Structured Review

Millipore m hepes
Crystals contain intact NG-domain complex. ( a ) SDS–PAGE demonstrates that both proteins are present in the crystal. The crystallization drop was supplemented with mother liquor containing 5 mg ml −1 lysozyme as a wash control (arrow). A crystal was removed using a 100 µm nylon loop, passed through successive lysozyme-free mother-liquor washes and dissolved in water. The dissolved crystal and a sample of the crystallization mother liquor were analyzed by SDS–PAGE. The expected products are the intact Ffh NG domain (~33 kDa) and the proteolysis products of <t>FtsY</t> NG, migrating at ~22 and ~6 kDa. The crystal used in the experiment shown had a ‘sheaf’ morphology and was grown at 7 mg ml −1 under 1.7 M ammonium sulfate, 0.1 M MES pH 6.5, 1 m M SrCl 2 crystallization conditions. Similar experiments with other crystals ( e.g. having the ‘needle’ morphology) gave the same result. ( b ) Gel filtration demonstrates that the proteins are associated in a bona fide complex. The crystals were grown using protein at 7 mg ml −1 and 1.7 M ammonium sulfate, 0.1 M MES pH 6.5 crystallization conditions. The mother liquor was removed from the drop and the crystals were first washed with protein-free mother liquor and then dissolved in 2 µl of a mobile phase buffer, 50 m M <t>HEPES,</t> 50 m M NaCl, 2 m M MgCl 2 , supplemented with 1 m M ).
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Images

1) Product Images from "Crystallization of the GMPPCP complex of the NG domains of Thermus aquaticus Ffh and FtsY"

Article Title: Crystallization of the GMPPCP complex of the NG domains of Thermus aquaticus Ffh and FtsY

Journal: Acta crystallographica. Section D, Biological crystallography

doi:

Crystals contain intact NG-domain complex. ( a ) SDS–PAGE demonstrates that both proteins are present in the crystal. The crystallization drop was supplemented with mother liquor containing 5 mg ml −1 lysozyme as a wash control (arrow). A crystal was removed using a 100 µm nylon loop, passed through successive lysozyme-free mother-liquor washes and dissolved in water. The dissolved crystal and a sample of the crystallization mother liquor were analyzed by SDS–PAGE. The expected products are the intact Ffh NG domain (~33 kDa) and the proteolysis products of FtsY NG, migrating at ~22 and ~6 kDa. The crystal used in the experiment shown had a ‘sheaf’ morphology and was grown at 7 mg ml −1 under 1.7 M ammonium sulfate, 0.1 M MES pH 6.5, 1 m M SrCl 2 crystallization conditions. Similar experiments with other crystals ( e.g. having the ‘needle’ morphology) gave the same result. ( b ) Gel filtration demonstrates that the proteins are associated in a bona fide complex. The crystals were grown using protein at 7 mg ml −1 and 1.7 M ammonium sulfate, 0.1 M MES pH 6.5 crystallization conditions. The mother liquor was removed from the drop and the crystals were first washed with protein-free mother liquor and then dissolved in 2 µl of a mobile phase buffer, 50 m M HEPES, 50 m M NaCl, 2 m M MgCl 2 , supplemented with 1 m M ).
Figure Legend Snippet: Crystals contain intact NG-domain complex. ( a ) SDS–PAGE demonstrates that both proteins are present in the crystal. The crystallization drop was supplemented with mother liquor containing 5 mg ml −1 lysozyme as a wash control (arrow). A crystal was removed using a 100 µm nylon loop, passed through successive lysozyme-free mother-liquor washes and dissolved in water. The dissolved crystal and a sample of the crystallization mother liquor were analyzed by SDS–PAGE. The expected products are the intact Ffh NG domain (~33 kDa) and the proteolysis products of FtsY NG, migrating at ~22 and ~6 kDa. The crystal used in the experiment shown had a ‘sheaf’ morphology and was grown at 7 mg ml −1 under 1.7 M ammonium sulfate, 0.1 M MES pH 6.5, 1 m M SrCl 2 crystallization conditions. Similar experiments with other crystals ( e.g. having the ‘needle’ morphology) gave the same result. ( b ) Gel filtration demonstrates that the proteins are associated in a bona fide complex. The crystals were grown using protein at 7 mg ml −1 and 1.7 M ammonium sulfate, 0.1 M MES pH 6.5 crystallization conditions. The mother liquor was removed from the drop and the crystals were first washed with protein-free mother liquor and then dissolved in 2 µl of a mobile phase buffer, 50 m M HEPES, 50 m M NaCl, 2 m M MgCl 2 , supplemented with 1 m M ).

Techniques Used: SDS Page, Crystallization Assay, Filtration

2) Product Images from "IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)"

Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt410

IGF2BP1 promotes LEF1 expression by preventing LEF1 mRNA degradation. ( A and B ) HEK293 cells were transfected with control (siC) or indicated IGF2BP1-directed (siI1-1, siI1-2) siRNAs for 72 h. Protein abundance on IGF2BP1 knockdown was determined relative to controls (siC) by western blotting using VCL and TUBA4A for cross-normalization, as indicated above panels. Representative western blots of three independent analyses are shown. ACTB and LEF1 mRNA levels were analyzed by qRT-PCR. Changes in RNA abundance on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) by the ΔΔC t -method using PPIA for normalization. ( C ) RNA decay was monitored in HEK293 cells transfected with indicated siRNAs for 72 h by blocking mRNA synthesis using ActD (5 µM) for indicated times. RNA levels were determined by qRT-PCR using normalization to PPIA by the ΔΔC t -method. RPLP0 served as a control. RNA decay is depicted in semi-logarithmic scale. Statistical significance determined over three independent analyses was analyzed by Student’s t -test, as shown in panels ( P -values). ( D and E ) The association of indicated mRNAs with IGF2BP1 in HEK293 cells was analyzed by RIP using formaldehyde fixation to stabilize mRNPs prior purification. Endogenous IGF2BP1 was immunopurified (I1) by a monoclonal antibody, as indicated by western blotting in the lower panel (IB). Co-purification of indicated mRNAs was analyzed relative to the input fraction (I, 10% of cell lysates) by semi-quantitative (D) as well as qRT-PCR (E). IgG-agarose served as a control (C) for unspecific mRNA binding. The enrichment of mRNAs by immunopurification of IGF2BP1 (I1) was determined relative to the input fraction by using the ΔC t -method (E). ( F ) Upper panel: Scheme of used Firefly reporters comprising the two alternative LEF1 3′-UTRs (A: Acc.No., NM_016269 /001130713/ 001166119; B: Acc.No., NM_001130714) or the vector-encoded BGH-3′UTR (C). Lower panel: HEK293 cells were transfected with control or indicated IGF2BP1-directed siRNAs for 48 h before the co-transfection of Firefly luciferase reporters (A–C: see scheme in upper panel) and Renilla luciferase control reporters for 24 h. Changes in Firefly luciferase reporter activities on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) on normalization by Renilla activities. Statistical significance was validated by Student’s t -test: * P
Figure Legend Snippet: IGF2BP1 promotes LEF1 expression by preventing LEF1 mRNA degradation. ( A and B ) HEK293 cells were transfected with control (siC) or indicated IGF2BP1-directed (siI1-1, siI1-2) siRNAs for 72 h. Protein abundance on IGF2BP1 knockdown was determined relative to controls (siC) by western blotting using VCL and TUBA4A for cross-normalization, as indicated above panels. Representative western blots of three independent analyses are shown. ACTB and LEF1 mRNA levels were analyzed by qRT-PCR. Changes in RNA abundance on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) by the ΔΔC t -method using PPIA for normalization. ( C ) RNA decay was monitored in HEK293 cells transfected with indicated siRNAs for 72 h by blocking mRNA synthesis using ActD (5 µM) for indicated times. RNA levels were determined by qRT-PCR using normalization to PPIA by the ΔΔC t -method. RPLP0 served as a control. RNA decay is depicted in semi-logarithmic scale. Statistical significance determined over three independent analyses was analyzed by Student’s t -test, as shown in panels ( P -values). ( D and E ) The association of indicated mRNAs with IGF2BP1 in HEK293 cells was analyzed by RIP using formaldehyde fixation to stabilize mRNPs prior purification. Endogenous IGF2BP1 was immunopurified (I1) by a monoclonal antibody, as indicated by western blotting in the lower panel (IB). Co-purification of indicated mRNAs was analyzed relative to the input fraction (I, 10% of cell lysates) by semi-quantitative (D) as well as qRT-PCR (E). IgG-agarose served as a control (C) for unspecific mRNA binding. The enrichment of mRNAs by immunopurification of IGF2BP1 (I1) was determined relative to the input fraction by using the ΔC t -method (E). ( F ) Upper panel: Scheme of used Firefly reporters comprising the two alternative LEF1 3′-UTRs (A: Acc.No., NM_016269 /001130713/ 001166119; B: Acc.No., NM_001130714) or the vector-encoded BGH-3′UTR (C). Lower panel: HEK293 cells were transfected with control or indicated IGF2BP1-directed siRNAs for 48 h before the co-transfection of Firefly luciferase reporters (A–C: see scheme in upper panel) and Renilla luciferase control reporters for 24 h. Changes in Firefly luciferase reporter activities on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) on normalization by Renilla activities. Statistical significance was validated by Student’s t -test: * P

Techniques Used: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Blocking Assay, Purification, Copurification, Binding Assay, Immu-Puri, Plasmid Preparation, Cotransfection, Luciferase

3) Product Images from "On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2"

Article Title: On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0120692

The 3D model of Atx—yPDI complex. (A) Covalent complex of sulfo-SBED-Atx and yPDI was digested with α-chymotrypsin and 10% of the reaction mixture (whole sample) was chromatographed on a Chrompack C18 (100 × 3.0 mm) HPLC column (red line) equilibrated in 0.1% (v/v) TFA and eluted with a gradient of 0.1% (v/v) TFA containing 90% (v/v) acetonitrile. From the rest of the whole sample biotin-containing peptides were isolated on avidin-beads and analysed on HPLC under identical conditions as the whole sample (blue line). Arrows point at fractions containing the major biotinylated peptides. (B) The major biotinylated peptides were identified by N-terminal sequencing. Their positions in primary structures of yPDI or Atx are marked by different colours. A colour designates yPDI and Atx peptides identified in the same fraction. Such peptides are covalently cross-linked and lie close together in the complex, at the rim of the interaction area between yPDI and Atx. (C) Modelling using rigid-body docking protocol Hex 5.1, HADDOCK procedure and experimentally defined structural restraints resulted in two solutions for Atx binding to yPDI. According to the first, Atx binds to yPDI between domains b and b’ (bb’-model or binding site), while according to the second, it binds at domains a’ and c of yPDI (a’c-model or binding site). Both solutions are displayed on the same molecule of yPDI using the PyMOL program in two views rotated by 180 o . Peptides cross-linked at Atx—yPDI interaction site mapping experiment are indicated by the same colours as in (B). Arrows point towards the active side of yPDI. Experimental details are in Materials and Methods.
Figure Legend Snippet: The 3D model of Atx—yPDI complex. (A) Covalent complex of sulfo-SBED-Atx and yPDI was digested with α-chymotrypsin and 10% of the reaction mixture (whole sample) was chromatographed on a Chrompack C18 (100 × 3.0 mm) HPLC column (red line) equilibrated in 0.1% (v/v) TFA and eluted with a gradient of 0.1% (v/v) TFA containing 90% (v/v) acetonitrile. From the rest of the whole sample biotin-containing peptides were isolated on avidin-beads and analysed on HPLC under identical conditions as the whole sample (blue line). Arrows point at fractions containing the major biotinylated peptides. (B) The major biotinylated peptides were identified by N-terminal sequencing. Their positions in primary structures of yPDI or Atx are marked by different colours. A colour designates yPDI and Atx peptides identified in the same fraction. Such peptides are covalently cross-linked and lie close together in the complex, at the rim of the interaction area between yPDI and Atx. (C) Modelling using rigid-body docking protocol Hex 5.1, HADDOCK procedure and experimentally defined structural restraints resulted in two solutions for Atx binding to yPDI. According to the first, Atx binds to yPDI between domains b and b’ (bb’-model or binding site), while according to the second, it binds at domains a’ and c of yPDI (a’c-model or binding site). Both solutions are displayed on the same molecule of yPDI using the PyMOL program in two views rotated by 180 o . Peptides cross-linked at Atx—yPDI interaction site mapping experiment are indicated by the same colours as in (B). Arrows point towards the active side of yPDI. Experimental details are in Materials and Methods.

Techniques Used: High Performance Liquid Chromatography, Isolation, Avidin-Biotin Assay, Sequencing, Binding Assay

4) Product Images from "Arc is a flexible modular protein capable of reversible self-oligomerization"

Article Title: Arc is a flexible modular protein capable of reversible self-oligomerization

Journal: Biochemical Journal

doi: 10.1042/BJ20141446

Visualization of hArc by EM and AFM ( A – C ) EM analyses of hArc. Samples of hArc (15 μg/ml; 0.33 μM) were prepared in water ( A ), in 20 mM sodium Hepes, pH 7.4 ( B ) and in the same buffer with 125 mM KCl ( C ). ( D – F ) AFM images of hArc. Samples were prepared in 10 mM potassium phosphate, pH 7.4, diffused on a mica surface. ( D ) Acquired height images of 2 μm, 500 nm and 200 nm scan sizes (from left to right). The rectangles mark the close-up of the following panel. In the right close-up panel, small images with blue and green framing present the phase image of the protein shown in the height image above. ( E ) Cross-section of the blue and green line in ( D ). ( F ) 3D representation of the 200 nm scan size image shown in ( D ).
Figure Legend Snippet: Visualization of hArc by EM and AFM ( A – C ) EM analyses of hArc. Samples of hArc (15 μg/ml; 0.33 μM) were prepared in water ( A ), in 20 mM sodium Hepes, pH 7.4 ( B ) and in the same buffer with 125 mM KCl ( C ). ( D – F ) AFM images of hArc. Samples were prepared in 10 mM potassium phosphate, pH 7.4, diffused on a mica surface. ( D ) Acquired height images of 2 μm, 500 nm and 200 nm scan sizes (from left to right). The rectangles mark the close-up of the following panel. In the right close-up panel, small images with blue and green framing present the phase image of the protein shown in the height image above. ( E ) Cross-section of the blue and green line in ( D ). ( F ) 3D representation of the 200 nm scan size image shown in ( D ).

Techniques Used:

Far-UV CD and DSF analysis of hArc ( A and B ) CD spectra of hArc (4.4 μM) in 10 mM potassium phosphate at ( A ) pH 5.2 (red), 6.8 (orange), 7.4 (green) and 8.2 (blue) at 20°C and at pH 7.4 at 100°C (burgundy); CD spectrum taken at 20°C after heating hArc to 100°C is similar to that shown by the green line; and at ( B ) pH 7.4 (green), with 5 μg/ml heparin (burgundy) or 20 μM calcium (blue). Thermal scans are shown in insets. ( C and D ) DSF experiments where 2 μM hArc was incubated with the indicated concentrations of heparin (units are μg/ml) and subjected to a thermal gradient from 20°C to 90°C in 20 mM sodium Hepes, pH 7.0, without KF salt ( C ) and with 150 mM KF salt ( D ). The thermal denaturations monitored by SYPRO Orange fluorescence ( λ ex =465 nm, λ em =610 nm) are shown in the main plots, whereas T m values obtained from the maximum first derivative of the data are shown in the insets.
Figure Legend Snippet: Far-UV CD and DSF analysis of hArc ( A and B ) CD spectra of hArc (4.4 μM) in 10 mM potassium phosphate at ( A ) pH 5.2 (red), 6.8 (orange), 7.4 (green) and 8.2 (blue) at 20°C and at pH 7.4 at 100°C (burgundy); CD spectrum taken at 20°C after heating hArc to 100°C is similar to that shown by the green line; and at ( B ) pH 7.4 (green), with 5 μg/ml heparin (burgundy) or 20 μM calcium (blue). Thermal scans are shown in insets. ( C and D ) DSF experiments where 2 μM hArc was incubated with the indicated concentrations of heparin (units are μg/ml) and subjected to a thermal gradient from 20°C to 90°C in 20 mM sodium Hepes, pH 7.0, without KF salt ( C ) and with 150 mM KF salt ( D ). The thermal denaturations monitored by SYPRO Orange fluorescence ( λ ex =465 nm, λ em =610 nm) are shown in the main plots, whereas T m values obtained from the maximum first derivative of the data are shown in the insets.

Techniques Used: Incubation, Fluorescence

5) Product Images from "The Store-operated Calcium Entry Pathways in Human Carcinoma A431 Cells"

Article Title: The Store-operated Calcium Entry Pathways in Human Carcinoma A431 Cells

Journal: The Journal of General Physiology

doi: 10.1085/jgp.200308815

Activation of I CRAC and I SOC currents in A431 cells by intracellular store-depletion. Whole-cell recordings were performed at 0-mV holding potential using ramp protocol (test potentials from −100 to 70 mV; duration of the ramp, 200 ms; interramp interval is 10 s). Pipette solution contained (in mM) 10 Cs-HEPES pH 7.3, 145 NMDG aspartate, 10 Cs-EGTA (pCa > 9.0), 1.5 MgCl 2 . Extracellular solution contained (in mM) 10 Cs-HEPES pH 7.3, 140 NMDG aspartate, 10 BaCl 2 . (A) The amplitudes of peak currents recorded at each ramp at −80 mV (filled circles), 30 mV (open circles) and 50 mV (filled triangles) test potentials are plotted as a function of time after break-in. (B) Current-voltage relationships recorded at 159 s (curve a) and 275 s (curve b). Data from the same experiment are shown in A and B. Ramps corresponding to curves a and b in B are indicated by arrows in A. The data are representative of 19 experiments.
Figure Legend Snippet: Activation of I CRAC and I SOC currents in A431 cells by intracellular store-depletion. Whole-cell recordings were performed at 0-mV holding potential using ramp protocol (test potentials from −100 to 70 mV; duration of the ramp, 200 ms; interramp interval is 10 s). Pipette solution contained (in mM) 10 Cs-HEPES pH 7.3, 145 NMDG aspartate, 10 Cs-EGTA (pCa > 9.0), 1.5 MgCl 2 . Extracellular solution contained (in mM) 10 Cs-HEPES pH 7.3, 140 NMDG aspartate, 10 BaCl 2 . (A) The amplitudes of peak currents recorded at each ramp at −80 mV (filled circles), 30 mV (open circles) and 50 mV (filled triangles) test potentials are plotted as a function of time after break-in. (B) Current-voltage relationships recorded at 159 s (curve a) and 275 s (curve b). Data from the same experiment are shown in A and B. Ramps corresponding to curves a and b in B are indicated by arrows in A. The data are representative of 19 experiments.

Techniques Used: Activation Assay, Mass Spectrometry, Transferring

6) Product Images from "Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress"

Article Title: Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress

Journal: Scientific Reports

doi: 10.1038/srep16529

Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p
Figure Legend Snippet: Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p

Techniques Used: Immunoprecipitation, Western Blot

7) Product Images from "Global proteomic assessment of the classical protein-tyrosine phosphatome and "redoxome""

Article Title: Global proteomic assessment of the classical protein-tyrosine phosphatome and "redoxome"

Journal: Cell

doi: 10.1016/j.cell.2011.07.020

Monitoring multiple PTP oxidation states by q-oxPTPome
Figure Legend Snippet: Monitoring multiple PTP oxidation states by q-oxPTPome

Techniques Used:

Development of qPTPome and q-oxPTPome
Figure Legend Snippet: Development of qPTPome and q-oxPTPome

Techniques Used:

8) Product Images from "Novel betulin derivative induces anti-proliferative activity by G2/M phase cell cycle arrest and apoptosis in Huh7 cells"

Article Title: Novel betulin derivative induces anti-proliferative activity by G2/M phase cell cycle arrest and apoptosis in Huh7 cells

Journal: Oncology Letters

doi: 10.3892/ol.2017.7575

Apoptosis is induced by 3,28-di-(2-nitroxy-acetyl)-oxy-betulin via the mitochondrial pathways in Huh7 cells. (A) Decrease in mitochondrial potential induced by 3,28-di-(2-nitroxy-acetyl)-oxy-betulin. Following treatment with 3,28-di-(2-nitroxy-acetyl)-oxy-betulin, the cells were stained with JC-1 and analyzed using flow cytometry. Changes in mitochondrial potential were determined by the ratio of red fluorescence with green fluorescence. (B) The total expression of Bcl-2 and Bax in Huh7 cells treated with or without 3,28-di-(2-nitroxy-acetyl)-oxy-betulin were analyzed using western blotting. Bcl-2 and Bax were identified to be decreased and increased, respectively, following treatment. β-actin was used as a loading control. (C) The cytosolic and mitochondrial levels of the pro-apoptotic proteins, cytochrome c and Bax, in Huh7 cells with or without 3,28-di-(2-nitroxy-acetyl)-oxy-betulin, determined using western blot analysis. β-actin was used as the loading control. Following treatment, the cytosolic cytochrome c level was markedly increased and the translocation of Bax from the cytosol to the mitochondria was increased. Represented images are presented from two independent experiments. JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetramethyl benzimidazolyl-carbocyanine iodide; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; VDAC, voltage-dependent anion channel.
Figure Legend Snippet: Apoptosis is induced by 3,28-di-(2-nitroxy-acetyl)-oxy-betulin via the mitochondrial pathways in Huh7 cells. (A) Decrease in mitochondrial potential induced by 3,28-di-(2-nitroxy-acetyl)-oxy-betulin. Following treatment with 3,28-di-(2-nitroxy-acetyl)-oxy-betulin, the cells were stained with JC-1 and analyzed using flow cytometry. Changes in mitochondrial potential were determined by the ratio of red fluorescence with green fluorescence. (B) The total expression of Bcl-2 and Bax in Huh7 cells treated with or without 3,28-di-(2-nitroxy-acetyl)-oxy-betulin were analyzed using western blotting. Bcl-2 and Bax were identified to be decreased and increased, respectively, following treatment. β-actin was used as a loading control. (C) The cytosolic and mitochondrial levels of the pro-apoptotic proteins, cytochrome c and Bax, in Huh7 cells with or without 3,28-di-(2-nitroxy-acetyl)-oxy-betulin, determined using western blot analysis. β-actin was used as the loading control. Following treatment, the cytosolic cytochrome c level was markedly increased and the translocation of Bax from the cytosol to the mitochondria was increased. Represented images are presented from two independent experiments. JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetramethyl benzimidazolyl-carbocyanine iodide; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; VDAC, voltage-dependent anion channel.

Techniques Used: Staining, Flow Cytometry, Cytometry, Fluorescence, Expressing, Western Blot, Translocation Assay

9) Product Images from "The C-terminus of interferon gamma receptor beta chain (IFN?R2) has antiapoptotic activity as a Bax inhibitor"

Article Title: The C-terminus of interferon gamma receptor beta chain (IFN?R2) has antiapoptotic activity as a Bax inhibitor

Journal:

doi:

IFNγR2 interacts with Bax. (A and B) Co-immunoprecipitation of endogenous IFNγR2 and Bax. HEK293T cells were lysed using CHAPS or NP40 buffer as described in Materials and Methods. Immunoprecipitation (IP) was performed in the same buffer
Figure Legend Snippet: IFNγR2 interacts with Bax. (A and B) Co-immunoprecipitation of endogenous IFNγR2 and Bax. HEK293T cells were lysed using CHAPS or NP40 buffer as described in Materials and Methods. Immunoprecipitation (IP) was performed in the same buffer

Techniques Used: Immunoprecipitation

10) Product Images from "The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7"

Article Title: The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7

Journal: Molecular Microbiology

doi: 10.1111/mmi.12651

Deletion of adhE affects expression of the T3SS and flagella in E . coli O157:H7.A. The secreted protein profile of WT EHEC and Δ adhE bacteria following culture in MEM-HEPES media. Protein identity was confirmed by tandem MS analysis and the bands corresponding to Tir (1), FliC (2), EspD (3) and EspA (4) are indicated.B and C. (B) Immunoblot analysis of secreted proteins (EspA, Tir and FliC) and (C) cell lysates (EscJ, σ 28 and GroEL) from WT EHEC and Δ adhE strains.D. Immuofluorescence microscopy of WT and Δ adhE bacteria transformed an RFP expressing plasmid (pRFP, red) probed with αEspA and αH7 (FliC) antibodies and Alexaflour 488 conjugated secondary antibodies (green). Scale bars are 2 μM.
Figure Legend Snippet: Deletion of adhE affects expression of the T3SS and flagella in E . coli O157:H7.A. The secreted protein profile of WT EHEC and Δ adhE bacteria following culture in MEM-HEPES media. Protein identity was confirmed by tandem MS analysis and the bands corresponding to Tir (1), FliC (2), EspD (3) and EspA (4) are indicated.B and C. (B) Immunoblot analysis of secreted proteins (EspA, Tir and FliC) and (C) cell lysates (EscJ, σ 28 and GroEL) from WT EHEC and Δ adhE strains.D. Immuofluorescence microscopy of WT and Δ adhE bacteria transformed an RFP expressing plasmid (pRFP, red) probed with αEspA and αH7 (FliC) antibodies and Alexaflour 488 conjugated secondary antibodies (green). Scale bars are 2 μM.

Techniques Used: Expressing, Mass Spectrometry, Microscopy, Transformation Assay, Plasmid Preparation

11) Product Images from "The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7"

Article Title: The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7

Journal: Molecular Microbiology

doi: 10.1111/mmi.12651

Deletion of adhE affects expression of the T3SS and flagella in E . coli O157:H7.A. The secreted protein profile of WT EHEC and Δ adhE bacteria following culture in MEM-HEPES media. Protein identity was confirmed by tandem MS analysis and the bands corresponding to Tir (1), FliC (2), EspD (3) and EspA (4) are indicated.B and C. (B) Immunoblot analysis of secreted proteins (EspA, Tir and FliC) and (C) cell lysates (EscJ, σ 28 and GroEL) from WT EHEC and Δ adhE strains.D. Immuofluorescence microscopy of WT and Δ adhE bacteria transformed an RFP expressing plasmid (pRFP, red) probed with αEspA and αH7 (FliC) antibodies and Alexaflour 488 conjugated secondary antibodies (green). Scale bars are 2 μM.
Figure Legend Snippet: Deletion of adhE affects expression of the T3SS and flagella in E . coli O157:H7.A. The secreted protein profile of WT EHEC and Δ adhE bacteria following culture in MEM-HEPES media. Protein identity was confirmed by tandem MS analysis and the bands corresponding to Tir (1), FliC (2), EspD (3) and EspA (4) are indicated.B and C. (B) Immunoblot analysis of secreted proteins (EspA, Tir and FliC) and (C) cell lysates (EscJ, σ 28 and GroEL) from WT EHEC and Δ adhE strains.D. Immuofluorescence microscopy of WT and Δ adhE bacteria transformed an RFP expressing plasmid (pRFP, red) probed with αEspA and αH7 (FliC) antibodies and Alexaflour 488 conjugated secondary antibodies (green). Scale bars are 2 μM.

Techniques Used: Expressing, Mass Spectrometry, Microscopy, Transformation Assay, Plasmid Preparation

12) Product Images from "Scaffold dependent histone deacetylase (HDAC) inhibitor induced re-equilibration of the subcellular localization and post-translational modification state of class I HDACs"

Article Title: Scaffold dependent histone deacetylase (HDAC) inhibitor induced re-equilibration of the subcellular localization and post-translational modification state of class I HDACs

Journal: PLoS ONE

doi: 10.1371/journal.pone.0186620

HDAC1 re-equilibration induced by HDACi is subsequent to histone acetylation and is affected by mitogenic stimuli. A) Western blot analysis of cytosolic fractions of MCF-7 cells treated with indicated concentrations of trichostatin A for 2 hours. B) Densitometry analysis of western blots of chromatin fractions from MCF-7 cells treated with 10 μM trichostatin A for 2 and 12 hours; change in AcH3 was normalized to TBP. C) Western blot analysis of chromatin bound fractions from MCF-7 cells treated with indicated concentrations of HDACi for 12 hours. D) Laser scanning confocal microscopy of MCF-7 cells, grown with 10% serum and treated with DMSO for 12 hours. Fluorescence signal for HDAC1 is shown in green (left panels), DAPI staining is shown in blue (middle panels), and merged optical sections are shown in the right panels. Representative optical section from two independent experiments is shown. Arrows indicate mitotic cells where HDAC1 is dispersed off chromatin, as indicated by DAPI staining. * Statistically significant difference compared with DMSO control (Student’s t-test, P
Figure Legend Snippet: HDAC1 re-equilibration induced by HDACi is subsequent to histone acetylation and is affected by mitogenic stimuli. A) Western blot analysis of cytosolic fractions of MCF-7 cells treated with indicated concentrations of trichostatin A for 2 hours. B) Densitometry analysis of western blots of chromatin fractions from MCF-7 cells treated with 10 μM trichostatin A for 2 and 12 hours; change in AcH3 was normalized to TBP. C) Western blot analysis of chromatin bound fractions from MCF-7 cells treated with indicated concentrations of HDACi for 12 hours. D) Laser scanning confocal microscopy of MCF-7 cells, grown with 10% serum and treated with DMSO for 12 hours. Fluorescence signal for HDAC1 is shown in green (left panels), DAPI staining is shown in blue (middle panels), and merged optical sections are shown in the right panels. Representative optical section from two independent experiments is shown. Arrows indicate mitotic cells where HDAC1 is dispersed off chromatin, as indicated by DAPI staining. * Statistically significant difference compared with DMSO control (Student’s t-test, P

Techniques Used: Western Blot, Confocal Microscopy, Fluorescence, Staining

Potent HDACi alter the subcellular localization of HDAC1. MCF-7 cells were treated with indicated concentrations of panobinostat or trichostatin A for 12 hours. A) Western blot analysis of the abundance of HDAC1 in the cytosolic, nuclear soluble, and chromatin bound fractions. B) Densitometry analysis of the abundance of HDAC1 normalized to GAPDH (cytosolic fraction) or to TATA-binding protein (TBP, nuclear soluble and chromatin bound fractions). C) Western blot analysis of the total abundance of class I HDACs and the loading controls TBP, GAPDH, and histone H3 after treatment with indicated concentrations of panobinostat for 12 hours. * Statistically significant difference compared with DMSO control (Student’s t-test, P
Figure Legend Snippet: Potent HDACi alter the subcellular localization of HDAC1. MCF-7 cells were treated with indicated concentrations of panobinostat or trichostatin A for 12 hours. A) Western blot analysis of the abundance of HDAC1 in the cytosolic, nuclear soluble, and chromatin bound fractions. B) Densitometry analysis of the abundance of HDAC1 normalized to GAPDH (cytosolic fraction) or to TATA-binding protein (TBP, nuclear soluble and chromatin bound fractions). C) Western blot analysis of the total abundance of class I HDACs and the loading controls TBP, GAPDH, and histone H3 after treatment with indicated concentrations of panobinostat for 12 hours. * Statistically significant difference compared with DMSO control (Student’s t-test, P

Techniques Used: Western Blot, Binding Assay

Increase in cytosolic HDAC1 is irreversible up to 24 hours. MCF-7 cells were treated with 10 μM panobinostat for 12 hours, the compound was then removed and cells allowed to recover for 24 hours. A) Western blot analysis of the abundance of HDAC1 in the cytosolic, nuclear soluble, and chromatin bound fractions. B) Densitometry analysis of the abundance of HDAC1 normalized to GAPDH (cytosolic fraction) or to TATA-binding protein (TBP, nuclear soluble and chromatin bound fractions). * Statistically significant difference compared with DMSO control (Student’s t-test, P
Figure Legend Snippet: Increase in cytosolic HDAC1 is irreversible up to 24 hours. MCF-7 cells were treated with 10 μM panobinostat for 12 hours, the compound was then removed and cells allowed to recover for 24 hours. A) Western blot analysis of the abundance of HDAC1 in the cytosolic, nuclear soluble, and chromatin bound fractions. B) Densitometry analysis of the abundance of HDAC1 normalized to GAPDH (cytosolic fraction) or to TATA-binding protein (TBP, nuclear soluble and chromatin bound fractions). * Statistically significant difference compared with DMSO control (Student’s t-test, P

Techniques Used: Western Blot, Binding Assay

Potent HDACi increase the abundance of non-phosphorylated HDAC3. MCF-7 cells were treated with indicated concentrations of panobinostat, trichostatin A, or SAHA for 12 hours and then biochemically fractionated. A) The abundance of non-phosphorylated HDAC3 was characterized by Western blot analysis in the cytosolic fraction. B) Densitometry analysis of the abundance of non-phosphorylated HDAC3 normalized to GAPDH. * Statistically significant difference compared with DMSO control (Student’s t-test, P
Figure Legend Snippet: Potent HDACi increase the abundance of non-phosphorylated HDAC3. MCF-7 cells were treated with indicated concentrations of panobinostat, trichostatin A, or SAHA for 12 hours and then biochemically fractionated. A) The abundance of non-phosphorylated HDAC3 was characterized by Western blot analysis in the cytosolic fraction. B) Densitometry analysis of the abundance of non-phosphorylated HDAC3 normalized to GAPDH. * Statistically significant difference compared with DMSO control (Student’s t-test, P

Techniques Used: Western Blot

13) Product Images from "Xgrip109: A ? Tubulin-Associated Protein with an Essential Role in ? Tubulin Ring Complex (?TuRC) Assembly and Centrosome Function "

Article Title: Xgrip109: A ? Tubulin-Associated Protein with an Essential Role in ? Tubulin Ring Complex (?TuRC) Assembly and Centrosome Function

Journal: The Journal of Cell Biology

doi:

Xgrip109 interacts directly with γ tubulin. ( A ) γTuRC is dissociated in high salt. Clarified Xenopus egg extracts were first precipitated with 30% ammonium sulfate. The pellet ( 30% Asp ) was resuspended in either Hepes 1 M (refer to Materials and Methods) or Hepes 100. The resuspended proteins were fractionated on 5–40% sucrose gradients. The sucrose gradient standards used were bovine serum albumin ( 4.4S ), bovine liver catalase ( 11.3S ), and bovine thyroglobulin ( 19.4S ). The protein standards used (indicated at the bottom of each panel in A ) were dissolved in either Hepes 1 M or Hepes 100 and then fractionated under identical conditions. Gradient fractions were collected from the top and each fraction was analyzed by SDS-PAGE followed by Western blotting with XenC and 109-2 antibodies. ( B ) A fraction of the γ tubulin remains associated with Xgrip109 in high salt. Immunoprecipitations with XenC (lanes 1 and 3 ) or 109-2 antibodies (lanes 2 and 4 ) were carried out using 30% Asp resuspended in either Hepes 1 M (lanes 1 and 2 ) or Hepes 100 (lanes 3 and 4 ). The precipitated proteins were separated by SDS-PAGE and then stained by Coomassie blue. The γTuRC components ( Xgrips ) are indicated by their respective molecular masses.
Figure Legend Snippet: Xgrip109 interacts directly with γ tubulin. ( A ) γTuRC is dissociated in high salt. Clarified Xenopus egg extracts were first precipitated with 30% ammonium sulfate. The pellet ( 30% Asp ) was resuspended in either Hepes 1 M (refer to Materials and Methods) or Hepes 100. The resuspended proteins were fractionated on 5–40% sucrose gradients. The sucrose gradient standards used were bovine serum albumin ( 4.4S ), bovine liver catalase ( 11.3S ), and bovine thyroglobulin ( 19.4S ). The protein standards used (indicated at the bottom of each panel in A ) were dissolved in either Hepes 1 M or Hepes 100 and then fractionated under identical conditions. Gradient fractions were collected from the top and each fraction was analyzed by SDS-PAGE followed by Western blotting with XenC and 109-2 antibodies. ( B ) A fraction of the γ tubulin remains associated with Xgrip109 in high salt. Immunoprecipitations with XenC (lanes 1 and 3 ) or 109-2 antibodies (lanes 2 and 4 ) were carried out using 30% Asp resuspended in either Hepes 1 M (lanes 1 and 2 ) or Hepes 100 (lanes 3 and 4 ). The precipitated proteins were separated by SDS-PAGE and then stained by Coomassie blue. The γTuRC components ( Xgrips ) are indicated by their respective molecular masses.

Techniques Used: SDS Page, Western Blot, Staining

Xgrip109 is essential for the formation  of a functional centrosome. ( A ) Western analysis  of the immunodepleted extract, 30% Asp, and  30% ammonium sulfate supernatant probed with  XenC and 109-2. Lanes  1  and  2 : clarified extract  immunodepleted with either random IgG (lane   1 ) or XenC (lane  2 ). Judging by the absence of  γ tubulin and Xgrip109, XenC depleted the  γTuRC (lane  2 ). Lane  3 , 30% ammonium sulfate  supernatant that does not contain detectable  γTuRC as expected. Lane  4 , 30% Asp resuspended in Hepes 1 M. Lanes  5  and  6,  30% Asp  resuspended in Hepes 1 M and then immunodepleted using either random IgG (lane  5 ) or 109-2  (lane  6 ). Lanes  7  and  8  are the same as lanes  5   and  6 , respectively, except that the proteins were  desalted into Hepes 100, and concentrated ∼20-fold. Lanes  5–8  show that immunodepletion of  Xgrip109 in 1 M KCl removed only a fraction of  γ tubulin (compare γ-tubulin signals in lanes  5   and  6 ), whereas Xgrip109 is completely depleted.  ( B ) Quantitation of the complementation assay.   Red columns , percentages of sperm with microtubule asters nucleated from the assembled centrosomes;  blue columns , percentages of sperm  without microtubule asters;  white columns , percentages of sperm with assembled centrosomes  that nucleated only a few disorganized microtubules;  Ran. IgG , centrosome formation assays  carried out with clarified extracts that were immunodepleted with random IgG. Over 80% of  the sperm centrioles assembled into centrosomes.  XenC. IgG , centrosome formation assays carried out with clarified extracts that were  immunodepleted of γTuRC using XenC IgG.  The centrosome assembly activity was abolished.  + Asp Ran. IgG , 30% Asp that was resuspended  in Hepes 1 M and immunodepleted with random  IgG complemented the γTuRC-depleted extract  to assemble centrosomes. + Asp Xgrip109 , 30%  Asp that was resuspended in Hepes 1M and immunodepleted of Xgrip109 did not complement  the γTuRC-depleted extract to assemble centrosomes. ( C ) Representative sperm nuclei with or without a microtubule aster from the assays in  B  are shown. The microtubules were  labeled by the addition of a small amount of rhodamine-tubulin in the assays. γ Tubulin was detected using an anti–γ-tubulin monoclonal antibody GTU-88 ( Sigma Chemical Co. ) and a fluorescein-conjugated goat anti–mouse secondary antibody. γ Tubulin is not recruited to the centrosome in the absence of Xgrip109. The sperm DNA was stained by DAPI. Bar, 10 μm.
Figure Legend Snippet: Xgrip109 is essential for the formation of a functional centrosome. ( A ) Western analysis of the immunodepleted extract, 30% Asp, and 30% ammonium sulfate supernatant probed with XenC and 109-2. Lanes 1 and 2 : clarified extract immunodepleted with either random IgG (lane 1 ) or XenC (lane 2 ). Judging by the absence of γ tubulin and Xgrip109, XenC depleted the γTuRC (lane 2 ). Lane 3 , 30% ammonium sulfate supernatant that does not contain detectable γTuRC as expected. Lane 4 , 30% Asp resuspended in Hepes 1 M. Lanes 5 and 6, 30% Asp resuspended in Hepes 1 M and then immunodepleted using either random IgG (lane 5 ) or 109-2 (lane 6 ). Lanes 7 and 8 are the same as lanes 5 and 6 , respectively, except that the proteins were desalted into Hepes 100, and concentrated ∼20-fold. Lanes 5–8 show that immunodepletion of Xgrip109 in 1 M KCl removed only a fraction of γ tubulin (compare γ-tubulin signals in lanes 5 and 6 ), whereas Xgrip109 is completely depleted. ( B ) Quantitation of the complementation assay. Red columns , percentages of sperm with microtubule asters nucleated from the assembled centrosomes; blue columns , percentages of sperm without microtubule asters; white columns , percentages of sperm with assembled centrosomes that nucleated only a few disorganized microtubules; Ran. IgG , centrosome formation assays carried out with clarified extracts that were immunodepleted with random IgG. Over 80% of the sperm centrioles assembled into centrosomes. XenC. IgG , centrosome formation assays carried out with clarified extracts that were immunodepleted of γTuRC using XenC IgG. The centrosome assembly activity was abolished. + Asp Ran. IgG , 30% Asp that was resuspended in Hepes 1 M and immunodepleted with random IgG complemented the γTuRC-depleted extract to assemble centrosomes. + Asp Xgrip109 , 30% Asp that was resuspended in Hepes 1M and immunodepleted of Xgrip109 did not complement the γTuRC-depleted extract to assemble centrosomes. ( C ) Representative sperm nuclei with or without a microtubule aster from the assays in B are shown. The microtubules were labeled by the addition of a small amount of rhodamine-tubulin in the assays. γ Tubulin was detected using an anti–γ-tubulin monoclonal antibody GTU-88 ( Sigma Chemical Co. ) and a fluorescein-conjugated goat anti–mouse secondary antibody. γ Tubulin is not recruited to the centrosome in the absence of Xgrip109. The sperm DNA was stained by DAPI. Bar, 10 μm.

Techniques Used: Functional Assay, Western Blot, Quantitation Assay, Activity Assay, Labeling, Staining

The complementation assay for centrosome formation. ( A ) Western analysis of the immunodepleted extracts, 30% ammonium supernatant, the pellet was probed with 109-2 and XenC. Lanes 1–4 : clarified extract; clarified extract immunodepleted using random IgG; clarified extract immunodepleted using XenC; 30% ammonium sulfate supernatant (there is no detectable γ tubulin or Xgrip109), respectively. Lanes 5 and 6 : 30% Asp resuspended in Hepes 100 (lane 5 ) or Hepes 1 M (lane 6 ), desalted, and then concentrated ∼20-fold. ( B ) Quantitation of the complementation assays. Red columns , percentage of sperm with centrosomes that nucleated astral microtubules; blue columns, percentage of sperm without any microtubule nucleation from the tip of the sperm; green columns , percentage of sperm with centrosomes that nucleated a few disorganized microtubules; Ran. IgG, depletion with random IgG allowed over 80% of sperm centrioles to assemble into centrosomes; XenC IgG , depletion with XenC IgG completely abolished the centrosome assembly activity in the extract; + ASP.100mM , addition of 30% Asp resuspended in Hepes 100 to the γTuRC- depleted extract resulted in over 80% of sperm centrioles to assemble into centrosomes; + ASP.1M , addition of 1 M salt-treated and desalted 30% Asp to the γTuRC-depleted extract resulted in over 70% of sperm centrioles to assemble into centrosomes. The error bars were determined from four independent assays in each case. ( C ) Representative sperm nuclei with or without a microtubule aster from the assays in B are shown. The microtubules were labeled by the addition of a small amount of rhodamine- tubulin in the assays. Xgrip109 was detected using 109-c antibodies and a fluorescein conjugated goat anti–rabbit secondary antibody. The sperm DNA was stained by DAPI. Bar, 10 μm.
Figure Legend Snippet: The complementation assay for centrosome formation. ( A ) Western analysis of the immunodepleted extracts, 30% ammonium supernatant, the pellet was probed with 109-2 and XenC. Lanes 1–4 : clarified extract; clarified extract immunodepleted using random IgG; clarified extract immunodepleted using XenC; 30% ammonium sulfate supernatant (there is no detectable γ tubulin or Xgrip109), respectively. Lanes 5 and 6 : 30% Asp resuspended in Hepes 100 (lane 5 ) or Hepes 1 M (lane 6 ), desalted, and then concentrated ∼20-fold. ( B ) Quantitation of the complementation assays. Red columns , percentage of sperm with centrosomes that nucleated astral microtubules; blue columns, percentage of sperm without any microtubule nucleation from the tip of the sperm; green columns , percentage of sperm with centrosomes that nucleated a few disorganized microtubules; Ran. IgG, depletion with random IgG allowed over 80% of sperm centrioles to assemble into centrosomes; XenC IgG , depletion with XenC IgG completely abolished the centrosome assembly activity in the extract; + ASP.100mM , addition of 30% Asp resuspended in Hepes 100 to the γTuRC- depleted extract resulted in over 80% of sperm centrioles to assemble into centrosomes; + ASP.1M , addition of 1 M salt-treated and desalted 30% Asp to the γTuRC-depleted extract resulted in over 70% of sperm centrioles to assemble into centrosomes. The error bars were determined from four independent assays in each case. ( C ) Representative sperm nuclei with or without a microtubule aster from the assays in B are shown. The microtubules were labeled by the addition of a small amount of rhodamine- tubulin in the assays. Xgrip109 was detected using 109-c antibodies and a fluorescein conjugated goat anti–rabbit secondary antibody. The sperm DNA was stained by DAPI. Bar, 10 μm.

Techniques Used: Western Blot, Quantitation Assay, Activity Assay, Labeling, Staining

Xgrip109 is a component of γTuRC. ( A ) Xgrip109 antibodies. Lanes 1 and 2 show Western analysis of Xenopus egg extracts separated by 10% SDS-PAGE followed by immunoblotting with affinity-purified antibodies 109-2 and 109-c, respectively. Both antibodies specifically recognize a protein of 109 kD in the Xenopus egg extract. ( B ) Antibodies against Xgrip109 ( 109-2 ) and γ tubulin ( XenC ) both immunoprecipitated γTuRC components. Lanes 1 and 3 are immunoprecipitations carried out from the clarified egg extracts. Lanes 2 and 4 are immunoprecipitations carried out from 30% ammonium sulfate precipitates that were resuspended in low-salt buffer (refer to Materials and Methods). The immunoprecipitated proteins were separated by 10% SDS-PAGE and stained with Coomassie blue. Each of the γTuRC components (Xgrips) is indicated by its respective molecular mass. Immunoprecipitating antibodies are indicated at the bottom of the lanes. ( C ) Xgrip109 cosediments with γ tubulin. Xenopus egg extracts or extracts made from a Xenopus kidney cell line (XKL-WG cell line) were precipitated with 2.5% PEG, a treatment that precipitates more than 90% of the γ tubulin (data not shown). The pellet was resuspended in Hepes 100 and then sedimented on a 5–40% sucrose density gradient. Fractions were collected from the top of the gradients and then analyzed by Western blotting, probing with XenC and 109-2. The last fraction (fraction 17 ) in each sedimentation experiment included the pellet.
Figure Legend Snippet: Xgrip109 is a component of γTuRC. ( A ) Xgrip109 antibodies. Lanes 1 and 2 show Western analysis of Xenopus egg extracts separated by 10% SDS-PAGE followed by immunoblotting with affinity-purified antibodies 109-2 and 109-c, respectively. Both antibodies specifically recognize a protein of 109 kD in the Xenopus egg extract. ( B ) Antibodies against Xgrip109 ( 109-2 ) and γ tubulin ( XenC ) both immunoprecipitated γTuRC components. Lanes 1 and 3 are immunoprecipitations carried out from the clarified egg extracts. Lanes 2 and 4 are immunoprecipitations carried out from 30% ammonium sulfate precipitates that were resuspended in low-salt buffer (refer to Materials and Methods). The immunoprecipitated proteins were separated by 10% SDS-PAGE and stained with Coomassie blue. Each of the γTuRC components (Xgrips) is indicated by its respective molecular mass. Immunoprecipitating antibodies are indicated at the bottom of the lanes. ( C ) Xgrip109 cosediments with γ tubulin. Xenopus egg extracts or extracts made from a Xenopus kidney cell line (XKL-WG cell line) were precipitated with 2.5% PEG, a treatment that precipitates more than 90% of the γ tubulin (data not shown). The pellet was resuspended in Hepes 100 and then sedimented on a 5–40% sucrose density gradient. Fractions were collected from the top of the gradients and then analyzed by Western blotting, probing with XenC and 109-2. The last fraction (fraction 17 ) in each sedimentation experiment included the pellet.

Techniques Used: Western Blot, SDS Page, Affinity Purification, Immunoprecipitation, Staining, Sedimentation

Xgrip109 is required for the reformation of salt-disrupted γTuRC. Clarified Xenopus egg extracts were precipitated with 30% ammonium sulfate. The pellet fraction (30% Asp) was resuspended in either Hepes 100 as control or in Hepes 1 M. Sucrose gradients in A–E are all 5–40%. ( A ) 30% Asp resuspended in Hepes 1M was immunoprecipitated with random IgG and then analyzed on a sucrose gradient. ( B ) The same as in A , except that the anti-Xgrip109 antibody, 109-2, was used in the immunoprecipitation. ( C ) The same as in A , except that after immunoprecipitation, a desalting step was included before the sucrose gradient sedimentation. ( D ) The same as in B , except that after immunoprecipitation, a desalting step was included before the sucrose gradient sedimentation. ( E ) Control, 30% Asp resuspended in Hepes 100, desalted, and fractionated on a sucrose gradient. ( F ) SDS-PAGE separation followed by Coomassie blue staining of proteins that were immunoprecipitated with random IgG (lane 1 ) and 109-2 IgG (lane 2 ). ( G ) The same protein samples in F were analyzed by Western probing with XenC and 109-2. Because samples in A and B contained higher amounts of salt than that of the samples in C , D , and E (refer to Materials and Methods), the sucrose gradient fractions of A and B cannot be compared directly to that of C , D, and E . The molecular weight standards for A and B are indicated at the bottom of A ; C , D , and E are indicated at the bottom of E . Standards used are BSA ( 4.4S ), bovine liver catalase ( 11.3S ), and bovine thyroglobulin ( 19.4S ).
Figure Legend Snippet: Xgrip109 is required for the reformation of salt-disrupted γTuRC. Clarified Xenopus egg extracts were precipitated with 30% ammonium sulfate. The pellet fraction (30% Asp) was resuspended in either Hepes 100 as control or in Hepes 1 M. Sucrose gradients in A–E are all 5–40%. ( A ) 30% Asp resuspended in Hepes 1M was immunoprecipitated with random IgG and then analyzed on a sucrose gradient. ( B ) The same as in A , except that the anti-Xgrip109 antibody, 109-2, was used in the immunoprecipitation. ( C ) The same as in A , except that after immunoprecipitation, a desalting step was included before the sucrose gradient sedimentation. ( D ) The same as in B , except that after immunoprecipitation, a desalting step was included before the sucrose gradient sedimentation. ( E ) Control, 30% Asp resuspended in Hepes 100, desalted, and fractionated on a sucrose gradient. ( F ) SDS-PAGE separation followed by Coomassie blue staining of proteins that were immunoprecipitated with random IgG (lane 1 ) and 109-2 IgG (lane 2 ). ( G ) The same protein samples in F were analyzed by Western probing with XenC and 109-2. Because samples in A and B contained higher amounts of salt than that of the samples in C , D , and E (refer to Materials and Methods), the sucrose gradient fractions of A and B cannot be compared directly to that of C , D, and E . The molecular weight standards for A and B are indicated at the bottom of A ; C , D , and E are indicated at the bottom of E . Standards used are BSA ( 4.4S ), bovine liver catalase ( 11.3S ), and bovine thyroglobulin ( 19.4S ).

Techniques Used: Immunoprecipitation, Sedimentation, SDS Page, Staining, Western Blot, Molecular Weight

14) Product Images from "Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis"

Article Title: Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis

Journal: Blood

doi: 10.1182/blood-2009-10-246116

Perforin increases clathrin-mediated endocytosis . (A) Within 7 minutes of treatment, sublytic rPFN and SLO activate uptake of A488-GzmB, whereas ionomycin, even at the highest lytic concentration, does not. Mean fluorescence intensity (mean ±
Figure Legend Snippet: Perforin increases clathrin-mediated endocytosis . (A) Within 7 minutes of treatment, sublytic rPFN and SLO activate uptake of A488-GzmB, whereas ionomycin, even at the highest lytic concentration, does not. Mean fluorescence intensity (mean ±

Techniques Used: Concentration Assay, Fluorescence

15) Product Images from "Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis"

Article Title: Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis

Journal: Blood

doi: 10.1182/blood-2009-10-246116

Perforin increases clathrin-mediated endocytosis . (A) Within 7 minutes of treatment, sublytic rPFN and SLO activate uptake of A488-GzmB, whereas ionomycin, even at the highest lytic concentration, does not. Mean fluorescence intensity (mean ±
Figure Legend Snippet: Perforin increases clathrin-mediated endocytosis . (A) Within 7 minutes of treatment, sublytic rPFN and SLO activate uptake of A488-GzmB, whereas ionomycin, even at the highest lytic concentration, does not. Mean fluorescence intensity (mean ±

Techniques Used: Concentration Assay, Fluorescence

16) Product Images from "Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury"

Article Title: Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2008070798

Cytofluorimetric characterization of mesenchymal stem cell (MSC)-derived microvesicles (MVs). Representative FACS analyses of MVs (A) and MVs treated with RNase (B) showing the size (with 1-, 2- and −4-μm beads used as internal size standards) and the expression of CD44, CD29, α-4 integrin, α-5 integrin, CD73, α-6 integrin, and HLA-class I (thick lines) surface molecules. Dot lines indicate the isotypic controls. Ten different MV preparations were analyzed with similar results. In the CD44, CD29, α-4 integrin, α-5 integrin, and CD73 experiments, the Kolmogrov-Smirnov statistical analyses between relevant antibodies and the isotypic control was significant ( P
Figure Legend Snippet: Cytofluorimetric characterization of mesenchymal stem cell (MSC)-derived microvesicles (MVs). Representative FACS analyses of MVs (A) and MVs treated with RNase (B) showing the size (with 1-, 2- and −4-μm beads used as internal size standards) and the expression of CD44, CD29, α-4 integrin, α-5 integrin, CD73, α-6 integrin, and HLA-class I (thick lines) surface molecules. Dot lines indicate the isotypic controls. Ten different MV preparations were analyzed with similar results. In the CD44, CD29, α-4 integrin, α-5 integrin, and CD73 experiments, the Kolmogrov-Smirnov statistical analyses between relevant antibodies and the isotypic control was significant ( P

Techniques Used: Derivative Assay, FACS, Expressing

Incorporation of MVs in tubular epithelial cells (TECs). (A) Representative micrographs of internalization by TECs (30 min at 37 °C) of microvesicles (MVs) labeled with PKH26 preincubated or not with trypsin (0.5 mM) (Try); or with 100 μg/ml of sHA; or with 1 μg/ml blocking monoclonal antibody against CD44, CD29, and α4 integrin. Three experiments were performed with similar results. (B) Representative FACS analyses of internalization, after 30 min of incubation at 37 °C, by TECs of MVs labeled with PKH26 (black curves) preincubated or not with trypsin (Try) or with 100 μg/ml of sHA or with 1 μg/ml blocking monoclonal antibodies against CD44, CD29, and α4 integrin. Black curves indicate the internalization of untreated MVs. In the first panel, dot curve indicates the negative control (cells not incubated with MVs); red curve indicates the MVs treated with RNase and labeled with PKH26. In the other panels, dot curves indicate internalization of MVs after pretreatment with trypsin or incubation with blocking antibodies or sHA. Three experiments were performed with similar results.
Figure Legend Snippet: Incorporation of MVs in tubular epithelial cells (TECs). (A) Representative micrographs of internalization by TECs (30 min at 37 °C) of microvesicles (MVs) labeled with PKH26 preincubated or not with trypsin (0.5 mM) (Try); or with 100 μg/ml of sHA; or with 1 μg/ml blocking monoclonal antibody against CD44, CD29, and α4 integrin. Three experiments were performed with similar results. (B) Representative FACS analyses of internalization, after 30 min of incubation at 37 °C, by TECs of MVs labeled with PKH26 (black curves) preincubated or not with trypsin (Try) or with 100 μg/ml of sHA or with 1 μg/ml blocking monoclonal antibodies against CD44, CD29, and α4 integrin. Black curves indicate the internalization of untreated MVs. In the first panel, dot curve indicates the negative control (cells not incubated with MVs); red curve indicates the MVs treated with RNase and labeled with PKH26. In the other panels, dot curves indicate internalization of MVs after pretreatment with trypsin or incubation with blocking antibodies or sHA. Three experiments were performed with similar results.

Techniques Used: Labeling, Blocking Assay, FACS, Incubation, Negative Control

Renal cell proliferation in acute kidney injury (AKI) mice untreated or treated with MSCs or MVs. (A) Quantification of BrdU-positive cells/high power field (hpf). BrdU was injected intraperitoneally for 2 successive days before mice being killed. (B) Quantification of PCNA-positive cells/hpf. All quantitative data were obtained from eight different mice for experimental conditions. ANOVA with Dunnet's multicomparison test: * P
Figure Legend Snippet: Renal cell proliferation in acute kidney injury (AKI) mice untreated or treated with MSCs or MVs. (A) Quantification of BrdU-positive cells/high power field (hpf). BrdU was injected intraperitoneally for 2 successive days before mice being killed. (B) Quantification of PCNA-positive cells/hpf. All quantitative data were obtained from eight different mice for experimental conditions. ANOVA with Dunnet's multicomparison test: * P

Techniques Used: Mouse Assay, Injection

Effects of intravenous injection of microvesicles (MVs) or mesenchymal stem cells (MSCs) into acute kidney injury (AKI) mice. Mice were given intramuscular injection of 8 ml/kg of 50% glycerol on day 0, followed by intravenous injection of MVs or RNase-treated MVs or MSCs or vehicle as control on day 3. (A and B) Creatinine and blood urea nitrogen values at the beginning of the experiments and on day 3, 5, 8, and 15 after glycerol administration. ANOVA with Dunnet's multicomparison test: * P
Figure Legend Snippet: Effects of intravenous injection of microvesicles (MVs) or mesenchymal stem cells (MSCs) into acute kidney injury (AKI) mice. Mice were given intramuscular injection of 8 ml/kg of 50% glycerol on day 0, followed by intravenous injection of MVs or RNase-treated MVs or MSCs or vehicle as control on day 3. (A and B) Creatinine and blood urea nitrogen values at the beginning of the experiments and on day 3, 5, 8, and 15 after glycerol administration. ANOVA with Dunnet's multicomparison test: * P

Techniques Used: Injection, Mouse Assay

Schematic representation of the protocol of glycerol induced acute kidney injury (AKI) and treatment with mesenchymal stem cells (MSCs) or MSC-derived microvesicles (MVs). Glycerol was injected intramuscularly at time 0; the arrow at day 3 indicate the administration of 75,000 MSCs; or 15 μg of MSC-derived MVs; or MSC-derived MVs treated with RNase, trypsin, or sHA; or fibroblast-derived MVs; or vehicle alone; the subsequent arrows indicate the time of sacrifice.
Figure Legend Snippet: Schematic representation of the protocol of glycerol induced acute kidney injury (AKI) and treatment with mesenchymal stem cells (MSCs) or MSC-derived microvesicles (MVs). Glycerol was injected intramuscularly at time 0; the arrow at day 3 indicate the administration of 75,000 MSCs; or 15 μg of MSC-derived MVs; or MSC-derived MVs treated with RNase, trypsin, or sHA; or fibroblast-derived MVs; or vehicle alone; the subsequent arrows indicate the time of sacrifice.

Techniques Used: Derivative Assay, Injection

mRNA horizontal transfer and human protein expression in tubular epithelial cells (TECs) treated with mesenchymal stem cell (MSC)-derived microvesicles (MVs). (A) 1 × 10 5 TECs cultured in the absence (TEC) or in the presence (TEC+MV) of 30 μg MVs for 1 and 3 h were analyzed by RT-PCR for specific human mRNA. Bands of PCR products specific for human POLR2E and SUMO-1 of the expected size (90 bp) were detected in a 4% agarose gel electrophoresis. As positive control the extract of human bone marrow-derived MSCs (BM-MSC) was used. The asterisk indicates the control without cDNA. B: Representative micrographs showing the expression of human POLR2E and SUMO-1 proteins by TECs, cultured in the absence or in the presence of 30 μg MVs for 24 and 48 h. After 24 h, POLR2E protein was detected in the cytoplasm and SUMO-1 in the cytoplasm and nuclei of TECs. After 48 h, both proteins were translocated to the nucleus. Nuclei were counterstained with Hoechst dye. Independent experiments using four different MV preparations were performed with similar results. Original magnification: ×630.
Figure Legend Snippet: mRNA horizontal transfer and human protein expression in tubular epithelial cells (TECs) treated with mesenchymal stem cell (MSC)-derived microvesicles (MVs). (A) 1 × 10 5 TECs cultured in the absence (TEC) or in the presence (TEC+MV) of 30 μg MVs for 1 and 3 h were analyzed by RT-PCR for specific human mRNA. Bands of PCR products specific for human POLR2E and SUMO-1 of the expected size (90 bp) were detected in a 4% agarose gel electrophoresis. As positive control the extract of human bone marrow-derived MSCs (BM-MSC) was used. The asterisk indicates the control without cDNA. B: Representative micrographs showing the expression of human POLR2E and SUMO-1 proteins by TECs, cultured in the absence or in the presence of 30 μg MVs for 24 and 48 h. After 24 h, POLR2E protein was detected in the cytoplasm and SUMO-1 in the cytoplasm and nuclei of TECs. After 48 h, both proteins were translocated to the nucleus. Nuclei were counterstained with Hoechst dye. Independent experiments using four different MV preparations were performed with similar results. Original magnification: ×630.

Techniques Used: Expressing, Derivative Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control

17) Product Images from "Independent roles of the priming and the triggering of the NLRP3 inflammasome in the heart"

Article Title: Independent roles of the priming and the triggering of the NLRP3 inflammasome in the heart

Journal: Cardiovascular Research

doi: 10.1093/cvr/cvu259

Protein and mRNA analysis of the inflammasome components in the heart of wild-type mice with or without LPS low dose. ( A ) NLRP3, pro-caspase-1, and ASC protein expression were analysed by western blot and ( B ) were quantified through densitometric analysis.
Figure Legend Snippet: Protein and mRNA analysis of the inflammasome components in the heart of wild-type mice with or without LPS low dose. ( A ) NLRP3, pro-caspase-1, and ASC protein expression were analysed by western blot and ( B ) were quantified through densitometric analysis.

Techniques Used: Mouse Assay, Expressing, Western Blot

Catalytic activity and mRNA quantification of caspase-1 in spleen and heart of wt and Nlrp3-A350V/CreT mice. ( A ) Enzymatic activity of caspase-1 reported as relative fluorescence produced compared with controls ( n = 5 per group). Values are expressed
Figure Legend Snippet: Catalytic activity and mRNA quantification of caspase-1 in spleen and heart of wt and Nlrp3-A350V/CreT mice. ( A ) Enzymatic activity of caspase-1 reported as relative fluorescence produced compared with controls ( n = 5 per group). Values are expressed

Techniques Used: Activity Assay, Mouse Assay, Fluorescence, Produced

Effects of low-dose LPS in wild-type, Nlrp3-A350V , and KI Nlrp3-A350V/CreT mice. ( A ) Caspase-1 activity in the heart of wild type, Nlrp3-A350V , and Nlrp3-A350V/CreT treated with tamoxifen and then challenged with low-dose LPS or the vehicle solution for
Figure Legend Snippet: Effects of low-dose LPS in wild-type, Nlrp3-A350V , and KI Nlrp3-A350V/CreT mice. ( A ) Caspase-1 activity in the heart of wild type, Nlrp3-A350V , and Nlrp3-A350V/CreT treated with tamoxifen and then challenged with low-dose LPS or the vehicle solution for

Techniques Used: Mouse Assay, Activity Assay

18) Product Images from "Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma"

Article Title: Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma

Journal: Cancer Biology & Therapy

doi: 10.1080/15384047.2015.1017155

NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P
Figure Legend Snippet: NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P

Techniques Used: Concentration Assay, Caspase-3 Activity Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Expressing, Western Blot, Cell Culture

19) Product Images from "Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes"

Article Title: Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes

Journal: Blood

doi: 10.1182/blood-2004-11-4491

Cotransduction of human S-RCs with FeLV-C and GALV or RD114 and GALV-pseudotyped retrovirus particles. Human CD34 + peripheral blood CD34 + cells were cotransduced with FeLV-C and GALV or RD114 and GALV-pseudotyped virus preparations over a 4-day period and transplanted into fetal sheep. DNA was extracted from sheep peripheral blood cells 6 months after transplantation and analyzed for the presence of human cells and the integration of the FeLV-C or RD114 and GALV-pseudotyped viruses by PCR analysis of 0.4 μg DNA. (A) Sequences from the human β-globin gene were amplified to detect the presence of human cells. F1 and F2 indicate analysis of DNA from 2 lambs infused with human CD34 + cells exposed to FeLV-C (LacZ) and GaLV (nls-LacZ) pseudotyped particles; R1a, R1b, and R2, analysis of DNA from 3 lambs infused with human CD34 + cells exposed to RD114 and GALV-pseudotyped particles; R1a and R1b, DNA from twin lambs infused with the same population of cells; –C, DNA extracted from control sheep; and +C, DNA extracted from K562 cells. (B) The relative amounts of integrated FeLV-C, RD114, and GALV-pseudotyped vectors were measured using primers that span the nls in the MFGs-nlsLacZ vector. 1.0 nls-LacZ indicates 3T3-cell DNA containing a single copy of the MFGs-nlsLacZ vector; 1.0 LacZ, 3T3-cell DNA containing a single copy of the MFGs-LacZ vector; 0.5 LacZ, 3T3-cell DNA containing a 1:1 dilution of this 1.0 LacZ DNA with untransduced 3T3-cell DNA; and –C, DNA extracted from control sheep. For quantitation, only the β-globin band and the 289-bp MFGs-LacZ and 310-bp MFGs-nlsLacZ bands were analyzed.
Figure Legend Snippet: Cotransduction of human S-RCs with FeLV-C and GALV or RD114 and GALV-pseudotyped retrovirus particles. Human CD34 + peripheral blood CD34 + cells were cotransduced with FeLV-C and GALV or RD114 and GALV-pseudotyped virus preparations over a 4-day period and transplanted into fetal sheep. DNA was extracted from sheep peripheral blood cells 6 months after transplantation and analyzed for the presence of human cells and the integration of the FeLV-C or RD114 and GALV-pseudotyped viruses by PCR analysis of 0.4 μg DNA. (A) Sequences from the human β-globin gene were amplified to detect the presence of human cells. F1 and F2 indicate analysis of DNA from 2 lambs infused with human CD34 + cells exposed to FeLV-C (LacZ) and GaLV (nls-LacZ) pseudotyped particles; R1a, R1b, and R2, analysis of DNA from 3 lambs infused with human CD34 + cells exposed to RD114 and GALV-pseudotyped particles; R1a and R1b, DNA from twin lambs infused with the same population of cells; –C, DNA extracted from control sheep; and +C, DNA extracted from K562 cells. (B) The relative amounts of integrated FeLV-C, RD114, and GALV-pseudotyped vectors were measured using primers that span the nls in the MFGs-nlsLacZ vector. 1.0 nls-LacZ indicates 3T3-cell DNA containing a single copy of the MFGs-nlsLacZ vector; 1.0 LacZ, 3T3-cell DNA containing a single copy of the MFGs-LacZ vector; 0.5 LacZ, 3T3-cell DNA containing a 1:1 dilution of this 1.0 LacZ DNA with untransduced 3T3-cell DNA; and –C, DNA extracted from control sheep. For quantitation, only the β-globin band and the 289-bp MFGs-LacZ and 310-bp MFGs-nlsLacZ bands were analyzed.

Techniques Used: Transplantation Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Quantitation Assay

20) Product Images from "Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes"

Article Title: Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes

Journal: Blood

doi: 10.1182/blood-2004-11-4491

Cotransduction of human S-RCs with FeLV-C and GALV or RD114 and GALV-pseudotyped retrovirus particles. Human CD34 + peripheral blood CD34 + cells were cotransduced with FeLV-C and GALV or RD114 and GALV-pseudotyped virus preparations over a 4-day period and transplanted into fetal sheep. DNA was extracted from sheep peripheral blood cells 6 months after transplantation and analyzed for the presence of human cells and the integration of the FeLV-C or RD114 and GALV-pseudotyped viruses by PCR analysis of 0.4 μg DNA. (A) Sequences from the human β-globin gene were amplified to detect the presence of human cells. F1 and F2 indicate analysis of DNA from 2 lambs infused with human CD34 + cells exposed to FeLV-C (LacZ) and GaLV (nls-LacZ) pseudotyped particles; R1a, R1b, and R2, analysis of DNA from 3 lambs infused with human CD34 + cells exposed to RD114 and GALV-pseudotyped particles; R1a and R1b, DNA from twin lambs infused with the same population of cells; –C, DNA extracted from control sheep; and +C, DNA extracted from K562 cells. (B) The relative amounts of integrated FeLV-C, RD114, and GALV-pseudotyped vectors were measured using primers that span the nls in the MFGs-nlsLacZ vector. 1.0 nls-LacZ indicates 3T3-cell DNA containing a single copy of the MFGs-nlsLacZ vector; 1.0 LacZ, 3T3-cell DNA containing a single copy of the MFGs-LacZ vector; 0.5 LacZ, 3T3-cell DNA containing a 1:1 dilution of this 1.0 LacZ DNA with untransduced 3T3-cell DNA; and –C, DNA extracted from control sheep. For quantitation, only the β-globin band and the 289-bp MFGs-LacZ and 310-bp MFGs-nlsLacZ bands were analyzed.
Figure Legend Snippet: Cotransduction of human S-RCs with FeLV-C and GALV or RD114 and GALV-pseudotyped retrovirus particles. Human CD34 + peripheral blood CD34 + cells were cotransduced with FeLV-C and GALV or RD114 and GALV-pseudotyped virus preparations over a 4-day period and transplanted into fetal sheep. DNA was extracted from sheep peripheral blood cells 6 months after transplantation and analyzed for the presence of human cells and the integration of the FeLV-C or RD114 and GALV-pseudotyped viruses by PCR analysis of 0.4 μg DNA. (A) Sequences from the human β-globin gene were amplified to detect the presence of human cells. F1 and F2 indicate analysis of DNA from 2 lambs infused with human CD34 + cells exposed to FeLV-C (LacZ) and GaLV (nls-LacZ) pseudotyped particles; R1a, R1b, and R2, analysis of DNA from 3 lambs infused with human CD34 + cells exposed to RD114 and GALV-pseudotyped particles; R1a and R1b, DNA from twin lambs infused with the same population of cells; –C, DNA extracted from control sheep; and +C, DNA extracted from K562 cells. (B) The relative amounts of integrated FeLV-C, RD114, and GALV-pseudotyped vectors were measured using primers that span the nls in the MFGs-nlsLacZ vector. 1.0 nls-LacZ indicates 3T3-cell DNA containing a single copy of the MFGs-nlsLacZ vector; 1.0 LacZ, 3T3-cell DNA containing a single copy of the MFGs-LacZ vector; 0.5 LacZ, 3T3-cell DNA containing a 1:1 dilution of this 1.0 LacZ DNA with untransduced 3T3-cell DNA; and –C, DNA extracted from control sheep. For quantitation, only the β-globin band and the 289-bp MFGs-LacZ and 310-bp MFGs-nlsLacZ bands were analyzed.

Techniques Used: Transplantation Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Quantitation Assay

Cotransduction of K562 and human peripheral blood CD34 + cells with FeLV-C and GALV or RD114 and GALV-pseudotyped retrovirus particles. Slot blot analysis of 1.0 mL and 0.1 mL virus-containing medium was used to compare the titer of the different virus preparations (A). These titers were used to adjust the volumes of supernatants to ensure that equivalent numbers of each pseudotype were used. Human CD34 + peripheral blood CD34 + cells were cotransduced with FeLV-C and GALV or RD114 and GALV-pseudotyped virus preparations over a 4-day period. Parallel cultures of K562 cells were cotransduced with the same virus preparations each day (d1, d2, d3, d4). One culture was cotransduced over the 4-day period (d1-4). DNA was extracted from the K562 cultures and a portion of the CD34 + cells for analysis of the integration of the FeLV-C or RD114 and GALV-pseudotyped viruses by PCR using primers that span the nls in the MFG-nlsLacZ vector. (B) The analysis of FeLV-C/GALV- and RD114/GALV-transduced K562 cells is shown in the left and center panels. Sequences from the human β-globin were amplified in the same samples as a control. The analysis of FeLV-C/GALV (G/F)– and RD114/GALV (G/R)–transduced CD34 + cells used for transplantation into fetal sheep is shown in the right panel.
Figure Legend Snippet: Cotransduction of K562 and human peripheral blood CD34 + cells with FeLV-C and GALV or RD114 and GALV-pseudotyped retrovirus particles. Slot blot analysis of 1.0 mL and 0.1 mL virus-containing medium was used to compare the titer of the different virus preparations (A). These titers were used to adjust the volumes of supernatants to ensure that equivalent numbers of each pseudotype were used. Human CD34 + peripheral blood CD34 + cells were cotransduced with FeLV-C and GALV or RD114 and GALV-pseudotyped virus preparations over a 4-day period. Parallel cultures of K562 cells were cotransduced with the same virus preparations each day (d1, d2, d3, d4). One culture was cotransduced over the 4-day period (d1-4). DNA was extracted from the K562 cultures and a portion of the CD34 + cells for analysis of the integration of the FeLV-C or RD114 and GALV-pseudotyped viruses by PCR using primers that span the nls in the MFG-nlsLacZ vector. (B) The analysis of FeLV-C/GALV- and RD114/GALV-transduced K562 cells is shown in the left and center panels. Sequences from the human β-globin were amplified in the same samples as a control. The analysis of FeLV-C/GALV (G/F)– and RD114/GALV (G/R)–transduced CD34 + cells used for transplantation into fetal sheep is shown in the right panel.

Techniques Used: Dot Blot, Polymerase Chain Reaction, Plasmid Preparation, Amplification, Transplantation Assay

21) Product Images from "Three motAB Stator Gene Products in Bdellovibrio bacteriovorus Contribute to Motility of a Single Flagellum during Predatory and Prey-Independent Growth ▿ Contribute to Motility of a Single Flagellum during Predatory and Prey-Independent Growth ▿ †"

Article Title: Three motAB Stator Gene Products in Bdellovibrio bacteriovorus Contribute to Motility of a Single Flagellum during Predatory and Prey-Independent Growth ▿ Contribute to Motility of a Single Flagellum during Predatory and Prey-Independent Growth ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.00941-10

Bdellovibrio motility in the presence of phenamil, NaCl, and CCCP. (a) Mean swimming speed (± standard deviation) and percent motility in the presence of the sodium channel inhibitor phenamil. (b) Mean swimming speed in Ca-HEPES buffer at pH 8.2 with different NaCl concentrations. Error bars show the standard deviation around the mean. (c) Mean swimming speed (± standard deviation) and percent motility in the presence of the proton channel inhibitor CCCP.
Figure Legend Snippet: Bdellovibrio motility in the presence of phenamil, NaCl, and CCCP. (a) Mean swimming speed (± standard deviation) and percent motility in the presence of the sodium channel inhibitor phenamil. (b) Mean swimming speed in Ca-HEPES buffer at pH 8.2 with different NaCl concentrations. Error bars show the standard deviation around the mean. (c) Mean swimming speed (± standard deviation) and percent motility in the presence of the proton channel inhibitor CCCP.

Techniques Used: Standard Deviation

22) Product Images from "Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice"

Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.198001

Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
Figure Legend Snippet: Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

Techniques Used: Mouse Assay, Activation Assay

MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P
Figure Legend Snippet: MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

Techniques Used: Activity Assay, Expressing, Western Blot

23) Product Images from "Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle *"

Article Title: Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.735530

AUC SV and SEC/MALS. A , sedimentation coefficient distribution of KsHDH in 25 m m HEPES/KOH, pH 7.5, 25 m m KCl. The three peaks correspond to the HDH monomer (α, 5 S), the covalently bound homotrimer (α 3 , 10.97 S), and the non-covalently
Figure Legend Snippet: AUC SV and SEC/MALS. A , sedimentation coefficient distribution of KsHDH in 25 m m HEPES/KOH, pH 7.5, 25 m m KCl. The three peaks correspond to the HDH monomer (α, 5 S), the covalently bound homotrimer (α 3 , 10.97 S), and the non-covalently

Techniques Used: Size-exclusion Chromatography, Sedimentation

24) Product Images from "Transcript Analysis of Heat Shock Protein 72 in Vitrified 2-Cell Mouse Embryos and Subsequent In Vitro Development"

Article Title: Transcript Analysis of Heat Shock Protein 72 in Vitrified 2-Cell Mouse Embryos and Subsequent In Vitro Development

Journal: Cell Journal (Yakhteh)

doi:

The percentages of blastocyst formation of 2-cell embryos in three groups, cont; control (non-vitrified) group, vit1 ; vitrification with 7.5% DMSO and 7.5% EG, vit 2 ; vitrification with 15% DMSO and 15% EG. a, b and c indicate the significant differences among control, vit1 and vit 2 (p
Figure Legend Snippet: The percentages of blastocyst formation of 2-cell embryos in three groups, cont; control (non-vitrified) group, vit1 ; vitrification with 7.5% DMSO and 7.5% EG, vit 2 ; vitrification with 15% DMSO and 15% EG. a, b and c indicate the significant differences among control, vit1 and vit 2 (p

Techniques Used:

Mean of cleavage rates of 2-cell embryos (to morula) in three groups, cont; control (non-vitrified) group, vit1 ; vitrification with 7.5% DMSO and 7.5% EG, vit 2 ; vitrification with 15% DMSO and 15% EG. a, b and c indicate the significant differences among control, vit1 and vit 2 (p
Figure Legend Snippet: Mean of cleavage rates of 2-cell embryos (to morula) in three groups, cont; control (non-vitrified) group, vit1 ; vitrification with 7.5% DMSO and 7.5% EG, vit 2 ; vitrification with 15% DMSO and 15% EG. a, b and c indicate the significant differences among control, vit1 and vit 2 (p

Techniques Used:

The relative quantification of Hsp72 after normalization by Hprt1 in 2-cell embryo groups, cont; control (nonvitrified) group, vit1 ; vitrification with 7.5% DMSO and 7.5% EG, vit 2 ; vitrification with 15% DMSO and 15% EG. a, b and c indicate the significant differences among control, vit1 and vit 2 (p
Figure Legend Snippet: The relative quantification of Hsp72 after normalization by Hprt1 in 2-cell embryo groups, cont; control (nonvitrified) group, vit1 ; vitrification with 7.5% DMSO and 7.5% EG, vit 2 ; vitrification with 15% DMSO and 15% EG. a, b and c indicate the significant differences among control, vit1 and vit 2 (p

Techniques Used:

Mean inverse Ct values of Hprt1 as the relevant abundance of transcript 2-cell embryo groups, Ct; threshold cycle, cont; control (non-vitrified) group, vit1; vitrification with 7.5% DMSO and 7.5% EG, vit 2 ; vitrification with 15% DMSO and 15% EG. Bars are indicative of having no significant difference.
Figure Legend Snippet: Mean inverse Ct values of Hprt1 as the relevant abundance of transcript 2-cell embryo groups, Ct; threshold cycle, cont; control (non-vitrified) group, vit1; vitrification with 7.5% DMSO and 7.5% EG, vit 2 ; vitrification with 15% DMSO and 15% EG. Bars are indicative of having no significant difference.

Techniques Used:

25) Product Images from "Regulation of Human Papillomavirus Type 16 E7 Activity through Direct Protein Interaction with the E2 Transcriptional Activator"

Article Title: Regulation of Human Papillomavirus Type 16 E7 Activity through Direct Protein Interaction with the E2 Transcriptional Activator

Journal:

doi: 10.1128/JVI.80.4.1787-1797.2006

The transformation activity of E7 is inhibited in the presence of E2. Primary BRK cells from 9-day-old Wistar rats were transfected with 6 μg of plasmids expressing HPV-16 E7 (a) or adenovirus E1a (b) together with Ras as a cooperating oncogene
Figure Legend Snippet: The transformation activity of E7 is inhibited in the presence of E2. Primary BRK cells from 9-day-old Wistar rats were transfected with 6 μg of plasmids expressing HPV-16 E7 (a) or adenovirus E1a (b) together with Ras as a cooperating oncogene

Techniques Used: Transformation Assay, Activity Assay, Transfection, Expressing

26) Product Images from "Exosome complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis"

Article Title: Exosome complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis

Journal: eLife

doi: 10.7554/eLife.17877

Exosome complex disruption renders primary erythroid cells hypersensitive to limiting erythropoietin concentrations. ( A ) Flow cytometric analysis with Annexin V and the membrane-impermeable dye DRAQ7 to quantitate apoptosis with control and Exosc8 -knockdown primary erythroid cells expanded for 48 hr under Epo-limiting conditions. ( B ) Quantification of the percentage of primary erythroid cells in live, late and early apoptotic populations (mean ± SE, 4 biological replicates). Statistical analysis of control and treatment conditions was conducted with the Student’s T-test. *p
Figure Legend Snippet: Exosome complex disruption renders primary erythroid cells hypersensitive to limiting erythropoietin concentrations. ( A ) Flow cytometric analysis with Annexin V and the membrane-impermeable dye DRAQ7 to quantitate apoptosis with control and Exosc8 -knockdown primary erythroid cells expanded for 48 hr under Epo-limiting conditions. ( B ) Quantification of the percentage of primary erythroid cells in live, late and early apoptotic populations (mean ± SE, 4 biological replicates). Statistical analysis of control and treatment conditions was conducted with the Student’s T-test. *p

Techniques Used: Flow Cytometry

27) Product Images from "A CAF-1-PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage"

Article Title: A CAF-1-PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

Journal: Molecular and Cellular Biology

doi:

PCNA mediates chromatin assembly linked to single-strand break repair. (A) Analysis of supercoiling during single-strand break repair in the Drosophila cell-free system. Supercoiled circular plasmid DNA (lane 1) was treated with DNase I to induce single-strand breaks (lane 2). These DNA substrates were incubated for 3 h at 23°C with either nondepleted extract (lanes 3 and 4), extract depleted with GST alone (lanes 5 and 6), or extract depleted with GST-p150(1-244) (lanes 7 to 10). Recombinant human PCNA was added to the reactions in lanes 9 and 10 to a final concentration of 8 ng/μl. The migration of relaxed/nicked circular DNA (Ir/II) and supercoiled DNA (I) molecules is indicated. (B) Bead-linked DNA substrates containing DNase I-induced single-strand breaks or undamaged control molecules were incubated in the Drosophila cell-free system as described for panel A for 1 min at 23°C. Bead-linked DNA substrates were equilibrated and washed in a slightly higher ionic strength buffer (40 mM HEPES-KOH [pH 7.8], 100 mM KCl, 0.05% NP-40) for reactions with the Drosophila cell-free system. PCNA binding to the bead-linked DNA substrates was detected by Western blotting. The recombinant human PCNA containing a C-terminal histidine tag migrates more slowly than endogenous Drosophila PCNA.
Figure Legend Snippet: PCNA mediates chromatin assembly linked to single-strand break repair. (A) Analysis of supercoiling during single-strand break repair in the Drosophila cell-free system. Supercoiled circular plasmid DNA (lane 1) was treated with DNase I to induce single-strand breaks (lane 2). These DNA substrates were incubated for 3 h at 23°C with either nondepleted extract (lanes 3 and 4), extract depleted with GST alone (lanes 5 and 6), or extract depleted with GST-p150(1-244) (lanes 7 to 10). Recombinant human PCNA was added to the reactions in lanes 9 and 10 to a final concentration of 8 ng/μl. The migration of relaxed/nicked circular DNA (Ir/II) and supercoiled DNA (I) molecules is indicated. (B) Bead-linked DNA substrates containing DNase I-induced single-strand breaks or undamaged control molecules were incubated in the Drosophila cell-free system as described for panel A for 1 min at 23°C. Bead-linked DNA substrates were equilibrated and washed in a slightly higher ionic strength buffer (40 mM HEPES-KOH [pH 7.8], 100 mM KCl, 0.05% NP-40) for reactions with the Drosophila cell-free system. PCNA binding to the bead-linked DNA substrates was detected by Western blotting. The recombinant human PCNA containing a C-terminal histidine tag migrates more slowly than endogenous Drosophila PCNA.

Techniques Used: Plasmid Preparation, Incubation, Recombinant, Concentration Assay, Migration, Binding Assay, Western Blot

28) Product Images from "Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia"

Article Title: Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia

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

doi:

Expression of perlecan antisense leads to reduced perlecan secretion and loss of FGF-2 binding inhibition in endothelial cell-conditioned media. ( A ) Perlecan antisense construct. ( B ) Western blot of conditioned media from untransfected BAE cells, and clones transfected with the antiperlecan sequences (BAE-AP1–6) or the vector alone (BAE-NEO1–4). All samples were normalized for cell number (1 × 10 4 cells) and subjected to heparinase treatment before gel electrophoresis. ( C ) FGF-2 binding to smooth muscle cells is inhibited by conditioned media from BAE-NEO cells to a greater extent than by that from BAE-AP cells 125 ). VSMCs were plated (5 × 10 4 cells/well) in 24-well culture plates (Costar) in DMEM, 10% calf serum. When the cells reached confluence (day 3), the medium was removed, and the monolayers were washed with cold (0°C) binding buffer (DMEM/25 mM Hepes/0.05% gelatin), and binding of 125 I-FGF-2 was conducted in the presence of conditioned media (equivalent to the media conditioned by 3.5 × 10 4 cells) for 2.5 h at 4°C. The data shown are the averages ± SEM of triplicate determinations. Control binding represented the amount of 125 I-FGF-2 bound to cells in the absence of any conditioned media (100%).
Figure Legend Snippet: Expression of perlecan antisense leads to reduced perlecan secretion and loss of FGF-2 binding inhibition in endothelial cell-conditioned media. ( A ) Perlecan antisense construct. ( B ) Western blot of conditioned media from untransfected BAE cells, and clones transfected with the antiperlecan sequences (BAE-AP1–6) or the vector alone (BAE-NEO1–4). All samples were normalized for cell number (1 × 10 4 cells) and subjected to heparinase treatment before gel electrophoresis. ( C ) FGF-2 binding to smooth muscle cells is inhibited by conditioned media from BAE-NEO cells to a greater extent than by that from BAE-AP cells 125 ). VSMCs were plated (5 × 10 4 cells/well) in 24-well culture plates (Costar) in DMEM, 10% calf serum. When the cells reached confluence (day 3), the medium was removed, and the monolayers were washed with cold (0°C) binding buffer (DMEM/25 mM Hepes/0.05% gelatin), and binding of 125 I-FGF-2 was conducted in the presence of conditioned media (equivalent to the media conditioned by 3.5 × 10 4 cells) for 2.5 h at 4°C. The data shown are the averages ± SEM of triplicate determinations. Control binding represented the amount of 125 I-FGF-2 bound to cells in the absence of any conditioned media (100%).

Techniques Used: Expressing, Binding Assay, Inhibition, Construct, Western Blot, Clone Assay, Transfection, Plasmid Preparation, Nucleic Acid Electrophoresis

29) Product Images from "Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments"

Article Title: Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

Journal: eLife

doi: 10.7554/eLife.06585

Interactions between filamentous actin, GFP-Lpd (850–1250aa), and GFP-LZ-Lpd (850–1250aa) measured by cosedimentation at different buffer ionic strengths. ( A ) Monomeric GFP-Lpd 850−1250aa and dimeric GFP-LZ-Lpd 850−1250aa interact with filamentous actin in the presence of 50, 100, 150 mM KCl. SDS-PAGE from three experiments showing the cosedimentation of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa (monomeric protein concentration). Buffer composition is 20 mM HEPES [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl 2 , 0.5 mM EGTA. ( B ) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). ( C ) SDS-PAGE as in Figure 1H , showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). ( D ) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl 2 , and 0.5 mM EGTA. ( C , D ) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification. DOI: http://dx.doi.org/10.7554/eLife.06585.004
Figure Legend Snippet: Interactions between filamentous actin, GFP-Lpd (850–1250aa), and GFP-LZ-Lpd (850–1250aa) measured by cosedimentation at different buffer ionic strengths. ( A ) Monomeric GFP-Lpd 850−1250aa and dimeric GFP-LZ-Lpd 850−1250aa interact with filamentous actin in the presence of 50, 100, 150 mM KCl. SDS-PAGE from three experiments showing the cosedimentation of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa (monomeric protein concentration). Buffer composition is 20 mM HEPES [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl 2 , 0.5 mM EGTA. ( B ) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). ( C ) SDS-PAGE as in Figure 1H , showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). ( D ) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd 850−1250aa or GFP-LZ-Lpd 850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl 2 , and 0.5 mM EGTA. ( C , D ) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification. DOI: http://dx.doi.org/10.7554/eLife.06585.004

Techniques Used: SDS Page, Protein Concentration, Sedimentation, Concentration Assay, Incubation, Purification

30) Product Images from "Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E"

Article Title: Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

Journal: The EMBO Journal

doi: 10.15252/embj.201694639

The EHEC ‐specific small RNA Esr41 regulates iron uptake, storage and colicin resistance FACS analysis of constitutively expressed sfGFP fusions to chuA (A), cirA (B) or bfr (C) in the presence of Esr41 (orange), RyhB (blue) or a scrambled RNA control (red). The histogram shows GFP fluorescence for each sRNA–mRNA fusion. Esr41 confers resistance to colicins 1A and 1B in the sensitive background, E. coli DH5α. Top agar lawns of DH5α expressing a control scrambled RNA (pJV300), Esr41 (pZE12:: esr41 ) or RyhB (pZE12:: ryhB ) were spotted with colicins indicated (top). Zones of clearing indicate sensitivity to the tested colicin. Esr41 confers a competitive disadvantage on EHEC under iron‐limiting conditions. Wild‐type EHEC and isogenic ∆ esr41 strain (black), or EHEC ∆ esr41 and the chromosomally repaired strain EHEC ∆ esr41 :: esr41 were inoculated at equal densities and cultured in MEM‐HEPES media for 3 days. The proportion of each strain was determined at each time point (days) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness (see Appendix Supplementary Methods ).
Figure Legend Snippet: The EHEC ‐specific small RNA Esr41 regulates iron uptake, storage and colicin resistance FACS analysis of constitutively expressed sfGFP fusions to chuA (A), cirA (B) or bfr (C) in the presence of Esr41 (orange), RyhB (blue) or a scrambled RNA control (red). The histogram shows GFP fluorescence for each sRNA–mRNA fusion. Esr41 confers resistance to colicins 1A and 1B in the sensitive background, E. coli DH5α. Top agar lawns of DH5α expressing a control scrambled RNA (pJV300), Esr41 (pZE12:: esr41 ) or RyhB (pZE12:: ryhB ) were spotted with colicins indicated (top). Zones of clearing indicate sensitivity to the tested colicin. Esr41 confers a competitive disadvantage on EHEC under iron‐limiting conditions. Wild‐type EHEC and isogenic ∆ esr41 strain (black), or EHEC ∆ esr41 and the chromosomally repaired strain EHEC ∆ esr41 :: esr41 were inoculated at equal densities and cultured in MEM‐HEPES media for 3 days. The proportion of each strain was determined at each time point (days) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness (see Appendix Supplementary Methods ).

Techniques Used: FACS, Fluorescence, Expressing, Cell Culture

31) Product Images from "A Novel Lactate Dehydrogenase Inhibitor, 1-(Phenylseleno)-4-(Trifluoromethyl) Benzene, Suppresses Tumor Growth through Apoptotic Cell Death"

Article Title: A Novel Lactate Dehydrogenase Inhibitor, 1-(Phenylseleno)-4-(Trifluoromethyl) Benzene, Suppresses Tumor Growth through Apoptotic Cell Death

Journal: Scientific Reports

doi: 10.1038/s41598-019-40617-3

PSTMB inhibits LDHA activity in Michaelis-Menten and Lineweaver-Burk plots. To check LDHA enzyme kinetics, purified recombinant human LDHA protein (10 ng) were incubated in buffer containing 20 mM of HEPES-K + (pH 7.2), 30 μM of NADH with 0, 0.1, 0.5, 1 and 2 mM of pyruvate in the presence or absence of PSTMB (0, 0.1, 0.5 μM). for 10 min. The fluorescence of NADH was examined at wavelength of excitation at 340 nm and emission at 460 nm with spectrofluorometer. Michaelis-Menten curves ( A ) and Lineweaver-Burk plots ( B ) are shown to determine the inhibition mode of PSTMB. The values are averages of three separate experiments. The results are plotted as means ± SD.
Figure Legend Snippet: PSTMB inhibits LDHA activity in Michaelis-Menten and Lineweaver-Burk plots. To check LDHA enzyme kinetics, purified recombinant human LDHA protein (10 ng) were incubated in buffer containing 20 mM of HEPES-K + (pH 7.2), 30 μM of NADH with 0, 0.1, 0.5, 1 and 2 mM of pyruvate in the presence or absence of PSTMB (0, 0.1, 0.5 μM). for 10 min. The fluorescence of NADH was examined at wavelength of excitation at 340 nm and emission at 460 nm with spectrofluorometer. Michaelis-Menten curves ( A ) and Lineweaver-Burk plots ( B ) are shown to determine the inhibition mode of PSTMB. The values are averages of three separate experiments. The results are plotted as means ± SD.

Techniques Used: Activity Assay, Purification, Recombinant, Incubation, Fluorescence, Inhibition

32) Product Images from "Functional and Morphological Analysis of OFF Bipolar Cells"

Article Title: Functional and Morphological Analysis of OFF Bipolar Cells

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-7720-8_15

Preparation for the eye dissection. (A) A ventilated cart to place the animal cage inside for dark adaptation. (B) A handmade chopper with a razor blade (red arrow) inserted. A micrometer dial on the bottom moves the slicing chamber laterally during the dissection. (C) Grease pens are assembled from a 1 ml syringe and a pipetter tip (~200 µl). Vacuum grease is inserted into the back of the syringe. (D) A dark box for holding retinal preparations. The bottom is filled with water and oxygen is bubbled continuously. When the box is closed, the inside is completely dark and a moist, oxygenated environment is continually provided. Preparations sit on the shelf. (E) A dissecting chamber filled with HEPES solution. PE tubing from an oxygen source continuously delivers oxygen to the retinal tissue. (F) All tools and pipettes are arranged next to the dissecting microscope, with HEPES solution on ice nearby. (G) A slicing chamber to hold retina tissue (white arrow) attached to the Millipore filter paper (green arrow). (H) A plastic coverslip with parallel grease rails (yellow asterisks) and an attached retinal slice (white arrow) with filter paper (green arrow) set into the grease. (I) A micro-pipette filler and a 1 ml pipette for reference.
Figure Legend Snippet: Preparation for the eye dissection. (A) A ventilated cart to place the animal cage inside for dark adaptation. (B) A handmade chopper with a razor blade (red arrow) inserted. A micrometer dial on the bottom moves the slicing chamber laterally during the dissection. (C) Grease pens are assembled from a 1 ml syringe and a pipetter tip (~200 µl). Vacuum grease is inserted into the back of the syringe. (D) A dark box for holding retinal preparations. The bottom is filled with water and oxygen is bubbled continuously. When the box is closed, the inside is completely dark and a moist, oxygenated environment is continually provided. Preparations sit on the shelf. (E) A dissecting chamber filled with HEPES solution. PE tubing from an oxygen source continuously delivers oxygen to the retinal tissue. (F) All tools and pipettes are arranged next to the dissecting microscope, with HEPES solution on ice nearby. (G) A slicing chamber to hold retina tissue (white arrow) attached to the Millipore filter paper (green arrow). (H) A plastic coverslip with parallel grease rails (yellow asterisks) and an attached retinal slice (white arrow) with filter paper (green arrow) set into the grease. (I) A micro-pipette filler and a 1 ml pipette for reference.

Techniques Used: Dissection, Microscopy, Transferring

33) Product Images from "Effect of acute acid-base disturbances on the phosphorylation of phospholipase C-γ1 and Erk1/2 in the renal proximal tubule"

Article Title: Effect of acute acid-base disturbances on the phosphorylation of phospholipase C-γ1 and Erk1/2 in the renal proximal tubule

Journal: Physiological Reports

doi: 10.14814/phy2.12280

Quantification of p-Erk1/2 immunoreactivity—pT202/pY204 for Erk1 and pT185/pY187 for Erk2—after solution treatments of 5 min. We show densitometry data from western blots, like those in Figure 5 . For each treatment, we first normalize p-Erk1 and p-Erk2 immunoreactivity to total Erk1 or Erk2 immunoreactivity—that is, (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total)—in the same lane (reprobed blot). (A) (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total) for nominally CO 2 / -free treatment solutions, each normalized to (p-Erk1)/(Erk1.total) or (p-Erk2)/(Erk2.total) for the nominally CO 2 / -free solution (HEPES) on the same blot. (B) (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total) for HEPES and CO 2 / treatment solutions, each normalized to (p-Erk1)/(Erk1.total) or (p-Erk2)/(Erk2.total) for the Ctrl treatment (5% CO 2 /22 mmol/L ) on the same blot. (C) (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total) for treatments like those in panel B, but with the ErbB inhibitor PD168393, each normalized (p-Erk1)/(Erk1.total) or (p-Erk2)/(Erk2.total) for the equivalent treatment without PD168393 on the same blot. (D) (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total) for PD168393-treated PTs, as in panel C, but instead normalized to (p-Erk1)/(Erk1.total) or (p-Erk2)/(Erk2.total) for PD168393-treated PTs incubated in the Ctrl solution (5% CO 2 /22 mmol/L ) on the same blot. Sample size for each bar is shown beneath the legend for the panel. PD, PD168393. # Denotes a bar for which P
Figure Legend Snippet: Quantification of p-Erk1/2 immunoreactivity—pT202/pY204 for Erk1 and pT185/pY187 for Erk2—after solution treatments of 5 min. We show densitometry data from western blots, like those in Figure 5 . For each treatment, we first normalize p-Erk1 and p-Erk2 immunoreactivity to total Erk1 or Erk2 immunoreactivity—that is, (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total)—in the same lane (reprobed blot). (A) (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total) for nominally CO 2 / -free treatment solutions, each normalized to (p-Erk1)/(Erk1.total) or (p-Erk2)/(Erk2.total) for the nominally CO 2 / -free solution (HEPES) on the same blot. (B) (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total) for HEPES and CO 2 / treatment solutions, each normalized to (p-Erk1)/(Erk1.total) or (p-Erk2)/(Erk2.total) for the Ctrl treatment (5% CO 2 /22 mmol/L ) on the same blot. (C) (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total) for treatments like those in panel B, but with the ErbB inhibitor PD168393, each normalized (p-Erk1)/(Erk1.total) or (p-Erk2)/(Erk2.total) for the equivalent treatment without PD168393 on the same blot. (D) (p-Erk1)/(Erk1.total) and (p-Erk2)/(Erk2.total) for PD168393-treated PTs, as in panel C, but instead normalized to (p-Erk1)/(Erk1.total) or (p-Erk2)/(Erk2.total) for PD168393-treated PTs incubated in the Ctrl solution (5% CO 2 /22 mmol/L ) on the same blot. Sample size for each bar is shown beneath the legend for the panel. PD, PD168393. # Denotes a bar for which P

Techniques Used: Western Blot, Incubation

Quantification of PLC-γ1 immunoreactivity at pY783 after 5-min exposure of PTs to treatment solutions. We show densitometry data from western blots, like those in Figure 1 . For each treatment, we first normalize PLC-γ1.pY783 to total PLC-γ1 immunoreactivity in the same lane (reprobed blot). (A) PLC-γ1.pY783/(total PLC-γ1) for nominally CO 2 / -free treatment solutions, each normalized to PLC-γ1.pY783/(total PLC-γ1) for the nominally CO 2 / -free solution (HEPES) on the same blot. (B) PLC-γ1.pY783/(total PLC-γ1) for HEPES and CO 2 / treatment solutions, each normalized to PLC-γ1.pY783/(total PLC-γ1) for the Ctrl treatment (5% CO 2 /22 mmol/L ) on the same blot. (C) PLC-γ1.pY783/(total PLC-γ1) for treatments like those in panel B, but with and without the ErbB inhibitor PD168393, each normalized to PLC-γ1.pY783/(total PLC-γ1) for the equivalent treatment without PD168393 on the same blot. (D) PLC-γ1.pY783/(total PLC-γ1) for PD168393-treated PTs, as in panel C, but instead normalized to PLC-γ1.pY783/(total PLC-γ1) for PD168393-treated PTs incubated in the Ctrl solution (5% CO 2 /22 mmol/L ) on the same blot. Sample size for each bar is shown in parentheses beneath the legend. # Denotes a bar for which P
Figure Legend Snippet: Quantification of PLC-γ1 immunoreactivity at pY783 after 5-min exposure of PTs to treatment solutions. We show densitometry data from western blots, like those in Figure 1 . For each treatment, we first normalize PLC-γ1.pY783 to total PLC-γ1 immunoreactivity in the same lane (reprobed blot). (A) PLC-γ1.pY783/(total PLC-γ1) for nominally CO 2 / -free treatment solutions, each normalized to PLC-γ1.pY783/(total PLC-γ1) for the nominally CO 2 / -free solution (HEPES) on the same blot. (B) PLC-γ1.pY783/(total PLC-γ1) for HEPES and CO 2 / treatment solutions, each normalized to PLC-γ1.pY783/(total PLC-γ1) for the Ctrl treatment (5% CO 2 /22 mmol/L ) on the same blot. (C) PLC-γ1.pY783/(total PLC-γ1) for treatments like those in panel B, but with and without the ErbB inhibitor PD168393, each normalized to PLC-γ1.pY783/(total PLC-γ1) for the equivalent treatment without PD168393 on the same blot. (D) PLC-γ1.pY783/(total PLC-γ1) for PD168393-treated PTs, as in panel C, but instead normalized to PLC-γ1.pY783/(total PLC-γ1) for PD168393-treated PTs incubated in the Ctrl solution (5% CO 2 /22 mmol/L ) on the same blot. Sample size for each bar is shown in parentheses beneath the legend. # Denotes a bar for which P

Techniques Used: Planar Chromatography, Western Blot, Incubation

34) Product Images from "Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins"

Article Title: Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj458

Stretching curves for λ-DNA in the absence of protein (open circle), in the presence of 20 nM NCp7 (wild type) protein (closed circle), and for ssDNA with NC bound on it (closed triangle), all in FIM buffer (see Materials and Methods) and room temperature. For ssDNA with NC bound, data are from ref. ( 35 ), and was taken with 10 nM NCp7 in 25 mM NaCl, 10 mM HEPES, pH 7.5, room temperature. Stretching curves for dsDNA (open square) and ssDNA (open triangle), fit to standard polymer models, in the absence of protein ( 90 ) are also shown. The method used to determine Δ F , the force transition width, is shown schematically.
Figure Legend Snippet: Stretching curves for λ-DNA in the absence of protein (open circle), in the presence of 20 nM NCp7 (wild type) protein (closed circle), and for ssDNA with NC bound on it (closed triangle), all in FIM buffer (see Materials and Methods) and room temperature. For ssDNA with NC bound, data are from ref. ( 35 ), and was taken with 10 nM NCp7 in 25 mM NaCl, 10 mM HEPES, pH 7.5, room temperature. Stretching curves for dsDNA (open square) and ssDNA (open triangle), fit to standard polymer models, in the absence of protein ( 90 ) are also shown. The method used to determine Δ F , the force transition width, is shown schematically.

Techniques Used:

35) Product Images from "The Ferroxidase Activity of Yeast Frataxin"

Article Title: The Ferroxidase Activity of Yeast Frataxin

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M206711200

Production of H 2 O 2 during Fe(II) oxidation in mYfh1p A , reactions containing 96 μ M mYfh1p and 24 or 48 α M Fe(II) were incubated in 50 mM HEPES-KOH, pH 7.0, for 30 min at 30 °C in the presence of Amplex Red/HRP reagent. Immediately afterward, fluorescence intensity was recorded from 570 to 610 nm. The fluorescence intensity curves before background correction are shown ( bottom graph ). For the standard curve ( top graph ), H 2 O 2 standards (50 – 600 nM) were incubated in 50 mM HEPES-KOH, pH 7.0, for 30 min at 30 °C in the presence of Amplex Red/HRP reagent. The fluorescence intensity curves were recorded, and samples containing buffer plus Amplex Red/HRP were used for background corrections. The corrected fluorescence intensity curves were integrated, and a standard curve was constructed. The correlation coefficient of the fitted line to the data is 0.999. To determine the concentration of H 2 O 2 produced in the presence of mYfh1p (see “Results”), samples containing buffer plus 24 or 48 μ M Fe(II) and Amplex Red/HRP were used as blanks for background corrections. The corrected fluorescence intensity curves for mYfh1p were integrated, and the H 2 O 2 concentration was calculated from the standard curve. A.U. , arbitrary units. B , reactions containing 96 μ M mYfh1p were incubated for 30 min at 30 °C in the absence or presence of 48 μ M Fe(II) under the experimental conditions used in the Amplex Red/HRP assays described above. Following incubation with 2,4-dinitrophenylhy-drazine (DNPH) to derivatize carbonyl groups to DNP, the samples (7.5 μ g of total protein) were analyzed by SDS/PAGE and Western blotting using a polyclonal anti-DNP antiserum. C , 2 μ g of the purified mYfh1p monomer used in the experiments described above was analyzed by SDS/PAGE and Coomassie Blue staining ( lane 5 ). D , following immunodetection, the membrane was subjected to SYPRO Ruby protein blot staining. Arrows d , degradation products of mYfh1p; lane MW , molecular mass standards. E , a mixture of 48 μ M Fe(II), 24 μ M H 2 O 2 , and 5 mM 2-deoxyribose ( DOR ) was incubated in 10 mM HEPES-KOH, pH 7.0, in the absence or presence of 96 μ M mYfh1p for 30 min at 30 °C, and production of malondialdehyde-thiobarbituric acid ( MDA-TBA ) ( ε 532 = 1.54 × 10 5 M −1 cm −1 ). The indicated controls were analyzed at the same time and treated identically. The bars represent the means ± S.D. of four independent measurements.
Figure Legend Snippet: Production of H 2 O 2 during Fe(II) oxidation in mYfh1p A , reactions containing 96 μ M mYfh1p and 24 or 48 α M Fe(II) were incubated in 50 mM HEPES-KOH, pH 7.0, for 30 min at 30 °C in the presence of Amplex Red/HRP reagent. Immediately afterward, fluorescence intensity was recorded from 570 to 610 nm. The fluorescence intensity curves before background correction are shown ( bottom graph ). For the standard curve ( top graph ), H 2 O 2 standards (50 – 600 nM) were incubated in 50 mM HEPES-KOH, pH 7.0, for 30 min at 30 °C in the presence of Amplex Red/HRP reagent. The fluorescence intensity curves were recorded, and samples containing buffer plus Amplex Red/HRP were used for background corrections. The corrected fluorescence intensity curves were integrated, and a standard curve was constructed. The correlation coefficient of the fitted line to the data is 0.999. To determine the concentration of H 2 O 2 produced in the presence of mYfh1p (see “Results”), samples containing buffer plus 24 or 48 μ M Fe(II) and Amplex Red/HRP were used as blanks for background corrections. The corrected fluorescence intensity curves for mYfh1p were integrated, and the H 2 O 2 concentration was calculated from the standard curve. A.U. , arbitrary units. B , reactions containing 96 μ M mYfh1p were incubated for 30 min at 30 °C in the absence or presence of 48 μ M Fe(II) under the experimental conditions used in the Amplex Red/HRP assays described above. Following incubation with 2,4-dinitrophenylhy-drazine (DNPH) to derivatize carbonyl groups to DNP, the samples (7.5 μ g of total protein) were analyzed by SDS/PAGE and Western blotting using a polyclonal anti-DNP antiserum. C , 2 μ g of the purified mYfh1p monomer used in the experiments described above was analyzed by SDS/PAGE and Coomassie Blue staining ( lane 5 ). D , following immunodetection, the membrane was subjected to SYPRO Ruby protein blot staining. Arrows d , degradation products of mYfh1p; lane MW , molecular mass standards. E , a mixture of 48 μ M Fe(II), 24 μ M H 2 O 2 , and 5 mM 2-deoxyribose ( DOR ) was incubated in 10 mM HEPES-KOH, pH 7.0, in the absence or presence of 96 μ M mYfh1p for 30 min at 30 °C, and production of malondialdehyde-thiobarbituric acid ( MDA-TBA ) ( ε 532 = 1.54 × 10 5 M −1 cm −1 ). The indicated controls were analyzed at the same time and treated identically. The bars represent the means ± S.D. of four independent measurements.

Techniques Used: Incubation, Fluorescence, Construct, Concentration Assay, Produced, SDS Page, Western Blot, Purification, Staining, Immunodetection, Multiple Displacement Amplification

36) Product Images from "Comparison of the Effects of Surface Tension and Osmotic Pressure on the Interfacial Hydration of a Fluid Phospholipid Bilayer"

Article Title: Comparison of the Effects of Surface Tension and Osmotic Pressure on the Interfacial Hydration of a Fluid Phospholipid Bilayer

Journal: Biophysical Journal

doi:

Effects of increasing concentrations of betaine (▪), choline chloride (▴), and sucrose (•) on the GP of Laurdan ( X = 0.01) in POPC LUVs. Solutes were dissolved in buffer (5 mM HEPES and 0.1 mM EDTA, at pH 7.4) at concentrations corresponding to given increasing values of osmolarity Π, namely, 0.5, 1, and 2 osm/kg. Changes in fluorophore emission spectra were measured at 25°C. Total lipid concentration was 25 mM. Each data point represents the average of at least three separate measurements with the error bars indicating standard deviation.
Figure Legend Snippet: Effects of increasing concentrations of betaine (▪), choline chloride (▴), and sucrose (•) on the GP of Laurdan ( X = 0.01) in POPC LUVs. Solutes were dissolved in buffer (5 mM HEPES and 0.1 mM EDTA, at pH 7.4) at concentrations corresponding to given increasing values of osmolarity Π, namely, 0.5, 1, and 2 osm/kg. Changes in fluorophore emission spectra were measured at 25°C. Total lipid concentration was 25 mM. Each data point represents the average of at least three separate measurements with the error bars indicating standard deviation.

Techniques Used: Concentration Assay, Standard Deviation

37) Product Images from "Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system"

Article Title: Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system

Journal: Scientific Reports

doi: 10.1038/srep30357

Purification of PfRH5 protein variants. (A) Size exclusion chromatography (SEC) curve of the Ni-IMAC peak fraction pool run on Superdex200 in 20 mM HEPES pH 7.4, 150 mM NaCl. The PfRH5 peak and the shoulder of contaminant 38 kDa Drosophila S2 protein are shown. (B) Coomassie gel analysis of SEC fractions. 10 μL of the SEC fractions B1–B6 were resolved on a 12% Tris-Glycine gel and proteins were stained with InstantBlue. (C) Coomassie gel analysis of pools of PfRH5 SEC peak fractions showing product purified in the presence (+) or absence (−) of the Con A 4B Sepharose purification step. Proteins were resolved on a 12% Tris-HEPES gel and stained with InstantBlue. (D) Coomassie gel analysis of a representative purification run of PfRH5 v1.0. Proteins were separated on a Bio-Rad Any kD TGX gel and stained with SimplyBlue SafeStain. M: molecular weight markers; ST 0 : starting material; ST C : starting material after TFF and buffer exchange; FT: flow-through from HisTrap column; W: HisTrap 40 mM Imidazole wash; E: Elution from HisTrap with 400 mM Imidazole; FT cA : flow-through from Con A column; P: final product after SEC. (E ) Coomassie gel analysis of all purified PfRH5 protein variants run head-to-head (v1.0–v5.0). 1.25 μg each protein sample (DTT reduced and heat denatured) was separated on a Bio-Rad Any kD Criterion TGX gel and stained with InstantBlue.
Figure Legend Snippet: Purification of PfRH5 protein variants. (A) Size exclusion chromatography (SEC) curve of the Ni-IMAC peak fraction pool run on Superdex200 in 20 mM HEPES pH 7.4, 150 mM NaCl. The PfRH5 peak and the shoulder of contaminant 38 kDa Drosophila S2 protein are shown. (B) Coomassie gel analysis of SEC fractions. 10 μL of the SEC fractions B1–B6 were resolved on a 12% Tris-Glycine gel and proteins were stained with InstantBlue. (C) Coomassie gel analysis of pools of PfRH5 SEC peak fractions showing product purified in the presence (+) or absence (−) of the Con A 4B Sepharose purification step. Proteins were resolved on a 12% Tris-HEPES gel and stained with InstantBlue. (D) Coomassie gel analysis of a representative purification run of PfRH5 v1.0. Proteins were separated on a Bio-Rad Any kD TGX gel and stained with SimplyBlue SafeStain. M: molecular weight markers; ST 0 : starting material; ST C : starting material after TFF and buffer exchange; FT: flow-through from HisTrap column; W: HisTrap 40 mM Imidazole wash; E: Elution from HisTrap with 400 mM Imidazole; FT cA : flow-through from Con A column; P: final product after SEC. (E ) Coomassie gel analysis of all purified PfRH5 protein variants run head-to-head (v1.0–v5.0). 1.25 μg each protein sample (DTT reduced and heat denatured) was separated on a Bio-Rad Any kD Criterion TGX gel and stained with InstantBlue.

Techniques Used: Purification, Size-exclusion Chromatography, Staining, Molecular Weight, Buffer Exchange, Flow Cytometry

38) Product Images from "Ultrasensitive Detection of Proteins on Western Blots with Semiconducting Polymer Dots"

Article Title: Ultrasensitive Detection of Proteins on Western Blots with Semiconducting Polymer Dots

Journal: Macromolecular rapid communications

doi: 10.1002/marc.201200809

(A) Structure of CN-PPV polymer and the absorption (dashed) and emission (solid) spectra of CN-PPV Pdot (black) and CN-PPV Pdot-streptavidin conjugate (CNPPV-Strep, red) in a 0.1% PEG 20 mM HEPES buffer. (B) The absorption and emission spectra of Qdot
Figure Legend Snippet: (A) Structure of CN-PPV polymer and the absorption (dashed) and emission (solid) spectra of CN-PPV Pdot (black) and CN-PPV Pdot-streptavidin conjugate (CNPPV-Strep, red) in a 0.1% PEG 20 mM HEPES buffer. (B) The absorption and emission spectra of Qdot

Techniques Used:

39) Product Images from "Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps"

Article Title: Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00184

Extracellular acidosis leads to decreased phorbol myristate acetate (PMA)- and immobilized immune complex (iIC)-induced reactive oxygen species (ROS) production in N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)- and bicarbonate-buffered media . Neutrophils (2 × 10 6 /ml) were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with PMA, iIC, or left untreated. Real-time analysis of intra- and extracellular myeloperoxidase-dependent ROS was monitored by using the luminol-assay for 1 h at 37°C (under CO 2 for bicarbonate-buffered conditions). (A,B,D,E) Representative real-time kinetics and (C,F) area under the curve (AUC) values (mean ± SEM) of ROS-dependent chemiluminescence intensities (RLUs). n = 3–9 independent experiments (* p
Figure Legend Snippet: Extracellular acidosis leads to decreased phorbol myristate acetate (PMA)- and immobilized immune complex (iIC)-induced reactive oxygen species (ROS) production in N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)- and bicarbonate-buffered media . Neutrophils (2 × 10 6 /ml) were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with PMA, iIC, or left untreated. Real-time analysis of intra- and extracellular myeloperoxidase-dependent ROS was monitored by using the luminol-assay for 1 h at 37°C (under CO 2 for bicarbonate-buffered conditions). (A,B,D,E) Representative real-time kinetics and (C,F) area under the curve (AUC) values (mean ± SEM) of ROS-dependent chemiluminescence intensities (RLUs). n = 3–9 independent experiments (* p

Techniques Used:

40) Product Images from "Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells"

Article Title: Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

Journal: BMC Research Notes

doi: 10.1186/1756-0500-3-220

Effects of α-santalol on the distribution of A431 cells in the different phases of the cell cycle . A431 Cells were treated with α-santalol (0 μM-75 μM) for 6 h ( A and B ), 12 h ( C and D ) and 24 h ( E and F ). At the end of respective treatment, cells were harvested and digested with RNase. Cellular DNA was stained with propidium iodide and analyzed by flow cytometer as described in the Materials and Methods. Panels A , C and E are histograms representing different time treatment with α-santalol. Data in Panel B , D and F from the cell cycle distribution were summarized and presented as the mean ± SD of three observations. *, P
Figure Legend Snippet: Effects of α-santalol on the distribution of A431 cells in the different phases of the cell cycle . A431 Cells were treated with α-santalol (0 μM-75 μM) for 6 h ( A and B ), 12 h ( C and D ) and 24 h ( E and F ). At the end of respective treatment, cells were harvested and digested with RNase. Cellular DNA was stained with propidium iodide and analyzed by flow cytometer as described in the Materials and Methods. Panels A , C and E are histograms representing different time treatment with α-santalol. Data in Panel B , D and F from the cell cycle distribution were summarized and presented as the mean ± SD of three observations. *, P

Techniques Used: Staining, Flow Cytometry, Cytometry

41) Product Images from "Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction"

Article Title: Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

Journal: Adipocyte

doi: 10.1080/21623945.2020.1720480

Influence of a gradual decrease in the concentrations of insulin, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine (IBMX) and biotin in the induction medium to 30% and 10% of their original concentrations on lipid incorporation by bovine ASC. The ‘100% medium’ contained 10 µg/mL insulin, 1 µM dexamethasone, 20 µM rosiglitazone, 250 µM IBMX and 33 µM biotin in the recipes used by Riedel et al. [ 22 ]. The results are presented as means ± SEM of three independent experiments with two replicates. *Asterisks indicate an effect of factor day with P
Figure Legend Snippet: Influence of a gradual decrease in the concentrations of insulin, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine (IBMX) and biotin in the induction medium to 30% and 10% of their original concentrations on lipid incorporation by bovine ASC. The ‘100% medium’ contained 10 µg/mL insulin, 1 µM dexamethasone, 20 µM rosiglitazone, 250 µM IBMX and 33 µM biotin in the recipes used by Riedel et al. [ 22 ]. The results are presented as means ± SEM of three independent experiments with two replicates. *Asterisks indicate an effect of factor day with P

Techniques Used:

42) Product Images from "Activation of p53 signaling by MI-63 induces apoptosis in acute myelogenous leukemia cells"

Article Title: Activation of p53 signaling by MI-63 induces apoptosis in acute myelogenous leukemia cells

Journal: Leukemia & lymphoma

doi: 10.3109/10428191003731325

Annexin V positive fraction in leukemia progenitor cells (from patients with AML) treated with AraC and/or MI-63. Cells isolated from patients with AML, were treated with AraC and/or MI-63 at indicated concentrations and Annexin V positive fraction was
Figure Legend Snippet: Annexin V positive fraction in leukemia progenitor cells (from patients with AML) treated with AraC and/or MI-63. Cells isolated from patients with AML, were treated with AraC and/or MI-63 at indicated concentrations and Annexin V positive fraction was

Techniques Used: Isolation

43) Product Images from "Triggering of Suicidal Erythrocyte Death by Penta-O-galloyl-?-d-glucose"

Article Title: Triggering of Suicidal Erythrocyte Death by Penta-O-galloyl-?-d-glucose

Journal: Toxins

doi: 10.3390/toxins6010054

Effect of penta- O -galloyl-β- d -glucose (PGG) on erythrocyte forward scatter ( A ) Original histogram of forward scatter of erythrocytes following exposure for 48 h to Ringer solution without (grey) and with (black) the presence of 25 µM penta- O -galloyl-β- d -glucose; ( B ) Arithmetic means ± SEM ( n = 8) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) penta- O -galloyl-β- d -glucose (1–50 µM). * ( p
Figure Legend Snippet: Effect of penta- O -galloyl-β- d -glucose (PGG) on erythrocyte forward scatter ( A ) Original histogram of forward scatter of erythrocytes following exposure for 48 h to Ringer solution without (grey) and with (black) the presence of 25 µM penta- O -galloyl-β- d -glucose; ( B ) Arithmetic means ± SEM ( n = 8) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) penta- O -galloyl-β- d -glucose (1–50 µM). * ( p

Techniques Used: Incubation

Effect of penta- O -galloyl-β- d -glucose (PGG) on ceramide formation. ( A ) Original histogram of anti-ceramide FITC-fluorescence in erythrocytes following exposure for 48 h to Ringer solution without (grey) and with (black) the presence of 25 µM penta- O -galloyl-β- d -glucose; ( B ) Arithmetic means ± SEM ( n = 8) of ceramide abundance after a 48 h incubation in Ringer solution without (white bar) or with (black bars) penta- O -galloyl-β- d -glucose (25 µM). ** ( p
Figure Legend Snippet: Effect of penta- O -galloyl-β- d -glucose (PGG) on ceramide formation. ( A ) Original histogram of anti-ceramide FITC-fluorescence in erythrocytes following exposure for 48 h to Ringer solution without (grey) and with (black) the presence of 25 µM penta- O -galloyl-β- d -glucose; ( B ) Arithmetic means ± SEM ( n = 8) of ceramide abundance after a 48 h incubation in Ringer solution without (white bar) or with (black bars) penta- O -galloyl-β- d -glucose (25 µM). ** ( p

Techniques Used: Fluorescence, Incubation

Effect of penta- O -galloyl-β- d -glucose (PGG) on phosphatidylserine exposure ( A ) Confocal images of FITC-dependent fluorescence (upper panels) and light microscopy (lower panels) of human erthrocytes following exposure for 48 h to Ringer solution without (left panel) and with (right panel) the presence of 25 µM penta- O -galloyl-β- d -glucose; ( B ) Original histogram of annexin V binding of erythrocytes following exposure for 48 h to Ringer solution without (grey) and with (black) the presence of 25 µM penta- O -galloyl-β- d -glucose. ( C ) Arithmetic means ± SEM of erythrocyte annexin V binding ( n = 8) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) the presence of penta- O -galloyl-β- d -glucose (1–50 µM). * ( p
Figure Legend Snippet: Effect of penta- O -galloyl-β- d -glucose (PGG) on phosphatidylserine exposure ( A ) Confocal images of FITC-dependent fluorescence (upper panels) and light microscopy (lower panels) of human erthrocytes following exposure for 48 h to Ringer solution without (left panel) and with (right panel) the presence of 25 µM penta- O -galloyl-β- d -glucose; ( B ) Original histogram of annexin V binding of erythrocytes following exposure for 48 h to Ringer solution without (grey) and with (black) the presence of 25 µM penta- O -galloyl-β- d -glucose. ( C ) Arithmetic means ± SEM of erythrocyte annexin V binding ( n = 8) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) the presence of penta- O -galloyl-β- d -glucose (1–50 µM). * ( p

Techniques Used: Fluorescence, Light Microscopy, Binding Assay, Incubation

Effect of penta- O -galloyl-β- d -glucose (PGG) on hemolysis. Arithmetic means ± SEM of the percentage hemolytic erythrocytes ( n = 8) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) the presence of penta- O -galloyl-β- d -glucose (1–50 µM). *** ( p
Figure Legend Snippet: Effect of penta- O -galloyl-β- d -glucose (PGG) on hemolysis. Arithmetic means ± SEM of the percentage hemolytic erythrocytes ( n = 8) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) the presence of penta- O -galloyl-β- d -glucose (1–50 µM). *** ( p

Techniques Used: Incubation

Effect of penta- O -galloyl-β- d -glucose (PGG) on phosphatidylserine exposure in the presence and absence of extracellular Ca 2+ . Arithmetic means ± SEM ( n = 8) of the percentage of annexin V binding erythrocytes after a 48-h treatment with Ringer solution without (white bar) or with (black bars) 25 µM penta- O -galloyl-β- d -glucose in the presence (left bars, Plus Calcium) and absence (right bars, Minus Calcium) of calcium. *** ( p
Figure Legend Snippet: Effect of penta- O -galloyl-β- d -glucose (PGG) on phosphatidylserine exposure in the presence and absence of extracellular Ca 2+ . Arithmetic means ± SEM ( n = 8) of the percentage of annexin V binding erythrocytes after a 48-h treatment with Ringer solution without (white bar) or with (black bars) 25 µM penta- O -galloyl-β- d -glucose in the presence (left bars, Plus Calcium) and absence (right bars, Minus Calcium) of calcium. *** ( p

Techniques Used: Binding Assay

44) Product Images from "Rapid Maturation of Effector T Cells in Tumors, but Not Lymphoid Organs, during Tumor Regression"

Article Title: Rapid Maturation of Effector T Cells in Tumors, but Not Lymphoid Organs, during Tumor Regression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0000821

Increased cell death over time in tumor-infiltrating DUC18 CTL. Thy1.1 DUC 18 effector T cells were transferred into tumor-bearing recipients on day 0. On the indicated days, organs were harvested and stained for Thy1.1/Vβ8.3 + DUC18 T cells. A) The percentage of DUC18 T cells that were dead was determined by gating on the DUC18 T cell population and analyzing the PI bright percentage. Data were pooled from 9 mice in 3 independent experiments. B) Organs were harvested on day 6 post-T cell transfer, and samples were stained with Thy1.1, Vβ8.3, Annexin V Alexa 488 and 7AAD. Histograms are shown after gating on the DUC18 T cell population, and represent data from a single mouse. C) The percentage of Annexin V + DUC18 T cells is shown, based on the region shown in the histogram in B (p value for % apoptotic DUC18 T cells at day 6 in tumors versus dLNs = 0.0002 and for tumors versus spleens = 0.0007). Cumulative data from 3 independent experiments are shown.
Figure Legend Snippet: Increased cell death over time in tumor-infiltrating DUC18 CTL. Thy1.1 DUC 18 effector T cells were transferred into tumor-bearing recipients on day 0. On the indicated days, organs were harvested and stained for Thy1.1/Vβ8.3 + DUC18 T cells. A) The percentage of DUC18 T cells that were dead was determined by gating on the DUC18 T cell population and analyzing the PI bright percentage. Data were pooled from 9 mice in 3 independent experiments. B) Organs were harvested on day 6 post-T cell transfer, and samples were stained with Thy1.1, Vβ8.3, Annexin V Alexa 488 and 7AAD. Histograms are shown after gating on the DUC18 T cell population, and represent data from a single mouse. C) The percentage of Annexin V + DUC18 T cells is shown, based on the region shown in the histogram in B (p value for % apoptotic DUC18 T cells at day 6 in tumors versus dLNs = 0.0002 and for tumors versus spleens = 0.0007). Cumulative data from 3 independent experiments are shown.

Techniques Used: CTL Assay, Staining, Mouse Assay, Significance Assay

45) Product Images from "Ubiquitination of ?-Synuclein Is Not Required for Formation of Pathological Inclusions in ?-Synucleinopathies"

Article Title: Ubiquitination of ?-Synuclein Is Not Required for Formation of Pathological Inclusions in ?-Synucleinopathies

Journal: The American Journal of Pathology

doi:

Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.
Figure Legend Snippet: Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.

Techniques Used: Western Blot, Recombinant, Immunoprecipitation, Isolation, Modification, In Vitro

46) Product Images from "Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence"

Article Title: Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

Journal: Reproductive Biology and Endocrinology : RB & E

doi: 10.1186/1477-7827-9-45

In vitro incorporation of FITC-labeled Vtg derived from the bubble-eye goldfish by zebrafish oocytes . (A) Stability of FITC-labeled Vtg in the culture medium. The medium was subjected to SDS-PAGE after 48 h of incubation at 28°C. (B) Ovarian explant at 48 h of culture with FITC-labeled Vtg solution. (C) FITC fluorescence in the explant. (D) Enlargement of the ovary emitting FITC fluorescence. (E) Ovary explant cultured with purified FITC-labeled Vtg. Arrows show ovaries emitting FITC fluorescence. Scale bar = 200 μm.
Figure Legend Snippet: In vitro incorporation of FITC-labeled Vtg derived from the bubble-eye goldfish by zebrafish oocytes . (A) Stability of FITC-labeled Vtg in the culture medium. The medium was subjected to SDS-PAGE after 48 h of incubation at 28°C. (B) Ovarian explant at 48 h of culture with FITC-labeled Vtg solution. (C) FITC fluorescence in the explant. (D) Enlargement of the ovary emitting FITC fluorescence. (E) Ovary explant cultured with purified FITC-labeled Vtg. Arrows show ovaries emitting FITC fluorescence. Scale bar = 200 μm.

Techniques Used: In Vitro, Labeling, Derivative Assay, SDS Page, Incubation, Fluorescence, Cell Culture, Purification

Schematic representation of a system for monitoring Vtg incorporation into zebrafish oocytes using FITC-labeled Vtg prepared from bubble-eye goldfish .
Figure Legend Snippet: Schematic representation of a system for monitoring Vtg incorporation into zebrafish oocytes using FITC-labeled Vtg prepared from bubble-eye goldfish .

Techniques Used: Labeling

Preparation and subsequent monitoring of FITC-labeled Vtg accumulation in the ovary after abdominal injection . (A) SDS-PAGE analysis of eye-sac lymph proteins after FITC labeling (two leftmost lanes), and assessment of FITC-labeled Vtg stability in zebrafish body fluid (two rightmost lanes). Leftmost lane shows FITC-fluorescence, indicating strong emission from Vtg, and the next lane shows the same gel stained with Coomassie Brilliant Blue (CBB). Of the two rightmost lanes, the first shows FITC-fluorescence in zebrafish body fluid collected 24 h after injection of FITC-labeled Vtg solution into the abdominal cavity, and the next lane shows the same sample stained with CBB. (B-D) Fluorescent images of zebrafish with the left abdominal wall dissected at 1, 2 and 3 days after injection of FITC-labeled Vtg solution. (E) Section of the zebrafish ovary shown in D. (F) SDS-PAGE of purified FITC-labeled Vtg and its incorporation into oocytes. (G) SDS-PAGE of FITC-labeled male goldfish lymph, and zebrafish ovary 72 h after injection of this solution. (H) SDS-PAGE of FITC-labeled BSA, and zebrafish ovary 72 h after injection of this solution. Li: liver; Oc: oocyte; Ov: ovary. Scale bar = 2 mm in B-D and F-H, 200 μm in E.
Figure Legend Snippet: Preparation and subsequent monitoring of FITC-labeled Vtg accumulation in the ovary after abdominal injection . (A) SDS-PAGE analysis of eye-sac lymph proteins after FITC labeling (two leftmost lanes), and assessment of FITC-labeled Vtg stability in zebrafish body fluid (two rightmost lanes). Leftmost lane shows FITC-fluorescence, indicating strong emission from Vtg, and the next lane shows the same gel stained with Coomassie Brilliant Blue (CBB). Of the two rightmost lanes, the first shows FITC-fluorescence in zebrafish body fluid collected 24 h after injection of FITC-labeled Vtg solution into the abdominal cavity, and the next lane shows the same sample stained with CBB. (B-D) Fluorescent images of zebrafish with the left abdominal wall dissected at 1, 2 and 3 days after injection of FITC-labeled Vtg solution. (E) Section of the zebrafish ovary shown in D. (F) SDS-PAGE of purified FITC-labeled Vtg and its incorporation into oocytes. (G) SDS-PAGE of FITC-labeled male goldfish lymph, and zebrafish ovary 72 h after injection of this solution. (H) SDS-PAGE of FITC-labeled BSA, and zebrafish ovary 72 h after injection of this solution. Li: liver; Oc: oocyte; Ov: ovary. Scale bar = 2 mm in B-D and F-H, 200 μm in E.

Techniques Used: Labeling, Injection, SDS Page, Fluorescence, Staining, Purification

Development of zebrafish embryos that incorporated FITC-labeled Vtg, heterogeneously prepared from the bubble-eye goldfish, in the yolk during oocyte growth . Embryonic stage is given as days post fertilization (dpf). Scale bar = 200 μm.
Figure Legend Snippet: Development of zebrafish embryos that incorporated FITC-labeled Vtg, heterogeneously prepared from the bubble-eye goldfish, in the yolk during oocyte growth . Embryonic stage is given as days post fertilization (dpf). Scale bar = 200 μm.

Techniques Used: Labeling

47) Product Images from "Early protection to stress mediated by CDK-dependent PI3,5P2 signaling from the vacuole/lysosome"

Article Title: Early protection to stress mediated by CDK-dependent PI3,5P2 signaling from the vacuole/lysosome

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201611144

Phosphorylation of the Fab1 complex by Pho85–Pho80 leads to a change in its conformation and/or composition. (A) Schematic of Fab1-TEVr 1790 -HA 6 . Phosphorylation sites identified by alanine mutagenesis and TEVr 1790 are indicated. (B) A fab1Δ mutant expressing pFab1-TEVr 1790 -HA 6 or pFab1 8A -TEV 1790 -HA 6 exposed to 0.9 M NaCl for 0 min (Basal) or 5 min (Salt) and lysed with TEV buffer. After limited digestion with TEVp (++, 0.125 U/µl; +, 0.0625 U/µl) for 10 or 30 min at 4°C, reactions were terminated with an equal amount of 2× SDS sample buffer. Samples were separated by SDS-PAGE. Western blot: anti-HA or Pgk1 (control). Black arrowheads indicate full-length Fab1 or C termini of Fab1 generated by TEV cleavage. Blots are representative of three independent experiments. (C and D) Quantifications of band intensities from osmotic shock (C) and basal (D) experiments from 0.125 U/µl TEVp with 30 min treatment. Means ± SD are shown. n = 3. *, P
Figure Legend Snippet: Phosphorylation of the Fab1 complex by Pho85–Pho80 leads to a change in its conformation and/or composition. (A) Schematic of Fab1-TEVr 1790 -HA 6 . Phosphorylation sites identified by alanine mutagenesis and TEVr 1790 are indicated. (B) A fab1Δ mutant expressing pFab1-TEVr 1790 -HA 6 or pFab1 8A -TEV 1790 -HA 6 exposed to 0.9 M NaCl for 0 min (Basal) or 5 min (Salt) and lysed with TEV buffer. After limited digestion with TEVp (++, 0.125 U/µl; +, 0.0625 U/µl) for 10 or 30 min at 4°C, reactions were terminated with an equal amount of 2× SDS sample buffer. Samples were separated by SDS-PAGE. Western blot: anti-HA or Pgk1 (control). Black arrowheads indicate full-length Fab1 or C termini of Fab1 generated by TEV cleavage. Blots are representative of three independent experiments. (C and D) Quantifications of band intensities from osmotic shock (C) and basal (D) experiments from 0.125 U/µl TEVp with 30 min treatment. Means ± SD are shown. n = 3. *, P

Techniques Used: Mutagenesis, Expressing, SDS Page, Western Blot, Generated

Putative Pho85–Pho80 target sites in Fab1 and Vac7 are phosphorylated in vivo. (A and C) A fab1Δ mutant expressing pFab1-TEVr 1506 -HA 6 or pFab1 8A ( T1569A , T1583A , T1594A , T1691A , S1924A , T1953A , T1963A , and S2166A )–TEV 1506 -HA 6 (Basal; A) or incubated with 0.9 M NaCl for 5 min (Osmotic shock; C). Samples were then lysed with 10% TCA. Lysates were treated with TEVp in the presence or absence of PPase. 6% and 9% SDS-PAGE gels were used. Western blot: anti-HA or anti-Pgk1 (control). (B and D) Quantifications of data in A and C, respectively. (E and G) A vac7Δ mutant expressing pVac7-TEVr 572 -HA 3 , Vac7 5A ( S593A , S607A , T858A , S903A , and S912A )–TEVr 572 –HA 3 or Vac7 7A ( S593A , S607A , T858A , S903A , S912A , T1050A , and S1074A )–TEVr 572 -HA 3 (Basal; E) or treated with 0.9 M NaCl for 5 min (Osmotic shock; G). Samples were then lysed with 10% TCA. Lysates were treated with TEVp in the presence or absence of PPase. Western blot: anti-HA or anti-Pgk1 (control). Blots are representative of three independent experiments. (F and H) Quantifications of data in E and G, respectively. Means ± SD are shown. n = 3. *, P
Figure Legend Snippet: Putative Pho85–Pho80 target sites in Fab1 and Vac7 are phosphorylated in vivo. (A and C) A fab1Δ mutant expressing pFab1-TEVr 1506 -HA 6 or pFab1 8A ( T1569A , T1583A , T1594A , T1691A , S1924A , T1953A , T1963A , and S2166A )–TEV 1506 -HA 6 (Basal; A) or incubated with 0.9 M NaCl for 5 min (Osmotic shock; C). Samples were then lysed with 10% TCA. Lysates were treated with TEVp in the presence or absence of PPase. 6% and 9% SDS-PAGE gels were used. Western blot: anti-HA or anti-Pgk1 (control). (B and D) Quantifications of data in A and C, respectively. (E and G) A vac7Δ mutant expressing pVac7-TEVr 572 -HA 3 , Vac7 5A ( S593A , S607A , T858A , S903A , and S912A )–TEVr 572 –HA 3 or Vac7 7A ( S593A , S607A , T858A , S903A , S912A , T1050A , and S1074A )–TEVr 572 -HA 3 (Basal; E) or treated with 0.9 M NaCl for 5 min (Osmotic shock; G). Samples were then lysed with 10% TCA. Lysates were treated with TEVp in the presence or absence of PPase. Western blot: anti-HA or anti-Pgk1 (control). Blots are representative of three independent experiments. (F and H) Quantifications of data in E and G, respectively. Means ± SD are shown. n = 3. *, P

Techniques Used: In Vivo, Mutagenesis, Expressing, Incubation, SDS Page, Western Blot

48) Product Images from "Investigating the function and possible biological role of an acetylcholine-gated chloride channel subunit (ACC-1) from the parasitic nematode Haemonchus contortus"

Article Title: Investigating the function and possible biological role of an acetylcholine-gated chloride channel subunit (ACC-1) from the parasitic nematode Haemonchus contortus

Journal: International Journal for Parasitology: Drugs and Drug Resistance

doi: 10.1016/j.ijpddr.2018.10.010

Hco-ACC-1 and Hco-ACC-2 form a functional heteromeric receptor. A. Representative electrophysiological traces of the acetylcholine responses to oocytes expressing Hco-ACC-1 alone, Hco-ACC-2 alone and Hco-ACC-1 + 2. B. Does response curves comparing the sensitivities of the Hco-ACC-2 channel and the Hco-ACC-1/2 channel to acetylcholine and carbachol. Each data point is a mean ± SEM with n ≥ 4. C. Current/Voltage relationship of the Hco-ACC-1/2 channel comparing full Cl − ND96 (103.6 mM) partial Cl − ND96 (62.5 mM). D. Ligand docking of acetylcholine to the Hco-ACC1/2 receptor shown the distance of the ligand to W225. E. Ligand docking of carbachol to the Hco-ACC-1/2 receptor shown the distance of the ligand to W225.
Figure Legend Snippet: Hco-ACC-1 and Hco-ACC-2 form a functional heteromeric receptor. A. Representative electrophysiological traces of the acetylcholine responses to oocytes expressing Hco-ACC-1 alone, Hco-ACC-2 alone and Hco-ACC-1 + 2. B. Does response curves comparing the sensitivities of the Hco-ACC-2 channel and the Hco-ACC-1/2 channel to acetylcholine and carbachol. Each data point is a mean ± SEM with n ≥ 4. C. Current/Voltage relationship of the Hco-ACC-1/2 channel comparing full Cl − ND96 (103.6 mM) partial Cl − ND96 (62.5 mM). D. Ligand docking of acetylcholine to the Hco-ACC1/2 receptor shown the distance of the ligand to W225. E. Ligand docking of carbachol to the Hco-ACC-1/2 receptor shown the distance of the ligand to W225.

Techniques Used: Functional Assay, Expressing

49) Product Images from "Replication protein A promotes 5?- > 3? end processing during homology-dependent DNA double-strand break repair"

Article Title: Replication protein A promotes 5?- > 3? end processing during homology-dependent DNA double-strand break repair

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201005110

Functional and physical interactions between RPA and xDNA2. (A) The effect of RPA on xDNA2’s 5′→3′ exonuclease activity against two different single-stranded oligonucleotides. The substrates were labeled with 32 P-labeled dA (marked by the asterisks) and attached to Streptavidin paramagnetic beads via the 3′ biotin-dC. After incubation at room temperature for 1 h, the reactions were stopped with SDS-EDTA, boiled for 10 min, and separated on a 10% TAE-PAGE. The percentage of the substrate undegraded was relative to the total signal for each reaction. The sizes of the products were determined by separating on a sequencing gel (not depicted). (B) The effect of RPA and T4 gp32 on the nuclease activity of xDNA2. The substrate, 48mer-1 beads, was incubated with various proteins as indicated at room temperature for 1 h and analyzed similarly to that in A. (C) Coimmunoprecipitation of RPA and xDNA2. The immunoprecipitates were separated on an 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed for different proteins by Western blotting. For RPA, a rat antibody against the p70 subunit was used for Western blotting. Untreated cytosol was loaded at the indicated amounts to provide the standard for quantitation. White lines indicate that intervening lanes have been spliced out. (D) Interaction between the purified RPA and xDNA2. FLAG beads were precoated with either recombinant xDNA2 or BSA and then incubated with the purified RPA protein. The beads and supernatant fractions were analyzed similarly to that in C. xRPA, Xenopus RPA. Ab, antibody.
Figure Legend Snippet: Functional and physical interactions between RPA and xDNA2. (A) The effect of RPA on xDNA2’s 5′→3′ exonuclease activity against two different single-stranded oligonucleotides. The substrates were labeled with 32 P-labeled dA (marked by the asterisks) and attached to Streptavidin paramagnetic beads via the 3′ biotin-dC. After incubation at room temperature for 1 h, the reactions were stopped with SDS-EDTA, boiled for 10 min, and separated on a 10% TAE-PAGE. The percentage of the substrate undegraded was relative to the total signal for each reaction. The sizes of the products were determined by separating on a sequencing gel (not depicted). (B) The effect of RPA and T4 gp32 on the nuclease activity of xDNA2. The substrate, 48mer-1 beads, was incubated with various proteins as indicated at room temperature for 1 h and analyzed similarly to that in A. (C) Coimmunoprecipitation of RPA and xDNA2. The immunoprecipitates were separated on an 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed for different proteins by Western blotting. For RPA, a rat antibody against the p70 subunit was used for Western blotting. Untreated cytosol was loaded at the indicated amounts to provide the standard for quantitation. White lines indicate that intervening lanes have been spliced out. (D) Interaction between the purified RPA and xDNA2. FLAG beads were precoated with either recombinant xDNA2 or BSA and then incubated with the purified RPA protein. The beads and supernatant fractions were analyzed similarly to that in C. xRPA, Xenopus RPA. Ab, antibody.

Techniques Used: Functional Assay, Recombinase Polymerase Amplification, Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Sequencing, SDS Page, Western Blot, Quantitation Assay, Purification, Recombinant

50) Product Images from "Cordycepin Down-Regulates Multiple Drug Resistant (MDR)/HIF-1α through Regulating AMPK/mTORC1 Signaling in GBC-SD Gallbladder Cancer Cells"

Article Title: Cordycepin Down-Regulates Multiple Drug Resistant (MDR)/HIF-1α through Regulating AMPK/mTORC1 Signaling in GBC-SD Gallbladder Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms150712778

Cordycepin induces GBC-SD cell apoptosis. GBC-SD cells were treated with indicated cordycepin (10–100 μM) for 72 h, cell apoptosis was tested by Annexin V/PI FACS ( A – C ) and Caspase-3 activity assay ( D ); Note that for Annexin V FACS assay, the results of five sets of experiments were quantified ( B , C ); GBC-SD cells were treated with z-VAD-fmk (50 μM) for 1 h, followed by cordycepin (25/100 μM) stimulation, cells were further cultured for 72 h, and cell viability was tested ( E ); Experiments were repeated five times. * Stands for p
Figure Legend Snippet: Cordycepin induces GBC-SD cell apoptosis. GBC-SD cells were treated with indicated cordycepin (10–100 μM) for 72 h, cell apoptosis was tested by Annexin V/PI FACS ( A – C ) and Caspase-3 activity assay ( D ); Note that for Annexin V FACS assay, the results of five sets of experiments were quantified ( B , C ); GBC-SD cells were treated with z-VAD-fmk (50 μM) for 1 h, followed by cordycepin (25/100 μM) stimulation, cells were further cultured for 72 h, and cell viability was tested ( E ); Experiments were repeated five times. * Stands for p

Techniques Used: FACS, Caspase-3 Activity Assay, Cell Culture

51) Product Images from "Chemiluminescence Resonance Energy Transfer-based Detection for Microchip Electrophoresis"

Article Title: Chemiluminescence Resonance Energy Transfer-based Detection for Microchip Electrophoresis

Journal: Analytical chemistry

doi: 10.1021/ac9027643

CL spectra of the luminol-NaBrO-QD system in the presence of analytes. Peaks: a. GSH; b. Epinephrine; c. Ala; d. Estradiol; e. no analyte; CL reaction buffer (pH10.5) was 30 mM borate solution containing 1.5×10 −4 M luminol, 3.0×10
Figure Legend Snippet: CL spectra of the luminol-NaBrO-QD system in the presence of analytes. Peaks: a. GSH; b. Epinephrine; c. Ala; d. Estradiol; e. no analyte; CL reaction buffer (pH10.5) was 30 mM borate solution containing 1.5×10 −4 M luminol, 3.0×10

Techniques Used:

52) Product Images from "Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis"

Article Title: Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.005914

Exosite is critical for stabilization of putative protein-binding interface. A, regions identified by H/Dx-MS are displayed on the caspase-6 structure 3K7E. The NTD ( green ) containing the exosite patch ( blue ) and 240's regions ( red ) make contacts with the CTD ( orange ) on the reverse face of caspase-6. B, rotating caspase-6 90° reveals a potential binding interface that occurs in the tetrameric helical structures of caspase-6, incorporating all regions identified by H/Dx. C, ), containing large portions of buried surface area primarily consisting of buried hydrophobics. Residue label subscripts correspond to the chain of the crystal structure. D, fluorescence polarization assay of FITC-labeled caspase-6 C163S FL constructs of either the WT ( black ), R42–44A ( white ), or R44K ( gray ) variants incubated with the WT unlabeled protein as a substrate to assess K D apparent. E as in D except incubated with their respective unlabeled R42–44A C163S FL or R44K C163S FL variants. F, K D values of all labeled proteins incubated with the unlabeled WT substrate are not statistically significantly different, as compared to those incubated with R42–44A or R44K unlabeled proteins, which have a weaker K D value. In all cases, the caspase-6 variants are the ΔN D179CT form.
Figure Legend Snippet: Exosite is critical for stabilization of putative protein-binding interface. A, regions identified by H/Dx-MS are displayed on the caspase-6 structure 3K7E. The NTD ( green ) containing the exosite patch ( blue ) and 240's regions ( red ) make contacts with the CTD ( orange ) on the reverse face of caspase-6. B, rotating caspase-6 90° reveals a potential binding interface that occurs in the tetrameric helical structures of caspase-6, incorporating all regions identified by H/Dx. C, ), containing large portions of buried surface area primarily consisting of buried hydrophobics. Residue label subscripts correspond to the chain of the crystal structure. D, fluorescence polarization assay of FITC-labeled caspase-6 C163S FL constructs of either the WT ( black ), R42–44A ( white ), or R44K ( gray ) variants incubated with the WT unlabeled protein as a substrate to assess K D apparent. E as in D except incubated with their respective unlabeled R42–44A C163S FL or R44K C163S FL variants. F, K D values of all labeled proteins incubated with the unlabeled WT substrate are not statistically significantly different, as compared to those incubated with R42–44A or R44K unlabeled proteins, which have a weaker K D value. In all cases, the caspase-6 variants are the ΔN D179CT form.

Techniques Used: Protein Binding, Mass Spectrometry, Binding Assay, Fluorescence, Labeling, Construct, Incubation

POOL results show distal sites are important for biochemical function. POOL analysis performed on an ensemble of caspase-6 crystal structures in various activation states and conformations indicated by PDB codes in the figure. The top 12% of residues identified and predicted to be important for enzymatic function for each structure are highlighted in gray . Residues further clustered based on their conformational state: helical or strand, as well as their proximity to the caspase-6 active-site, 130's region, or distal positions.
Figure Legend Snippet: POOL results show distal sites are important for biochemical function. POOL analysis performed on an ensemble of caspase-6 crystal structures in various activation states and conformations indicated by PDB codes in the figure. The top 12% of residues identified and predicted to be important for enzymatic function for each structure are highlighted in gray . Residues further clustered based on their conformational state: helical or strand, as well as their proximity to the caspase-6 active-site, 130's region, or distal positions.

Techniques Used: Activation Assay

Disruption of the 42 RRR 44 exosite increases dynamics of caspase-6 variants in NTD, 240's regions. A and B, caspase-6, R42–44A, or R44K incubated in D 2 O and monitored for exchange over time. Difference plots generated by subtracting the deuterium uptake profiles of common peptides of WT caspase-6 from peptides from either R42–44A ( A ) or R44K ( B ) variants. Regions depicted in blue (negative) are less exchanging in the variant than in the WT, whereas regions in red (positive) were more exchanging in the variant than in WT based on the error of the data sets ±0.42 or ±0.73 for the R42–44A or R44K based on the 98 and 95% confidence interval, respectively. C and D, )) of the R42–44A ( C ) or R44K ( D ) variants. E, relative deuterium uptake difference scale. F–L, important peptides identified as significantly different upon exchange compared with the WT peptides. Experiments were repeated on 2 different days. Δ indicates the difference, wherein the data from WT is always subtracted from the data from the indicated variant. In all cases, the caspase-6 variants are the ΔN D179CT form.
Figure Legend Snippet: Disruption of the 42 RRR 44 exosite increases dynamics of caspase-6 variants in NTD, 240's regions. A and B, caspase-6, R42–44A, or R44K incubated in D 2 O and monitored for exchange over time. Difference plots generated by subtracting the deuterium uptake profiles of common peptides of WT caspase-6 from peptides from either R42–44A ( A ) or R44K ( B ) variants. Regions depicted in blue (negative) are less exchanging in the variant than in the WT, whereas regions in red (positive) were more exchanging in the variant than in WT based on the error of the data sets ±0.42 or ±0.73 for the R42–44A or R44K based on the 98 and 95% confidence interval, respectively. C and D, )) of the R42–44A ( C ) or R44K ( D ) variants. E, relative deuterium uptake difference scale. F–L, important peptides identified as significantly different upon exchange compared with the WT peptides. Experiments were repeated on 2 different days. Δ indicates the difference, wherein the data from WT is always subtracted from the data from the indicated variant. In all cases, the caspase-6 variants are the ΔN D179CT form.

Techniques Used: Incubation, Generated, Variant Assay

42 RRR 44 exosite functions as a hinge between NTD and core, stabilizing caspase-6. A–C, differential scanning fluorimetry was used to observe how substitutions in the 42 RRR 44 hinge impact the stability of caspase-6 ( A ), R44K ( B ), or R42–44A ( C ) in the unliganded state or liganded to the caspase-6 cognate inhibitor Ac-VEID-cho state. D–F, changes in the observed thermal stability may be attributed to sequential loss of interactions as side chains are removed. D, WT; E, 42 RRK 44 (R44K), and F, 42 AAA 44 (R42–44A). Models ( E and F ) were produced using the PyMOL mutagenesis wizard to demonstrate potential loss in contacts. In all cases the caspase-6 variants are the ΔN D179CT form.
Figure Legend Snippet: 42 RRR 44 exosite functions as a hinge between NTD and core, stabilizing caspase-6. A–C, differential scanning fluorimetry was used to observe how substitutions in the 42 RRR 44 hinge impact the stability of caspase-6 ( A ), R44K ( B ), or R42–44A ( C ) in the unliganded state or liganded to the caspase-6 cognate inhibitor Ac-VEID-cho state. D–F, changes in the observed thermal stability may be attributed to sequential loss of interactions as side chains are removed. D, WT; E, 42 RRK 44 (R44K), and F, 42 AAA 44 (R42–44A). Models ( E and F ) were produced using the PyMOL mutagenesis wizard to demonstrate potential loss in contacts. In all cases the caspase-6 variants are the ΔN D179CT form.

Techniques Used: Produced, Mutagenesis

53) Product Images from "Critical Role of S1PR1 and Integrin ?4 in HGF/c-Met-mediated Increases in Vascular Integrity"

Article Title: Critical Role of S1PR1 and Integrin ?4 in HGF/c-Met-mediated Increases in Vascular Integrity

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.404780

c-Met/S1PR1/ITGB4 complex modulation by HGF and S1P. A , HPAEC were treated with either S1P (1 μ m ) or HGF (25 ng/ml) for 1 and 5 min. Cell lysates were subjected to immunoprecipitation with an anti-ITGB4 antibody and tyrosine or threonine phosphorylation
Figure Legend Snippet: c-Met/S1PR1/ITGB4 complex modulation by HGF and S1P. A , HPAEC were treated with either S1P (1 μ m ) or HGF (25 ng/ml) for 1 and 5 min. Cell lysates were subjected to immunoprecipitation with an anti-ITGB4 antibody and tyrosine or threonine phosphorylation

Techniques Used: Immunoprecipitation

54) Product Images from "Alternating Electric Fields (TTFields) Activate Cav1.2 Channels in Human Glioblastoma Cells"

Article Title: Alternating Electric Fields (TTFields) Activate Cav1.2 Channels in Human Glioblastoma Cells

Journal: Cancers

doi: 10.3390/cancers11010110

TTFields induce a dihydropyridine-sensitive Ca 2+ -entry in human glioblastoma cells. ( A ) Time course of TTFields (200 kHz, 2.5 V/cm, 3 min)-induced changes of mean (±SE; n = 8–20) fura-2 340/380 nm fluorescence ratio as recorded in T98G (left) and U251 (right) cells with Ca 2+ (1 mM)-containing NaCl-solution (closed triangles) and EGTA (0.6 mM)-buffered Ca 2+ -free NaCl-solution (open circles). ( B ) Mean (±SE; n = 13–20) fura-2 340/380 nm fluorescence ratio recorded in T98G (left) and U251 cells (right) before, during, and after application of TTFields (200 kHz, 2.5 V/cm, 3 min) during continuous superfusion with Ca 2+ -containing NaCl-solution (open circles), Ca 2+ -containing NaCl solution further containing 1 µM benidipine (red triangles) or 1 µM nifedipine (blue diamonds). ( C ) Mean (±SE; n = 13–39) fura-2 340/380 nm fluorescence ratio recorded in T98G (left) and U251 cells (right) before, during, and after application of TTFields (200 kHz, 2.5 V/cm, 3 min) during continuous superfusion with benidipine (1 µM, top, red symbols) or nifedipine (1 µM, bottom, blue symbols)-containing NaCl-solution and after wash-out of the inhibitors (open symbols). ( D ) Mean (±SE; n = 34–63) slope (as indicated by white lines in ( C )) of the fura-2 340/380 nm fluorescence ratio changes before (control), at the end and shortly after TTF-application (middle) both in the presence of benidipine (red) or nifedipine (blue) as well as after wash-out of the inhibitors. * and *** in ( D ) indicate 3 p ≤ 0.05 and 3 p ≤ 0.01, respectively, (Welch)-corrected t -test and Bonferroni correction for 3 pairwise comparisons.
Figure Legend Snippet: TTFields induce a dihydropyridine-sensitive Ca 2+ -entry in human glioblastoma cells. ( A ) Time course of TTFields (200 kHz, 2.5 V/cm, 3 min)-induced changes of mean (±SE; n = 8–20) fura-2 340/380 nm fluorescence ratio as recorded in T98G (left) and U251 (right) cells with Ca 2+ (1 mM)-containing NaCl-solution (closed triangles) and EGTA (0.6 mM)-buffered Ca 2+ -free NaCl-solution (open circles). ( B ) Mean (±SE; n = 13–20) fura-2 340/380 nm fluorescence ratio recorded in T98G (left) and U251 cells (right) before, during, and after application of TTFields (200 kHz, 2.5 V/cm, 3 min) during continuous superfusion with Ca 2+ -containing NaCl-solution (open circles), Ca 2+ -containing NaCl solution further containing 1 µM benidipine (red triangles) or 1 µM nifedipine (blue diamonds). ( C ) Mean (±SE; n = 13–39) fura-2 340/380 nm fluorescence ratio recorded in T98G (left) and U251 cells (right) before, during, and after application of TTFields (200 kHz, 2.5 V/cm, 3 min) during continuous superfusion with benidipine (1 µM, top, red symbols) or nifedipine (1 µM, bottom, blue symbols)-containing NaCl-solution and after wash-out of the inhibitors (open symbols). ( D ) Mean (±SE; n = 34–63) slope (as indicated by white lines in ( C )) of the fura-2 340/380 nm fluorescence ratio changes before (control), at the end and shortly after TTF-application (middle) both in the presence of benidipine (red) or nifedipine (blue) as well as after wash-out of the inhibitors. * and *** in ( D ) indicate 3 p ≤ 0.05 and 3 p ≤ 0.01, respectively, (Welch)-corrected t -test and Bonferroni correction for 3 pairwise comparisons.

Techniques Used: Fluorescence

55) Product Images from "Kaposi Sarcoma-associated Herpesvirus vIRF-3 Protein Binds to F-box of Skp2 Protein and Acts as a Regulator of c-Myc Protein Function and Stability *"

Article Title: Kaposi Sarcoma-associated Herpesvirus vIRF-3 Protein Binds to F-box of Skp2 Protein and Acts as a Regulator of c-Myc Protein Function and Stability *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.335216

vIRF-3 interacts with F-box of Skp2. A , schematic representation of Skp2 deletion mutants tagged with Myc tag. The shaded box indicates the vIRF-3-binding domain. LRR , leucine-rich region. B , co-immunoprecipitation of Skp2 and vIRF-3 in PEL cells, BC-3
Figure Legend Snippet: vIRF-3 interacts with F-box of Skp2. A , schematic representation of Skp2 deletion mutants tagged with Myc tag. The shaded box indicates the vIRF-3-binding domain. LRR , leucine-rich region. B , co-immunoprecipitation of Skp2 and vIRF-3 in PEL cells, BC-3

Techniques Used: Binding Assay, Immunoprecipitation

56) Product Images from "Localization of the Blue-Light Receptor Phototropin to the Flagella of the Green Alga Chlamydomonas reinhardtii"

Article Title: Localization of the Blue-Light Receptor Phototropin to the Flagella of the Green Alga Chlamydomonas reinhardtii

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-01-0010

Association of phototropin with the axoneme fraction of the flagella. (A) Flagella isolated by the pH shock method were separated into an M+M fraction and an axoneme (A) fraction by using 0.05 or 0.5% NP-40 as described in MATERIALS AND METHODS. To 50 μlof flagella suspension (protein concentration 1–2 μg/μl), 50 μl of HMDEK buffer with 2 times detergent was added, and the mixture was centrifuged. The supernatant represents the M+M fraction. The pellet was resuspended in 100 μl of HMDEK buffer with detergent, resulting in the axoneme fraction. One hundred microliters each of the M+M and A fractions as well as 50 μl of total flagella proteins (F) were separated by SDS-PAGE and analyzed by Western blot (note that the amount of protein in these three samples was not the same). In addition to the phototropin-specific antibody, antibodies directed against the major flagellar membrane glycoprotein, FMG-1, RSP1 (one of the radial spoke proteins), and FLA10 (kinesin-II motor protein) were used. (B) Flagella were isolated from cells grown in the light or in the dark. The flagella were fractionated with 0.5% NP-40 in the presence of 10 mM ATP. The same amount of protein (7 μg) from each sample was separated by SDS-PAGE and used for immunoblotting (bottom). Top, gel stained with Coomassie Blue. The positions of marker proteins are shown on the right side.
Figure Legend Snippet: Association of phototropin with the axoneme fraction of the flagella. (A) Flagella isolated by the pH shock method were separated into an M+M fraction and an axoneme (A) fraction by using 0.05 or 0.5% NP-40 as described in MATERIALS AND METHODS. To 50 μlof flagella suspension (protein concentration 1–2 μg/μl), 50 μl of HMDEK buffer with 2 times detergent was added, and the mixture was centrifuged. The supernatant represents the M+M fraction. The pellet was resuspended in 100 μl of HMDEK buffer with detergent, resulting in the axoneme fraction. One hundred microliters each of the M+M and A fractions as well as 50 μl of total flagella proteins (F) were separated by SDS-PAGE and analyzed by Western blot (note that the amount of protein in these three samples was not the same). In addition to the phototropin-specific antibody, antibodies directed against the major flagellar membrane glycoprotein, FMG-1, RSP1 (one of the radial spoke proteins), and FLA10 (kinesin-II motor protein) were used. (B) Flagella were isolated from cells grown in the light or in the dark. The flagella were fractionated with 0.5% NP-40 in the presence of 10 mM ATP. The same amount of protein (7 μg) from each sample was separated by SDS-PAGE and used for immunoblotting (bottom). Top, gel stained with Coomassie Blue. The positions of marker proteins are shown on the right side.

Techniques Used: Isolation, Protein Concentration, SDS Page, Western Blot, Staining, Marker

Fractionation of flagella by the freeze/thaw method. One freeze-thaw cycle in HMDEK buffer in the absence of ATP was performed. After centrifugation, a supernatant fraction (soluble fraction, S) was obtained. The pellet was resuspended in HMDEK buffer containing 1% NP-40 and 10 mM ATP. Recentrifugation of this fraction yielded the membrane fraction (M) in the supernatant and the axoneme fraction (A) in the pellet. The same amount of protein (9 μg) was loaded per lane. For a control, antibodies FMG-1 (directed against the major flagellar membrane glycoprotein), RSP1 (one of the radical spoke proteins), and FLA10 (kinesin-II motor protein) were used.
Figure Legend Snippet: Fractionation of flagella by the freeze/thaw method. One freeze-thaw cycle in HMDEK buffer in the absence of ATP was performed. After centrifugation, a supernatant fraction (soluble fraction, S) was obtained. The pellet was resuspended in HMDEK buffer containing 1% NP-40 and 10 mM ATP. Recentrifugation of this fraction yielded the membrane fraction (M) in the supernatant and the axoneme fraction (A) in the pellet. The same amount of protein (9 μg) was loaded per lane. For a control, antibodies FMG-1 (directed against the major flagellar membrane glycoprotein), RSP1 (one of the radical spoke proteins), and FLA10 (kinesin-II motor protein) were used.

Techniques Used: Fractionation, Centrifugation

57) Product Images from "Colorimetric in situ assay of membrane-bound enzyme based on lipid bilayer inhibition of ion transport"

Article Title: Colorimetric in situ assay of membrane-bound enzyme based on lipid bilayer inhibition of ion transport

Journal: Theranostics

doi: 10.7150/thno.25123

(A) Schematic illustration for the mechanism of α-Glu assay via inhibition of lipid bilayer on iron release. (B) UV-vis spectra of the mixtures prepared by separate addition of ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 1, black curve), pAPG (20 μL, 20 μM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 2, red curve), α-Glu (10 μL, 1 U/mL) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 3, blue curve), α-Glu (10 μL, 1 U/mL) + pAPG (20 μL, 20 μM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 4, green curve), Fe 2+ (5 μL, 25 mM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 5, pink curve), pAPG (20 μL, 20 μM) + Fe 2+ (5 μL, 25 mM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 6, yellow curve), α-Glu (10 μL, 1 U/mL) + Fe 2+ (5 μL, 25 mM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 7, navy curve), APBA/AMNSs + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 8, wine curve), pAPG/APBA/AMNSs + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 9, Rose red curve), LB/pAPG/APBA/AMNSs + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 10, palegreen curve), and LB/pAPG/APBA/AMNSs + α-Glu (10 μL, 1 U/mL) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 11, dark blue curve) into a tube with the final volume of 140 μL by adding 100 mM MES buffer solution. Inset: the corresponding magnified spectra.
Figure Legend Snippet: (A) Schematic illustration for the mechanism of α-Glu assay via inhibition of lipid bilayer on iron release. (B) UV-vis spectra of the mixtures prepared by separate addition of ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 1, black curve), pAPG (20 μL, 20 μM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 2, red curve), α-Glu (10 μL, 1 U/mL) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 3, blue curve), α-Glu (10 μL, 1 U/mL) + pAPG (20 μL, 20 μM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 4, green curve), Fe 2+ (5 μL, 25 mM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 5, pink curve), pAPG (20 μL, 20 μM) + Fe 2+ (5 μL, 25 mM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 6, yellow curve), α-Glu (10 μL, 1 U/mL) + Fe 2+ (5 μL, 25 mM) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 7, navy curve), APBA/AMNSs + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 8, wine curve), pAPG/APBA/AMNSs + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 9, Rose red curve), LB/pAPG/APBA/AMNSs + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 10, palegreen curve), and LB/pAPG/APBA/AMNSs + α-Glu (10 μL, 1 U/mL) + ABTS (20 μL, 5 mM) + H 2 O 2 (20 μL, 4 mM) (vial 11, dark blue curve) into a tube with the final volume of 140 μL by adding 100 mM MES buffer solution. Inset: the corresponding magnified spectra.

Techniques Used: Inhibition

(A) UV-vis spectra and photographs (inset) of the mixtures prepared by the addition of various numbers of Caco-2 cells. Inset, from vial 1 to vial 15: the cell numbers are 0, 0.5×10 2 , 0.8×10 2 , 1.0×10 2 , 0.5×10 3 , 1.0×10 3 , 0.5×10 4 , 1.0×10 4 , 0.5×10 5 , 1.0×10 5 , 0.5×10 6 , 1.0×10 6 , 0.5×10 7 , 1.0×10 7 , and 0.5×10 8 cells/mL. (B) UV-vis spectra and photographs (inset) of mixtures, (C) absorbance values, and (D) inhibition ratios following the addition of various concentrations of GA to Caco-2 cells. Inset (B), vial 1 to vial 9: the GA concentrations are 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, and 1.6 mM. (E) UV-vis spectra and photographs (inset) of mixtures, (F) absorbance values, and (G) inhibition ratios following the addition of various concentrations of Que to Caco-2 cells. Inset (E), vial 1 to vial 9: the Que concentrations are 0.2, 0.9, 1.8, 3.6, 5.4, 7.2, 9, 10.8, 12.6, 14.4, and 16.2 μM. (H) IC 50 values of GA and Que on purified α-Glu (PG) and membrane-bound α-Glu (MBG). Cell numbers are 5×10 7 cells/mL. Error bars indicate standard deviations (n = 3).
Figure Legend Snippet: (A) UV-vis spectra and photographs (inset) of the mixtures prepared by the addition of various numbers of Caco-2 cells. Inset, from vial 1 to vial 15: the cell numbers are 0, 0.5×10 2 , 0.8×10 2 , 1.0×10 2 , 0.5×10 3 , 1.0×10 3 , 0.5×10 4 , 1.0×10 4 , 0.5×10 5 , 1.0×10 5 , 0.5×10 6 , 1.0×10 6 , 0.5×10 7 , 1.0×10 7 , and 0.5×10 8 cells/mL. (B) UV-vis spectra and photographs (inset) of mixtures, (C) absorbance values, and (D) inhibition ratios following the addition of various concentrations of GA to Caco-2 cells. Inset (B), vial 1 to vial 9: the GA concentrations are 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, and 1.6 mM. (E) UV-vis spectra and photographs (inset) of mixtures, (F) absorbance values, and (G) inhibition ratios following the addition of various concentrations of Que to Caco-2 cells. Inset (E), vial 1 to vial 9: the Que concentrations are 0.2, 0.9, 1.8, 3.6, 5.4, 7.2, 9, 10.8, 12.6, 14.4, and 16.2 μM. (H) IC 50 values of GA and Que on purified α-Glu (PG) and membrane-bound α-Glu (MBG). Cell numbers are 5×10 7 cells/mL. Error bars indicate standard deviations (n = 3).

Techniques Used: Inhibition, Purification

58) Product Images from "CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling"

Article Title: CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200705090

CD98hc–integrin interaction mediates Fn assembly. (A) CD98hc-deficient MEFs reconstituted with each chimera depicted in B (C98T69E98, C69T98E98, or C98T98E69) were treated as described in Fig. 3 A . Fixed cells were stained for Fn (green), actin (red), and nucleus (blue). Only C98T98E69 rescued CD98hc-deficient cells' ability to assemble Fn fibrils. Bars, 25 μm. (B) Expression and schematic of the chimeras used in A. CD98hc protein is depicted in black and CD69 in gray. Each chimera is defined by its cytoplasmic (C), transmembrane (T), or extracellular (E) domain derived from either CD98hc (98) or CD69 (69). CD98hc extracellular domain is necessary and sufficient for amino acid transport, whereas the intracellular and transmembrane domains are required for interactions with integrins ( Fenczik et al., 2001 ). Flow cytometry analysis (right) of cell surface expression of exogenous chimeras in CD98hc-deficient cells (filled histogram) is shown. Control staining (empty histogram) was performed with irrelevant IgG. (C) CD98–integrin association is required for contraction of the extracellular matrix. WT, CD98hc-deficient, and CD98hc-deficient MEFs reconstituted with C98T98E69 were mixed with 200 μl of Fn-containing platelet-poor plasma, 200 μl of 28 mM CaCl 2 , and 5 U/ml human thrombin in Hepes-DME. Tubes were incubated for 1 h at 37°C. Depicted are digital pictures of clots, as well as the calculated percentage of clot contraction (see Materials and methods). Values represent the mean ± SEM of triplicate determinations.
Figure Legend Snippet: CD98hc–integrin interaction mediates Fn assembly. (A) CD98hc-deficient MEFs reconstituted with each chimera depicted in B (C98T69E98, C69T98E98, or C98T98E69) were treated as described in Fig. 3 A . Fixed cells were stained for Fn (green), actin (red), and nucleus (blue). Only C98T98E69 rescued CD98hc-deficient cells' ability to assemble Fn fibrils. Bars, 25 μm. (B) Expression and schematic of the chimeras used in A. CD98hc protein is depicted in black and CD69 in gray. Each chimera is defined by its cytoplasmic (C), transmembrane (T), or extracellular (E) domain derived from either CD98hc (98) or CD69 (69). CD98hc extracellular domain is necessary and sufficient for amino acid transport, whereas the intracellular and transmembrane domains are required for interactions with integrins ( Fenczik et al., 2001 ). Flow cytometry analysis (right) of cell surface expression of exogenous chimeras in CD98hc-deficient cells (filled histogram) is shown. Control staining (empty histogram) was performed with irrelevant IgG. (C) CD98–integrin association is required for contraction of the extracellular matrix. WT, CD98hc-deficient, and CD98hc-deficient MEFs reconstituted with C98T98E69 were mixed with 200 μl of Fn-containing platelet-poor plasma, 200 μl of 28 mM CaCl 2 , and 5 U/ml human thrombin in Hepes-DME. Tubes were incubated for 1 h at 37°C. Depicted are digital pictures of clots, as well as the calculated percentage of clot contraction (see Materials and methods). Values represent the mean ± SEM of triplicate determinations.

Techniques Used: Staining, Expressing, Derivative Assay, Flow Cytometry, Cytometry, Incubation

CD98hc mediates cellular traction forces on the extracellular matrix. (A) WT and CD98hc-deficient MEFs were mixed with 200 μl of Fn-containing platelet-poor plasma, 200 μl of 28 mM CaCl 2 , and 5 U/ml human thrombin in Hepes-DME. Tubes were incubated for 2 h at 37°C. Depicted are digital images of WT and CD98hc-null clots. (B) Quantification of the percentage of clot contraction is presented (see Materials and methods). Values represent the mean ± SEM of triplicate determinations. The assay was repeated three times with similar results. (C) Decreased cellular traction forces in CD98-deficient MEFs. WT and CD98hc-deficient MEFs were plated on a 120-kD fragment of Fn-coated polyacrylamide sheets, in which fluorescent beads were embedded, as described in Materials and methods. Strain maps (green) overlaid with the brightfield images of WT (top) and CD98hc-null (bottom) cells plated on 120-kD Fn-coated polyacrylamide substrate (gray). CD98hc-deficient cells demonstrate reduced traction forces compared with WT cells. Bars, 10 μm.
Figure Legend Snippet: CD98hc mediates cellular traction forces on the extracellular matrix. (A) WT and CD98hc-deficient MEFs were mixed with 200 μl of Fn-containing platelet-poor plasma, 200 μl of 28 mM CaCl 2 , and 5 U/ml human thrombin in Hepes-DME. Tubes were incubated for 2 h at 37°C. Depicted are digital images of WT and CD98hc-null clots. (B) Quantification of the percentage of clot contraction is presented (see Materials and methods). Values represent the mean ± SEM of triplicate determinations. The assay was repeated three times with similar results. (C) Decreased cellular traction forces in CD98-deficient MEFs. WT and CD98hc-deficient MEFs were plated on a 120-kD fragment of Fn-coated polyacrylamide sheets, in which fluorescent beads were embedded, as described in Materials and methods. Strain maps (green) overlaid with the brightfield images of WT (top) and CD98hc-null (bottom) cells plated on 120-kD Fn-coated polyacrylamide substrate (gray). CD98hc-deficient cells demonstrate reduced traction forces compared with WT cells. Bars, 10 μm.

Techniques Used: Incubation

59) Product Images from "Modified FGF4 Signal Peptide Inhibits Entry of Herpes Simplex Virus Type 1"

Article Title: Modified FGF4 Signal Peptide Inhibits Entry of Herpes Simplex Virus Type 1

Journal: Journal of Virology

doi: 10.1128/JVI.75.6.2634-2645.2001

Entry phase and dose response. (A) Cell cultures (2 × 10 5 cells/well) in 96-well strip plates were switched to HEPES-buffered serum-supplemented DMEM, cooled on ice for 30 min, and infected at 4°C with 6 × 10 3 PFU of hr R3 per well. Cells were kept at 4°C for an additional 1 h before they were warmed to 23°C for 30 min and 37°C for the rest of the experiment. At 1-h intervals immediately following infection, strips of wells were treated for 1-h periods with 50 μM EB in serum-free DMEM and returned to regular medium. Before and after each treatment, the cells were rinsed three times with serum-free DMEM and serum-supplemented DMEM, respectively. The number of lacZ + cells was scored 11 h postinfection (●) and normalized to the number counted in mock-treated controls (maximally 187 ± 20 [ n = 3]). Separate strips of infected control cells were fixed and stained immediately following each mock treatment to monitor β-galactosidase expression over time (○). Data points represent means of triplicate measurements with standard errors of the means. (B) Cell cultures (2 × 10 5 cells/well) were switched to serum-free DMEM, cooled on ice, and infected at 4°C with 2.4 × 10 3 PFU of hr R3 per well. After infection, the cells were rinsed and treated with EB (●) or EBX (○) for 1 h at 4°C and for additional 30-min periods at 23 and 37°C. Triplicate counts of lacZ + cells were performed 6 h later (all points are means and standard errors of means; control score, 141 ± 5.9).
Figure Legend Snippet: Entry phase and dose response. (A) Cell cultures (2 × 10 5 cells/well) in 96-well strip plates were switched to HEPES-buffered serum-supplemented DMEM, cooled on ice for 30 min, and infected at 4°C with 6 × 10 3 PFU of hr R3 per well. Cells were kept at 4°C for an additional 1 h before they were warmed to 23°C for 30 min and 37°C for the rest of the experiment. At 1-h intervals immediately following infection, strips of wells were treated for 1-h periods with 50 μM EB in serum-free DMEM and returned to regular medium. Before and after each treatment, the cells were rinsed three times with serum-free DMEM and serum-supplemented DMEM, respectively. The number of lacZ + cells was scored 11 h postinfection (●) and normalized to the number counted in mock-treated controls (maximally 187 ± 20 [ n = 3]). Separate strips of infected control cells were fixed and stained immediately following each mock treatment to monitor β-galactosidase expression over time (○). Data points represent means of triplicate measurements with standard errors of the means. (B) Cell cultures (2 × 10 5 cells/well) were switched to serum-free DMEM, cooled on ice, and infected at 4°C with 2.4 × 10 3 PFU of hr R3 per well. After infection, the cells were rinsed and treated with EB (●) or EBX (○) for 1 h at 4°C and for additional 30-min periods at 23 and 37°C. Triplicate counts of lacZ + cells were performed 6 h later (all points are means and standard errors of means; control score, 141 ± 5.9).

Techniques Used: Stripping Membranes, Infection, Staining, Expressing

Effect of pretreating cells with EB on subsequent virus infection. Cell cultures (2 × 10 5 cells/well) in microtiter wells were switched to serum-free HEPES-buffered DMEM and incubated for 30 min at 37°C. A first set of cells were then pretreated for 1 h with EB, rinsed three times, and infected for 1 h with 7,400 PFU of hr R3 per well before they were returned to regular medium. Rinses and infections were carried out in the absence of EB in either serum-free (Δ) or serum-supplemented (▴) DMEM. A second set of cells was pretreated for 1 h with (●) or without (○) EB and infected for 1 h with 7,400 PFU of hr R3 per well in the presence of EB before they were returned to regular medium. Triplicate counts of lacZ + cells were performed 8 h postinfection (all points are means with standard errors of the means; control score, 265 ± 13 [ n = 3]).
Figure Legend Snippet: Effect of pretreating cells with EB on subsequent virus infection. Cell cultures (2 × 10 5 cells/well) in microtiter wells were switched to serum-free HEPES-buffered DMEM and incubated for 30 min at 37°C. A first set of cells were then pretreated for 1 h with EB, rinsed three times, and infected for 1 h with 7,400 PFU of hr R3 per well before they were returned to regular medium. Rinses and infections were carried out in the absence of EB in either serum-free (Δ) or serum-supplemented (▴) DMEM. A second set of cells was pretreated for 1 h with (●) or without (○) EB and infected for 1 h with 7,400 PFU of hr R3 per well in the presence of EB before they were returned to regular medium. Triplicate counts of lacZ + cells were performed 8 h postinfection (all points are means with standard errors of the means; control score, 265 ± 13 [ n = 3]).

Techniques Used: Infection, Incubation

60) Product Images from "Role of ?-arrestin1/ERK MAP kinase pathway in regulating adenosine A1 receptor desensitization and recovery"

Article Title: Role of ?-arrestin1/ERK MAP kinase pathway in regulating adenosine A1 receptor desensitization and recovery

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00190.2009

Desensitization of A 1 adenosine receptor (A 1 AR) after agonist treatment. A : DDT 1 MF-2 cells were treated with 1 μM R -phenylisopropyladenosine ( R -PIA) for different time periods, and A 1 AR antagonist radioligand [ 3 H-labeled 8-cyclopentyl-1,3-di[2′,3′]propylxanthine (DPCPX)] binding was performed on membrane preparations. *Statistically significant difference from control ( P
Figure Legend Snippet: Desensitization of A 1 adenosine receptor (A 1 AR) after agonist treatment. A : DDT 1 MF-2 cells were treated with 1 μM R -phenylisopropyladenosine ( R -PIA) for different time periods, and A 1 AR antagonist radioligand [ 3 H-labeled 8-cyclopentyl-1,3-di[2′,3′]propylxanthine (DPCPX)] binding was performed on membrane preparations. *Statistically significant difference from control ( P

Techniques Used: Labeling, Binding Assay

61) Product Images from "Functional and structural similarity of human DNA primase [4Fe4S] cluster domain constructs"

Article Title: Functional and structural similarity of human DNA primase [4Fe4S] cluster domain constructs

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209345

p58C 266-464 participates in redox switching on DNA. The cartoon (top left) depicts p58C DNA binding and redox switching on an Au electrode. (Bottom left) CV scan of electrochemically unaltered p58C 266-464 . (Right) bulk oxidation (above) of p58C 266-464 and subsequent CV scans (below). This construct displays similar electrochemical behavior to p58C 272-464 . All electrochemistry was performed in anaerobic conditions with 40 μM [4Fe4S] p58C 266-464 in 20 mM HEPES (pH 7.2), 75 mM NaCl. CV was performed at 100 mV/s scan rate.
Figure Legend Snippet: p58C 266-464 participates in redox switching on DNA. The cartoon (top left) depicts p58C DNA binding and redox switching on an Au electrode. (Bottom left) CV scan of electrochemically unaltered p58C 266-464 . (Right) bulk oxidation (above) of p58C 266-464 and subsequent CV scans (below). This construct displays similar electrochemical behavior to p58C 272-464 . All electrochemistry was performed in anaerobic conditions with 40 μM [4Fe4S] p58C 266-464 in 20 mM HEPES (pH 7.2), 75 mM NaCl. CV was performed at 100 mV/s scan rate.

Techniques Used: Binding Assay, Construct

62) Product Images from "A Short Half-Life αIIbβ3 Antagonist ANTP266 Reduces Thrombus Formation"

Article Title: A Short Half-Life αIIbβ3 Antagonist ANTP266 Reduces Thrombus Formation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19082306

Effects of ANTP266 on the P-selectin expression and platelet spreading. ( A ) Effect of ANTP266 on the P-selectin expression. Platelet-rich plasma was preincubated with ANTP266 (50 μM) or the vehicle for 5 min at 37 °C. Activation was initiated by the addition of 20 μM ADP, 1 μg/mL collagen, 0.25 U/mL thrombin, or 2 μM U46619 for 5 min at 37 °C. P-selectin expression on platelet surface was detected by using flow cytometry. ( B ) Effect of ANTP266 on platelet spreading. Washed human platelets were allowed to adhere and spread on immobilized fibrinogen in a tissue culture incubator. After washing, the adherent platelets were fixed, permeabilized, stained, and captured under a fluorescence microscope. Spreading areas of adherent platelets were quantified using Metamorph software. Representative images fromat least three independent experiments with similar results. ( C ) Quantification of spreading areas (pixel number) of three random fields per experiment. Data are presented as mean ± SD ( n = 3). **** p
Figure Legend Snippet: Effects of ANTP266 on the P-selectin expression and platelet spreading. ( A ) Effect of ANTP266 on the P-selectin expression. Platelet-rich plasma was preincubated with ANTP266 (50 μM) or the vehicle for 5 min at 37 °C. Activation was initiated by the addition of 20 μM ADP, 1 μg/mL collagen, 0.25 U/mL thrombin, or 2 μM U46619 for 5 min at 37 °C. P-selectin expression on platelet surface was detected by using flow cytometry. ( B ) Effect of ANTP266 on platelet spreading. Washed human platelets were allowed to adhere and spread on immobilized fibrinogen in a tissue culture incubator. After washing, the adherent platelets were fixed, permeabilized, stained, and captured under a fluorescence microscope. Spreading areas of adherent platelets were quantified using Metamorph software. Representative images fromat least three independent experiments with similar results. ( C ) Quantification of spreading areas (pixel number) of three random fields per experiment. Data are presented as mean ± SD ( n = 3). **** p

Techniques Used: Expressing, Activation Assay, Flow Cytometry, Cytometry, Staining, Fluorescence, Microscopy, Software

63) Product Images from "ATP-Dependent Recruitment of Export Factor Aly/REF onto Intronless mRNAs by RNA Helicase UAP56 ▿"

Article Title: ATP-Dependent Recruitment of Export Factor Aly/REF onto Intronless mRNAs by RNA Helicase UAP56 ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01341-07

Recruitment of Aly/REF onto intronless mRNAs. (A) A mixture of 32 P-labeled in vitro-transcribed RNAs containing two intronless mRNAs (DHFR and Ftz mRNAs), CTE, U1ΔSm, and U6Δss snRNAs was injected into the nuclei of Xenopus ) or control antibody (anti-Myc, 9E10; Sigma). RNA precipitated with each antibody was recovered and analyzed by denaturing PAGE and autoradiography. The input lanes contained 10% of the input mixture. (B) The same 32 P-labeled RNA mixture as for panel A, except that tRNA Phe was supplemented, was incubated in 7.5% HNE, 12 mM HEPES-KOH (pH 7.9), 60 mM KCl, 1.6 mM MgCl 2 , 0.1 mM EDTA, 6% glycerol at 30°C for 30 min in the absence (−) or presence (+) of 2 mM ATP. After the incubation, immunoprecipitation was performed and the coprecipitated RNA was analyzed as described for panel A.
Figure Legend Snippet: Recruitment of Aly/REF onto intronless mRNAs. (A) A mixture of 32 P-labeled in vitro-transcribed RNAs containing two intronless mRNAs (DHFR and Ftz mRNAs), CTE, U1ΔSm, and U6Δss snRNAs was injected into the nuclei of Xenopus ) or control antibody (anti-Myc, 9E10; Sigma). RNA precipitated with each antibody was recovered and analyzed by denaturing PAGE and autoradiography. The input lanes contained 10% of the input mixture. (B) The same 32 P-labeled RNA mixture as for panel A, except that tRNA Phe was supplemented, was incubated in 7.5% HNE, 12 mM HEPES-KOH (pH 7.9), 60 mM KCl, 1.6 mM MgCl 2 , 0.1 mM EDTA, 6% glycerol at 30°C for 30 min in the absence (−) or presence (+) of 2 mM ATP. After the incubation, immunoprecipitation was performed and the coprecipitated RNA was analyzed as described for panel A.

Techniques Used: Labeling, In Vitro, Injection, Polyacrylamide Gel Electrophoresis, Autoradiography, Incubation, Immunoprecipitation

64) Product Images from "Evaluation of Skin Permeation and Retention of Topical Dapsone in Murine Cutaneous Leishmaniasis Lesions"

Article Title: Evaluation of Skin Permeation and Retention of Topical Dapsone in Murine Cutaneous Leishmaniasis Lesions

Journal: Pharmaceutics

doi: 10.3390/pharmaceutics11110607

Percentage of L. major or L. braziliensis amastigote reduction at different concentrations of DAP after 48 h of treatment. Results are expressed as mean ± SD ( n = 6).
Figure Legend Snippet: Percentage of L. major or L. braziliensis amastigote reduction at different concentrations of DAP after 48 h of treatment. Results are expressed as mean ± SD ( n = 6).

Techniques Used:

65) Product Images from "The C-terminus of interferon gamma receptor beta chain (IFN?R2) has antiapoptotic activity as a Bax inhibitor"

Article Title: The C-terminus of interferon gamma receptor beta chain (IFN?R2) has antiapoptotic activity as a Bax inhibitor

Journal:

doi:

IFNγR2 interacts with Bax. (A and B) Co-immunoprecipitation of endogenous IFNγR2 and Bax. HEK293T cells were lysed using CHAPS or NP40 buffer as described in Materials and Methods. Immunoprecipitation (IP) was performed in the same buffer
Figure Legend Snippet: IFNγR2 interacts with Bax. (A and B) Co-immunoprecipitation of endogenous IFNγR2 and Bax. HEK293T cells were lysed using CHAPS or NP40 buffer as described in Materials and Methods. Immunoprecipitation (IP) was performed in the same buffer

Techniques Used: Immunoprecipitation

66) Product Images from "Dopamine Receptors in Human Lymphocytes: Radioligand Binding and Quantitative RT-PCR Assays"

Article Title: Dopamine Receptors in Human Lymphocytes: Radioligand Binding and Quantitative RT-PCR Assays

Journal:

doi: 10.1016/j.jneumeth.2008.07.018

Binding of [ 3 H]SCH 23390 with PBMC in the assay buffers of different composition. Cells (3.8×10 6 ) were incubated with 0.8 nM of [ 3 H]SCH 23390 for 60 min at 23°C in Buffer 1 (170 mM Tris-HCl), Buffer 2 (50 mM Tris-HCL), or Buffer 3 (HBSS/HEPES).
Figure Legend Snippet: Binding of [ 3 H]SCH 23390 with PBMC in the assay buffers of different composition. Cells (3.8×10 6 ) were incubated with 0.8 nM of [ 3 H]SCH 23390 for 60 min at 23°C in Buffer 1 (170 mM Tris-HCl), Buffer 2 (50 mM Tris-HCL), or Buffer 3 (HBSS/HEPES).

Techniques Used: Binding Assay, Incubation

67) Product Images from "Residue Ionization and Ion Transport through OmpF Channels"

Article Title: Residue Ionization and Ion Transport through OmpF Channels

Journal: Biophysical Journal

doi:

Typical tracks of ion conductance for single trimeric OmpF channels reconstituted into planar lipid membranes at pH 3.9 ( A ), pH 5.5 ( B ), and pH 8.0 ( C ) demonstrate that voltage-induced channel closure is facilitated by low pH. The membrane was formed from DPhPC, the membrane bathing solutions contained 1 M KCl and 5 mM HEPES or MES. Time averaging was 100 ms. The dashed lines show zero current levels, the dotted lines in A designate a fully open state ( L3 , all thee monomers open), and two partially closed states ( L2 , two monomers open; L1 , one monomer open). Note the presence of residual conductance ( L0 ) after the total three-step closure ( A , B ).
Figure Legend Snippet: Typical tracks of ion conductance for single trimeric OmpF channels reconstituted into planar lipid membranes at pH 3.9 ( A ), pH 5.5 ( B ), and pH 8.0 ( C ) demonstrate that voltage-induced channel closure is facilitated by low pH. The membrane was formed from DPhPC, the membrane bathing solutions contained 1 M KCl and 5 mM HEPES or MES. Time averaging was 100 ms. The dashed lines show zero current levels, the dotted lines in A designate a fully open state ( L3 , all thee monomers open), and two partially closed states ( L2 , two monomers open; L1 , one monomer open). Note the presence of residual conductance ( L0 ) after the total three-step closure ( A , B ).

Techniques Used: Mass Spectrometry

68) Product Images from ""

Article Title:

Journal: The Journal of Pharmacology and Experimental Therapeutics

doi: 10.1124/jpet.117.244095

D3- β Arr is a functionally selective TSHR agonist of β -Arr 1-mediated signaling and does not antagonize TSH-induced cAMP production. (A) DiscoverX1 cells were exposed to 0.18 μ M TSH or 10 μ M D3- β Arr in HBSS with 1 mM IBMX for 1 hour. cAMP levels were measured by ELISA. In contrast to TSH, D3- β Arr-induced activation of the TSHR does not lead to cAMP production. The data are from three experiments with duplicate samples. (B and C) TSH does not antagonize TSHR stimulation of cAMP production. (B) DiscoverX1 cells were pretreated with DMSO (Basal) or 10 μ M D3- β Arr in HBSS for 30 minutes. Subsequently, cells were exposed to 18 nM TSH (EC 50 ) with or without 10 μ M D3- β Arr in HBSS with 1 mM IBMX for 1 hour. cAMP levels were measured by ELISA. The data are from three experiments with duplicate samples. (C) Primary cultures of human thyrocytes from five different donors were studied. Cells were pretreated with DMSO (basal) or 10 µ M D3- β Arr in HBSS/HEPES for 1 hour. Subsequently, cells were exposed to 90 nM TSH (EC 50 for cAMP in human thyrocytes) with or without 10 μ M D3- β Arr in HBSS with 1 mM IBMX for 2 hours at 37°C. Incubations were stopped, the cells were lysed, and cAMP levels were measured by ELISA. (A–C) The data are presented as means ± S.D. Statistical significance is determined by unpaired two-tailed t test (**** P
Figure Legend Snippet: D3- β Arr is a functionally selective TSHR agonist of β -Arr 1-mediated signaling and does not antagonize TSH-induced cAMP production. (A) DiscoverX1 cells were exposed to 0.18 μ M TSH or 10 μ M D3- β Arr in HBSS with 1 mM IBMX for 1 hour. cAMP levels were measured by ELISA. In contrast to TSH, D3- β Arr-induced activation of the TSHR does not lead to cAMP production. The data are from three experiments with duplicate samples. (B and C) TSH does not antagonize TSHR stimulation of cAMP production. (B) DiscoverX1 cells were pretreated with DMSO (Basal) or 10 μ M D3- β Arr in HBSS for 30 minutes. Subsequently, cells were exposed to 18 nM TSH (EC 50 ) with or without 10 μ M D3- β Arr in HBSS with 1 mM IBMX for 1 hour. cAMP levels were measured by ELISA. The data are from three experiments with duplicate samples. (C) Primary cultures of human thyrocytes from five different donors were studied. Cells were pretreated with DMSO (basal) or 10 µ M D3- β Arr in HBSS/HEPES for 1 hour. Subsequently, cells were exposed to 90 nM TSH (EC 50 for cAMP in human thyrocytes) with or without 10 μ M D3- β Arr in HBSS with 1 mM IBMX for 2 hours at 37°C. Incubations were stopped, the cells were lysed, and cAMP levels were measured by ELISA. (A–C) The data are presented as means ± S.D. Statistical significance is determined by unpaired two-tailed t test (**** P

Techniques Used: Enzyme-linked Immunosorbent Assay, Activation Assay, Two Tailed Test

69) Product Images from "Cryopreservation in liquid nitrogen of gonocytes from neonatal porcine testes stored at 4°C"

Article Title: Cryopreservation in liquid nitrogen of gonocytes from neonatal porcine testes stored at 4°C

Journal: Reproductive Medicine and Biology

doi: 10.1111/j.1447-0578.2008.00215.x

Lectin Dolichos biflorus agglutinin (DBA) staining of cultured testicular germ cells after storage at 4°C and after storage in liquid nitrogen. Cells were isolated from the testes after 2 h of storage at 4°C (a,c,e,g) or after 24 h of storage in Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) supplemented with HEPES at 4°C (b,d,f). Isolated cells were frozen in basic medium containing 10% glycerol (a,b), in basic medium containing 10% dimethylsulfoxide (DMSO) (c,d), in basic medium containing 10% DMSO and 0.07 mol/L sucrose (e,f,g) and then cultured. Cells were stained with DBA after 7 days (a–f) or 12 days (g) of culture. The gonocytes were recognized as the DBA‐positive cells (arrows). Bars = 50 µm.
Figure Legend Snippet: Lectin Dolichos biflorus agglutinin (DBA) staining of cultured testicular germ cells after storage at 4°C and after storage in liquid nitrogen. Cells were isolated from the testes after 2 h of storage at 4°C (a,c,e,g) or after 24 h of storage in Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) supplemented with HEPES at 4°C (b,d,f). Isolated cells were frozen in basic medium containing 10% glycerol (a,b), in basic medium containing 10% dimethylsulfoxide (DMSO) (c,d), in basic medium containing 10% DMSO and 0.07 mol/L sucrose (e,f,g) and then cultured. Cells were stained with DBA after 7 days (a–f) or 12 days (g) of culture. The gonocytes were recognized as the DBA‐positive cells (arrows). Bars = 50 µm.

Techniques Used: Staining, Cell Culture, Isolation, Modification

70) Product Images from "Good’s buffers as a basis for developing self-buffering and biocompatible ionic liquids for biological research †"

Article Title: Good’s buffers as a basis for developing self-buffering and biocompatible ionic liquids for biological research †

Journal: Green chemistry : an international journal and green chemistry resource : GC

doi: 10.1039/C4GC00328D

(a) The pH profiles of the investigated [N 1111 ][GB] in pure water at 20 ± 1 °C. (b) The pH profiles of the universal buffers; GB refers to a mixture of MES, HEPES, and CHES, whereas GB-ILs refer to [N 1111 ][MES], [N 1111 ][HEPES], and [N
Figure Legend Snippet: (a) The pH profiles of the investigated [N 1111 ][GB] in pure water at 20 ± 1 °C. (b) The pH profiles of the universal buffers; GB refers to a mixture of MES, HEPES, and CHES, whereas GB-ILs refer to [N 1111 ][MES], [N 1111 ][HEPES], and [N

Techniques Used:

Ternary phase diagrams for the systems composed of IL + K 3 C 6 H 5 O 7 + water at 25 °C and atmospheric pressure: (○) [N 4444 ]-[Tricine], (◆) [N 4444 ][MES], (■) [N 4444 ][TES], (●) [N 4444 ][HEPES], (—) [N 4444 ][CHES],
Figure Legend Snippet: Ternary phase diagrams for the systems composed of IL + K 3 C 6 H 5 O 7 + water at 25 °C and atmospheric pressure: (○) [N 4444 ]-[Tricine], (◆) [N 4444 ][MES], (■) [N 4444 ][TES], (●) [N 4444 ][HEPES], (—) [N 4444 ][CHES],

Techniques Used:

71) Product Images from "Lipid Membrane Adsorption Determines Photodynamic Efficiency of β-Imidazolyl-Substituted Porphyrins"

Article Title: Lipid Membrane Adsorption Determines Photodynamic Efficiency of β-Imidazolyl-Substituted Porphyrins

Journal: Biomolecules

doi: 10.3390/biom9120853

Dependence of the boundary potential due to adsorption of porphyrins (filled symbols, scale on the left) and the oxidation rate R of di-4-ANEPPS (blank symbols, scale on the right) on the pH. The measurements of Δφ b are presented for the all three β-imidazolyl porphyrin derivatives, but the oxidation rate—only for 1Zn. Measurements were performed in a buffer solution containing 20 mM KCl, 2 mM HEPES, 2 mM Tris and 2 mM citrate. The pH value was changed by adding HCl or KOH to the solutions on both sides of the membrane.
Figure Legend Snippet: Dependence of the boundary potential due to adsorption of porphyrins (filled symbols, scale on the left) and the oxidation rate R of di-4-ANEPPS (blank symbols, scale on the right) on the pH. The measurements of Δφ b are presented for the all three β-imidazolyl porphyrin derivatives, but the oxidation rate—only for 1Zn. Measurements were performed in a buffer solution containing 20 mM KCl, 2 mM HEPES, 2 mM Tris and 2 mM citrate. The pH value was changed by adding HCl or KOH to the solutions on both sides of the membrane.

Techniques Used: Adsorption

UV-VIS spectra of 5 μM solutions of β-imidazolium porphyrin derivatives in ethanol ( A ) and in water containing 20 mM KCl, 2 mM HEPES, 2 mM citrate and 2 mM Tris at various pH (1Zn—( B ); 1H2—( C ); and 1In—( D )).
Figure Legend Snippet: UV-VIS spectra of 5 μM solutions of β-imidazolium porphyrin derivatives in ethanol ( A ) and in water containing 20 mM KCl, 2 mM HEPES, 2 mM citrate and 2 mM Tris at various pH (1Zn—( B ); 1H2—( C ); and 1In—( D )).

Techniques Used:

Dependence of the oxidation rate ( R ) of di-4-ANEPPS on the concentration of porphyrins in the solution. The target molecules of di-4-ANEPPS were adsorbed either on the cis side (solid symbols) or on the trans side (blank symbols) of the membrane. The solution contained 20 mM KCl, 2 mM HEPES, pH 7. Di-4-ANEPPS was added in a concentration of 0.3 μM.
Figure Legend Snippet: Dependence of the oxidation rate ( R ) of di-4-ANEPPS on the concentration of porphyrins in the solution. The target molecules of di-4-ANEPPS were adsorbed either on the cis side (solid symbols) or on the trans side (blank symbols) of the membrane. The solution contained 20 mM KCl, 2 mM HEPES, pH 7. Di-4-ANEPPS was added in a concentration of 0.3 μM.

Techniques Used: Concentration Assay

Adsorption of porphyrins on the lipid membrane. The boundary potential changes due to the adsorption of three porphyrins were determined either by the inner field compensation (IFC) method (solid symbols), or as the ζ-potential change of liposomes (blank symbols), or from the nonactin-induced change of conductance (crosses). Measurements were performed in 20 mM KCl, 2 mM HEPES, pH 7.0 buffer solutions.
Figure Legend Snippet: Adsorption of porphyrins on the lipid membrane. The boundary potential changes due to the adsorption of three porphyrins were determined either by the inner field compensation (IFC) method (solid symbols), or as the ζ-potential change of liposomes (blank symbols), or from the nonactin-induced change of conductance (crosses). Measurements were performed in 20 mM KCl, 2 mM HEPES, pH 7.0 buffer solutions.

Techniques Used: Adsorption

72) Product Images from "T-lymphocyte Kv1.3 channel activation triggers the NLRP3 inflammasome signaling pathway in hypertensive patients"

Article Title: T-lymphocyte Kv1.3 channel activation triggers the NLRP3 inflammasome signaling pathway in hypertensive patients

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2017.4490

Current density changes of peripheral blood T-lymphocyte K v 1.3 channel from the hypertensive subjects in the (A) control and (B) following 4-AP intervention. Subsequent to perfusion by the 4-AP at a final concentration of 3 mmol/l, the current density of the T-lymphocyte membrane K v 1.3 channel in the 4-AP perfusion group was significantly lower, compared with the blank control group. 4-AP, 4-aminopyridine; K v 1.3, voltage-gated K + channel 1.3.
Figure Legend Snippet: Current density changes of peripheral blood T-lymphocyte K v 1.3 channel from the hypertensive subjects in the (A) control and (B) following 4-AP intervention. Subsequent to perfusion by the 4-AP at a final concentration of 3 mmol/l, the current density of the T-lymphocyte membrane K v 1.3 channel in the 4-AP perfusion group was significantly lower, compared with the blank control group. 4-AP, 4-aminopyridine; K v 1.3, voltage-gated K + channel 1.3.

Techniques Used: Concentration Assay

73) Product Images from "Biogenic Polyphosphate Nanoparticles from a Marine Cyanobacterium Synechococcus sp. PCC 7002: Production, Characterization, and Anti-Inflammatory Properties In Vitro"

Article Title: Biogenic Polyphosphate Nanoparticles from a Marine Cyanobacterium Synechococcus sp. PCC 7002: Production, Characterization, and Anti-Inflammatory Properties In Vitro

Journal: Marine Drugs

doi: 10.3390/md16090322

Characterization of biogenic polyphosphate nanoparticles (BPNPs) within the algae cells: ( a ) appearance of the Synechococcus 7002 culture, ( b ) typical transmission electron microscopic (TEM) image of thin sections of Synechococcus 7002 cells, ( c ) energy dispersive spectroscopy (EDS) analysis of the yellow circle region in panel b, and ( d ) fluorescence microscope image of Synechococcus 7002 cells stained with 4′,6-diamidino-2-phenylindole (DAPI, ×400).
Figure Legend Snippet: Characterization of biogenic polyphosphate nanoparticles (BPNPs) within the algae cells: ( a ) appearance of the Synechococcus 7002 culture, ( b ) typical transmission electron microscopic (TEM) image of thin sections of Synechococcus 7002 cells, ( c ) energy dispersive spectroscopy (EDS) analysis of the yellow circle region in panel b, and ( d ) fluorescence microscope image of Synechococcus 7002 cells stained with 4′,6-diamidino-2-phenylindole (DAPI, ×400).

Techniques Used: Transmission Assay, Transmission Electron Microscopy, Spectroscopy, Fluorescence, Microscopy, Staining

74) Product Images from "Interferon-γ regulates growth and controls Fcγ receptor expression and activation in human intestinal mast cells"

Article Title: Interferon-γ regulates growth and controls Fcγ receptor expression and activation in human intestinal mast cells

Journal: BMC Immunology

doi: 10.1186/1471-2172-15-27

Histamine and LTC 4 release from intestinal MCs after IgG-crosslinking. MCs were cultured with SCF and with or without IFN-γ (100 ng/ml) for 2 days. The last 24 h plasma IgG (10 μg/ml) was added to the culture medium if indicated. Then, MCs were washed and challenged with an anti-IgG mAb or and isotype control mAb for 60 min. FcϵRI-crosslinking induced by mAb 22E7 served as positive control and non-treated MCs as negative control. (A) Histamine (n = 6) and (B) LTC 4 (n = 4) were measured in the supernatants. Means ± SEM are shown. *p
Figure Legend Snippet: Histamine and LTC 4 release from intestinal MCs after IgG-crosslinking. MCs were cultured with SCF and with or without IFN-γ (100 ng/ml) for 2 days. The last 24 h plasma IgG (10 μg/ml) was added to the culture medium if indicated. Then, MCs were washed and challenged with an anti-IgG mAb or and isotype control mAb for 60 min. FcϵRI-crosslinking induced by mAb 22E7 served as positive control and non-treated MCs as negative control. (A) Histamine (n = 6) and (B) LTC 4 (n = 4) were measured in the supernatants. Means ± SEM are shown. *p

Techniques Used: Cell Culture, Positive Control, Negative Control

Binding of IgG subtypes on intestinal MCs. MCs were cultured in the presence of SCF with or without addition of IFN-γ (100 ng/ml) for the last 3 days. Then, MCs were incubated with IgG1-4 (500 μg/ml), respectively, or buffer control for 60 min. Binding of IgGs was analyzed by FACS with mAbs specific for the respective IgG subtype (dark gray peak). Isotype control (bright gray peak). One experiment out of three is shown.
Figure Legend Snippet: Binding of IgG subtypes on intestinal MCs. MCs were cultured in the presence of SCF with or without addition of IFN-γ (100 ng/ml) for the last 3 days. Then, MCs were incubated with IgG1-4 (500 μg/ml), respectively, or buffer control for 60 min. Binding of IgGs was analyzed by FACS with mAbs specific for the respective IgG subtype (dark gray peak). Isotype control (bright gray peak). One experiment out of three is shown.

Techniques Used: Binding Assay, Cell Culture, Incubation, FACS

75) Product Images from "Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation"

Article Title: Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.8b11912

Model protein tagging with TPAC. (a) Structure and mass difference of the histidine modification by TPAC. (b) Deconvoluted mass spectra showing ribonuclease A labeled by TPAC with concentration ranging from 0 to 2 mM. Single and double TPAC modified RNaseA: expected mass 13 817, 13 951 Da, found 13 816, 13 950 Da. Its crystal structure (PDB 1bel) is shown in the inset highlighting its major modified histidine. (c) Deconvoluted mass spectra showing ribonuclease A labeled with 1.5 mM of TPAC with different buffered pH. (d) Calmodulin, (e) myoglobin and (f) lysozyme are also labeled by TPAC as shown by their deconvoluted mass spectra. TPAC-modified calmodulin: expected mass 16 924 Da, found 16 924 Da; single, double and triple TPAC modified myoglobin: expected mass 17 085, 17 219, and 17 353 Da, found 17 085, 17 218, and 17 352 Da; single and double TPAC modified lysozyme: expected mass 14 439, 14 573 Da, found 14438, 14 572 Da. Their crystal structures (PDB 2o60, 1bje, and 193l) are shown as insets highlighting the major modified histidines. Conditions: 20 μ M protein, 2 mM TPAC in 25 mM HEPES with pH 8.5, room temperature, 1 h unless otherwise noted.
Figure Legend Snippet: Model protein tagging with TPAC. (a) Structure and mass difference of the histidine modification by TPAC. (b) Deconvoluted mass spectra showing ribonuclease A labeled by TPAC with concentration ranging from 0 to 2 mM. Single and double TPAC modified RNaseA: expected mass 13 817, 13 951 Da, found 13 816, 13 950 Da. Its crystal structure (PDB 1bel) is shown in the inset highlighting its major modified histidine. (c) Deconvoluted mass spectra showing ribonuclease A labeled with 1.5 mM of TPAC with different buffered pH. (d) Calmodulin, (e) myoglobin and (f) lysozyme are also labeled by TPAC as shown by their deconvoluted mass spectra. TPAC-modified calmodulin: expected mass 16 924 Da, found 16 924 Da; single, double and triple TPAC modified myoglobin: expected mass 17 085, 17 219, and 17 353 Da, found 17 085, 17 218, and 17 352 Da; single and double TPAC modified lysozyme: expected mass 14 439, 14 573 Da, found 14438, 14 572 Da. Their crystal structures (PDB 2o60, 1bje, and 193l) are shown as insets highlighting the major modified histidines. Conditions: 20 μ M protein, 2 mM TPAC in 25 mM HEPES with pH 8.5, room temperature, 1 h unless otherwise noted.

Techniques Used: Modification, Labeling, Concentration Assay

Related Articles

Clone Assay:

Article Title: Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system
Article Snippet: A section of the basigin gene encoding immunoglobin domains 1 and 2 of the short isoform (αα 22–205) was cloned with an N-terminal His6 tag followed by a tobacco etch virus (TEV) protease cleavage site. .. Experiments were performed at 20 °C in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 2 mg/mL dextran, and 1 mg/mL salmon sperm DNA (Sigma).

Dissection:

Article Title: Effect of acute acid-base disturbances on the phosphorylation of phospholipase C-γ1 and Erk1/2 in the renal proximal tubule
Article Snippet: Media for PT suspension For renal cortex dissection and PT enrichment, our base solution was half Dulbecco's modified Eagle's medium and half Ham's F-12 medium (DMEM-F12 50/50), purchased as a powder lacking l -glutamine and NaHCO3 (Catalog #90-091-PB, Corning, Cellgro® Mediatech Inc., Manassas, VA). .. We supplemented this DMEM-F12 50/50 media with 15 mmol/L HEPES, 2 mmol/L l -glutamine, 2 mmol/L heptanoic acid (Catalog #75190; Sigma-Aldrich, St. Louis, MO), and 2 mmol/L l -Lactic acid (30% in water by weight, Sigma-Aldrich, Catalog #L1875), and then titrated to pH 7.40.

Article Title: Functional and Morphological Analysis of OFF Bipolar Cells
Article Snippet: Paragraph title: 3.2. Retinal dissection ... Immediately after the solution is removed, a halved piece of Millipore filter paper is placed on top of the retinal slab and a drop of HEPES solution is placed on top of the Millipore filter paper.

Lambda DNA Preparation:

Article Title: Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins
Article Snippet: .. Lambda DNA, dNTPs (dATP, dTTP, dGTP), enzymes and buffers were purchased from Promega, the biotin-11-dCTP from Enzo, HEPES, NaCl and NaOH from Sigma, and the streptavidin-coated beads from Bang Laboratories. ..

Blocking Assay:

Article Title: Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments
Article Snippet: Ultimately, the assay quality will depend on your ability to block defects on the biotin-PEG functionalized glass surfaces. .. Actin was then combined with 2× TIRF imaging buffer (20 µl volume) and 4× protein (i.e., VASP, profilin, Lpd) (10 µl volume) resulting in a final buffer composition of 20 mM HEPES [pH 7], 50–100 mM KCl, 1 mM MgCl2 , 1 mM EGTA, 0.2% methylcellulose cP400, 1 mg/ml BSA, 1 mM ATP, 20 mM BME, 1 mM Trolox (Sigma, Cat# 238813), 20 mM glucose, 125 µg/ml glucose oxidase (Serva, #22780.01 Aspergillus niger ), and 20 µg/ml catalase (Sigma, #C40-100MG Bovine Liver).

Incubation:

Article Title:
Article Snippet: .. For measurement of agonistic response, the cells were incubated for 60 minutes in HBSS/10 mM HEPES with 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma Aldrich) and 0.18 μ M TSH or 10 μ M D3- β Arr in a humidified 5% CO2 incubator at 37°C. .. For measurement of antagonistic response, the cells were incubated with 10 μ M D3- β Arr for 20 minutes in HBSS/10 mM HEPES.

BIA-KA:

Article Title: Dopamine Receptors in Human Lymphocytes: Radioligand Binding and Quantitative RT-PCR Assays
Article Snippet: .. The following reagents were used: [3 H] , 69 Ci/mmol (Amersham); (+)-Butaclamol, pepstatin A, leupeptin hydrochloride, PMFS (phenylmethanesulfonyl fluoride), EDTA, HEPES, Tris-HCl, Histopaque-1077, PEI (polyethylenimine), ascorbic acid, and NaCL physiological solution (Sigma); HBSS and PBS buffers, and Trizol-LS reagent (Invitrogen); Ready-Safe scintillation cocktail (Beckman); BCA protein assay reagent kit (Pierce); RNEasy Mini Kit (Qiagen); Superscript II (Gibco BRL); RNAsin (Promega); DNA-free Kit and human brain total RNA (Ambion), dNTP set (Gene Choice). .. Primers and fluorescence-labeled probes (assays–on–demand) for D1 (ref: Hs00265245_s1), D2 (ref: Hs00241436_m1), D3 (ref: Hs00168045_m1), D4 (ref: Hs00609526_m1), D5 (ref: Hs00361234_s1) receptors and human GAPDH control reagents (with included forward and reverse primers and probes) were ordered from Applied Biosystems.

Modification:

Article Title: Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia
Article Snippet: 125 I-FGF-2 was prepared by a modification of the Bolton–Hunter procedure ( ). .. Heparinase III, Chondroitinase ABC, heparan sulfate from bovine kidney, gelatin, Hepes, and trypsin were from Sigma.

Article Title: Effect of acute acid-base disturbances on the phosphorylation of phospholipase C-γ1 and Erk1/2 in the renal proximal tubule
Article Snippet: Media for PT suspension For renal cortex dissection and PT enrichment, our base solution was half Dulbecco's modified Eagle's medium and half Ham's F-12 medium (DMEM-F12 50/50), purchased as a powder lacking l -glutamine and NaHCO3 (Catalog #90-091-PB, Corning, Cellgro® Mediatech Inc., Manassas, VA). .. We supplemented this DMEM-F12 50/50 media with 15 mmol/L HEPES, 2 mmol/L l -glutamine, 2 mmol/L heptanoic acid (Catalog #75190; Sigma-Aldrich, St. Louis, MO), and 2 mmol/L l -Lactic acid (30% in water by weight, Sigma-Aldrich, Catalog #L1875), and then titrated to pH 7.40.

Western Blot:

Article Title: Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia
Article Snippet: Heparinase III, Chondroitinase ABC, heparan sulfate from bovine kidney, gelatin, Hepes, and trypsin were from Sigma. .. Antiheparan sulfate proteoglycan (Upstate Biotechnology) served as the primary perlecan antibody, whereas anti-rat IgG, horseradish peroxidase-linked whole antibody from sheep (Amersham) served as the secondary antibody for Western blots ( ).

Flow Cytometry:

Article Title: Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments
Article Snippet: Actin was then combined with 2× TIRF imaging buffer (20 µl volume) and 4× protein (i.e., VASP, profilin, Lpd) (10 µl volume) resulting in a final buffer composition of 20 mM HEPES [pH 7], 50–100 mM KCl, 1 mM MgCl2 , 1 mM EGTA, 0.2% methylcellulose cP400, 1 mg/ml BSA, 1 mM ATP, 20 mM BME, 1 mM Trolox (Sigma, Cat# 238813), 20 mM glucose, 125 µg/ml glucose oxidase (Serva, #22780.01 Aspergillus niger ), and 20 µg/ml catalase (Sigma, #C40-100MG Bovine Liver). .. A final reaction volume of 40 µl (10 µl actin, 20 µl 2× TIRF buffer, 10 µl VASP/Lpd/etc) was flowed through a PEG/biotin-PEG TIRF flow cell, sealed with VALAP (1:1:1 mixture of Vaseline, lanoline, and paraffin wax), and imaged at 23°C.

Concentration Assay:

Article Title: Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system
Article Snippet: Experiments were performed at 20 °C in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 2 mg/mL dextran, and 1 mg/mL salmon sperm DNA (Sigma). .. A concentration series of each PfRH5 variant protein (8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 μM) was injected over the basigin-coated chip for 120 s at 30 μL/min, followed by a 300 s dissociation time.

Article Title: Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins
Article Snippet: Lambda DNA, dNTPs (dATP, dTTP, dGTP), enzymes and buffers were purchased from Promega, the biotin-11-dCTP from Enzo, HEPES, NaCl and NaOH from Sigma, and the streptavidin-coated beads from Bang Laboratories. .. To measure the effect of the proteins on this transition, a buffer solution containing a fixed concentration of protein dissolved in FIM buffer was added to the experimental cell until the buffer surrounding the captured DNA molecule was completely exchanged.

Transferring:

Article Title: Functional and Morphological Analysis of OFF Bipolar Cells
Article Snippet: Using a small transfer pipette, slowly remove the HEPES solution around the slab. .. Immediately after the solution is removed, a halved piece of Millipore filter paper is placed on top of the retinal slab and a drop of HEPES solution is placed on top of the Millipore filter paper.

Inhibition:

Article Title:
Article Snippet: After 24 hours, the cells were assayed for activation (agonist) or inhibition (antagonist) of TSHR signaling in response to added ligands. .. For measurement of agonistic response, the cells were incubated for 60 minutes in HBSS/10 mM HEPES with 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma Aldrich) and 0.18 μ M TSH or 10 μ M D3- β Arr in a humidified 5% CO2 incubator at 37°C.

Imaging:

Article Title: Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments
Article Snippet: .. Actin was then combined with 2× TIRF imaging buffer (20 µl volume) and 4× protein (i.e., VASP, profilin, Lpd) (10 µl volume) resulting in a final buffer composition of 20 mM HEPES [pH 7], 50–100 mM KCl, 1 mM MgCl2 , 1 mM EGTA, 0.2% methylcellulose cP400, 1 mg/ml BSA, 1 mM ATP, 20 mM BME, 1 mM Trolox (Sigma, Cat# 238813), 20 mM glucose, 125 µg/ml glucose oxidase (Serva, #22780.01 Aspergillus niger ), and 20 µg/ml catalase (Sigma, #C40-100MG Bovine Liver). .. A final reaction volume of 40 µl (10 µl actin, 20 µl 2× TIRF buffer, 10 µl VASP/Lpd/etc) was flowed through a PEG/biotin-PEG TIRF flow cell, sealed with VALAP (1:1:1 mixture of Vaseline, lanoline, and paraffin wax), and imaged at 23°C.

Protein Concentration:

Article Title: Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins
Article Snippet: All proteins were aliquoted in volumes of 10 µl and 10 or 25 µM protein concentration in specific NC-storage buffer (20 mM HEPES, 5 mM β-mercaptoethanol, 0.1 mM TCEP, pH 7.5), and stored at −80°C before use. .. Lambda DNA, dNTPs (dATP, dTTP, dGTP), enzymes and buffers were purchased from Promega, the biotin-11-dCTP from Enzo, HEPES, NaCl and NaOH from Sigma, and the streptavidin-coated beads from Bang Laboratories.

Injection:

Article Title: Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system
Article Snippet: Experiments were performed at 20 °C in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 2 mg/mL dextran, and 1 mg/mL salmon sperm DNA (Sigma). .. A concentration series of each PfRH5 variant protein (8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 μM) was injected over the basigin-coated chip for 120 s at 30 μL/min, followed by a 300 s dissociation time.

Recombinant:

Article Title: Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system
Article Snippet: Surface plasmon resonance (SPR) The production of recombinant basigin in Origami B (DE3) E. coli has been previously described . .. Experiments were performed at 20 °C in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 2 mg/mL dextran, and 1 mg/mL salmon sperm DNA (Sigma).

Article Title: Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia
Article Snippet: Human recombinant FGF-2 was from Scios-Nova (Mountain View, CA). .. Heparinase III, Chondroitinase ABC, heparan sulfate from bovine kidney, gelatin, Hepes, and trypsin were from Sigma.

Article Title: The Ferroxidase Activity of Yeast Frataxin
Article Snippet: HEPES, ferrous ammonium sulfate, 2-de-oxyribose, thiobarbituric acid, and α - α ′-bipyridine were from Sigma, and beef liver catalase was from Roche Molecular Biochemicals. .. Recombinant mYfh1p was expressed in E. coli ( ) and purified as previously described ( ).

Fluorescence:

Article Title: Dopamine Receptors in Human Lymphocytes: Radioligand Binding and Quantitative RT-PCR Assays
Article Snippet: The following reagents were used: [3 H] , 69 Ci/mmol (Amersham); (+)-Butaclamol, pepstatin A, leupeptin hydrochloride, PMFS (phenylmethanesulfonyl fluoride), EDTA, HEPES, Tris-HCl, Histopaque-1077, PEI (polyethylenimine), ascorbic acid, and NaCL physiological solution (Sigma); HBSS and PBS buffers, and Trizol-LS reagent (Invitrogen); Ready-Safe scintillation cocktail (Beckman); BCA protein assay reagent kit (Pierce); RNEasy Mini Kit (Qiagen); Superscript II (Gibco BRL); RNAsin (Promega); DNA-free Kit and human brain total RNA (Ambion), dNTP set (Gene Choice). .. Primers and fluorescence-labeled probes (assays–on–demand) for D1 (ref: Hs00265245_s1), D2 (ref: Hs00241436_m1), D3 (ref: Hs00168045_m1), D4 (ref: Hs00609526_m1), D5 (ref: Hs00361234_s1) receptors and human GAPDH control reagents (with included forward and reverse primers and probes) were ordered from Applied Biosystems.

Isolation:

Article Title: Cryopreservation in liquid nitrogen of gonocytes from neonatal porcine testes stored at 4°C
Article Snippet: Paragraph title: Isolation of gonocytes from the testes ... The testes were thoroughly minced using fine scissors and digested in an enzyme solution containing 2 mg/mL collagenase (type IV) (Sigma‐Aldrich, St Louis, MO, USA) and 50 g/mL Deoxyribonuclease I (DNase I; Sigma‐Aldrich) in DMEM/F12 supplemented with 15 mmol/L HEPES, 100 IU/mL penicillin (Sigma‐Aldrich), 50 µg/mL streptomycin (Sigma‐Aldrich) and 40 µg/mL gentamycin (Sigma‐Aldrich) at 37°C for 60 min by shaking at 140 cycles/min in a water bath.

Microscopy:

Article Title: Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments
Article Snippet: Because Lpd850−1250aa and human VASP are highly basic proteins, poor coverslip functionalization will result in a tremendous amount of non-specific protein absorption to the glass substrates used for TIRF microscopy. .. Actin was then combined with 2× TIRF imaging buffer (20 µl volume) and 4× protein (i.e., VASP, profilin, Lpd) (10 µl volume) resulting in a final buffer composition of 20 mM HEPES [pH 7], 50–100 mM KCl, 1 mM MgCl2 , 1 mM EGTA, 0.2% methylcellulose cP400, 1 mg/ml BSA, 1 mM ATP, 20 mM BME, 1 mM Trolox (Sigma, Cat# 238813), 20 mM glucose, 125 µg/ml glucose oxidase (Serva, #22780.01 Aspergillus niger ), and 20 µg/ml catalase (Sigma, #C40-100MG Bovine Liver).

Purification:

Article Title: The Ferroxidase Activity of Yeast Frataxin
Article Snippet: HEPES, ferrous ammonium sulfate, 2-de-oxyribose, thiobarbituric acid, and α - α ′-bipyridine were from Sigma, and beef liver catalase was from Roche Molecular Biochemicals. .. Recombinant mYfh1p was expressed in E. coli ( ) and purified as previously described ( ).

Labeling:

Article Title: Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments
Article Snippet: For experiments involving dynamic single actin filament elongation, actin polymerization was initiated by combining 1 µl of 10× ME (10× ME contains 0.5 mM MgCl2 , 2 mM EGTA) with 9 µl of 4.44 µM monomeric actin (5–20% Cy5 labeled and diluted in G-buffer: 2 mM Tris [pH 8.0], 0.5 mM TCEP, 0.1 mM CaCl2 , 0.2 mM ATP, 0.01% azide). .. Actin was then combined with 2× TIRF imaging buffer (20 µl volume) and 4× protein (i.e., VASP, profilin, Lpd) (10 µl volume) resulting in a final buffer composition of 20 mM HEPES [pH 7], 50–100 mM KCl, 1 mM MgCl2 , 1 mM EGTA, 0.2% methylcellulose cP400, 1 mg/ml BSA, 1 mM ATP, 20 mM BME, 1 mM Trolox (Sigma, Cat# 238813), 20 mM glucose, 125 µg/ml glucose oxidase (Serva, #22780.01 Aspergillus niger ), and 20 µg/ml catalase (Sigma, #C40-100MG Bovine Liver).

Chromatin Immunoprecipitation:

Article Title: Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system
Article Snippet: Experiments were performed at 20 °C in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 2 mg/mL dextran, and 1 mg/mL salmon sperm DNA (Sigma). .. Basigin was immobilized on a CM5 chip (GE Healthcare) by amine coupling (GE Healthcare kit) to a total of 850 Response Units (RU).

SPR Assay:

Article Title: Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system
Article Snippet: Paragraph title: Surface plasmon resonance (SPR) ... Experiments were performed at 20 °C in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 2 mg/mL dextran, and 1 mg/mL salmon sperm DNA (Sigma).

Binding Assay:

Article Title: Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments
Article Snippet: Actin was then combined with 2× TIRF imaging buffer (20 µl volume) and 4× protein (i.e., VASP, profilin, Lpd) (10 µl volume) resulting in a final buffer composition of 20 mM HEPES [pH 7], 50–100 mM KCl, 1 mM MgCl2 , 1 mM EGTA, 0.2% methylcellulose cP400, 1 mg/ml BSA, 1 mM ATP, 20 mM BME, 1 mM Trolox (Sigma, Cat# 238813), 20 mM glucose, 125 µg/ml glucose oxidase (Serva, #22780.01 Aspergillus niger ), and 20 µg/ml catalase (Sigma, #C40-100MG Bovine Liver). .. To calculate the dissociation rate constant (Kd ) for GFP-Lpd850−1250aa binding to single actin filaments ( ) we used TIRF microscopy to image the density of GFP-Lpd850−1250aa bound to phalloidin stabilized actin filaments (20% Cy5 labeled).

Activation Assay:

Article Title:
Article Snippet: After 24 hours, the cells were assayed for activation (agonist) or inhibition (antagonist) of TSHR signaling in response to added ligands. .. For measurement of agonistic response, the cells were incubated for 60 minutes in HBSS/10 mM HEPES with 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma Aldrich) and 0.18 μ M TSH or 10 μ M D3- β Arr in a humidified 5% CO2 incubator at 37°C.

Lysis:

Article Title:
Article Snippet: For measurement of agonistic response, the cells were incubated for 60 minutes in HBSS/10 mM HEPES with 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma Aldrich) and 0.18 μ M TSH or 10 μ M D3- β Arr in a humidified 5% CO2 incubator at 37°C. .. After 60 minutes, the incubation was terminated by aspiration of the incubation medium and addition of lysis buffer of the cAMP-Screen Direct System (Life Technologies Corporation, Grand Island, NY). cAMP content was determined as described by the manufacturer.

Variant Assay:

Article Title: Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system
Article Snippet: Experiments were performed at 20 °C in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, 2 mg/mL dextran, and 1 mg/mL salmon sperm DNA (Sigma). .. A concentration series of each PfRH5 variant protein (8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 μM) was injected over the basigin-coated chip for 120 s at 30 μL/min, followed by a 300 s dissociation time.

other:

Article Title: Comparison of the Effects of Surface Tension and Osmotic Pressure on the Interfacial Hydration of a Fluid Phospholipid Bilayer
Article Snippet: Choline chloride, EDTA, HEPES, POPC, and sucrose were from Sigma (St. Louis, MO).

Article Title: Residue Ionization and Ion Transport through OmpF Channels
Article Snippet: The following chemical reagents were used in this study: KCl, KOH, and HCl (Mallinckrodt, St. Louis, MO); 2M CaCl2 (Quality Biological, Gaithersburg, MD); ultrol grade MES or HEPES (Calbiochem, San Diego, CA); “purum” hexadecane (Fluka, Buchs, Switzerland); 1,2-diphytanoyl- sn -glycero-3-phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL); pentane (Burdick and Jackson, Muskegon, MI); agarose (Bethesda Research Laboratory, Gaithersburg, MD).

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  • 99
    Millipore immunoprecipitation buffer
    Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for <t>immunoprecipitation</t> (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p
    Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore caspase assay buffer
    NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by <t>caspase-3</t> activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P
    Caspase Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore calpain reaction buffer
    Schematic diagram showing the relationship of <t>calpain/NF-κB/inflammation/NVU</t> damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
    Calpain Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore m hepes koh
    AUC SV and SEC/MALS. A , sedimentation coefficient distribution of KsHDH in 25 m m <t>HEPES/KOH,</t> pH 7.5, 25 m m KCl. The three peaks correspond to the HDH monomer (α, 5 S), the covalently bound homotrimer (α 3 , 10.97 S), and the non-covalently
    M Hepes Koh, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p

    Journal: Scientific Reports

    Article Title: Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress

    doi: 10.1038/srep16529

    Figure Lengend Snippet: Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p

    Article Snippet: Immunoprecipitation and Western blotting For immunoprecipitation, cell lysates prepared from EC were incubated in immunoprecipitation buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2 , 1% Nonidet P-40, 0.4 mM Na3 VO4 , 40 mM NaF, 50 μM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, and Calbiochem protease inhibitor mixture III at 1:250 dilution).

    Techniques: Immunoprecipitation, Western Blot

    NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P

    Journal: Cancer Biology & Therapy

    Article Title: Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma

    doi: 10.1080/15384047.2015.1017155

    Figure Lengend Snippet: NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P

    Article Snippet: Twenty μg of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (15 μg/ml) (Calbiochem, Darmstadt, Germany) as the substrate.

    Techniques: Concentration Assay, Caspase-3 Activity Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Expressing, Western Blot, Cell Culture

    Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Mouse Assay, Activation Assay

    MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Activity Assay, Expressing, Western Blot

    AUC SV and SEC/MALS. A , sedimentation coefficient distribution of KsHDH in 25 m m HEPES/KOH, pH 7.5, 25 m m KCl. The three peaks correspond to the HDH monomer (α, 5 S), the covalently bound homotrimer (α 3 , 10.97 S), and the non-covalently

    Journal: The Journal of Biological Chemistry

    Article Title: Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle *

    doi: 10.1074/jbc.M116.735530

    Figure Lengend Snippet: AUC SV and SEC/MALS. A , sedimentation coefficient distribution of KsHDH in 25 m m HEPES/KOH, pH 7.5, 25 m m KCl. The three peaks correspond to the HDH monomer (α, 5 S), the covalently bound homotrimer (α 3 , 10.97 S), and the non-covalently

    Article Snippet: Fractions of the peak eluting at 11 ± 1 ml were pooled, concentrated, and buffer-exchanged to 25 m m HEPES-KOH, pH 7.5, 25 m m KCl using a 100 kDa molecular mass cut-off Amicon ultrafiltration device (Millipore Bioscience, Schwalbach, Germany).

    Techniques: Size-exclusion Chromatography, Sedimentation