cacl2 Millipore Search Results


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  • 99
    Millipore mgcl2
    Effect of divalent metal ions on methylation activity of HP0593 MTase. A . Histogram showing methylation activity of 250 nM HP0593 MTase in the absence of any metal ions (-Me 2+ ) and in the presence of cobalt chloride (Co 2+ ), magnesium chloride (Mg 2+ ), manganese chloride (Mn 2+ ), calcium chloride (Ca 2+ ), zinc chloride (Zn 2+ ), and nickel chloride (Ni 2+ ). 1 = 0.4 mM, 2 = 0.5 mM, 3 = 1.0 mM, and 4 = 5.0 mM. Kinetics of DNA binding. HP0593 MTase was injected for 120 sec over streptavidin chip containing immobilized duplex 18 DNA at a flow rate of 20 µl/min followed by dissociation phase of 120 sec. The global fit of the data was used to calculate the binding constants.  B.  SPR sensorgram displaying the response of increasing HP0593 MTase concentrations (25–100 nM) in presence of 1.0 mM MnCl 2 . ( Inset ) kinetic constants.  C.  SPR sensorgram displaying the response of increasing HP0593 MTase concentrations (100–200 nM) in absence of metal. ( Inset ) kinetic constants.
    Mgcl2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hepes
    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 <t>KCl.</t> 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 <t>HEPES</t> [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
    Hepes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50050 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore triton x 100
    Characterization of trypsin (–) and trypsin (+) SW480 EVs. (a) Schematic workflow shows the overall processes of the EV isolation and trypsin treatment before quantitative proteomics. (b) The size distributions of EVs were measured by dynamic light scattering analyses indicating an average diameter of 182.57 ± 19.48 nm for trypsin (–) EVs and 180.43 ± 11.33 nm for trypsin (+) EVs. (c) TEM revealed that the EVs preserved the intact morphology similar to the EVs before trypsin treatment. (d) Trypsin (–) and trypsin (+) EVs were analysed by Western blotting with antibodies against ICAM1, an integral membrane protein with a large extracellular domain [  21 ], and actin, a cytosolic intravesicular protein [  22 ]. Three independent biological replicates of Western blotting are shown with ICAM1 and actin for trypsin (–) and trypsin (+) EVs in Figure S1. (e) Dynamic light scattering analysis shows the disruption of EVs by 0.1% Triton X-100 treatment for 10 min at room temperature. (f) Western blotting of SW480 EVs with or without pre-treatment of membrane disrupting 0.1% Triton X-100 for 10 min at room temperature showed the susceptibility of intravesicular actin and tubulin by trypsin.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bsa
    Selected bacteria reside in LC3-positive phagolysosomes.A and B. Long-term infected <t>(LTI)</t> macrophages for at least 2 years and recently infected (RI) with BCG-GFP ancestral strain for 24 h were stained with Lysotracker (A, left panel) or <t>DQ-BSA</t> (B, right panel).C and D. Quantitative analysis of the fluorescence intensity of Lysotracker (C) and DQ-BSA (D) associated with phagosomes in LTI macrophages compared with RI cells. Around 80 phagosomes were quantified in Lysotracker or DQ-BSA stained macrophages from three independent experiments.E and F. Recently infected macrophages (RI) during 24 h (E) and LTI (F) macrophages (2 years old) were subjected to immunofluorescence and endogenous LC3 and LAMP-2 were detected using specific antibodies. Insets depict colocalization with single markers.G and H. Quantitative analysis of the fluorescence intensity of LC3 (G) and LAMP-2 (H) associated with phagosomes of RI and LTI macrophages. Around 130 phagosomes were quantified for LC3 detection and 190 phagosomes for LAMP-2 detection from three independent experiments.Data represent the mean ± S.E.M., (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001 from two-tailed Student's t -test. Nuclei were stained with Hoechst 33258. Scale bars: 10 μm.
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 45765 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bovine serum albumin
    Selected bacteria reside in LC3-positive phagolysosomes.A and B. Long-term infected <t>(LTI)</t> macrophages for at least 2 years and recently infected (RI) with BCG-GFP ancestral strain for 24 h were stained with Lysotracker (A, left panel) or <t>DQ-BSA</t> (B, right panel).C and D. Quantitative analysis of the fluorescence intensity of Lysotracker (C) and DQ-BSA (D) associated with phagosomes in LTI macrophages compared with RI cells. Around 80 phagosomes were quantified in Lysotracker or DQ-BSA stained macrophages from three independent experiments.E and F. Recently infected macrophages (RI) during 24 h (E) and LTI (F) macrophages (2 years old) were subjected to immunofluorescence and endogenous LC3 and LAMP-2 were detected using specific antibodies. Insets depict colocalization with single markers.G and H. Quantitative analysis of the fluorescence intensity of LC3 (G) and LAMP-2 (H) associated with phagosomes of RI and LTI macrophages. Around 130 phagosomes were quantified for LC3 detection and 190 phagosomes for LAMP-2 detection from three independent experiments.Data represent the mean ± S.E.M., (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001 from two-tailed Student's t -test. Nuclei were stained with Hoechst 33258. Scale bars: 10 μm.
    Bovine Serum Albumin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 96597 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore collagenase
    Selected bacteria reside in LC3-positive phagolysosomes.A and B. Long-term infected <t>(LTI)</t> macrophages for at least 2 years and recently infected (RI) with BCG-GFP ancestral strain for 24 h were stained with Lysotracker (A, left panel) or <t>DQ-BSA</t> (B, right panel).C and D. Quantitative analysis of the fluorescence intensity of Lysotracker (C) and DQ-BSA (D) associated with phagosomes in LTI macrophages compared with RI cells. Around 80 phagosomes were quantified in Lysotracker or DQ-BSA stained macrophages from three independent experiments.E and F. Recently infected macrophages (RI) during 24 h (E) and LTI (F) macrophages (2 years old) were subjected to immunofluorescence and endogenous LC3 and LAMP-2 were detected using specific antibodies. Insets depict colocalization with single markers.G and H. Quantitative analysis of the fluorescence intensity of LC3 (G) and LAMP-2 (H) associated with phagosomes of RI and LTI macrophages. Around 130 phagosomes were quantified for LC3 detection and 190 phagosomes for LAMP-2 detection from three independent experiments.Data represent the mean ± S.E.M., (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001 from two-tailed Student's t -test. Nuclei were stained with Hoechst 33258. Scale bars: 10 μm.
    Collagenase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20723 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ethylenediaminetetraacetic acid
    Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with <t>ethylenediaminetetraacetic</t> acid shows absence of activity. (C)
    Ethylenediaminetetraacetic Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4351 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore protease inhibitor cocktail
    Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with <t>ethylenediaminetetraacetic</t> acid shows absence of activity. (C)
    Protease Inhibitor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 138030 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trypsin
    Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with <t>ethylenediaminetetraacetic</t> acid shows absence of activity. (C)
    Trypsin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 24572 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tris
    Soluble rCTD is a dimer in equilibrium and the <t>CTD</t> cleaved off natively expressed proRgpB spontaneously dimerizes. ( A ) rCTD at 1, 0.5 and 0.1 mg ml −1 was treated with glutaraldehyde and analysed by SDS-PAGE. ( B ) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM <t>Tris,</t> 150 mM NaCl, 2.5 mM CaCl 2 , 0.02% NaN 3 pH 7.5 ( C ) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.
    Tris, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 38479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore thrombin
    Soluble rCTD is a dimer in equilibrium and the <t>CTD</t> cleaved off natively expressed proRgpB spontaneously dimerizes. ( A ) rCTD at 1, 0.5 and 0.1 mg ml −1 was treated with glutaraldehyde and analysed by SDS-PAGE. ( B ) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM <t>Tris,</t> 150 mM NaCl, 2.5 mM CaCl 2 , 0.02% NaN 3 pH 7.5 ( C ) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.
    Thrombin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dnase i
    <t>DNase</t> I footprinting assay of the ykoL promoter using PhoP and PhoP∼P. A PCR fragment corresponding to the ykoL promoter region (−153 to +60 relative to the transcriptional start site of ykoL ) was used as a DNA probe. Coding strand footprinting 32 P-labeled YkoL-FOR primer and the noncoding strand footprinting 32 P-labeled YkoL-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The black vertical lines show the regions that were bound by PhoP and PhoP∼P. The thick black lines show the regions where PhoP and PhoP∼P bound with a higher affinity. The numbers indicate the positions of the PhoP binding sites relative to the transcriptional start site M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the ykoL promoter the translational start site, RBS, transcriptional start site (+1), and corresponding −35 and −10 sequences of the promoter are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of PhoP dimer (5 ± 2-bp spacer and a maximum of two mismatches). The positions of YkoL-FOR and YkoL-REV primers are shown by thick arrows and are labeled.
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore propidium iodide
    Image-based cell death assays. Example images of cells 24 h after TNFα treatment by the fluorescence assays under evaluation. For each assay, the relevant channels were merged, pseudocolored, and adjusted for brightness and contrast. (A) Cells stained with <t>propidium</t> iodide (PI, red ), Annexin V Alexa Fluor 488 (AnnV, green ), and DRAQ5 ( blue ). (B) Cells stained with tetramethylrhodamine, ethyl ester (TMRE; red ), and DRAQ5 ( blue ). (C) Intensity weighted ratio images of cells expressing the caspase sensor showing monomeric Cerulean (mCer; cyan ) and stimulated emission of Venus (Venus, yellow ). (D) Cells stained with nonyl acridine orange (NAO; green ) and DRAQ5 ( blue ). Scale bar = 20 μm.
    Propidium Iodide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 69523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore protease
    Image-based cell death assays. Example images of cells 24 h after TNFα treatment by the fluorescence assays under evaluation. For each assay, the relevant channels were merged, pseudocolored, and adjusted for brightness and contrast. (A) Cells stained with <t>propidium</t> iodide (PI, red ), Annexin V Alexa Fluor 488 (AnnV, green ), and DRAQ5 ( blue ). (B) Cells stained with tetramethylrhodamine, ethyl ester (TMRE; red ), and DRAQ5 ( blue ). (C) Intensity weighted ratio images of cells expressing the caspase sensor showing monomeric Cerulean (mCer; cyan ) and stimulated emission of Venus (Venus, yellow ). (D) Cells stained with nonyl acridine orange (NAO; green ) and DRAQ5 ( blue ). Scale bar = 20 μm.
    Protease, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4908 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dtt
    USP7S interacts with Mule and prevents its self-ubiquitylation in vitro. ( A ) Equal amounts (2.5 pmol) of purified recombinant His-tagged USP7S and GST-tagged Mule were incubated in buffer containing 50 mM Tris–HCl, pH 8.0, 50 mM <t>KCl,</t> 1 mM EDTA, 1 mM <t>DTT</t> for 30 min at room temperature with shaking. USP7S and Mule were then pulled down using either magnetic beads as a control (Mock PD, 100%) or using magnetic Ni-NTA agarose beads (His PD, 100%) or GST agarose beads (GST PD, 100%), respectively. The beads were boiled in SDS–PAGE sample buffer for 5 min, and proteins were separated by 10% SDS–PAGE and analysed using USP7S and Mule antibodies (10% of the input is loaded). ( B ) HeLa cells were simultaneously transfected with USP7S and Mule (1 pmol each) expression plasmids, whole-cell extracts were prepared and used to pull down HA-tagged Mule or Flag-tagged USP7S using either magnetic beads as a control (Mock PD, 100%), HA- (HA PD, 100%) or Flag-agarose (Flag PD, 100%) beads. Proteins were separated by 4–20% SDS–PAGE and analysed using USP7S and Mule antibodies (15% of the input is loaded). ( C ) In vitro deubiquitylation of self-ubiquitylated Mule HECT-domain by recombinant wild-type USP7S protein (wtUSP7S) or its inactive mutant (C223S USP7S) analysed by western blotting. Equal loading of the enzyme is demonstrated using USP7S antibodies.
    Dtt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pbs
    Western blot analysis of p-IκB-α and p-ERK1/2 protein levels from lungs of <t>PBS-challenged</t> mice pretreated with vehicle (PBS), from ovalbumin (OVA)-challenged mice pretreated with vehicle (OVA), <t>Ang-(1–7)</t> and A779 plus Ang-(1–7).
    Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cacl2
    Western blot analysis of p-IκB-α and p-ERK1/2 protein levels from lungs of <t>PBS-challenged</t> mice pretreated with vehicle (PBS), from ovalbumin (OVA)-challenged mice pretreated with vehicle (OVA), <t>Ang-(1–7)</t> and A779 plus Ang-(1–7).
    Cacl2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20049 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lysis buffer
    Western blot analysis of p-IκB-α and p-ERK1/2 protein levels from lungs of <t>PBS-challenged</t> mice pretreated with vehicle (PBS), from ovalbumin (OVA)-challenged mice pretreated with vehicle (OVA), <t>Ang-(1–7)</t> and A779 plus Ang-(1–7).
    Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 95438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore atp
    Role of the C2 domain on translocation properties of PKCα. HEK293 cells were cotransfected with plasmids coding for PKCα(C2-mut)-EGFP and PKCα(WT)-mRFP. (A) Depicts results in cells stimulated with 100 μM <t>ATP</t> (A, b) and 1 μM <t>PMA</t> (A, c). Green (EGFP fusion protein) and red (mRFP fusion protein) traces depict the time course of fluorescence derived from the plasma membrane (top traces) and cytosol (bottom traces) during the stimulation regimes. Similar results were obtained in 16 independent experiments ( n = 51 cells). (B) Translocation responses of WT and C2-mutated PKCs to flash photolytic increases in Ca 2+ . Cells were loaded with caged Ca 2+ NPEGTA-AM. B (a) depicts the resting fluorescence of the two PKC constructs, as indicated. B (c) shows the time course of the plasma membrane (top traces) and cytosolic (bottom traces) fluorescence changes of the EGFP construct containing the mutated C2 domain (green) and the WT-mRFP (red). The time points of the UV flashes are indicated by the two blue vertical dashed lines. For the three time points depicted in B (c) by the dashed black arrows, the relative changes of the fluorescence have been replotted in B (b). The color-coded images (see color wedge attached) for the WT (top row) and mutated PKC (bottom traces) are shown. Similar results were obtained in 14 independent experiments ( n = 33 cells).
    Atp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore micrococcal nuclease
    Role of the C2 domain on translocation properties of PKCα. HEK293 cells were cotransfected with plasmids coding for PKCα(C2-mut)-EGFP and PKCα(WT)-mRFP. (A) Depicts results in cells stimulated with 100 μM <t>ATP</t> (A, b) and 1 μM <t>PMA</t> (A, c). Green (EGFP fusion protein) and red (mRFP fusion protein) traces depict the time course of fluorescence derived from the plasma membrane (top traces) and cytosol (bottom traces) during the stimulation regimes. Similar results were obtained in 16 independent experiments ( n = 51 cells). (B) Translocation responses of WT and C2-mutated PKCs to flash photolytic increases in Ca 2+ . Cells were loaded with caged Ca 2+ NPEGTA-AM. B (a) depicts the resting fluorescence of the two PKC constructs, as indicated. B (c) shows the time course of the plasma membrane (top traces) and cytosolic (bottom traces) fluorescence changes of the EGFP construct containing the mutated C2 domain (green) and the WT-mRFP (red). The time points of the UV flashes are indicated by the two blue vertical dashed lines. For the three time points depicted in B (c) by the dashed black arrows, the relative changes of the fluorescence have been replotted in B (b). The color-coded images (see color wedge attached) for the WT (top row) and mutated PKC (bottom traces) are shown. Similar results were obtained in 14 independent experiments ( n = 33 cells).
    Micrococcal Nuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1897 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pronase e
    Coexpression and faithful trafficking of tandem CLAG3 isoforms. (A) Indirect immunofluorescence images showing colocalization of Dd2-HA and 3D7-miniSOG-FLAG CLAG3 isoforms in TFLC3 parasites at the schizont stage and in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (B and C) Immunoblot assays performed using TFLC3 cell lysates and indicated epitope tag antibodies without or with extracellular <t>pronase</t> E treatment prior to cell lysis (− and +, respectively). (D and E) Immunoblot assays using anti-CLAG3 antibody and indicated parasites, without and with pronase E treatment (− and +, respectively). While two cleavage products are detected in TFLC3 , the C3attB parent produces a single fragment.
    Pronase E, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1674 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pmsf
    Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with <t>DMSO</t> (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor <t>PMSF</t> (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.
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    Effect of divalent metal ions on methylation activity of HP0593 MTase. A . Histogram showing methylation activity of 250 nM HP0593 MTase in the absence of any metal ions (-Me 2+ ) and in the presence of cobalt chloride (Co 2+ ), magnesium chloride (Mg 2+ ), manganese chloride (Mn 2+ ), calcium chloride (Ca 2+ ), zinc chloride (Zn 2+ ), and nickel chloride (Ni 2+ ). 1 = 0.4 mM, 2 = 0.5 mM, 3 = 1.0 mM, and 4 = 5.0 mM. Kinetics of DNA binding. HP0593 MTase was injected for 120 sec over streptavidin chip containing immobilized duplex 18 DNA at a flow rate of 20 µl/min followed by dissociation phase of 120 sec. The global fit of the data was used to calculate the binding constants.  B.  SPR sensorgram displaying the response of increasing HP0593 MTase concentrations (25–100 nM) in presence of 1.0 mM MnCl 2 . ( Inset ) kinetic constants.  C.  SPR sensorgram displaying the response of increasing HP0593 MTase concentrations (100–200 nM) in absence of metal. ( Inset ) kinetic constants.

    Journal: PLoS ONE

    Article Title: Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

    doi: 10.1371/journal.pone.0016810

    Figure Lengend Snippet: Effect of divalent metal ions on methylation activity of HP0593 MTase. A . Histogram showing methylation activity of 250 nM HP0593 MTase in the absence of any metal ions (-Me 2+ ) and in the presence of cobalt chloride (Co 2+ ), magnesium chloride (Mg 2+ ), manganese chloride (Mn 2+ ), calcium chloride (Ca 2+ ), zinc chloride (Zn 2+ ), and nickel chloride (Ni 2+ ). 1 = 0.4 mM, 2 = 0.5 mM, 3 = 1.0 mM, and 4 = 5.0 mM. Kinetics of DNA binding. HP0593 MTase was injected for 120 sec over streptavidin chip containing immobilized duplex 18 DNA at a flow rate of 20 µl/min followed by dissociation phase of 120 sec. The global fit of the data was used to calculate the binding constants. B. SPR sensorgram displaying the response of increasing HP0593 MTase concentrations (25–100 nM) in presence of 1.0 mM MnCl 2 . ( Inset ) kinetic constants. C. SPR sensorgram displaying the response of increasing HP0593 MTase concentrations (100–200 nM) in absence of metal. ( Inset ) kinetic constants.

    Article Snippet: Chloramphenicol, bovine serum albumin, MgCl2 , MnCl2 , CoCl2 , CaCl2 , NiCl2 , ZnCl2 , ampicillin, imidazole, Coomassie Brilliant Blue R-250, RNase A, glutaraldehyde, and S -adenosyl-L-homocysteine (AdoHcy) were procured from Sigma-Aldrich Co., USA.

    Techniques: Methylation, Activity Assay, Binding Assay, Injection, Size-exclusion Chromatography, Chromatin Immunoprecipitation, Flow Cytometry, SPR Assay

    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

    Journal: eLife

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

    doi: 10.7554/eLife.06585

    Figure Lengend 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

    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).

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

    Characterization of trypsin (–) and trypsin (+) SW480 EVs. (a) Schematic workflow shows the overall processes of the EV isolation and trypsin treatment before quantitative proteomics. (b) The size distributions of EVs were measured by dynamic light scattering analyses indicating an average diameter of 182.57 ± 19.48 nm for trypsin (–) EVs and 180.43 ± 11.33 nm for trypsin (+) EVs. (c) TEM revealed that the EVs preserved the intact morphology similar to the EVs before trypsin treatment. (d) Trypsin (–) and trypsin (+) EVs were analysed by Western blotting with antibodies against ICAM1, an integral membrane protein with a large extracellular domain [  21 ], and actin, a cytosolic intravesicular protein [  22 ]. Three independent biological replicates of Western blotting are shown with ICAM1 and actin for trypsin (–) and trypsin (+) EVs in Figure S1. (e) Dynamic light scattering analysis shows the disruption of EVs by 0.1% Triton X-100 treatment for 10 min at room temperature. (f) Western blotting of SW480 EVs with or without pre-treatment of membrane disrupting 0.1% Triton X-100 for 10 min at room temperature showed the susceptibility of intravesicular actin and tubulin by trypsin.

    Journal: Journal of Extracellular Vesicles

    Article Title: Quantitative proteomic analysis of trypsin-treated extracellular vesicles to identify the real-vesicular proteins

    doi: 10.1080/20013078.2020.1757209

    Figure Lengend Snippet: Characterization of trypsin (–) and trypsin (+) SW480 EVs. (a) Schematic workflow shows the overall processes of the EV isolation and trypsin treatment before quantitative proteomics. (b) The size distributions of EVs were measured by dynamic light scattering analyses indicating an average diameter of 182.57 ± 19.48 nm for trypsin (–) EVs and 180.43 ± 11.33 nm for trypsin (+) EVs. (c) TEM revealed that the EVs preserved the intact morphology similar to the EVs before trypsin treatment. (d) Trypsin (–) and trypsin (+) EVs were analysed by Western blotting with antibodies against ICAM1, an integral membrane protein with a large extracellular domain [ 21 ], and actin, a cytosolic intravesicular protein [ 22 ]. Three independent biological replicates of Western blotting are shown with ICAM1 and actin for trypsin (–) and trypsin (+) EVs in Figure S1. (e) Dynamic light scattering analysis shows the disruption of EVs by 0.1% Triton X-100 treatment for 10 min at room temperature. (f) Western blotting of SW480 EVs with or without pre-treatment of membrane disrupting 0.1% Triton X-100 for 10 min at room temperature showed the susceptibility of intravesicular actin and tubulin by trypsin.

    Article Snippet: For Triton X-100 treatment, the pelleted EVs (20 µg) were resuspended and incubated in HBS with 0.1% (v/v) Triton X-100 (Sigma) or no Triton X-100 control for 10 min at room temperature as described in previous study [ ].

    Techniques: Isolation, Transmission Electron Microscopy, Western Blot

    Quantitative proteomic analyses of trypsin (–) and trypsin (+) SW480 EVs. (a) Using the APEX tool [  20 ], the relative abundances of vesicular proteins were calculated and given in arbitrary units. The scatter plot showed the relative enrichment of EV proteins in trypsin (–) and trypsin (+) EVs. The identification count in EVpedia database ( http://evpedia.info ) was indicated as size. Trypsin (–) EV- and trypsin (+) EV-enriched proteins were indicated as grey filled and empty dots, respectively. Black filled dots indicate proteins identified both in trypsin (−) and trypsin (+) EVs with less than 1.5-fold enrichment. Validated proteins by Western blotting were indicated as arrows with gene symbols. Note that proteins identified only in trypsin (–) and trypsin (+) SW480 EVs are plotted on the x-axis and y-axis, respectively. (b) Table showed the number of proteins categorized in each subgroup. (c) Western blotting analyses showed that calnexin and RPL14, trypsin-sensitive vesicular proteins, are undetectable after trypsin treatment but CD81 and GAPDH, trypsin-resistant vesicular proteins, are not affected after trypsin treatment. Three independent biological replicates of Western blotting are shown with CD81, GAPDH, calnexin, and RPL14 for trypsin (–) and trypsin (+) EVs in Figure S2. (d) Western blotting shows that CD81 was not affected by trypsin with and without Triton X-100 treatment, suggesting the intrinsic trypsin resistant property of CD81.

    Journal: Journal of Extracellular Vesicles

    Article Title: Quantitative proteomic analysis of trypsin-treated extracellular vesicles to identify the real-vesicular proteins

    doi: 10.1080/20013078.2020.1757209

    Figure Lengend Snippet: Quantitative proteomic analyses of trypsin (–) and trypsin (+) SW480 EVs. (a) Using the APEX tool [ 20 ], the relative abundances of vesicular proteins were calculated and given in arbitrary units. The scatter plot showed the relative enrichment of EV proteins in trypsin (–) and trypsin (+) EVs. The identification count in EVpedia database ( http://evpedia.info ) was indicated as size. Trypsin (–) EV- and trypsin (+) EV-enriched proteins were indicated as grey filled and empty dots, respectively. Black filled dots indicate proteins identified both in trypsin (−) and trypsin (+) EVs with less than 1.5-fold enrichment. Validated proteins by Western blotting were indicated as arrows with gene symbols. Note that proteins identified only in trypsin (–) and trypsin (+) SW480 EVs are plotted on the x-axis and y-axis, respectively. (b) Table showed the number of proteins categorized in each subgroup. (c) Western blotting analyses showed that calnexin and RPL14, trypsin-sensitive vesicular proteins, are undetectable after trypsin treatment but CD81 and GAPDH, trypsin-resistant vesicular proteins, are not affected after trypsin treatment. Three independent biological replicates of Western blotting are shown with CD81, GAPDH, calnexin, and RPL14 for trypsin (–) and trypsin (+) EVs in Figure S2. (d) Western blotting shows that CD81 was not affected by trypsin with and without Triton X-100 treatment, suggesting the intrinsic trypsin resistant property of CD81.

    Article Snippet: For Triton X-100 treatment, the pelleted EVs (20 µg) were resuspended and incubated in HBS with 0.1% (v/v) Triton X-100 (Sigma) or no Triton X-100 control for 10 min at room temperature as described in previous study [ ].

    Techniques: Western Blot

    Selected bacteria reside in LC3-positive phagolysosomes.A and B. Long-term infected (LTI) macrophages for at least 2 years and recently infected (RI) with BCG-GFP ancestral strain for 24 h were stained with Lysotracker (A, left panel) or DQ-BSA (B, right panel).C and D. Quantitative analysis of the fluorescence intensity of Lysotracker (C) and DQ-BSA (D) associated with phagosomes in LTI macrophages compared with RI cells. Around 80 phagosomes were quantified in Lysotracker or DQ-BSA stained macrophages from three independent experiments.E and F. Recently infected macrophages (RI) during 24 h (E) and LTI (F) macrophages (2 years old) were subjected to immunofluorescence and endogenous LC3 and LAMP-2 were detected using specific antibodies. Insets depict colocalization with single markers.G and H. Quantitative analysis of the fluorescence intensity of LC3 (G) and LAMP-2 (H) associated with phagosomes of RI and LTI macrophages. Around 130 phagosomes were quantified for LC3 detection and 190 phagosomes for LAMP-2 detection from three independent experiments.Data represent the mean ± S.E.M., (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001 from two-tailed Student's t -test. Nuclei were stained with Hoechst 33258. Scale bars: 10 μm.

    Journal: Cellular Microbiology

    Article Title: Experimental selection of long-term intracellular mycobacteria

    doi: 10.1111/cmi.12303

    Figure Lengend Snippet: Selected bacteria reside in LC3-positive phagolysosomes.A and B. Long-term infected (LTI) macrophages for at least 2 years and recently infected (RI) with BCG-GFP ancestral strain for 24 h were stained with Lysotracker (A, left panel) or DQ-BSA (B, right panel).C and D. Quantitative analysis of the fluorescence intensity of Lysotracker (C) and DQ-BSA (D) associated with phagosomes in LTI macrophages compared with RI cells. Around 80 phagosomes were quantified in Lysotracker or DQ-BSA stained macrophages from three independent experiments.E and F. Recently infected macrophages (RI) during 24 h (E) and LTI (F) macrophages (2 years old) were subjected to immunofluorescence and endogenous LC3 and LAMP-2 were detected using specific antibodies. Insets depict colocalization with single markers.G and H. Quantitative analysis of the fluorescence intensity of LC3 (G) and LAMP-2 (H) associated with phagosomes of RI and LTI macrophages. Around 130 phagosomes were quantified for LC3 detection and 190 phagosomes for LAMP-2 detection from three independent experiments.Data represent the mean ± S.E.M., (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001 from two-tailed Student's t -test. Nuclei were stained with Hoechst 33258. Scale bars: 10 μm.

    Article Snippet: To analyse cell death, LTI or RI macrophages were scraped and resuspended in PBS with 1% BSA (Albumin bovine serum, Sigma, Germany).

    Techniques: Infection, Staining, Fluorescence, Immunofluorescence, Two Tailed Test

    ELISA results for interfering antigens to test the specificity of anti-keratin primary antibody: (a) PBS; (b) negative control; (c) BSA; (d) HSA; (e) ovalbumin; (f) collagen; (g) sericin; (h) fibroin; (i) cotton extraction; (j) hemp extraction; (k) wool extraction; (l) keratin. Each column of the figure represents the mean ± SD (standard deviation) of n =5 assays. The dashed line shows the test criterion (the mean OD PBS plus three times the corresponding standard deviation). If OD 450 nm is above the dashed line, the result is positive, and vice versa.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: Development of an Enzyme-Linked Immunosorbent Assay and Gold-Labelled Immunochromatographic Strip Assay for the Detection of Ancient Wool

    doi: 10.1155/2018/2641624

    Figure Lengend Snippet: ELISA results for interfering antigens to test the specificity of anti-keratin primary antibody: (a) PBS; (b) negative control; (c) BSA; (d) HSA; (e) ovalbumin; (f) collagen; (g) sericin; (h) fibroin; (i) cotton extraction; (j) hemp extraction; (k) wool extraction; (l) keratin. Each column of the figure represents the mean ± SD (standard deviation) of n =5 assays. The dashed line shows the test criterion (the mean OD PBS plus three times the corresponding standard deviation). If OD 450 nm is above the dashed line, the result is positive, and vice versa.

    Article Snippet: Bovine serum albumin (BSA), human serum albumin (HSA), chicken ovalbumin, and collagen were purchased from Sigma-Aldrich.

    Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation

    Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with ethylenediaminetetraacetic acid shows absence of activity. (C)

    Journal: Tissue Engineering. Part A

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue

    doi: 10.1089/ten.tea.2018.0036

    Figure Lengend Snippet: Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with ethylenediaminetetraacetic acid shows absence of activity. (C)

    Article Snippet: Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Techniques: Expressing, Activity Assay, RNA Sequencing Assay, Zymography, Recombinant, Inhibition

    Soluble rCTD is a dimer in equilibrium and the CTD cleaved off natively expressed proRgpB spontaneously dimerizes. ( A ) rCTD at 1, 0.5 and 0.1 mg ml −1 was treated with glutaraldehyde and analysed by SDS-PAGE. ( B ) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl 2 , 0.02% NaN 3 pH 7.5 ( C ) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.

    Journal: Scientific Reports

    Article Title: The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

    doi: 10.1038/srep23123

    Figure Lengend Snippet: Soluble rCTD is a dimer in equilibrium and the CTD cleaved off natively expressed proRgpB spontaneously dimerizes. ( A ) rCTD at 1, 0.5 and 0.1 mg ml −1 was treated with glutaraldehyde and analysed by SDS-PAGE. ( B ) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl 2 , 0.02% NaN 3 pH 7.5 ( C ) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.

    Article Snippet: Limited proteolysis investigations Different rIgSF-CTD variants were incubated with tested proteases in 20 mM Tris, 50 mM NaCl, 5 mM CaCl2 , pH 7.6 (bovine factor Xa (Sigma-Aldrich, St. Louis, MO, USA)), 25 mM Tris, 1 mM EDTA, 0.02% NaN3 , pH 8.0 (Lys-C endopeptidase and V8 protease (Promega, Fitchburg, WI, USA)) or 0.1 M Tris, 150 mM NaCl, 5 mM EDTA, 0.05% Tween 20, 0.02% NaN3 , pH 7.5 (porcine pancreatic elastase (Promega)) and human neutrophil elastase (BioCentrum, Krakow, Poland)) for up to 72 h at different weight ratios.

    Techniques: SDS Page, Recombinant, Size-exclusion Chromatography, Western Blot

    DNase I footprinting assay of the ykoL promoter using PhoP and PhoP∼P. A PCR fragment corresponding to the ykoL promoter region (−153 to +60 relative to the transcriptional start site of ykoL ) was used as a DNA probe. Coding strand footprinting 32 P-labeled YkoL-FOR primer and the noncoding strand footprinting 32 P-labeled YkoL-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The black vertical lines show the regions that were bound by PhoP and PhoP∼P. The thick black lines show the regions where PhoP and PhoP∼P bound with a higher affinity. The numbers indicate the positions of the PhoP binding sites relative to the transcriptional start site M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the ykoL promoter the translational start site, RBS, transcriptional start site (+1), and corresponding −35 and −10 sequences of the promoter are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of PhoP dimer (5 ± 2-bp spacer and a maximum of two mismatches). The positions of YkoL-FOR and YkoL-REV primers are shown by thick arrows and are labeled.

    Journal: Journal of Bacteriology

    Article Title: Transcriptional Regulation of the phoPR Operon in Bacillus subtilis

    doi: 10.1128/JB.186.4.1182-1190.2004

    Figure Lengend Snippet: DNase I footprinting assay of the ykoL promoter using PhoP and PhoP∼P. A PCR fragment corresponding to the ykoL promoter region (−153 to +60 relative to the transcriptional start site of ykoL ) was used as a DNA probe. Coding strand footprinting 32 P-labeled YkoL-FOR primer and the noncoding strand footprinting 32 P-labeled YkoL-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The black vertical lines show the regions that were bound by PhoP and PhoP∼P. The thick black lines show the regions where PhoP and PhoP∼P bound with a higher affinity. The numbers indicate the positions of the PhoP binding sites relative to the transcriptional start site M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the ykoL promoter the translational start site, RBS, transcriptional start site (+1), and corresponding −35 and −10 sequences of the promoter are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of PhoP dimer (5 ± 2-bp spacer and a maximum of two mismatches). The positions of YkoL-FOR and YkoL-REV primers are shown by thick arrows and are labeled.

    Article Snippet: The reactions were stopped with DNase I stop solution (0.4 M Na-acetate, 50 μg of calf thymus DNA [Sigma] per ml, and 2.5 mM EDTA).

    Techniques: Footprinting, Polymerase Chain Reaction, Labeling, Incubation, Binding Assay, Sequencing

    DNase I footprinting assay of the phoPR promoter by using PhoP and PhoP∼P. A PCR fragment corresponding to the phoPR promoter region (−205 to +16 relative to the translational start site of phoP ) was used as the DNA probe. Coding strand footprinting 32 P-labeled PhoP-FOR primer and the noncoding strand footprinting 32 P-labeled PhoP-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The thick black vertical lines show the regions where PhoP and PhoP∼P bound. The numbers indicate the positions of the PhoP binding sites relative to the translational start site. M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the phoPR promoter (lower part of the figure), the translational start site, ribosome binding site (RBS), transcriptional start sites (TS), and corresponding −35 and −10 sequences of the P 1 and P 2 promoters are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of the PhoP dimer (5 ± 2-bp spacer and maximum of two mismatches). The positions of PhoP-FOR and PhoP-REV primers are shown by thick arrows and are labeled.

    Journal: Journal of Bacteriology

    Article Title: Transcriptional Regulation of the phoPR Operon in Bacillus subtilis

    doi: 10.1128/JB.186.4.1182-1190.2004

    Figure Lengend Snippet: DNase I footprinting assay of the phoPR promoter by using PhoP and PhoP∼P. A PCR fragment corresponding to the phoPR promoter region (−205 to +16 relative to the translational start site of phoP ) was used as the DNA probe. Coding strand footprinting 32 P-labeled PhoP-FOR primer and the noncoding strand footprinting 32 P-labeled PhoP-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The thick black vertical lines show the regions where PhoP and PhoP∼P bound. The numbers indicate the positions of the PhoP binding sites relative to the translational start site. M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the phoPR promoter (lower part of the figure), the translational start site, ribosome binding site (RBS), transcriptional start sites (TS), and corresponding −35 and −10 sequences of the P 1 and P 2 promoters are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of the PhoP dimer (5 ± 2-bp spacer and maximum of two mismatches). The positions of PhoP-FOR and PhoP-REV primers are shown by thick arrows and are labeled.

    Article Snippet: The reactions were stopped with DNase I stop solution (0.4 M Na-acetate, 50 μg of calf thymus DNA [Sigma] per ml, and 2.5 mM EDTA).

    Techniques: Footprinting, Polymerase Chain Reaction, Labeling, Incubation, Binding Assay, Sequencing

    Image-based cell death assays. Example images of cells 24 h after TNFα treatment by the fluorescence assays under evaluation. For each assay, the relevant channels were merged, pseudocolored, and adjusted for brightness and contrast. (A) Cells stained with propidium iodide (PI, red ), Annexin V Alexa Fluor 488 (AnnV, green ), and DRAQ5 ( blue ). (B) Cells stained with tetramethylrhodamine, ethyl ester (TMRE; red ), and DRAQ5 ( blue ). (C) Intensity weighted ratio images of cells expressing the caspase sensor showing monomeric Cerulean (mCer; cyan ) and stimulated emission of Venus (Venus, yellow ). (D) Cells stained with nonyl acridine orange (NAO; green ) and DRAQ5 ( blue ). Scale bar = 20 μm.

    Journal: Assay and Drug Development Technologies

    Article Title: A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis

    doi: 10.1089/adt.2015.661

    Figure Lengend Snippet: Image-based cell death assays. Example images of cells 24 h after TNFα treatment by the fluorescence assays under evaluation. For each assay, the relevant channels were merged, pseudocolored, and adjusted for brightness and contrast. (A) Cells stained with propidium iodide (PI, red ), Annexin V Alexa Fluor 488 (AnnV, green ), and DRAQ5 ( blue ). (B) Cells stained with tetramethylrhodamine, ethyl ester (TMRE; red ), and DRAQ5 ( blue ). (C) Intensity weighted ratio images of cells expressing the caspase sensor showing monomeric Cerulean (mCer; cyan ) and stimulated emission of Venus (Venus, yellow ). (D) Cells stained with nonyl acridine orange (NAO; green ) and DRAQ5 ( blue ). Scale bar = 20 μm.

    Article Snippet: Cell Staining Cells were incubated under culture conditions with fluorescent dyes for 30 min before image acquisition, using either 100 nM NAO (Sigma-Aldrich) or 10 nM tetramethylrhodamine, ethyl ester (TMRE; Life Technologies) in α-MEM, or 1 μM propidium iodide (PI; Sigma-Aldrich) and 1:100 Annexin V Alexa Fluor 488 (Life Technologies) in 0.01 M HEPES pH 7.4, 0.14 M NaCl, and 2.5 mM CaCl2 .

    Techniques: Fluorescence, Staining, Expressing

    USP7S interacts with Mule and prevents its self-ubiquitylation in vitro. ( A ) Equal amounts (2.5 pmol) of purified recombinant His-tagged USP7S and GST-tagged Mule were incubated in buffer containing 50 mM Tris–HCl, pH 8.0, 50 mM KCl, 1 mM EDTA, 1 mM DTT for 30 min at room temperature with shaking. USP7S and Mule were then pulled down using either magnetic beads as a control (Mock PD, 100%) or using magnetic Ni-NTA agarose beads (His PD, 100%) or GST agarose beads (GST PD, 100%), respectively. The beads were boiled in SDS–PAGE sample buffer for 5 min, and proteins were separated by 10% SDS–PAGE and analysed using USP7S and Mule antibodies (10% of the input is loaded). ( B ) HeLa cells were simultaneously transfected with USP7S and Mule (1 pmol each) expression plasmids, whole-cell extracts were prepared and used to pull down HA-tagged Mule or Flag-tagged USP7S using either magnetic beads as a control (Mock PD, 100%), HA- (HA PD, 100%) or Flag-agarose (Flag PD, 100%) beads. Proteins were separated by 4–20% SDS–PAGE and analysed using USP7S and Mule antibodies (15% of the input is loaded). ( C ) In vitro deubiquitylation of self-ubiquitylated Mule HECT-domain by recombinant wild-type USP7S protein (wtUSP7S) or its inactive mutant (C223S USP7S) analysed by western blotting. Equal loading of the enzyme is demonstrated using USP7S antibodies.

    Journal: Nucleic Acids Research

    Article Title: USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair

    doi: 10.1093/nar/gks1359

    Figure Lengend Snippet: USP7S interacts with Mule and prevents its self-ubiquitylation in vitro. ( A ) Equal amounts (2.5 pmol) of purified recombinant His-tagged USP7S and GST-tagged Mule were incubated in buffer containing 50 mM Tris–HCl, pH 8.0, 50 mM KCl, 1 mM EDTA, 1 mM DTT for 30 min at room temperature with shaking. USP7S and Mule were then pulled down using either magnetic beads as a control (Mock PD, 100%) or using magnetic Ni-NTA agarose beads (His PD, 100%) or GST agarose beads (GST PD, 100%), respectively. The beads were boiled in SDS–PAGE sample buffer for 5 min, and proteins were separated by 10% SDS–PAGE and analysed using USP7S and Mule antibodies (10% of the input is loaded). ( B ) HeLa cells were simultaneously transfected with USP7S and Mule (1 pmol each) expression plasmids, whole-cell extracts were prepared and used to pull down HA-tagged Mule or Flag-tagged USP7S using either magnetic beads as a control (Mock PD, 100%), HA- (HA PD, 100%) or Flag-agarose (Flag PD, 100%) beads. Proteins were separated by 4–20% SDS–PAGE and analysed using USP7S and Mule antibodies (15% of the input is loaded). ( C ) In vitro deubiquitylation of self-ubiquitylated Mule HECT-domain by recombinant wild-type USP7S protein (wtUSP7S) or its inactive mutant (C223S USP7S) analysed by western blotting. Equal loading of the enzyme is demonstrated using USP7S antibodies.

    Article Snippet: Ubiquitin and E2 were removed from the reaction mixture by filtration and buffer exchange into deubiquitylation buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol and 2 mM DTT using Amicon Ultra 30K units (Millipore).

    Techniques: In Vitro, Purification, Recombinant, Incubation, Magnetic Beads, SDS Page, Transfection, Expressing, Mutagenesis, Western Blot

    Western blot analysis of p-IκB-α and p-ERK1/2 protein levels from lungs of PBS-challenged mice pretreated with vehicle (PBS), from ovalbumin (OVA)-challenged mice pretreated with vehicle (OVA), Ang-(1–7) and A779 plus Ang-(1–7).

    Journal: British Journal of Pharmacology

    Article Title: Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-?B-dependent pathways

    doi: 10.1111/j.1476-5381.2012.01905.x

    Figure Lengend Snippet: Western blot analysis of p-IκB-α and p-ERK1/2 protein levels from lungs of PBS-challenged mice pretreated with vehicle (PBS), from ovalbumin (OVA)-challenged mice pretreated with vehicle (OVA), Ang-(1–7) and A779 plus Ang-(1–7).

    Article Snippet: Chemicals used included ovalbumin (grade V), halothane, absolute ethanol (Merck KGaA, Darmstadt, Germany), formaldehyde (Surechem Products LTD, Suffolk, UK), Alu-Gel-S (SERVA Electrophoresis GmbH, Heidelberg, Germany), isotone II diluent solution (Beckman Coulter Inc., Krefeld, Germany), Zap-OGLOBIN (Coulter Electronics LTD, Buckinghamshire, UK), Diff-Quik (Baxter Dade AG, Dudingen, Switzerland), PBS (Sigma-Aldrich, St Louis, MO, USA), Ang-(1–7), A779, Tris-base, NaCl, Na4 P2 O7 , NaF, CaCl2 , MgCl2 , Glycerol, NP4 O, Na3 VO4 , PMSF, protease inhibitor cocktail, Ponceau 2R (Sigma-Aldrich), phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) xylene, ethanol (Honeywell Riedel-de Haën®, Seelze, Germany), haematoxylin, eosin, glacial acetic acid, ferric chloride, HCl, periodic acid, DMSO (BDH Laboratory Supplies, Poole, UK), Dpx mountant, picric acid, mercuric chloride, phosphomolybdic acid, light green SF yellowish, Schiff's reagent (Sigma-Aldrich), acid fuchsin (Gurr Microscopy Materials; BDH Ltd., Poole, UK), rabbit monoclonal antibodies for total and phosphorylated IκB-α and rabbit polyclonal antibody for phosphorylated ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-actin antibody, phenanthroline (Sigma-Aldrich), nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany).

    Techniques: Western Blot, Mouse Assay

    Histological examination of PAS stain of ovalbumin-challenged/vehicle-pretreated mice shows significant bronchial mucus production and goblet cell hyper/metaplasia in mice (B) ( n = 6) compared with PBS-challenged mice (A) ( n = 6). Treatment with Ang-(1–7)

    Journal: British Journal of Pharmacology

    Article Title: Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-?B-dependent pathways

    doi: 10.1111/j.1476-5381.2012.01905.x

    Figure Lengend Snippet: Histological examination of PAS stain of ovalbumin-challenged/vehicle-pretreated mice shows significant bronchial mucus production and goblet cell hyper/metaplasia in mice (B) ( n = 6) compared with PBS-challenged mice (A) ( n = 6). Treatment with Ang-(1–7)

    Article Snippet: Chemicals used included ovalbumin (grade V), halothane, absolute ethanol (Merck KGaA, Darmstadt, Germany), formaldehyde (Surechem Products LTD, Suffolk, UK), Alu-Gel-S (SERVA Electrophoresis GmbH, Heidelberg, Germany), isotone II diluent solution (Beckman Coulter Inc., Krefeld, Germany), Zap-OGLOBIN (Coulter Electronics LTD, Buckinghamshire, UK), Diff-Quik (Baxter Dade AG, Dudingen, Switzerland), PBS (Sigma-Aldrich, St Louis, MO, USA), Ang-(1–7), A779, Tris-base, NaCl, Na4 P2 O7 , NaF, CaCl2 , MgCl2 , Glycerol, NP4 O, Na3 VO4 , PMSF, protease inhibitor cocktail, Ponceau 2R (Sigma-Aldrich), phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) xylene, ethanol (Honeywell Riedel-de Haën®, Seelze, Germany), haematoxylin, eosin, glacial acetic acid, ferric chloride, HCl, periodic acid, DMSO (BDH Laboratory Supplies, Poole, UK), Dpx mountant, picric acid, mercuric chloride, phosphomolybdic acid, light green SF yellowish, Schiff's reagent (Sigma-Aldrich), acid fuchsin (Gurr Microscopy Materials; BDH Ltd., Poole, UK), rabbit monoclonal antibodies for total and phosphorylated IκB-α and rabbit polyclonal antibody for phosphorylated ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-actin antibody, phenanthroline (Sigma-Aldrich), nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany).

    Techniques: Staining, Mouse Assay

    Masson's Trichrome staining of lung samples from ovalbumin-challenged/vehicle-treated mice show significant peribronchial and perivascular fibrosis (B) ( n = 6) compared with PBS-challenged mice (A) ( n = 6). Treatment with Ang-(1–7) (0.3 mg·kg

    Journal: British Journal of Pharmacology

    Article Title: Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-?B-dependent pathways

    doi: 10.1111/j.1476-5381.2012.01905.x

    Figure Lengend Snippet: Masson's Trichrome staining of lung samples from ovalbumin-challenged/vehicle-treated mice show significant peribronchial and perivascular fibrosis (B) ( n = 6) compared with PBS-challenged mice (A) ( n = 6). Treatment with Ang-(1–7) (0.3 mg·kg

    Article Snippet: Chemicals used included ovalbumin (grade V), halothane, absolute ethanol (Merck KGaA, Darmstadt, Germany), formaldehyde (Surechem Products LTD, Suffolk, UK), Alu-Gel-S (SERVA Electrophoresis GmbH, Heidelberg, Germany), isotone II diluent solution (Beckman Coulter Inc., Krefeld, Germany), Zap-OGLOBIN (Coulter Electronics LTD, Buckinghamshire, UK), Diff-Quik (Baxter Dade AG, Dudingen, Switzerland), PBS (Sigma-Aldrich, St Louis, MO, USA), Ang-(1–7), A779, Tris-base, NaCl, Na4 P2 O7 , NaF, CaCl2 , MgCl2 , Glycerol, NP4 O, Na3 VO4 , PMSF, protease inhibitor cocktail, Ponceau 2R (Sigma-Aldrich), phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) xylene, ethanol (Honeywell Riedel-de Haën®, Seelze, Germany), haematoxylin, eosin, glacial acetic acid, ferric chloride, HCl, periodic acid, DMSO (BDH Laboratory Supplies, Poole, UK), Dpx mountant, picric acid, mercuric chloride, phosphomolybdic acid, light green SF yellowish, Schiff's reagent (Sigma-Aldrich), acid fuchsin (Gurr Microscopy Materials; BDH Ltd., Poole, UK), rabbit monoclonal antibodies for total and phosphorylated IκB-α and rabbit polyclonal antibody for phosphorylated ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-actin antibody, phenanthroline (Sigma-Aldrich), nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany).

    Techniques: Staining, Mouse Assay

    Representative low-magnification light photomicrographs display H E staining of whole lung samples from (A) PBS vehicle ( n = 6), (B) ovalbumin (OVA)-challenged ( n = 6), (C) OVA-challenged, Ang-(1–7) (0.3 mg·kg −1 ; i.p.)

    Journal: British Journal of Pharmacology

    Article Title: Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-?B-dependent pathways

    doi: 10.1111/j.1476-5381.2012.01905.x

    Figure Lengend Snippet: Representative low-magnification light photomicrographs display H E staining of whole lung samples from (A) PBS vehicle ( n = 6), (B) ovalbumin (OVA)-challenged ( n = 6), (C) OVA-challenged, Ang-(1–7) (0.3 mg·kg −1 ; i.p.)

    Article Snippet: Chemicals used included ovalbumin (grade V), halothane, absolute ethanol (Merck KGaA, Darmstadt, Germany), formaldehyde (Surechem Products LTD, Suffolk, UK), Alu-Gel-S (SERVA Electrophoresis GmbH, Heidelberg, Germany), isotone II diluent solution (Beckman Coulter Inc., Krefeld, Germany), Zap-OGLOBIN (Coulter Electronics LTD, Buckinghamshire, UK), Diff-Quik (Baxter Dade AG, Dudingen, Switzerland), PBS (Sigma-Aldrich, St Louis, MO, USA), Ang-(1–7), A779, Tris-base, NaCl, Na4 P2 O7 , NaF, CaCl2 , MgCl2 , Glycerol, NP4 O, Na3 VO4 , PMSF, protease inhibitor cocktail, Ponceau 2R (Sigma-Aldrich), phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) xylene, ethanol (Honeywell Riedel-de Haën®, Seelze, Germany), haematoxylin, eosin, glacial acetic acid, ferric chloride, HCl, periodic acid, DMSO (BDH Laboratory Supplies, Poole, UK), Dpx mountant, picric acid, mercuric chloride, phosphomolybdic acid, light green SF yellowish, Schiff's reagent (Sigma-Aldrich), acid fuchsin (Gurr Microscopy Materials; BDH Ltd., Poole, UK), rabbit monoclonal antibodies for total and phosphorylated IκB-α and rabbit polyclonal antibody for phosphorylated ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-actin antibody, phenanthroline (Sigma-Aldrich), nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany).

    Techniques: Staining

    Role of the C2 domain on translocation properties of PKCα. HEK293 cells were cotransfected with plasmids coding for PKCα(C2-mut)-EGFP and PKCα(WT)-mRFP. (A) Depicts results in cells stimulated with 100 μM ATP (A, b) and 1 μM PMA (A, c). Green (EGFP fusion protein) and red (mRFP fusion protein) traces depict the time course of fluorescence derived from the plasma membrane (top traces) and cytosol (bottom traces) during the stimulation regimes. Similar results were obtained in 16 independent experiments ( n = 51 cells). (B) Translocation responses of WT and C2-mutated PKCs to flash photolytic increases in Ca 2+ . Cells were loaded with caged Ca 2+ NPEGTA-AM. B (a) depicts the resting fluorescence of the two PKC constructs, as indicated. B (c) shows the time course of the plasma membrane (top traces) and cytosolic (bottom traces) fluorescence changes of the EGFP construct containing the mutated C2 domain (green) and the WT-mRFP (red). The time points of the UV flashes are indicated by the two blue vertical dashed lines. For the three time points depicted in B (c) by the dashed black arrows, the relative changes of the fluorescence have been replotted in B (b). The color-coded images (see color wedge attached) for the WT (top row) and mutated PKC (bottom traces) are shown. Similar results were obtained in 14 independent experiments ( n = 33 cells).

    Journal: The Journal of Cell Biology

    Article Title: PKC?: a versatile key for decoding the cellular calcium toolkit

    doi: 10.1083/jcb.200604033

    Figure Lengend Snippet: Role of the C2 domain on translocation properties of PKCα. HEK293 cells were cotransfected with plasmids coding for PKCα(C2-mut)-EGFP and PKCα(WT)-mRFP. (A) Depicts results in cells stimulated with 100 μM ATP (A, b) and 1 μM PMA (A, c). Green (EGFP fusion protein) and red (mRFP fusion protein) traces depict the time course of fluorescence derived from the plasma membrane (top traces) and cytosol (bottom traces) during the stimulation regimes. Similar results were obtained in 16 independent experiments ( n = 51 cells). (B) Translocation responses of WT and C2-mutated PKCs to flash photolytic increases in Ca 2+ . Cells were loaded with caged Ca 2+ NPEGTA-AM. B (a) depicts the resting fluorescence of the two PKC constructs, as indicated. B (c) shows the time course of the plasma membrane (top traces) and cytosolic (bottom traces) fluorescence changes of the EGFP construct containing the mutated C2 domain (green) and the WT-mRFP (red). The time points of the UV flashes are indicated by the two blue vertical dashed lines. For the three time points depicted in B (c) by the dashed black arrows, the relative changes of the fluorescence have been replotted in B (b). The color-coded images (see color wedge attached) for the WT (top row) and mutated PKC (bottom traces) are shown. Similar results were obtained in 14 independent experiments ( n = 33 cells).

    Article Snippet: ATP (Sigma-Aldrich) and PMA (Calbiochem) were dissolved as stock solutions in the appropriate solvents and diluted in the external salt solution at the given concentrations before each experiment.

    Techniques: Translocation Assay, Fluorescence, Derivative Assay, Construct

    Coexpression and faithful trafficking of tandem CLAG3 isoforms. (A) Indirect immunofluorescence images showing colocalization of Dd2-HA and 3D7-miniSOG-FLAG CLAG3 isoforms in TFLC3 parasites at the schizont stage and in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (B and C) Immunoblot assays performed using TFLC3 cell lysates and indicated epitope tag antibodies without or with extracellular pronase E treatment prior to cell lysis (− and +, respectively). (D and E) Immunoblot assays using anti-CLAG3 antibody and indicated parasites, without and with pronase E treatment (− and +, respectively). While two cleavage products are detected in TFLC3 , the C3attB parent produces a single fragment.

    Journal: mBio

    Article Title: CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane

    doi: 10.1128/mBio.02293-17

    Figure Lengend Snippet: Coexpression and faithful trafficking of tandem CLAG3 isoforms. (A) Indirect immunofluorescence images showing colocalization of Dd2-HA and 3D7-miniSOG-FLAG CLAG3 isoforms in TFLC3 parasites at the schizont stage and in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (B and C) Immunoblot assays performed using TFLC3 cell lysates and indicated epitope tag antibodies without or with extracellular pronase E treatment prior to cell lysis (− and +, respectively). (D and E) Immunoblot assays using anti-CLAG3 antibody and indicated parasites, without and with pronase E treatment (− and +, respectively). While two cleavage products are detected in TFLC3 , the C3attB parent produces a single fragment.

    Article Snippet: Enriched trophozoite-infected erythrocytes were suspended in phosphate-buffered saline (PBS) supplemented with 0.6 mM CaCl2 and 1 mM MgCl2 (PBS2) at 5% hematocrit prior to treatment with 1 mg/ml pronase E (Sigma-Aldrich) for 1 h at 37°C.

    Techniques: Immunofluorescence, Lysis

    CLAG3 merodiploids reveal a stoichiometric contribution to PSAC-mediated nutrient uptake. (A) Schematic showing the minimum distinct combinations of host membrane RhopH complexes in the TFLC3 parasite. CLAG3 is shown as blue ribbon dimers embedded in the host membrane; note the permutations of the two C-terminal epitope tags (gold circles and pink ellipses). RhopH2 and RhopH3 are represented in green. (B) Sorbitol uptake kinetics for indicated parasites with addition of 0, 0.3, 0.6, 1.8, 5, and 15 µM ISPA-28 (top to bottom traces in each panel, respectively). Inhibition in TFLC3 and miniSOG2 is intermediate between those observed for 3D7 and Dd2. (C) Ribbon diagrams showing expression of two CLAG3 isoforms in TFLC3 and miniSOG2 . (D) Immunofluorescence images showing trafficking of the 3D7-miniSOG-FLAG CLAG3 isoform in miniSOG2 . This protein, expressed under the msp2 promoter, colocalizes with RhopH3 in schizonts and is exported normally in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (E and F) Immunoblot assays using membranes from miniSOG2 probed with anti-FLAG (E) and anti-CLAG3 (F) antibodies. Proteins were separated after cells were incubated without and with pronase E treatment (− and +, respectively). Both CLAG3 proteins expressed in this parasite are integral to the erythrocyte membrane. (G) ISPA-28 dose responses from experiments as in panel B. Symbols represent mean ± SEM for wild-type parasites (3D7, white circles; Dd2, black circles) and merodiploid transfectants ( TFLC3 , blue triangles; miniSOG2 , red triangles).

    Journal: mBio

    Article Title: CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane

    doi: 10.1128/mBio.02293-17

    Figure Lengend Snippet: CLAG3 merodiploids reveal a stoichiometric contribution to PSAC-mediated nutrient uptake. (A) Schematic showing the minimum distinct combinations of host membrane RhopH complexes in the TFLC3 parasite. CLAG3 is shown as blue ribbon dimers embedded in the host membrane; note the permutations of the two C-terminal epitope tags (gold circles and pink ellipses). RhopH2 and RhopH3 are represented in green. (B) Sorbitol uptake kinetics for indicated parasites with addition of 0, 0.3, 0.6, 1.8, 5, and 15 µM ISPA-28 (top to bottom traces in each panel, respectively). Inhibition in TFLC3 and miniSOG2 is intermediate between those observed for 3D7 and Dd2. (C) Ribbon diagrams showing expression of two CLAG3 isoforms in TFLC3 and miniSOG2 . (D) Immunofluorescence images showing trafficking of the 3D7-miniSOG-FLAG CLAG3 isoform in miniSOG2 . This protein, expressed under the msp2 promoter, colocalizes with RhopH3 in schizonts and is exported normally in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (E and F) Immunoblot assays using membranes from miniSOG2 probed with anti-FLAG (E) and anti-CLAG3 (F) antibodies. Proteins were separated after cells were incubated without and with pronase E treatment (− and +, respectively). Both CLAG3 proteins expressed in this parasite are integral to the erythrocyte membrane. (G) ISPA-28 dose responses from experiments as in panel B. Symbols represent mean ± SEM for wild-type parasites (3D7, white circles; Dd2, black circles) and merodiploid transfectants ( TFLC3 , blue triangles; miniSOG2 , red triangles).

    Article Snippet: Enriched trophozoite-infected erythrocytes were suspended in phosphate-buffered saline (PBS) supplemented with 0.6 mM CaCl2 and 1 mM MgCl2 (PBS2) at 5% hematocrit prior to treatment with 1 mg/ml pronase E (Sigma-Aldrich) for 1 h at 37°C.

    Techniques: Inhibition, Expressing, Immunofluorescence, Incubation

    Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with DMSO (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor PMSF (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1

    doi:

    Figure Lengend Snippet: Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with DMSO (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor PMSF (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.

    Article Snippet: Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2 ) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [ ]), EDTA (10 mM), ortho -phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem).

    Techniques: Expressing, Cell Culture, Transfection, Incubation