cell lines were lysed with <t>Bj/EDTA,</t> immunoprecipitated with 5A6 mAb and the proteins revealed by Western blotting with HRP-conjugated streptavidin. The values on the right are molecular sizes in kilodaltons. C, EWI-2wint production is directly connected to EWI-2 expression. Daudi cells interfered with negative siRNA or EWI-2 siRNA were <t>biotinylated,</t> lysed in Bj/EDTA, immunoprecipitated with the anti-CD81 5A6 mAb or an anti-EWI-2 mAb (8A12) and blotted sequentially with HRP-conjugated streptavidin and 5A6 mAb.
    Bj Edta, supplied by Roche, 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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    Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Journal: PLoS ONE

    Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile

    doi: 10.1371/journal.pone.0055636

    Figure Lengend Snippet: Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells. A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Article Snippet: Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

    Techniques: FACS, Selection, Staining, Isolation, Expressing, Whisker Assay

    Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Journal: PLoS ONE

    Article Title: DNA Methylation Analysis in the Intestinal Epithelium--Effect of Cell Separation on Gene Expression and Methylation Profile

    doi: 10.1371/journal.pone.0055636

    Figure Lengend Snippet: Influence of isolation methods on gene expression profiles. A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3 rd and 97 th percentile of the data. Significance testing was performed using paired Students T-test and values for p

    Article Snippet: Four pooled biopsy samples were then subjected to either chemical digestion using a combination of EDTA and DTT, or enzymatic digestion with Liberase (a blend of collagenase I/II and a neutral protease; Roche Applied Science) and hyaluronidase.

    Techniques: Isolation, Expressing, Whisker Assay

    Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

    Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Immunohistochemistry, Staining

    In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: In Situ Hybridization, Labeling, Expressing

    Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Staining

    Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Electrophoresis, Isolation

    Localization of Dsg2 and Cav-1 to membrane lipid rafts. ( A ) A431 cells were treated with MβCD (10 mM) for 1 hr and extracted in a Tris-NaCl-EDTA buffer containing Tx. Proteins were subjected to a discontinous (5-35%) sucrose-gradient separation, resolved over SDS-PAGE and immunoblotted for Cav-1, Cav-2, Flo1, Flo2, β-Cat, γ-Cat, actin, E-Cad and Dsg2. Immunoblotting revealed that Cav-1 localized predominantly to low-density fractions 4 and 5 (top left panel), corresponding to lipid rafts. Dsg2 was distributed through all fractions from 4-12. Treatment with MβCD (10 mM) for 1 hr, disrupted lipid rafts, and shifted both Cav-1 and Dsg2 to the more dense fractions. In addition to the 160 kDa Dsg2 full-length protein, we observed a 65 kDa band in the lipid raft fraction # 4 (vertical arrow). Accumulation of this fragment was enhanced and shifted to the denser fractions in the presence of MβCD (arrow head). We note that β-Cat, γ-Cat, E-Cad and actin fractioned to the lower, denser fractions, and remained relatively unchanged in the presence of MβCD. ( B ) Proteins from fractions 4 (lipid raft fraction) and 12 (high molecular weight, non-raft fraction) from above were resolved over SDS-PAGE and immunoblotted for Dsg2 using two different antibodies, 10D2 and DG3.10. Treatment with MβCD increased the level of the 65-kDa Dsg2 fragment as detected by DG3.10, but not 10D2.

    Journal: Oncogene

    Article Title: A role for caveolin-1 in desmoglein binding and desmosome dynamics

    doi: 10.1038/onc.2011.346

    Figure Lengend Snippet: Localization of Dsg2 and Cav-1 to membrane lipid rafts. ( A ) A431 cells were treated with MβCD (10 mM) for 1 hr and extracted in a Tris-NaCl-EDTA buffer containing Tx. Proteins were subjected to a discontinous (5-35%) sucrose-gradient separation, resolved over SDS-PAGE and immunoblotted for Cav-1, Cav-2, Flo1, Flo2, β-Cat, γ-Cat, actin, E-Cad and Dsg2. Immunoblotting revealed that Cav-1 localized predominantly to low-density fractions 4 and 5 (top left panel), corresponding to lipid rafts. Dsg2 was distributed through all fractions from 4-12. Treatment with MβCD (10 mM) for 1 hr, disrupted lipid rafts, and shifted both Cav-1 and Dsg2 to the more dense fractions. In addition to the 160 kDa Dsg2 full-length protein, we observed a 65 kDa band in the lipid raft fraction # 4 (vertical arrow). Accumulation of this fragment was enhanced and shifted to the denser fractions in the presence of MβCD (arrow head). We note that β-Cat, γ-Cat, E-Cad and actin fractioned to the lower, denser fractions, and remained relatively unchanged in the presence of MβCD. ( B ) Proteins from fractions 4 (lipid raft fraction) and 12 (high molecular weight, non-raft fraction) from above were resolved over SDS-PAGE and immunoblotted for Dsg2 using two different antibodies, 10D2 and DG3.10. Treatment with MβCD increased the level of the 65-kDa Dsg2 fragment as detected by DG3.10, but not 10D2.

    Article Snippet: For Tx-soluble proteins, cells were incubated on ice for 20 min in a 1% Tx-containing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA), complete with protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma).

    Techniques: SDS Page, Molecular Weight

    The interaction between HCV glycoproteins and CD81 is blocked when CD81 interacts with partner protein(s). A, HCV E1E2 heterodimers immobilized onto anti-E2 coated beads interacted with CD81 in all cells lysed in Triton X-100 (TX). However, the interaction of HCV-E1E2 with CD81 was blocked in Daudi and Ramos cells lysed in Brij (Bj). Maintenance of CD81 with other tetraspanins (T) and partners (P) is disrupted by the indicated lysis conditions, as diagrammed. Anti-CD81 and irrelevant (Cont) mAbs were used in immunoprecipitations as controls. Precipitation of CD81 was revealed by western blotting with the anti-CD81 5A6 mAb. CD81-LEL corresponds to the large extracellular loop of CD81 fused to the glutathione-S transferase. B, After cell surface biotinylation, the indicated cell lines were lysed with Bj/EDTA, immunoprecipitated with 5A6 mAb and the proteins revealed by Western blotting with HRP-conjugated streptavidin. The values on the right are molecular sizes in kilodaltons. C, EWI-2wint production is directly connected to EWI-2 expression. Daudi cells interfered with negative siRNA or EWI-2 siRNA were biotinylated, lysed in Bj/EDTA, immunoprecipitated with the anti-CD81 5A6 mAb or an anti-EWI-2 mAb (8A12) and blotted sequentially with HRP-conjugated streptavidin and 5A6 mAb.

    Journal: PLoS ONE

    Article Title: The CD81 Partner EWI-2wint Inhibits Hepatitis C Virus Entry

    doi: 10.1371/journal.pone.0001866

    Figure Lengend Snippet: The interaction between HCV glycoproteins and CD81 is blocked when CD81 interacts with partner protein(s). A, HCV E1E2 heterodimers immobilized onto anti-E2 coated beads interacted with CD81 in all cells lysed in Triton X-100 (TX). However, the interaction of HCV-E1E2 with CD81 was blocked in Daudi and Ramos cells lysed in Brij (Bj). Maintenance of CD81 with other tetraspanins (T) and partners (P) is disrupted by the indicated lysis conditions, as diagrammed. Anti-CD81 and irrelevant (Cont) mAbs were used in immunoprecipitations as controls. Precipitation of CD81 was revealed by western blotting with the anti-CD81 5A6 mAb. CD81-LEL corresponds to the large extracellular loop of CD81 fused to the glutathione-S transferase. B, After cell surface biotinylation, the indicated cell lines were lysed with Bj/EDTA, immunoprecipitated with 5A6 mAb and the proteins revealed by Western blotting with HRP-conjugated streptavidin. The values on the right are molecular sizes in kilodaltons. C, EWI-2wint production is directly connected to EWI-2 expression. Daudi cells interfered with negative siRNA or EWI-2 siRNA were biotinylated, lysed in Bj/EDTA, immunoprecipitated with the anti-CD81 5A6 mAb or an anti-EWI-2 mAb (8A12) and blotted sequentially with HRP-conjugated streptavidin and 5A6 mAb.

    Article Snippet: Detection of cell surface biotinylated proteins Cells were biotinylated as previously described with 0.2 mg/ml EZ-link-Sulpho-NHS-LC-biotin (Pierce), and lysed into Bj/EDTA or TX/EDTA containing protease inhibitors (Complete, Roche).

    Techniques: Lysis, Western Blot, Immunoprecipitation, Expressing