tet podocytes  (Thermo Fisher)


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    Structured Review

    Thermo Fisher tet podocytes
    UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney <t>podocytes.</t> A . UCH-L1 <t>tet-on</t> podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. *** p
    Tet Podocytes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 6142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis"

    Article Title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-11-76

    UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney podocytes. A . UCH-L1 tet-on podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. *** p
    Figure Legend Snippet: UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney podocytes. A . UCH-L1 tet-on podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. *** p

    Techniques Used: Staining

    2) Product Images from "UCP-3 uncoupling protein confers hypoxia resistance to renal epithelial cells and is upregulated in renal cell carcinoma"

    Article Title: UCP-3 uncoupling protein confers hypoxia resistance to renal epithelial cells and is upregulated in renal cell carcinoma

    Journal: Scientific Reports

    doi: 10.1038/srep13450

    H/R-adapted cultures exhibit after H/R stress less hyperpolarization of the inner mitochondrial membrane potential (ΔΨ m ) than control cultures. ( A,B ) Dot plots ( A ) and histograms ( B ) showing forward scatter and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) fluorescence as a measure of cell size and ΔΨ m , respectively. Depicted are a control (left) and a H/R-adapted PT culture (right) recorded by flow cytometry under normoxic conditions (black lines in ( B )) and after H/R stress (48 h hypoxia/24 h reoxygenation; ( A ) and red histograms in ( B )). Cell populations with dissipated ΔΨ m (low ΔΨ m ) are indicated by gate and marker in A and B, respectively. ( C,D ) Mean percentage of control (open bars) and H/R-adapted cells (closed bars) with broken-down ΔΨm ( C ) and ( D ) mean TMRE fluorescence intensity of the cell population with high ΔΨ m (±SE, n = 9 from 3 cultures each determined in triplicate) recorded as in ( B ) under normoxic conditions (left), after H/R stress (48 h hypoxia/24 h reoxygenation, middle), or after pharmacological break-down of ΔΨ m by the proton ionophore carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 1 μM). * and ** indicate p ≤ 0.05 and p ≤ 0.01, respectively (ANOVA).
    Figure Legend Snippet: H/R-adapted cultures exhibit after H/R stress less hyperpolarization of the inner mitochondrial membrane potential (ΔΨ m ) than control cultures. ( A,B ) Dot plots ( A ) and histograms ( B ) showing forward scatter and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) fluorescence as a measure of cell size and ΔΨ m , respectively. Depicted are a control (left) and a H/R-adapted PT culture (right) recorded by flow cytometry under normoxic conditions (black lines in ( B )) and after H/R stress (48 h hypoxia/24 h reoxygenation; ( A ) and red histograms in ( B )). Cell populations with dissipated ΔΨ m (low ΔΨ m ) are indicated by gate and marker in A and B, respectively. ( C,D ) Mean percentage of control (open bars) and H/R-adapted cells (closed bars) with broken-down ΔΨm ( C ) and ( D ) mean TMRE fluorescence intensity of the cell population with high ΔΨ m (±SE, n = 9 from 3 cultures each determined in triplicate) recorded as in ( B ) under normoxic conditions (left), after H/R stress (48 h hypoxia/24 h reoxygenation, middle), or after pharmacological break-down of ΔΨ m by the proton ionophore carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 1 μM). * and ** indicate p ≤ 0.05 and p ≤ 0.01, respectively (ANOVA).

    Techniques Used: Fluorescence, Flow Cytometry, Cytometry, Marker

    3) Product Images from "Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats"

    Article Title: Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022181

    Fluorescence micrographs demonstrating the double-labelling of TUNEL with other metabolic markers. ( A2–H2 ) Double labelling of TUNEL with: ( A1 , E1 ) calpain assay, ( B1 , F1 ) PARP assay, ( C1 , G1 ) avidin and ( D1 , H1 ) caspase-3 in ( A–D ) P23H retina at PN15 and ( E–H ) S334ter retina at PN12 transgenic rats. ( A3–H3 ) Merged pictures. ( A4–H4 ) Right panel indicates the percentages of co-labelled cells in the ONL. In both mutants calpain and PARP activity co-localized with TUNEL in ∼30–40% of cells, while avidin-binding co-localized in 12–13% with TUNEL. Caspase-3 co-localization was observed in almost 47% of TUNEL-positive cells in S334ter but only in 4% in P23H retinas. Scale bar = 25 µm.
    Figure Legend Snippet: Fluorescence micrographs demonstrating the double-labelling of TUNEL with other metabolic markers. ( A2–H2 ) Double labelling of TUNEL with: ( A1 , E1 ) calpain assay, ( B1 , F1 ) PARP assay, ( C1 , G1 ) avidin and ( D1 , H1 ) caspase-3 in ( A–D ) P23H retina at PN15 and ( E–H ) S334ter retina at PN12 transgenic rats. ( A3–H3 ) Merged pictures. ( A4–H4 ) Right panel indicates the percentages of co-labelled cells in the ONL. In both mutants calpain and PARP activity co-localized with TUNEL in ∼30–40% of cells, while avidin-binding co-localized in 12–13% with TUNEL. Caspase-3 co-localization was observed in almost 47% of TUNEL-positive cells in S334ter but only in 4% in P23H retinas. Scale bar = 25 µm.

    Techniques Used: Fluorescence, TUNEL Assay, Avidin-Biotin Assay, Transgenic Assay, Activity Assay, Binding Assay

    Progression of metabolic cell death markers during 1st postnatal month. Percentage of labelled ONL cells in ( A ) P23H and ( B ) S334ter transgenic rats. While in both RP animal models, most markers analysed peaked together with cell death as evidenced by the TUNEL assay, activation of caspase-3 was absent in P23H retina but present in S334ter retina. In both mutants, calpain activity showed a delayed regression after the peak of cell death. Values are mean ± SD from at least three different animals. All mean ± SD and P values are consigned in the table S1 .
    Figure Legend Snippet: Progression of metabolic cell death markers during 1st postnatal month. Percentage of labelled ONL cells in ( A ) P23H and ( B ) S334ter transgenic rats. While in both RP animal models, most markers analysed peaked together with cell death as evidenced by the TUNEL assay, activation of caspase-3 was absent in P23H retina but present in S334ter retina. In both mutants, calpain activity showed a delayed regression after the peak of cell death. Values are mean ± SD from at least three different animals. All mean ± SD and P values are consigned in the table S1 .

    Techniques Used: Transgenic Assay, TUNEL Assay, Activation Assay, Activity Assay

    Differential regulation of cell death markers in (1) wt and (2) rhodopsin transgenic rats. ( A–B ) TUNEL assay for dying cells, ( C–D ) caspase-3 immunostaining, ( E–F ) calpain 3 immunostaining, ( G–H ) in situ calpain activity assay, ( I–J ) avidin binding and ( K–L ) in situ PARP activity assay. Left panels ( A , C , E , G and I ) correspond to PN15 animals and right panels ( B , D , F , H and J ) to PN12. All stainings showed large numbers of positive cells in P23H and especially in S334ter ONL, but not in wt retina. Scale bar = 50 µm.
    Figure Legend Snippet: Differential regulation of cell death markers in (1) wt and (2) rhodopsin transgenic rats. ( A–B ) TUNEL assay for dying cells, ( C–D ) caspase-3 immunostaining, ( E–F ) calpain 3 immunostaining, ( G–H ) in situ calpain activity assay, ( I–J ) avidin binding and ( K–L ) in situ PARP activity assay. Left panels ( A , C , E , G and I ) correspond to PN15 animals and right panels ( B , D , F , H and J ) to PN12. All stainings showed large numbers of positive cells in P23H and especially in S334ter ONL, but not in wt retina. Scale bar = 50 µm.

    Techniques Used: Transgenic Assay, TUNEL Assay, Immunostaining, In Situ, Activity Assay, Avidin-Biotin Assay, Binding Assay

    4) Product Images from "Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection"

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    Journal: Nature

    doi: 10.1038/nature19802

    Dependence of RNA targeting on crRNA variants, temperature and point mutations a, LbuC2c2 ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.
    Figure Legend Snippet: Dependence of RNA targeting on crRNA variants, temperature and point mutations a, LbuC2c2 ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.

    Techniques Used: Cleavage Assay, Activity Assay

    Binding data for LbuC2c2 to mature crRNA and target ssRNA a, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GG to LbuC2c2-WT, LbuC2c2-dHEPN1, LbuC2c2-dHEPN2, or LbuC2c2-dHEPN1/dHEPN2. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 27.1 ± 7.5 nM (LbuC2c2-WT), 15.2 ± 3.2 nM (LbuC2c2-dHEPN1), 11.5 ± 2.5 nM (LbuC2c2-dHEPN2), and 43.3 ± 11.5 nM (LbuC2c2- dHEPN1/dHEPN2). b, Representative electrophoretic mobility shift assay for binding reactions between LbuC2c2-dHEPN1/dHEPN2: crRNA-A_GG and either ‘on-target’ A ssRNA or ‘off-target’ B ssRNA, as indicated. Three independent experiments were conducted as described in the Methods. The gel was cropped for clarity. c, Quantified binding data from (b) were fitted to standard binding isoforms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 1.62 ± 0.43 nM for ssRNA A and N.D (≫10 nM) for ssRNA B. d, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GA to LbuC2c2-WT and LbuC2c2-R1079A. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 4.65 ± 0.6 nM (LbuC2c2-WT) and 2.52 ± 0.5 nM (LbuC2c2-R1079A). It is of note that these binding affinities differ from panel a. This difference is accounted for in a slight difference in the 5′ sequence of the guide with panel a guides beginning with a 5′-G G CCA… and panel d 5′-G A CCA. While the native sequence guide (5′-G A CCA) binds tighter to LbuC2c2, no difference is seen in the RNA targeting efficiencies of these guide variants (). Extended Data Fig. 6c
    Figure Legend Snippet: Binding data for LbuC2c2 to mature crRNA and target ssRNA a, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GG to LbuC2c2-WT, LbuC2c2-dHEPN1, LbuC2c2-dHEPN2, or LbuC2c2-dHEPN1/dHEPN2. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 27.1 ± 7.5 nM (LbuC2c2-WT), 15.2 ± 3.2 nM (LbuC2c2-dHEPN1), 11.5 ± 2.5 nM (LbuC2c2-dHEPN2), and 43.3 ± 11.5 nM (LbuC2c2- dHEPN1/dHEPN2). b, Representative electrophoretic mobility shift assay for binding reactions between LbuC2c2-dHEPN1/dHEPN2: crRNA-A_GG and either ‘on-target’ A ssRNA or ‘off-target’ B ssRNA, as indicated. Three independent experiments were conducted as described in the Methods. The gel was cropped for clarity. c, Quantified binding data from (b) were fitted to standard binding isoforms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 1.62 ± 0.43 nM for ssRNA A and N.D (≫10 nM) for ssRNA B. d, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GA to LbuC2c2-WT and LbuC2c2-R1079A. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 4.65 ± 0.6 nM (LbuC2c2-WT) and 2.52 ± 0.5 nM (LbuC2c2-R1079A). It is of note that these binding affinities differ from panel a. This difference is accounted for in a slight difference in the 5′ sequence of the guide with panel a guides beginning with a 5′-G G CCA… and panel d 5′-G A CCA. While the native sequence guide (5′-G A CCA) binds tighter to LbuC2c2, no difference is seen in the RNA targeting efficiencies of these guide variants (). Extended Data Fig. 6c

    Techniques Used: Binding Assay, Standard Deviation, Electrophoretic Mobility Shift Assay, Sequencing

    C2c2 provides sensitive detection of transcripts in complex mixtures a , Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b , Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c ,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β -actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β -actin null negative control). (mean ± s.d., n = 3) d , Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e , Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.
    Figure Legend Snippet: C2c2 provides sensitive detection of transcripts in complex mixtures a , Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b , Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c ,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β -actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β -actin null negative control). (mean ± s.d., n = 3) d , Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e , Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

    Techniques Used: RNA Detection, Fluorescence, Generated, Negative Control, CRISPR

    5) Product Images from "Three distinct developmental pathways for adaptive and two IFN-γ-producing γδ T subsets in adult thymus"

    Article Title: Three distinct developmental pathways for adaptive and two IFN-γ-producing γδ T subsets in adult thymus

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01963-w

    Progression through the D, E and F populations is induced by TCR signalling. a – c Changes in surface marker expression of TCRδ + cells from sorted population A to G cells after 2 days of culture on OP9-DL1 monolayers in the presence or absence of immobilised anti-CD3. The data are visualised as a histograms and b bar plots of the percentage of cells positive for each individual marker as well as c gated into the A to G populations. Bars depict the mean ± SEM from three independent experiments with cells sorted from four to eight mice. Statistical analyses were performed using the paired two-sided Student’s t test, with significance defined as * p
    Figure Legend Snippet: Progression through the D, E and F populations is induced by TCR signalling. a – c Changes in surface marker expression of TCRδ + cells from sorted population A to G cells after 2 days of culture on OP9-DL1 monolayers in the presence or absence of immobilised anti-CD3. The data are visualised as a histograms and b bar plots of the percentage of cells positive for each individual marker as well as c gated into the A to G populations. Bars depict the mean ± SEM from three independent experiments with cells sorted from four to eight mice. Statistical analyses were performed using the paired two-sided Student’s t test, with significance defined as * p

    Techniques Used: Marker, Expressing, Mouse Assay

    6) Product Images from "Kindlin-2 regulates hemostasis by controlling endothelial cell–surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism"

    Article Title: Kindlin-2 regulates hemostasis by controlling endothelial cell–surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism

    Journal: Blood

    doi: 10.1182/blood-2013-04-497669

    Defective clathrin-dependent trafficking of CD39 and CD73 in kindlin-2 +/ − ECs. (A) Western blot analysis of WT and kindlin-2 +/− EC lysates probed with Abs to CD39 and CD73 reveal similar total content of these enzymes in both mouse strains
    Figure Legend Snippet: Defective clathrin-dependent trafficking of CD39 and CD73 in kindlin-2 +/ − ECs. (A) Western blot analysis of WT and kindlin-2 +/− EC lysates probed with Abs to CD39 and CD73 reveal similar total content of these enzymes in both mouse strains

    Techniques Used: Western Blot

    Kindlin-2 +/ − ECs show enhanced inhibition of platelet aggregation. (A) Representative tracings of platelet aggregation induced by ADP (10 μM) in the absence or presence of increasing numbers of WT or kindlin-2 +/− ECs. Under the
    Figure Legend Snippet: Kindlin-2 +/ − ECs show enhanced inhibition of platelet aggregation. (A) Representative tracings of platelet aggregation induced by ADP (10 μM) in the absence or presence of increasing numbers of WT or kindlin-2 +/− ECs. Under the

    Techniques Used: Inhibition

    CD39 and CD73 expression levels and enzymatic activities are significantly increased on the surface of kindlin-2 +/ − ECs. (A) Representative FACS analysis of WT and kindlin-2 +/− ECs stained with PE-conjugated Abs to CD39 and CD73. Gray-filled
    Figure Legend Snippet: CD39 and CD73 expression levels and enzymatic activities are significantly increased on the surface of kindlin-2 +/ − ECs. (A) Representative FACS analysis of WT and kindlin-2 +/− ECs stained with PE-conjugated Abs to CD39 and CD73. Gray-filled

    Techniques Used: Expressing, FACS, Staining

    Reduction of kindlin-2 expression in WT ECs results in enhanced CD39 and CD73 expression and activities. WT and kindlin-2 +/− ECs were either untreated or treated with control or kindlin-2 siRNA. Untreated kindlin-2 +/− ECs served as control.
    Figure Legend Snippet: Reduction of kindlin-2 expression in WT ECs results in enhanced CD39 and CD73 expression and activities. WT and kindlin-2 +/− ECs were either untreated or treated with control or kindlin-2 siRNA. Untreated kindlin-2 +/− ECs served as control.

    Techniques Used: Expressing

    Clathrin interacts with clathrin box sequence within the F3 domain of kindlin-2. (A) Representative binding of 1 concentration (1.6 μM) of the following full-length kindlin-2 (1-680) and kindlin-2 fragments: (1-105), (95-680), and (281-541) to
    Figure Legend Snippet: Clathrin interacts with clathrin box sequence within the F3 domain of kindlin-2. (A) Representative binding of 1 concentration (1.6 μM) of the following full-length kindlin-2 (1-680) and kindlin-2 fragments: (1-105), (95-680), and (281-541) to

    Techniques Used: Sequencing, Binding Assay, Concentration Assay

    Prolonged bleeding and blood vessel occlusion time in kindlin-2 +/ − mice. (A) Total blood loss upon tail tip clip resection as measured by released hemoglobin using Drabkin reagent (n = 10). (B) Tail bleeding time as estimated after tail tip resection
    Figure Legend Snippet: Prolonged bleeding and blood vessel occlusion time in kindlin-2 +/ − mice. (A) Total blood loss upon tail tip clip resection as measured by released hemoglobin using Drabkin reagent (n = 10). (B) Tail bleeding time as estimated after tail tip resection

    Techniques Used: Mouse Assay, Cross-linking Immunoprecipitation

    7) Product Images from "Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein"

    Article Title: Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky875

    DNA binding affinity of mtSSB and Escherichia coli SSB. Changes in fluorescence anisotropy of a fluorescein-conjugated 90-nt oligonucleotide substrate were measured in response to the step-wise addition of ( A ) mtSSB or ( B ) E. coli SSB proteins, as described in ‘Materials and Methods’ section. Binding buffer contained 30 mM HEPES-KOH (pH 7.5), 1 mM 2-mercaptoethanol, 5 mM MgCl 2 , 0.01% NP-40, 50 mM NaCl, 20 nM oligonucleotide and the indicated amounts of proteins. Protein concentrations are expressed as tetramers. Error bars are standard deviations of triplicate determinations. ( C ) DNA binding cooperativity was estimated by replotting binding data for mtSSB (blue circles) and E. coli SSB (red squares) as a Hill plot.
    Figure Legend Snippet: DNA binding affinity of mtSSB and Escherichia coli SSB. Changes in fluorescence anisotropy of a fluorescein-conjugated 90-nt oligonucleotide substrate were measured in response to the step-wise addition of ( A ) mtSSB or ( B ) E. coli SSB proteins, as described in ‘Materials and Methods’ section. Binding buffer contained 30 mM HEPES-KOH (pH 7.5), 1 mM 2-mercaptoethanol, 5 mM MgCl 2 , 0.01% NP-40, 50 mM NaCl, 20 nM oligonucleotide and the indicated amounts of proteins. Protein concentrations are expressed as tetramers. Error bars are standard deviations of triplicate determinations. ( C ) DNA binding cooperativity was estimated by replotting binding data for mtSSB (blue circles) and E. coli SSB (red squares) as a Hill plot.

    Techniques Used: Binding Assay, Fluorescence

    8) Product Images from "Clonal B cells in patients with hepatitis C virus-associated mixed cryoglobulinemia contain an expanded anergic CD21low B-cell subset"

    Article Title: Clonal B cells in patients with hepatitis C virus-associated mixed cryoglobulinemia contain an expanded anergic CD21low B-cell subset

    Journal: Blood

    doi: 10.1182/blood-2010-10-312942

    G6 + B cells are prone to apoptosis and cell death, and the apoptotic cells are disproportionately CD21 low . PBMCs were incubated for 6 hours in the presence of media alone, mouse IgG 1 (isotype control), or 1 μg/mL anti-CD95 with 2 μg/mL protein G. Cells were then stained with annexin V, 7-AAD, anti-CD20, anti-CD21, and G6 mAbs and were analyzed by flow cytometry. Analyses of G6 + and G6 − CD20 + B cells are shown. (A) Annexin V and 7-AAD staining of cells at baseline and after 6 hours of stimulation. (B) Analysis of surface CD21 and CD20 expression on cells incubated for 6 hours with α-CD95/protein G; annexin V + 7-AAD + , annexin V + 7-AAD − , and annexin V − 7-AAD − subsets. (C) Cell surface anti-CD20 (of total lymphocytes), G6 (of total B cells), and anti-CD21 (of G6 + and G6 − B cells) staining at baseline, compared with 6 hours, of stimulation. PBMCs from LDU 125 were collected 10 months after the cells collected for the microarray and primary immunophenotyping experiments. Data are representative of 3 independent experiments.
    Figure Legend Snippet: G6 + B cells are prone to apoptosis and cell death, and the apoptotic cells are disproportionately CD21 low . PBMCs were incubated for 6 hours in the presence of media alone, mouse IgG 1 (isotype control), or 1 μg/mL anti-CD95 with 2 μg/mL protein G. Cells were then stained with annexin V, 7-AAD, anti-CD20, anti-CD21, and G6 mAbs and were analyzed by flow cytometry. Analyses of G6 + and G6 − CD20 + B cells are shown. (A) Annexin V and 7-AAD staining of cells at baseline and after 6 hours of stimulation. (B) Analysis of surface CD21 and CD20 expression on cells incubated for 6 hours with α-CD95/protein G; annexin V + 7-AAD + , annexin V + 7-AAD − , and annexin V − 7-AAD − subsets. (C) Cell surface anti-CD20 (of total lymphocytes), G6 (of total B cells), and anti-CD21 (of G6 + and G6 − B cells) staining at baseline, compared with 6 hours, of stimulation. PBMCs from LDU 125 were collected 10 months after the cells collected for the microarray and primary immunophenotyping experiments. Data are representative of 3 independent experiments.

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

    9) Product Images from "Superoxide mediates tight junction complex dissociation in cyclically stretched lung slices"

    Article Title: Superoxide mediates tight junction complex dissociation in cyclically stretched lung slices

    Journal: Journal of biomechanics

    doi: 10.1016/j.jbiomech.2015.10.032

    Percent Image Field Area of FITC tagged streptavidin in rat alveolar epithelial cell monolayer (RAEC) during cyclic stretch (37% ΔSA, 10min, 0.25Hz). Mito-Tempo (500μM) and Tiron (10mM) are treated for 2hrs prior to stretch. VC=DMSO vehicle
    Figure Legend Snippet: Percent Image Field Area of FITC tagged streptavidin in rat alveolar epithelial cell monolayer (RAEC) during cyclic stretch (37% ΔSA, 10min, 0.25Hz). Mito-Tempo (500μM) and Tiron (10mM) are treated for 2hrs prior to stretch. VC=DMSO vehicle

    Techniques Used:

    10) Product Images from "Activation of mannan-binding lectin-associated serine proteases leads to generation of a fibrin clot"

    Article Title: Activation of mannan-binding lectin-associated serine proteases leads to generation of a fibrin clot

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2009.03200.x

    Cleavage of a synthetic peptide FGR-AMC by L-FCN-MASPs in mono Q eluates. The top panel shows the amount of 35-kDa L-FCN present in fractions eluted from a mono Q column, as indicated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The chart shows the corresponding proteolytic activity of the L-FCN-associated MASP, following capture of the L-ficolin on an N -acetyl bovine serum albumin (BSA) surface. MASP activity in the individual fractions was quantified by measuring the fluorescence increase after cleavage of the peptide derivative FGR-AMC (Presanis et al. , 2004). The results indicate that the L-FCN detected by SDS-PAGE was indeed complexed with active MASPs.
    Figure Legend Snippet: Cleavage of a synthetic peptide FGR-AMC by L-FCN-MASPs in mono Q eluates. The top panel shows the amount of 35-kDa L-FCN present in fractions eluted from a mono Q column, as indicated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The chart shows the corresponding proteolytic activity of the L-FCN-associated MASP, following capture of the L-ficolin on an N -acetyl bovine serum albumin (BSA) surface. MASP activity in the individual fractions was quantified by measuring the fluorescence increase after cleavage of the peptide derivative FGR-AMC (Presanis et al. , 2004). The results indicate that the L-FCN detected by SDS-PAGE was indeed complexed with active MASPs.

    Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Activity Assay, Fluorescence

    Fibrinopeptide release by ficolin MASPs trapped on various matrices. Fibrinopeptides were released following cleavage of fibrinogen by L-FCN-MASPs as described in the Materials and methods.
    Figure Legend Snippet: Fibrinopeptide release by ficolin MASPs trapped on various matrices. Fibrinopeptides were released following cleavage of fibrinogen by L-FCN-MASPs as described in the Materials and methods.

    Techniques Used:

    Effect of prothrombin addition to purified L-FCN-MASPs in fibrinopeptide release. Fibrinopeptides were released following cleavage of fibrinogen by L-FCN-MASP in the presence and absence of prothrombin, as described in the Materials and methods.
    Figure Legend Snippet: Effect of prothrombin addition to purified L-FCN-MASPs in fibrinopeptide release. Fibrinopeptides were released following cleavage of fibrinogen by L-FCN-MASP in the presence and absence of prothrombin, as described in the Materials and methods.

    Techniques Used: Purification

    Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) pattern of L-FCN-MASPs. Purified L-FCN-MASPs were separated under reducing (lane 1) and non-reducing (lane 2) conditions. Markers: lane 3. Gels were stained with Coomassie brilliant blue.
    Figure Legend Snippet: Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) pattern of L-FCN-MASPs. Purified L-FCN-MASPs were separated under reducing (lane 1) and non-reducing (lane 2) conditions. Markers: lane 3. Gels were stained with Coomassie brilliant blue.

    Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Staining

    (a) Fibrin polymerization and lysis. The top panel shows polypeptide organization of fibrinogen. The area with horizontal lines is the coiled coil region. The area with vertical lines is fibrinopeptide A. The area with diagonal lines is fibrinopeptide B. Double arrows indicate the thrombin cleavage site. Single arrows indicate plasmin cleavage sites. The middle panel shows the domain organization of fibrinogen. The bottom panel shows cross-linking of the fibrin monomer. The E domain binds to the holes on up to four D domains, forming a long fibrous latticework. The clot is then stabilized through cross-linking. The clot can be degraded, yielding different degradation products if it has been cross-linked. The D fragment is released when the clot is not cross-linked by factor XIIIIa. (b) Fibrinogen cleavage by purified lectin-MASPs. Fibrinogen was incubated with various concentrations of purified lectin-MASPs and subjected to reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Arrows indicate (1) the γ-dimer, (2) the α-chain, (3) the β-chain, (4) the γ-chain, (5) L-FCN and (6) mannan-binding lectin (MBL). Panels (a) and (b) show the results obtained with L-FCN-MASPs and MBL-MASPs, respectively. (a) Lane 1, fibrinogen (5 μg) incubated without L-FCN-MASPs at 37° for 14 hr; lanes 2–8, with 3·5, 1·5, 0·75, 0·4, 0·2, 0·1,0 05 and 0·025 μg of L-FCN-MASPs; lane 9, with 1·25 μg of L-FCN-MASPs; lane 10, marker. (b) Lane 1, MBL-MASPs (1 ug); lane 2, fibrinogen (1 ug); lanes 3–9, fibrinogen (1 ug) incubated with 3·0, 1·5, 0·75, 0·375, 0·18, 0·09 and 0·046 ug of MBL-MASPs; lane 10, marker. The results show that both L-FCN and MBL exhibit a dose-dependent cross-linking of the fibrinogen γ-chains, resulting in the generation of the γ-dimer as well as degradation of the α-chain. Additionally, a small change in the size of the β-chain could be observed, indicating the release of fibrinopeptide B.
    Figure Legend Snippet: (a) Fibrin polymerization and lysis. The top panel shows polypeptide organization of fibrinogen. The area with horizontal lines is the coiled coil region. The area with vertical lines is fibrinopeptide A. The area with diagonal lines is fibrinopeptide B. Double arrows indicate the thrombin cleavage site. Single arrows indicate plasmin cleavage sites. The middle panel shows the domain organization of fibrinogen. The bottom panel shows cross-linking of the fibrin monomer. The E domain binds to the holes on up to four D domains, forming a long fibrous latticework. The clot is then stabilized through cross-linking. The clot can be degraded, yielding different degradation products if it has been cross-linked. The D fragment is released when the clot is not cross-linked by factor XIIIIa. (b) Fibrinogen cleavage by purified lectin-MASPs. Fibrinogen was incubated with various concentrations of purified lectin-MASPs and subjected to reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Arrows indicate (1) the γ-dimer, (2) the α-chain, (3) the β-chain, (4) the γ-chain, (5) L-FCN and (6) mannan-binding lectin (MBL). Panels (a) and (b) show the results obtained with L-FCN-MASPs and MBL-MASPs, respectively. (a) Lane 1, fibrinogen (5 μg) incubated without L-FCN-MASPs at 37° for 14 hr; lanes 2–8, with 3·5, 1·5, 0·75, 0·4, 0·2, 0·1,0 05 and 0·025 μg of L-FCN-MASPs; lane 9, with 1·25 μg of L-FCN-MASPs; lane 10, marker. (b) Lane 1, MBL-MASPs (1 ug); lane 2, fibrinogen (1 ug); lanes 3–9, fibrinogen (1 ug) incubated with 3·0, 1·5, 0·75, 0·375, 0·18, 0·09 and 0·046 ug of MBL-MASPs; lane 10, marker. The results show that both L-FCN and MBL exhibit a dose-dependent cross-linking of the fibrinogen γ-chains, resulting in the generation of the γ-dimer as well as degradation of the α-chain. Additionally, a small change in the size of the β-chain could be observed, indicating the release of fibrinopeptide B.

    Techniques Used: Lysis, Purification, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Binding Assay, Marker

    Factor XIII cleavage by purified lectin mannan-binding lectin-associated serine proteases (MASPs). Factor XIII was incubated with L-FCN-MASPs and mannan-binding lectin (MBL) MASPs for different times at 37° and subjected to reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Panels (a) and (b) show the results obtained with L-FCN-MASPs and MBL-MASPs, respectively. Arrows indicate (1) the B chain, (2) the A chain, (3) the cleaved A chain. (a) Lane 1, L-FCN-MASPs; lanes 2–8, factor XIII + L-FCN-MASPs incubated for 0 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr and 8 hr, respectively; lane 9, factor XIII alone incubated for 8 hr. (b) Factor XIII (1 μg) incubated with 2·5 μg of purified MBL-MASPs for different time intervals at 37° and subjected to SDS-PAGE under reducing conditions. Lane 1, MBL-MASPs; lanes 2–8, factor XIII + MBL-MASPs incubated for 0 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr and 8 hr; lane 9, factor XIII incubated for 8 hr. Arrows indicate (1) the B chain, (2) the A chain and (3) the cleaved A chain.
    Figure Legend Snippet: Factor XIII cleavage by purified lectin mannan-binding lectin-associated serine proteases (MASPs). Factor XIII was incubated with L-FCN-MASPs and mannan-binding lectin (MBL) MASPs for different times at 37° and subjected to reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Panels (a) and (b) show the results obtained with L-FCN-MASPs and MBL-MASPs, respectively. Arrows indicate (1) the B chain, (2) the A chain, (3) the cleaved A chain. (a) Lane 1, L-FCN-MASPs; lanes 2–8, factor XIII + L-FCN-MASPs incubated for 0 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr and 8 hr, respectively; lane 9, factor XIII alone incubated for 8 hr. (b) Factor XIII (1 μg) incubated with 2·5 μg of purified MBL-MASPs for different time intervals at 37° and subjected to SDS-PAGE under reducing conditions. Lane 1, MBL-MASPs; lanes 2–8, factor XIII + MBL-MASPs incubated for 0 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr and 8 hr; lane 9, factor XIII incubated for 8 hr. Arrows indicate (1) the B chain, (2) the A chain and (3) the cleaved A chain.

    Techniques Used: Purification, Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page

    11) Product Images from "Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells"

    Article Title: Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells

    Journal: Bioorganic & medicinal chemistry

    doi: 10.1016/j.bmc.2018.04.016

    Selectivity data of antagonists in PAR1 (TFLLRN-NH 2 )- and PAR2 (SLIGKV-NH 2 )-driven i Ca 2+ mobilization a a ML161, RR90, Q94, TJF5 (Fairlie’s PAR2 antagonist) were used at 10 μM; Vorapaxar, Atopaxar, and RWJ-58259 were at 0.316 μM. TFLLRN-NH 2 and SLIGKV-NH 2 were used at 3.16 μM; Vehicle (V) = 10% DMSO-HBSS/HEPES.
    Figure Legend Snippet: Selectivity data of antagonists in PAR1 (TFLLRN-NH 2 )- and PAR2 (SLIGKV-NH 2 )-driven i Ca 2+ mobilization a a ML161, RR90, Q94, TJF5 (Fairlie’s PAR2 antagonist) were used at 10 μM; Vorapaxar, Atopaxar, and RWJ-58259 were at 0.316 μM. TFLLRN-NH 2 and SLIGKV-NH 2 were used at 3.16 μM; Vehicle (V) = 10% DMSO-HBSS/HEPES.

    Techniques Used:

    12) Product Images from "GRK2: a Novel Cell Specific Regulator of Severity and Duration of Inflammatory Pain"

    Article Title: GRK2: a Novel Cell Specific Regulator of Severity and Duration of Inflammatory Pain

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.5752-09.2010

    Spinal cord microglial/macrophage GRK2 levels after SNT (a) The sensitivity to mechanical stimulation was determined in sham-operated or rats subjected to a unilateral L5 spinal nerve transection (SNT) two weeks after surgery (n=8 per group). (b) Scatter plot and bar graph of GRK2 levels in OX-42 + microglia/macrophages isolated from ipsi- and contralateral lumbar spinal cord of sham-operated and SNT rats at day 14 after surgery. GRK2 expression was quantified in 50–100 cells on three separate slides containing cells isolated from 2–3 rats per slide. (c) Representative pictures of GRK2, OX-42 and DAPI-staining of isolated spinal cord microglia/macrophages from ipsi- and contralateral lumbar spinal cord of sham-operated and SNT rats (first 4 columns). Specificity of GRK2 staining was determined by administration of a specific blocking peptide to the primary antibody and for OX-42 by using an isotype control antibody and is shown in the 2 columns at the right. Data are expressed as mean ± SEM. *** p
    Figure Legend Snippet: Spinal cord microglial/macrophage GRK2 levels after SNT (a) The sensitivity to mechanical stimulation was determined in sham-operated or rats subjected to a unilateral L5 spinal nerve transection (SNT) two weeks after surgery (n=8 per group). (b) Scatter plot and bar graph of GRK2 levels in OX-42 + microglia/macrophages isolated from ipsi- and contralateral lumbar spinal cord of sham-operated and SNT rats at day 14 after surgery. GRK2 expression was quantified in 50–100 cells on three separate slides containing cells isolated from 2–3 rats per slide. (c) Representative pictures of GRK2, OX-42 and DAPI-staining of isolated spinal cord microglia/macrophages from ipsi- and contralateral lumbar spinal cord of sham-operated and SNT rats (first 4 columns). Specificity of GRK2 staining was determined by administration of a specific blocking peptide to the primary antibody and for OX-42 by using an isotype control antibody and is shown in the 2 columns at the right. Data are expressed as mean ± SEM. *** p

    Techniques Used: Isolation, Expressing, Staining, Blocking Assay

    13) Product Images from "1,4-dihydroxy-2-naphthoic Acid Induces Apoptosis in Human Keratinocyte: Potential Application for Psoriasis Treatment"

    Article Title: 1,4-dihydroxy-2-naphthoic Acid Induces Apoptosis in Human Keratinocyte: Potential Application for Psoriasis Treatment

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/792840

    Phosphatidylserine externalization is increased in DHNA-treated HaCaT cells. (a, b) Density plots and (c, d) bar chart showing effects of the DHNA on distribution of viable (lower left) and early/late apoptotic (lower/upper right) HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only or various concentration of DHNA for 9 (a, c) and 24 h (b, d) and then analyzed by Annexin V/PI and flow cytometry. (e) DHNA-induced apoptosis in HaCaT cells was reduced by pan-caspase inhibitor. HaCaT cells were pretreated with 40  μ M pan-caspase inhibitor Z-VAD-FMK for 1 h followed by DHNA treatment for 24 h. Three independent experiments with triplicates each time were performed with similar results. Data are expressed as mean ± SEM from one representative experiment and significant difference at * P
    Figure Legend Snippet: Phosphatidylserine externalization is increased in DHNA-treated HaCaT cells. (a, b) Density plots and (c, d) bar chart showing effects of the DHNA on distribution of viable (lower left) and early/late apoptotic (lower/upper right) HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only or various concentration of DHNA for 9 (a, c) and 24 h (b, d) and then analyzed by Annexin V/PI and flow cytometry. (e) DHNA-induced apoptosis in HaCaT cells was reduced by pan-caspase inhibitor. HaCaT cells were pretreated with 40  μ M pan-caspase inhibitor Z-VAD-FMK for 1 h followed by DHNA treatment for 24 h. Three independent experiments with triplicates each time were performed with similar results. Data are expressed as mean ± SEM from one representative experiment and significant difference at * P

    Techniques Used: Concentration Assay, Flow Cytometry, Cytometry

    14) Product Images from "Anergy Induction by Dimeric TCR Ligands"

    Article Title: Anergy Induction by Dimeric TCR Ligands

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi:

    Survival and proliferation of anergic T cells in the presence of rIL-2. T cells from clone Ob.2F3 were pretreated with 20 μ g/ml of soluble DR2/MBP-IgG for 96 h and labeled with 0.5 μ M CFSE for 1 h at 37°C. T cells were washed three times with PBS, resuspended in medium with rIL-2, and plated on 96-well plates in the presence of peptide-pulsed DR2 + B cells. After 24, 60, or 108 h, T cells were stained with Alexa 594-labeled annexin V and analyzed by FACS. In the presence of IL-2, the anergic T cells were viable at all three time points, indicated by the fact that they remained annexin V negative. Proliferation in response to the rIL-2 was most apparent at 108 h, as indicated by a stepwise loss of CFSE labeling.
    Figure Legend Snippet: Survival and proliferation of anergic T cells in the presence of rIL-2. T cells from clone Ob.2F3 were pretreated with 20 μ g/ml of soluble DR2/MBP-IgG for 96 h and labeled with 0.5 μ M CFSE for 1 h at 37°C. T cells were washed three times with PBS, resuspended in medium with rIL-2, and plated on 96-well plates in the presence of peptide-pulsed DR2 + B cells. After 24, 60, or 108 h, T cells were stained with Alexa 594-labeled annexin V and analyzed by FACS. In the presence of IL-2, the anergic T cells were viable at all three time points, indicated by the fact that they remained annexin V negative. Proliferation in response to the rIL-2 was most apparent at 108 h, as indicated by a stepwise loss of CFSE labeling.

    Techniques Used: Labeling, Staining, FACS

    Anergic T cells become susceptible to late apoptosis following stimulation with B cells and peptide. T cells (clone Ob.2F3) were pretreated with 20 μ g/ml of soluble DR2/MBP-IgG ( A ), peptide-pulsed B cells ( B ), or 20 μ g/ml of soluble DR2/MBP-IgG plus 10 μ g/ml of soluble anti-CD28 ( C ). After 96 h, T cells were labeled with CSFE and cocultured with peptide-pulsed B cells. Following 24, 60, and 108 h, T cells were stained with Alexa 594-labeled annexin V, followed by FACS analysis. The gate was set such that cellular debris was excluded from analysis. A , After 24 h of stimulation with peptide-pulsed B cells, the majority of anergic T cells were viable (91.7% annexin V negative). However, after 60 and 108 h, large numbers of anergic T cells were apoptotic. The annexin V-positive population comprised 38% and 53.1% of gated T cells at 60 and 108 h, respectively. B , In contrast, T cells that had been pretreated with peptide-pulsed B cells proliferated vigorously following re-stimulation. Proliferation was already apparent at 24 h by a loss of CFSE-staining intensity. Significant cell division was also evident at 60 h, and T cells remained viable throughout the experiment. C , T cells pretreated with soluble DR2/MBP-IgG in the presence of soluble anti-CD28 (clone 3D10) were not anergic and showed extensive proliferation and little apoptosis.
    Figure Legend Snippet: Anergic T cells become susceptible to late apoptosis following stimulation with B cells and peptide. T cells (clone Ob.2F3) were pretreated with 20 μ g/ml of soluble DR2/MBP-IgG ( A ), peptide-pulsed B cells ( B ), or 20 μ g/ml of soluble DR2/MBP-IgG plus 10 μ g/ml of soluble anti-CD28 ( C ). After 96 h, T cells were labeled with CSFE and cocultured with peptide-pulsed B cells. Following 24, 60, and 108 h, T cells were stained with Alexa 594-labeled annexin V, followed by FACS analysis. The gate was set such that cellular debris was excluded from analysis. A , After 24 h of stimulation with peptide-pulsed B cells, the majority of anergic T cells were viable (91.7% annexin V negative). However, after 60 and 108 h, large numbers of anergic T cells were apoptotic. The annexin V-positive population comprised 38% and 53.1% of gated T cells at 60 and 108 h, respectively. B , In contrast, T cells that had been pretreated with peptide-pulsed B cells proliferated vigorously following re-stimulation. Proliferation was already apparent at 24 h by a loss of CFSE-staining intensity. Significant cell division was also evident at 60 h, and T cells remained viable throughout the experiment. C , T cells pretreated with soluble DR2/MBP-IgG in the presence of soluble anti-CD28 (clone 3D10) were not anergic and showed extensive proliferation and little apoptosis.

    Techniques Used: Labeling, Staining, FACS

    15) Product Images from "Bioengineering functional smooth muscle with spontaneous rhythmic contraction in vitro"

    Article Title: Bioengineering functional smooth muscle with spontaneous rhythmic contraction in vitro

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31992-4

    Maintenance of intestinal smooth muscle cell mixture (ISMC Mix) and vascular aortic smooth muscle cell line (MOVAS cells) cultured on STO cells in vitro . Non-sorted cells from enzymatically digested intestinal muscle strips were used as ISMC Mix. All ISMC Mix and MOVAS cells were cultured separately in FBS medium for the first 4 days followed by FBS or F12 medium. ( a – c ) 100 k ISMC Mix were cultured on STO cells or Ge with FBS or F12 medium for up to 3 weeks. ( a ) Immunofluorescence of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), and neuronal marker β-tubulin (green) at day 7. Scale bar, 100 µm. ( b ) GFP + ISMC Mix were analyzed for mRNA expression ( Kit, Myh11, Tubb 3 , Kitl , and Acta2 ) and gfp DNA ( n = 3 ). ( c ) Spontaneous contraction demonstrated the functionality of ISMC Mix cultured on STO cells with FBS or F12 medium (Supplementary Video 2 and 3 ) and frequency was measured ( n ≥ 5 ). ( d ) MOVAS cells were analyzed for mRNA expression ( Myh11 ) at day 7 ( n = 3 ; triplicate samples). FBS = 15% FBS in DMEM. F12 = advanced DMEM/F12. *Samples were normalized to de-epithelialized intestine. **Samples were normalized to 100 k cells from day 0. Error bars, s.d. ***P
    Figure Legend Snippet: Maintenance of intestinal smooth muscle cell mixture (ISMC Mix) and vascular aortic smooth muscle cell line (MOVAS cells) cultured on STO cells in vitro . Non-sorted cells from enzymatically digested intestinal muscle strips were used as ISMC Mix. All ISMC Mix and MOVAS cells were cultured separately in FBS medium for the first 4 days followed by FBS or F12 medium. ( a – c ) 100 k ISMC Mix were cultured on STO cells or Ge with FBS or F12 medium for up to 3 weeks. ( a ) Immunofluorescence of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), and neuronal marker β-tubulin (green) at day 7. Scale bar, 100 µm. ( b ) GFP + ISMC Mix were analyzed for mRNA expression ( Kit, Myh11, Tubb 3 , Kitl , and Acta2 ) and gfp DNA ( n = 3 ). ( c ) Spontaneous contraction demonstrated the functionality of ISMC Mix cultured on STO cells with FBS or F12 medium (Supplementary Video 2 and 3 ) and frequency was measured ( n ≥ 5 ). ( d ) MOVAS cells were analyzed for mRNA expression ( Myh11 ) at day 7 ( n = 3 ; triplicate samples). FBS = 15% FBS in DMEM. F12 = advanced DMEM/F12. *Samples were normalized to de-epithelialized intestine. **Samples were normalized to 100 k cells from day 0. Error bars, s.d. ***P

    Techniques Used: Cell Culture, In Vitro, Immunofluorescence, Immunocytochemistry, Marker, Expressing

    Engineering aligned intestinal smooth muscle with periodic contraction over 10 weeks in vitro . 100 k ISMC Mix were cultured on STO cells seeded ePCL scaffolds in FBS medium for the first 4 days before changing to F12 medium. ( a ) Confocal images of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), neuronal marker β-tubulin (green), and GFP (green) at 10 weeks. Scale bar, 100 µm. ( b ) GFP + ISMC Mix were analyzed for mRNA expression ( Kit, Myh11, Tubb3, Kitl , and Acta2 ) ( n = 5 ) and gfp DNA ( n = 4 ). The dashed line indicates the seeding density. ( c – e ) Relaxed and contracted state comparison of engineered intestinal smooth muscle. ( c ) Images of GFP expression from ISMC Mix seeded ePCL scaffold were extracted from a video recording and outlined (relax, black; contract, gray) for comparison. Scale bar, 2 mm (Supplementary Video 4 ). ( d) To show the degree of the periodic contraction, area of contracted ePCL scaffolds were normalized to that of relaxed ePCL scaffolds ( n = 10 ). ( e ) To show the directionality of the periodic constriction, height and width of contracted ePCL scaffolds were normalized to those of relaxed ePCL scaffolds ( n = 10 ). ( f ) Frequency of ePCL rhythmic contractions ( n = 4 ). The dark gray dashed line indicates the mean frequency over time and light gray dashed lines indicate its s.d. *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P
    Figure Legend Snippet: Engineering aligned intestinal smooth muscle with periodic contraction over 10 weeks in vitro . 100 k ISMC Mix were cultured on STO cells seeded ePCL scaffolds in FBS medium for the first 4 days before changing to F12 medium. ( a ) Confocal images of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), neuronal marker β-tubulin (green), and GFP (green) at 10 weeks. Scale bar, 100 µm. ( b ) GFP + ISMC Mix were analyzed for mRNA expression ( Kit, Myh11, Tubb3, Kitl , and Acta2 ) ( n = 5 ) and gfp DNA ( n = 4 ). The dashed line indicates the seeding density. ( c – e ) Relaxed and contracted state comparison of engineered intestinal smooth muscle. ( c ) Images of GFP expression from ISMC Mix seeded ePCL scaffold were extracted from a video recording and outlined (relax, black; contract, gray) for comparison. Scale bar, 2 mm (Supplementary Video 4 ). ( d) To show the degree of the periodic contraction, area of contracted ePCL scaffolds were normalized to that of relaxed ePCL scaffolds ( n = 10 ). ( e ) To show the directionality of the periodic constriction, height and width of contracted ePCL scaffolds were normalized to those of relaxed ePCL scaffolds ( n = 10 ). ( f ) Frequency of ePCL rhythmic contractions ( n = 4 ). The dark gray dashed line indicates the mean frequency over time and light gray dashed lines indicate its s.d. *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P

    Techniques Used: In Vitro, Cell Culture, Immunocytochemistry, Marker, Expressing

    MACS+ cells’ role in cultured ISMC Mix with rhythmic contractions in vitro . ( a – c ) 100 k MACS0 (passaged ISMC Mix: mixture of MACS+ cells, SMCs and neuronal cells) or MACS− cells (ISMC Mix without MACS+ cells: mostly SMCs and neuronal cells) were cultured on STO cells for 3 weeks. Cells were seeded and cultured in FBS medium for the first 4 days before changing to F12 medium. ( d – f ) 100 k MACS− were seeded on STO cells for 5 weeks, with (MACS−/+) or without (MACS−) addition of 60 k MACS+ cells on day 5. Cells were cultured in FBS medium for the first 7 days before changing to F12 medium. ( a,d ) Confocal images of ICC markers, Kit (red), Ano1 (green), co-localization (yellow). Scale bar, 100 µm. ( b,e ) Frequency of cultured cells motility due to spontaneous contraction ( b : wk2 n = 2 , wk3 n = 4 ; ( e ) wk5 n = 4 ). ( c,f ) Cultured cells were analyzed for mRNA expression ( Kit, Myh11, Tubb3 ) at week 2 ( c ) or week 5 ( f ) ( n = 4 ). *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P
    Figure Legend Snippet: MACS+ cells’ role in cultured ISMC Mix with rhythmic contractions in vitro . ( a – c ) 100 k MACS0 (passaged ISMC Mix: mixture of MACS+ cells, SMCs and neuronal cells) or MACS− cells (ISMC Mix without MACS+ cells: mostly SMCs and neuronal cells) were cultured on STO cells for 3 weeks. Cells were seeded and cultured in FBS medium for the first 4 days before changing to F12 medium. ( d – f ) 100 k MACS− were seeded on STO cells for 5 weeks, with (MACS−/+) or without (MACS−) addition of 60 k MACS+ cells on day 5. Cells were cultured in FBS medium for the first 7 days before changing to F12 medium. ( a,d ) Confocal images of ICC markers, Kit (red), Ano1 (green), co-localization (yellow). Scale bar, 100 µm. ( b,e ) Frequency of cultured cells motility due to spontaneous contraction ( b : wk2 n = 2 , wk3 n = 4 ; ( e ) wk5 n = 4 ). ( c,f ) Cultured cells were analyzed for mRNA expression ( Kit, Myh11, Tubb3 ) at week 2 ( c ) or week 5 ( f ) ( n = 4 ). *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P

    Techniques Used: Magnetic Cell Separation, Cell Culture, In Vitro, Immunocytochemistry, Expressing

    16) Product Images from "Cymbopogon citratus and Camellia sinensis extracts selectively induce apoptosis in cancer cells and reduce growth of lymphoma xenografts in vivo"

    Article Title: Cymbopogon citratus and Camellia sinensis extracts selectively induce apoptosis in cancer cells and reduce growth of lymphoma xenografts in vivo

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22502

    Lemongrass extract is dependent on the production of oxidative stress to induce apoptosis ( A ) MV-4-11, E6-1, and U-937 cells were treated with piperlongumine (PL), LG, or WT with or without the antioxidant NAC for 48 hours. Following treatment, cells were stained for Annexin V and PI. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (yellow), or negative for both Annexin V and PI (blue). Values are expressed as a mean ± SD from three independent experiments. ( B ) U-937 and E6-1 cells were treated with H2DCFDA following treatments with paraquat (PQ), LG, or WT with or without the antioxidant NAC for 3 hours. Results were obtained using the image-based cytometry with the Y-axis representative of percent of cells positive for DCF. Values are expressed as a mean ± SD from three independent experiments. ( C ) MV-4-11, E6-1, and U-937 cells were plated and allowed to incubate overnight. Following overnight incubation, cells were treated for 48 hours with or without NAC. To monitor mitochondria potential cells were incubated with JC-1 for 30 minutes before analysis. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for JC-1 expressed as a mean ± SD from three independent experiments. ( D ) U-937 micrographs at 48 hours. Top: Fluorescent images of cells without NAC stained with JC-1 (red) and Hoechst (blue) at 400x magnification. Bottom: Fluorescent images of cells with NAC stained with JC-1 (red) and Hoechst (blue) at 400x magnification. Scale bar is 25 microns. Images are representative of three independent experiments. ( E ) E6-1 cells were treated for 3 hours with LG with or without NAC, lysed, and subjected to SDS-PAGE. Cells were then transferred to a PVDF membrane and probed for the specific proteins. Bands were visualized with a chemiluminescence reagent. Statistical calculations were performed using Two-Way ANOVA multiple comparison for (A) and One-Way ANOVA multiple comparison for (B–C). ** p
    Figure Legend Snippet: Lemongrass extract is dependent on the production of oxidative stress to induce apoptosis ( A ) MV-4-11, E6-1, and U-937 cells were treated with piperlongumine (PL), LG, or WT with or without the antioxidant NAC for 48 hours. Following treatment, cells were stained for Annexin V and PI. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (yellow), or negative for both Annexin V and PI (blue). Values are expressed as a mean ± SD from three independent experiments. ( B ) U-937 and E6-1 cells were treated with H2DCFDA following treatments with paraquat (PQ), LG, or WT with or without the antioxidant NAC for 3 hours. Results were obtained using the image-based cytometry with the Y-axis representative of percent of cells positive for DCF. Values are expressed as a mean ± SD from three independent experiments. ( C ) MV-4-11, E6-1, and U-937 cells were plated and allowed to incubate overnight. Following overnight incubation, cells were treated for 48 hours with or without NAC. To monitor mitochondria potential cells were incubated with JC-1 for 30 minutes before analysis. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for JC-1 expressed as a mean ± SD from three independent experiments. ( D ) U-937 micrographs at 48 hours. Top: Fluorescent images of cells without NAC stained with JC-1 (red) and Hoechst (blue) at 400x magnification. Bottom: Fluorescent images of cells with NAC stained with JC-1 (red) and Hoechst (blue) at 400x magnification. Scale bar is 25 microns. Images are representative of three independent experiments. ( E ) E6-1 cells were treated for 3 hours with LG with or without NAC, lysed, and subjected to SDS-PAGE. Cells were then transferred to a PVDF membrane and probed for the specific proteins. Bands were visualized with a chemiluminescence reagent. Statistical calculations were performed using Two-Way ANOVA multiple comparison for (A) and One-Way ANOVA multiple comparison for (B–C). ** p

    Techniques Used: Staining, Cytometry, Incubation, SDS Page

    Lemongrass and white tea extracts induce apoptosis in several lymphoma cell lines; following treatment with specified doses, cells were stained for annexin V and PI ( A ) Lymphoma cell lines tested at 48 hours. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (yellow), or negative for both Annexin V and PI (blue). Values are expressed as a mean ± SD from three independent experiments. ( B ) U-937 micrographs at 24 hours. Top: Bright field and fluorescent merged images at 400x magnification. Bottom: Fluorescent images stained with Annexin V (green), PI (red), and Hoechst (blue) at 400× magnification. Scale bar is 50 microns. Images are representative of three independent experiments. Values are expressed as a mean ± SD from three independent experiments. Statistical calculations were performed using Two-Way ANOVA multiple comparison. * p
    Figure Legend Snippet: Lemongrass and white tea extracts induce apoptosis in several lymphoma cell lines; following treatment with specified doses, cells were stained for annexin V and PI ( A ) Lymphoma cell lines tested at 48 hours. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (yellow), or negative for both Annexin V and PI (blue). Values are expressed as a mean ± SD from three independent experiments. ( B ) U-937 micrographs at 24 hours. Top: Bright field and fluorescent merged images at 400x magnification. Bottom: Fluorescent images stained with Annexin V (green), PI (red), and Hoechst (blue) at 400× magnification. Scale bar is 50 microns. Images are representative of three independent experiments. Values are expressed as a mean ± SD from three independent experiments. Statistical calculations were performed using Two-Way ANOVA multiple comparison. * p

    Techniques Used: Staining, Cytometry

    Lemongrass and white tea extracts do not induce apoptosis in non-cancerous cells ( A ) Normal human skin fibroblasts and ( B ) peripheral blood nuclear cells (from healthy individuals) were tested at 48 hours. Following treatment with specified doses, cells were stained for Annexin V and PI. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (yellow), or negative for both Annexin V and PI (blue). Values are expressed as a mean ± SD from three independent experiments. Statistical calculations were performed using Two-Way ANOVA multiple comparison. **** p
    Figure Legend Snippet: Lemongrass and white tea extracts do not induce apoptosis in non-cancerous cells ( A ) Normal human skin fibroblasts and ( B ) peripheral blood nuclear cells (from healthy individuals) were tested at 48 hours. Following treatment with specified doses, cells were stained for Annexin V and PI. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (yellow), or negative for both Annexin V and PI (blue). Values are expressed as a mean ± SD from three independent experiments. Statistical calculations were performed using Two-Way ANOVA multiple comparison. **** p

    Techniques Used: Staining, Cytometry

    Functioning FADD protein is required to induce apoptosis in cancer cells treated with lemongrass extract ( A ) U-937 and ( B ) DN FADD Jurkat cells were treated for 12 hours and 48 hours, respectively, with the specified treatments, lysed, and subjected to SDS-PAGE. Cells were then transferred to a PVDF membrane and probed for the specific proteins. Bands were visualized with a chemiluminescence reagent. ( C ) DN FADD Jurkat cells were treated for 48 hours with the specified doses and stained with Annexin V and PI. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (yellow), or negative for both Annexin V and PI (blue). Values are expressed as a mean ± SD from three independent experiments. ( D ) DN FADD Jurkat cells were treated with H2DCFDA following treatments with paraquat (PQ), LG, or WT for 3 hours. Results were obtained using the image-based cytometry with the Y-axis representative of percent of cells positive for DCF. Values are expressed as a mean ± SD from three independent experiments. Statistical calculations were performed using Two-Way ANOVA multiple comparison for (C) and One-Way ANOVA multiple comparison for (D). *** p
    Figure Legend Snippet: Functioning FADD protein is required to induce apoptosis in cancer cells treated with lemongrass extract ( A ) U-937 and ( B ) DN FADD Jurkat cells were treated for 12 hours and 48 hours, respectively, with the specified treatments, lysed, and subjected to SDS-PAGE. Cells were then transferred to a PVDF membrane and probed for the specific proteins. Bands were visualized with a chemiluminescence reagent. ( C ) DN FADD Jurkat cells were treated for 48 hours with the specified doses and stained with Annexin V and PI. Results were obtained using image-based cytometry with the Y-axis representative of percent of cells positive for Annexin V (green), PI (red), Annexin V and PI (yellow), or negative for both Annexin V and PI (blue). Values are expressed as a mean ± SD from three independent experiments. ( D ) DN FADD Jurkat cells were treated with H2DCFDA following treatments with paraquat (PQ), LG, or WT for 3 hours. Results were obtained using the image-based cytometry with the Y-axis representative of percent of cells positive for DCF. Values are expressed as a mean ± SD from three independent experiments. Statistical calculations were performed using Two-Way ANOVA multiple comparison for (C) and One-Way ANOVA multiple comparison for (D). *** p

    Techniques Used: SDS Page, Staining, Cytometry

    17) Product Images from "Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis"

    Article Title: Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

    Journal: The Scientific World Journal

    doi: 10.1155/2016/2597376

    Construction of Nod2-nodosome containing the BS/EOS-associated mutation in a cell-free system. Synthetic protein-protein interactions were detected by pull-down assay and amplified luminescent proximity homogeneous assay (ALPHA). (a) Biotinylated-Nod2-WT (Nod2-WT-Btn) and 1 μ g FLAG-tagged RICK-WT (FLAG-RICK-WT) lysed in 300 μ L NP-40 buffer were precipitated with 20 μ L streptavidin-conjugated agarose beads with or without MDP. The precipitations were subjected to SDS-PAGE and immunoblotting. Detection on the blotting membranes was performed using anti-FLAG mAb M2 or anti-Nod2 mAb. (b) A total of 100 ng of each protein indicated was incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL MDP or 5 mg/mL N-Acetylmuramyl-D-Alanyl-D-Isoglutamine (MDP-D-isomer). Responses (counts) were measured using the EnSpire ™ Multimode Plate Reader. (c) Activation of Nod2-nodosome in a cell-free system by MDP degradation components. Interactions between Nod2-WT-Btn and FLAG-RICK-WT were detected by ALPHA. A total of 100 ng of Nod2-WT-Btn and FLAG-RICK-WT were incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, without or with 5.33 mg/mL MDP, 5.33 mg/mL MDP (D-isomer), 5.33 mg/mL N-acetylglucosamine (GlcNAc), 5.33 mg/mL L-alanine (L-Ala), or 5.33 mg/mL D-isoglutamine (D-isoGlu). Responses (counts) were measured using the EnSpire Multimode Plate Reader. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. (d) A total of 100 ng of Nod2-WT-Btn, or Nod2-R334W-Btn, or Nod2-N670K-Btn with FLAG-RICK-WT was incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL or 13.33 mg/mL MDP or 13.33 mg/mL MDP-D-isomer. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. CARD, caspase recruitment domain; MDP, muramyl dipeptide; WT, wild-type; P, precipitation; WB, western blot. ∗ p value
    Figure Legend Snippet: Construction of Nod2-nodosome containing the BS/EOS-associated mutation in a cell-free system. Synthetic protein-protein interactions were detected by pull-down assay and amplified luminescent proximity homogeneous assay (ALPHA). (a) Biotinylated-Nod2-WT (Nod2-WT-Btn) and 1 μ g FLAG-tagged RICK-WT (FLAG-RICK-WT) lysed in 300 μ L NP-40 buffer were precipitated with 20 μ L streptavidin-conjugated agarose beads with or without MDP. The precipitations were subjected to SDS-PAGE and immunoblotting. Detection on the blotting membranes was performed using anti-FLAG mAb M2 or anti-Nod2 mAb. (b) A total of 100 ng of each protein indicated was incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL MDP or 5 mg/mL N-Acetylmuramyl-D-Alanyl-D-Isoglutamine (MDP-D-isomer). Responses (counts) were measured using the EnSpire ™ Multimode Plate Reader. (c) Activation of Nod2-nodosome in a cell-free system by MDP degradation components. Interactions between Nod2-WT-Btn and FLAG-RICK-WT were detected by ALPHA. A total of 100 ng of Nod2-WT-Btn and FLAG-RICK-WT were incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, without or with 5.33 mg/mL MDP, 5.33 mg/mL MDP (D-isomer), 5.33 mg/mL N-acetylglucosamine (GlcNAc), 5.33 mg/mL L-alanine (L-Ala), or 5.33 mg/mL D-isoglutamine (D-isoGlu). Responses (counts) were measured using the EnSpire Multimode Plate Reader. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. (d) A total of 100 ng of Nod2-WT-Btn, or Nod2-R334W-Btn, or Nod2-N670K-Btn with FLAG-RICK-WT was incubated with 5 μ g/mL anti-FLAG mAb M2, 16.67 μ g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 μ g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL or 13.33 mg/mL MDP or 13.33 mg/mL MDP-D-isomer. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. CARD, caspase recruitment domain; MDP, muramyl dipeptide; WT, wild-type; P, precipitation; WB, western blot. ∗ p value

    Techniques Used: Mutagenesis, Pull Down Assay, Amplification, SDS Page, Incubation, Activation Assay, Standard Deviation, Western Blot

    18) Product Images from "Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A"

    Article Title: Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A

    Journal: Blood

    doi: 10.1182/blood-2010-03-272419

    Immunostaining of hFVIII . Localization of transgene protein expression was determined by immunofluorescent confocal microscopy. Endothelial cells were isolated from lung (A) and heart (B) of neonatal mice by immunoselection using Dynabeads M-450 sheep anti-rat IgG beads coated with purified rat anti-mouse CD31 (PECAM-1) antibody. Cells were cultured in vitro for 4 days and then immunostained for either hFVIII using Conan-Alexa 488 or mouse VWF using Dako-Alexa 568. Transgene protein hFVIII was detected in endothelial cells from both lung (Ai-vi) and heart (Bi-vi) of T2F8 Tg mice and was colocalized with mouse VWF in Weibel-Parade bodies as shown in yellow in merge images (iii, vi, xi, and xv). No hFVIII was detected in the endothelial cells from FVIII null mice (Avii-ix,Bvii-x) These results demonstrate that targeting FVIII expression to endothelial cells results in FVIII storage together with VWF in endothelial cell WPB.
    Figure Legend Snippet: Immunostaining of hFVIII . Localization of transgene protein expression was determined by immunofluorescent confocal microscopy. Endothelial cells were isolated from lung (A) and heart (B) of neonatal mice by immunoselection using Dynabeads M-450 sheep anti-rat IgG beads coated with purified rat anti-mouse CD31 (PECAM-1) antibody. Cells were cultured in vitro for 4 days and then immunostained for either hFVIII using Conan-Alexa 488 or mouse VWF using Dako-Alexa 568. Transgene protein hFVIII was detected in endothelial cells from both lung (Ai-vi) and heart (Bi-vi) of T2F8 Tg mice and was colocalized with mouse VWF in Weibel-Parade bodies as shown in yellow in merge images (iii, vi, xi, and xv). No hFVIII was detected in the endothelial cells from FVIII null mice (Avii-ix,Bvii-x) These results demonstrate that targeting FVIII expression to endothelial cells results in FVIII storage together with VWF in endothelial cell WPB.

    Techniques Used: Immunostaining, Expressing, Confocal Microscopy, Isolation, Mouse Assay, Purification, Cell Culture, In Vitro

    19) Product Images from "Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation"

    Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

    Journal: Molecular Genetics & Genomic Medicine

    doi: 10.1002/mgg3.3

    Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.
    Figure Legend Snippet: Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.

    Techniques Used: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.
    Figure Legend Snippet: Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.

    Techniques Used: Activity Assay, Concentration Assay, Gas Chromatography

    Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L
    Figure Legend Snippet: Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L

    Techniques Used: Incubation, Activity Assay, Gas Chromatography

    Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).
    Figure Legend Snippet: Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).

    Techniques Used: Ligand Binding Assay, Purification, Incubation, SDS Page, Gas Chromatography, Staining

    Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.
    Figure Legend Snippet: Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.

    Techniques Used: Ligand Binding Assay, Size-exclusion Chromatography, Gas Chromatography, Chromatography

    Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).
    Figure Legend Snippet: Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).

    Techniques Used: Activity Assay, Concentration Assay, Gas Chromatography

    Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).
    Figure Legend Snippet: Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).

    Techniques Used: Activity Assay, Protein Concentration, Gas Chromatography, Concentration Assay

    20) Product Images from "Kindlin-2 regulates hemostasis by controlling endothelial cell–surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism"

    Article Title: Kindlin-2 regulates hemostasis by controlling endothelial cell–surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism

    Journal: Blood

    doi: 10.1182/blood-2013-04-497669

    Defective clathrin-dependent trafficking of CD39 and CD73 in kindlin-2 +/ − ECs. (A) Western blot analysis of WT and kindlin-2 +/− EC lysates probed with Abs to CD39 and CD73 reveal similar total content of these enzymes in both mouse strains
    Figure Legend Snippet: Defective clathrin-dependent trafficking of CD39 and CD73 in kindlin-2 +/ − ECs. (A) Western blot analysis of WT and kindlin-2 +/− EC lysates probed with Abs to CD39 and CD73 reveal similar total content of these enzymes in both mouse strains

    Techniques Used: Western Blot

    CD39 and CD73 expression levels and enzymatic activities are significantly increased on the surface of kindlin-2 +/ − ECs. (A) Representative FACS analysis of WT and kindlin-2 +/− ECs stained with PE-conjugated Abs to CD39 and CD73. Gray-filled
    Figure Legend Snippet: CD39 and CD73 expression levels and enzymatic activities are significantly increased on the surface of kindlin-2 +/ − ECs. (A) Representative FACS analysis of WT and kindlin-2 +/− ECs stained with PE-conjugated Abs to CD39 and CD73. Gray-filled

    Techniques Used: Expressing, FACS, Staining

    Reduction of kindlin-2 expression in WT ECs results in enhanced CD39 and CD73 expression and activities. WT and kindlin-2 +/− ECs were either untreated or treated with control or kindlin-2 siRNA. Untreated kindlin-2 +/− ECs served as control.
    Figure Legend Snippet: Reduction of kindlin-2 expression in WT ECs results in enhanced CD39 and CD73 expression and activities. WT and kindlin-2 +/− ECs were either untreated or treated with control or kindlin-2 siRNA. Untreated kindlin-2 +/− ECs served as control.

    Techniques Used: Expressing

    21) Product Images from "Bacteria-induced egg hatching differs for Trichuris muris and Trichuris suis"

    Article Title: Bacteria-induced egg hatching differs for Trichuris muris and Trichuris suis

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-015-0986-z

    Trichuris muris eggs incubated with Gram-positive bacteria in RPMI-1640 without antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. muris (Milli-Q or H 2 SO 4 pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteri (n = 15), L. amylovorus (n = 15), and L. murinus (n = 10). Controls represent T. muris eggs incubated without the addition of bacteria (n = 25)
    Figure Legend Snippet: Trichuris muris eggs incubated with Gram-positive bacteria in RPMI-1640 without antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. muris (Milli-Q or H 2 SO 4 pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteri (n = 15), L. amylovorus (n = 15), and L. murinus (n = 10). Controls represent T. muris eggs incubated without the addition of bacteria (n = 25)

    Techniques Used: Incubation

    Trichuris muris eggs incubated with Gram-positive bacteria in RPMI-1640 with antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. muris (Milli-Q or H 2 SO 4 pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteri with T. muris in Milli-Q (n = 20), Lactobacillus reuteri with T. muris in H 2 SO 4 pH1 (n = 15), L. murinus (n = 10), and L. amylovorus (n = 15). Controls represent T. muris eggs without the addition of bacteria (n = 25)
    Figure Legend Snippet: Trichuris muris eggs incubated with Gram-positive bacteria in RPMI-1640 with antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. muris (Milli-Q or H 2 SO 4 pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteri with T. muris in Milli-Q (n = 20), Lactobacillus reuteri with T. muris in H 2 SO 4 pH1 (n = 15), L. murinus (n = 10), and L. amylovorus (n = 15). Controls represent T. muris eggs without the addition of bacteria (n = 25)

    Techniques Used: Incubation

    Trichuris suis eggs incubated with Gram-positive bacteria in RPMI-1640 without antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. suis (Milli-Q or H 2 SO 4 pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteri with T. suis in Milli-Q (n = 15), L. reuteri with T. suis in H 2 SO 4 pH1 (n = 20) , L. amylovorus (n = 15), and L. murinus (n = 10). Controls represent T. suis eggs without the addition of bacteria (n = 25)
    Figure Legend Snippet: Trichuris suis eggs incubated with Gram-positive bacteria in RPMI-1640 without antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. suis (Milli-Q or H 2 SO 4 pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteri with T. suis in Milli-Q (n = 15), L. reuteri with T. suis in H 2 SO 4 pH1 (n = 20) , L. amylovorus (n = 15), and L. murinus (n = 10). Controls represent T. suis eggs without the addition of bacteria (n = 25)

    Techniques Used: Incubation

    22) Product Images from "MERIT40 deficiency expands hematopoietic stem cell pools by regulating thrombopoietin receptor signaling"

    Article Title: MERIT40 deficiency expands hematopoietic stem cell pools by regulating thrombopoietin receptor signaling

    Journal: Blood

    doi: 10.1182/blood-2014-07-588145

    Purified M40 −/− HSCs exhibit enhanced repopulation and self-renewal ability. (A) Fifty CD34 − CD150 + CD48 − LSK cells from WT and M40 −/− mice were mixed with 0.4 × 10 6 Sca1-depleted competitor BM cells
    Figure Legend Snippet: Purified M40 −/− HSCs exhibit enhanced repopulation and self-renewal ability. (A) Fifty CD34 − CD150 + CD48 − LSK cells from WT and M40 −/− mice were mixed with 0.4 × 10 6 Sca1-depleted competitor BM cells

    Techniques Used: Purification, Mouse Assay

    M40 −/− HSCs show decelerated cell cycle progression, increased quiescence, and downregulation of gene expression associated with cell division. (A) BrdU incorporation analysis in SLAM LSK cells. WT and M40 −/− mice were
    Figure Legend Snippet: M40 −/− HSCs show decelerated cell cycle progression, increased quiescence, and downregulation of gene expression associated with cell division. (A) BrdU incorporation analysis in SLAM LSK cells. WT and M40 −/− mice were

    Techniques Used: Expressing, BrdU Incorporation Assay, Mouse Assay

    M40 deficiency leads to an expansion of phenotypic and functional HSCs. (A) Frequency of LT-HSCs in WT and M40 −/− mice as determined by flow cytometry. LT-HSC is defined as CD34 − Flk2 − CD150 + LSK, and representative plots
    Figure Legend Snippet: M40 deficiency leads to an expansion of phenotypic and functional HSCs. (A) Frequency of LT-HSCs in WT and M40 −/− mice as determined by flow cytometry. LT-HSC is defined as CD34 − Flk2 − CD150 + LSK, and representative plots

    Techniques Used: Functional Assay, Mouse Assay, Flow Cytometry, Cytometry

    23) Product Images from "The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis"

    Article Title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-11-76

    UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney podocytes. A . UCH-L1 tet-on podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. *** p
    Figure Legend Snippet: UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney podocytes. A . UCH-L1 tet-on podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. *** p

    Techniques Used: Staining

    24) Product Images from "Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila"

    Article Title: Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.02064

    Cohesin binds multiple sites in the cut regulatory region in Kc cells. Chromatin immunoprecipitation was performed with pre-immune and immune serum for Smc1 and Stromalin, and PCR amplicons spaced 1 kbp apart starting 0.6 kbp upstream of the wing margin enhancer (salmon-colored bar) extending into the transcribed region (blue-green bar) 2.8 kbp downstream of the transcription start site. Enrichment of each amplicon is plotted as the ratio of the amount of PCR product obtained with the immune serum relative to the amount obtained with the pre-immune serum. Most points represent the average of two or three measurements. Enrichment by Smc1 immune serum is plotted in blue, enrichment by Stromalin serum in red, and the black line is the average of the Smc1 and Stromalin values. This reveals cohesin-binding sites at 0.5, 4, 30.5, and 68 kbp upstream of the promoter. These peaks are recognized by an increase in the immune to pre-immune ratio relative to the baseline, which as expected, is close to 1.
    Figure Legend Snippet: Cohesin binds multiple sites in the cut regulatory region in Kc cells. Chromatin immunoprecipitation was performed with pre-immune and immune serum for Smc1 and Stromalin, and PCR amplicons spaced 1 kbp apart starting 0.6 kbp upstream of the wing margin enhancer (salmon-colored bar) extending into the transcribed region (blue-green bar) 2.8 kbp downstream of the transcription start site. Enrichment of each amplicon is plotted as the ratio of the amount of PCR product obtained with the immune serum relative to the amount obtained with the pre-immune serum. Most points represent the average of two or three measurements. Enrichment by Smc1 immune serum is plotted in blue, enrichment by Stromalin serum in red, and the black line is the average of the Smc1 and Stromalin values. This reveals cohesin-binding sites at 0.5, 4, 30.5, and 68 kbp upstream of the promoter. These peaks are recognized by an increase in the immune to pre-immune ratio relative to the baseline, which as expected, is close to 1.

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Binding Assay

    25) Product Images from "Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles"

    Article Title: Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles

    Journal:

    doi: 10.1016/j.virol.2008.01.018

    MHV replicase protein localization in icv-infected HeLa-MHVR cells using antibodies to the mitochondrial protein pyruvate dehydrogenase (PDH). Two sets of HELA-MHVR cells were infected with WT-A59 icv or Alb ts6 icv at an MOI of 1.0 and incubated at 33°C.
    Figure Legend Snippet: MHV replicase protein localization in icv-infected HeLa-MHVR cells using antibodies to the mitochondrial protein pyruvate dehydrogenase (PDH). Two sets of HELA-MHVR cells were infected with WT-A59 icv or Alb ts6 icv at an MOI of 1.0 and incubated at 33°C.

    Techniques Used: Infection, Incubation

    26) Product Images from "Oligo-guanosine nucleotide induces neuropilin-1 internalization in endothelial cells and inhibits angiogenesis"

    Article Title: Oligo-guanosine nucleotide induces neuropilin-1 internalization in endothelial cells and inhibits angiogenesis

    Journal: Blood

    doi: 10.1182/blood-2010-01-265801

    G18 promotes internalization of NRP1 and associates with NRP1 in LAMP2-marked structures . (A) Cells were incubated in medium alone or with 16 μg/mL sG16 at 37°C for 15 and 60 minutes. After fixation and permeabilization, cells were stained with anti-NRP1 primary mAb followed by Alexa 488–conjugated anti–mouse IgG Ab. Nuclei were stained with DAPI. (B) Cells were incubated with medium alone (top panels) or 16 μg/mL biotin-G18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with Alexa Fluor 546–conjugated streptavidin (to visualize biotin-G18) and with anti-NRP1 primary mAb followed by Alexa Fluor 488–conjugated anti–mouse IgG Ab (to visualize NRP1). (C) Cells were incubated with medium only (top panels) or 16 μg/mL biotin-G18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with Alexa Fluor 488–conjugated streptavidin (to visualize biotin-G18) and with anti-LAMP2 primary Ab followed by Alexa Fluor 594–conjugated anti–rabbit IgG Ab (to visualize LAMP2). (D) Cells were incubated with medium only (top panels) or 16 μg/mL sG18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with anti-NRP1 primary mAb followed by Alexa Fluor 488–conjugated anti–mouse IgG Ab (to visualize NRP1) and with anti-LAMP2 primary Ab followed by Alexa Fluor 594–conjugated anti–rabbit IgG Ab (to visualize LAMP2). Cells were examined by confocal microscopy. Images were imported into Adobe Photoshop 6.0 for processing. Scale bar, 20 μm.
    Figure Legend Snippet: G18 promotes internalization of NRP1 and associates with NRP1 in LAMP2-marked structures . (A) Cells were incubated in medium alone or with 16 μg/mL sG16 at 37°C for 15 and 60 minutes. After fixation and permeabilization, cells were stained with anti-NRP1 primary mAb followed by Alexa 488–conjugated anti–mouse IgG Ab. Nuclei were stained with DAPI. (B) Cells were incubated with medium alone (top panels) or 16 μg/mL biotin-G18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with Alexa Fluor 546–conjugated streptavidin (to visualize biotin-G18) and with anti-NRP1 primary mAb followed by Alexa Fluor 488–conjugated anti–mouse IgG Ab (to visualize NRP1). (C) Cells were incubated with medium only (top panels) or 16 μg/mL biotin-G18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with Alexa Fluor 488–conjugated streptavidin (to visualize biotin-G18) and with anti-LAMP2 primary Ab followed by Alexa Fluor 594–conjugated anti–rabbit IgG Ab (to visualize LAMP2). (D) Cells were incubated with medium only (top panels) or 16 μg/mL sG18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with anti-NRP1 primary mAb followed by Alexa Fluor 488–conjugated anti–mouse IgG Ab (to visualize NRP1) and with anti-LAMP2 primary Ab followed by Alexa Fluor 594–conjugated anti–rabbit IgG Ab (to visualize LAMP2). Cells were examined by confocal microscopy. Images were imported into Adobe Photoshop 6.0 for processing. Scale bar, 20 μm.

    Techniques Used: Incubation, Staining, Confocal Microscopy

    27) Product Images from "gp96, an endoplasmic reticulum master chaperone for integrins and Toll-like receptors, selectively regulates early T and B lymphopoiesis"

    Article Title: gp96, an endoplasmic reticulum master chaperone for integrins and Toll-like receptors, selectively regulates early T and B lymphopoiesis

    Journal: Blood

    doi: 10.1182/blood-2009-07-233031

    gp96 null hematopoietic progenitors fail to proliferate and differentiate in BM stromal cell cultures . Purified WT and gp96 KO BM Lin − c-kit + progenitor cells were cultured on BM stromal cell OP9 or OP9-DL1 for 3 weeks and analyzed for cell growth
    Figure Legend Snippet: gp96 null hematopoietic progenitors fail to proliferate and differentiate in BM stromal cell cultures . Purified WT and gp96 KO BM Lin − c-kit + progenitor cells were cultured on BM stromal cell OP9 or OP9-DL1 for 3 weeks and analyzed for cell growth

    Techniques Used: Purification, Cell Culture

    28) Product Images from "Characterization of DNase activity and gene in Streptococcus suis and evidence for a role as virulence factor"

    Article Title: Characterization of DNase activity and gene in Streptococcus suis and evidence for a role as virulence factor

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-7-424

    Electrophoretic analysis of DNA degradation by S. suis P1/7 cells. Lanes 1: DNA markers, 2: Control linear DNA (lambda DNA), 3: Linear DNA + S. suis P1/7, 4: Linear DNA + S. suis DNase deficient mutant (M2D), 5: Control plasmid DNA, 6: Plasmid DNA + S. suis P1/7, 7: Plasmid DNA + S. suis DNase deficient mutant (M2D).
    Figure Legend Snippet: Electrophoretic analysis of DNA degradation by S. suis P1/7 cells. Lanes 1: DNA markers, 2: Control linear DNA (lambda DNA), 3: Linear DNA + S. suis P1/7, 4: Linear DNA + S. suis DNase deficient mutant (M2D), 5: Control plasmid DNA, 6: Plasmid DNA + S. suis P1/7, 7: Plasmid DNA + S. suis DNase deficient mutant (M2D).

    Techniques Used: Lambda DNA Preparation, Mutagenesis, Plasmid Preparation

    Comparative analysis of the ssnA gene according to STs. Nucleic acid and predicted amino acid sequences of ssnA gene (from position 1108 to 1173) of S. suis belonging to ST1, ST25, and ST28 were compared in order to identify the origin of the loss of DNase activity in ST25.
    Figure Legend Snippet: Comparative analysis of the ssnA gene according to STs. Nucleic acid and predicted amino acid sequences of ssnA gene (from position 1108 to 1173) of S. suis belonging to ST1, ST25, and ST28 were compared in order to identify the origin of the loss of DNase activity in ST25.

    Techniques Used: Activity Assay

    Quantification of pro-inflammatory cytokines and MMP-9 produced by macrophages stimulated with S. suis P1/7 and M2D. Secretion of IL-6 (panel A) , CXCL8 (panel B) , TNF-α (panel C) and MMP-9 (panel D) by macrophages stimulated with cells of S. suis P1/7 and its DNase-deficient mutant M2D at MOIs of 10, 50, and 100. *: p
    Figure Legend Snippet: Quantification of pro-inflammatory cytokines and MMP-9 produced by macrophages stimulated with S. suis P1/7 and M2D. Secretion of IL-6 (panel A) , CXCL8 (panel B) , TNF-α (panel C) and MMP-9 (panel D) by macrophages stimulated with cells of S. suis P1/7 and its DNase-deficient mutant M2D at MOIs of 10, 50, and 100. *: p

    Techniques Used: Produced, Mutagenesis

    29) Product Images from "A Systematic Analysis of Host Factors Reveals a Med23-Interferon-? Regulatory Axis against Herpes Simplex Virus Type 1 Replication"

    Article Title: A Systematic Analysis of Host Factors Reveals a Med23-Interferon-? Regulatory Axis against Herpes Simplex Virus Type 1 Replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003514

    Med23 induces IFN-λ by interacting with the transcription factor IRF7. (a) Med23 directly interacts with IRFs. Med23 was overexpressed in HEK cells as a myc-tagged fusion protein individually with a range of HA-tagged IRFs. Protein amounts were quantified and equal amounts (325 µg) were immunoprecipitated (IP) with anti-HA or anti-myc antibody before western blot analysis and staining with anti-HA (WB) to confirm protein expression (IP; anti-HA IP) and identify interaction partners (Co-IP; anti-myc IP) (b) Med23 synergistically induces IRF7-responsive promoters. A549 cells were transfected with IFN-λ1-, IFN-β- or ISRE-responsive luciferase reporter constructs with IRF7 alone or in addition to Med23. Promoter activity was determined by measurement of Firefly luciferase activity 33 hr post-transfection, and normalized to Renilla luciferase and pCR3-transfected cells. Error bars represent the mean of at least three independent experiments. Statistical significance of the synergistic increase in IFN-λ induction by Med23 with IRF7 over IFN-β induction was determined by unpaired t-tests for unequal variances. * = p-value 0.02.
    Figure Legend Snippet: Med23 induces IFN-λ by interacting with the transcription factor IRF7. (a) Med23 directly interacts with IRFs. Med23 was overexpressed in HEK cells as a myc-tagged fusion protein individually with a range of HA-tagged IRFs. Protein amounts were quantified and equal amounts (325 µg) were immunoprecipitated (IP) with anti-HA or anti-myc antibody before western blot analysis and staining with anti-HA (WB) to confirm protein expression (IP; anti-HA IP) and identify interaction partners (Co-IP; anti-myc IP) (b) Med23 synergistically induces IRF7-responsive promoters. A549 cells were transfected with IFN-λ1-, IFN-β- or ISRE-responsive luciferase reporter constructs with IRF7 alone or in addition to Med23. Promoter activity was determined by measurement of Firefly luciferase activity 33 hr post-transfection, and normalized to Renilla luciferase and pCR3-transfected cells. Error bars represent the mean of at least three independent experiments. Statistical significance of the synergistic increase in IFN-λ induction by Med23 with IRF7 over IFN-β induction was determined by unpaired t-tests for unequal variances. * = p-value 0.02.

    Techniques Used: Immunoprecipitation, Western Blot, Staining, Expressing, Co-Immunoprecipitation Assay, Transfection, Luciferase, Construct, Activity Assay

    Med23 inhibits HSV-1 by inducing a Type III interferon (IFN-λ) response. (a) Med23 depletion or over-expression has no effect in A549-V cells deficient in Jak/Stat signalling. A549 cells and derivative A549-V cells were transfected with Med23 siRNA SMARTpool (Med23 KD) or a pCR3-Med23 overexpression plasmid (Med23+) 48 h (siRNA) or 24 h (pCR3) before infection with HSV-1-eGFP C12. Replication was monitored, and slopes calculated and normalized to controls (RSCF siRNA or pCR3). Error bars represent the mean of at least three independent experiments. (b) Pre-treatment with Type III interferons prevents Med23-mediated enhancement of HSV-1 replication. A549 cells were mock-transfected or transfected with Med23 siRNA. After 48 h cells were untreated or pre-treated with 50 ng/ml IFN-α, IFN-β, IFN-γ or 100 ng/ml IFN-λ1 or IFN-λ2/3 for 6 h, before infecting with HSV-1-eGFP C12. Replication was monitored and slopes calculated and normalized to unstimulated, mock-transfected cells. (c) Overexpression of Med23 preferentially induces type III interferons. pCR3 or Med23 were overexpressed in A549 cells and induction of type I (IFN-β) and type III (IFN-λ1, IFN-λ2/3) was measured by qRT-PCR. mRNA levels were normalized to HPRT and calibrated to mock transfected cells (control). Error bars represent the mean of technical replicates and is representative of multiple experiments. * = p -value 0.003; ** = p -value 0.002 (unpaired t-tests for unequal variances). (d) Overexpression of Med23 induces IFN-λ secretion. A range of cell types were transfected with pCR3 or Med23, supernatant harvested 120 h post-transfection and IFN-λ3 levels measured by ELISA. Chart shows the mean and standard deviation of duplicates over two experiments. (e) siRNA depletion of Med23 inhibits the induction of IFN-λ2/3 following HSV-1 infection. A549 cells were transfected with control (RSCF) or Med23-specific siRNA (Med23 KD) before infecting with HSV-1-eGFP C12 (MOI 0.5). RNA was harvested 0 or 8 h post-infection and IFN-λ2/3 mRNA levels measured by qRT-PCR. Expression was normalized as above, and calibrated to RSCF-transfected cells at 0 h post-infection. Error bars represent the mean of technical replicates and is representative of multiple experiments.
    Figure Legend Snippet: Med23 inhibits HSV-1 by inducing a Type III interferon (IFN-λ) response. (a) Med23 depletion or over-expression has no effect in A549-V cells deficient in Jak/Stat signalling. A549 cells and derivative A549-V cells were transfected with Med23 siRNA SMARTpool (Med23 KD) or a pCR3-Med23 overexpression plasmid (Med23+) 48 h (siRNA) or 24 h (pCR3) before infection with HSV-1-eGFP C12. Replication was monitored, and slopes calculated and normalized to controls (RSCF siRNA or pCR3). Error bars represent the mean of at least three independent experiments. (b) Pre-treatment with Type III interferons prevents Med23-mediated enhancement of HSV-1 replication. A549 cells were mock-transfected or transfected with Med23 siRNA. After 48 h cells were untreated or pre-treated with 50 ng/ml IFN-α, IFN-β, IFN-γ or 100 ng/ml IFN-λ1 or IFN-λ2/3 for 6 h, before infecting with HSV-1-eGFP C12. Replication was monitored and slopes calculated and normalized to unstimulated, mock-transfected cells. (c) Overexpression of Med23 preferentially induces type III interferons. pCR3 or Med23 were overexpressed in A549 cells and induction of type I (IFN-β) and type III (IFN-λ1, IFN-λ2/3) was measured by qRT-PCR. mRNA levels were normalized to HPRT and calibrated to mock transfected cells (control). Error bars represent the mean of technical replicates and is representative of multiple experiments. * = p -value 0.003; ** = p -value 0.002 (unpaired t-tests for unequal variances). (d) Overexpression of Med23 induces IFN-λ secretion. A range of cell types were transfected with pCR3 or Med23, supernatant harvested 120 h post-transfection and IFN-λ3 levels measured by ELISA. Chart shows the mean and standard deviation of duplicates over two experiments. (e) siRNA depletion of Med23 inhibits the induction of IFN-λ2/3 following HSV-1 infection. A549 cells were transfected with control (RSCF) or Med23-specific siRNA (Med23 KD) before infecting with HSV-1-eGFP C12 (MOI 0.5). RNA was harvested 0 or 8 h post-infection and IFN-λ2/3 mRNA levels measured by qRT-PCR. Expression was normalized as above, and calibrated to RSCF-transfected cells at 0 h post-infection. Error bars represent the mean of technical replicates and is representative of multiple experiments.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation, Expressing

    30) Product Images from "The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis"

    Article Title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-11-76

    UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney podocytes. A . UCH-L1 tet-on podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. *** p
    Figure Legend Snippet: UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney podocytes. A . UCH-L1 tet-on podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. *** p

    Techniques Used: Staining

    Inhibition of serine proteases, but not metalloproteases, cathepsin or calpain/cysteine proteases protects from TNF-induced necroptosis. A . Cells were stimulated or not with 100 ng/ml TNF for 5 (L929Ts), 16 (NIH3T3, HT-29), or 20 h (Jurkat) with optional addition of 20 (L929Ts, NIH3T3, HT-29) or 50 μM (Jurkat) of the broad-spectrum caspase inhibitor zVAD-fmk to prevent apoptosis, 2 (Jurkat) or 5 μg/ml (HT-29) cycloheximide (CHX) to sensitize for necroptosis [ 14 ] and 50 (L929Ts, NIH3T3) and 25 μM (Jurkat, HT-29) TPCK, or 50 μM of the necroptosis inhibitor necrostatin-1 (Nec-1, to confirm necroptosis). Subsequently, the cells were analyzed for loss of membrane integrity as a marker for cell death by PI staining and flow cytometry. Asterisks indicate statistical significance (t-test), * p
    Figure Legend Snippet: Inhibition of serine proteases, but not metalloproteases, cathepsin or calpain/cysteine proteases protects from TNF-induced necroptosis. A . Cells were stimulated or not with 100 ng/ml TNF for 5 (L929Ts), 16 (NIH3T3, HT-29), or 20 h (Jurkat) with optional addition of 20 (L929Ts, NIH3T3, HT-29) or 50 μM (Jurkat) of the broad-spectrum caspase inhibitor zVAD-fmk to prevent apoptosis, 2 (Jurkat) or 5 μg/ml (HT-29) cycloheximide (CHX) to sensitize for necroptosis [ 14 ] and 50 (L929Ts, NIH3T3) and 25 μM (Jurkat, HT-29) TPCK, or 50 μM of the necroptosis inhibitor necrostatin-1 (Nec-1, to confirm necroptosis). Subsequently, the cells were analyzed for loss of membrane integrity as a marker for cell death by PI staining and flow cytometry. Asterisks indicate statistical significance (t-test), * p

    Techniques Used: Inhibition, Marker, Staining, Flow Cytometry, Cytometry, T-Test

    31) Product Images from "STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense"

    Article Title: STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense

    Journal: Blood

    doi: 10.1182/blood-2012-08-450403

    Stim1 − / − neutrophils show defective SOCE in response to soluble agonists and thapsigargin. (A) WT neutrophils were labeled with Indo-1 AM and then stimulated with the indicated doses of fMLF (left panel) or MIP- 2 (right panel), and calcium
    Figure Legend Snippet: Stim1 − / − neutrophils show defective SOCE in response to soluble agonists and thapsigargin. (A) WT neutrophils were labeled with Indo-1 AM and then stimulated with the indicated doses of fMLF (left panel) or MIP- 2 (right panel), and calcium

    Techniques Used: Labeling

    32) Product Images from "Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes"

    Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02655-1

    In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1
    Figure Legend Snippet: In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

    Techniques Used: In Vitro, Activity Assay, Fluorescence, Purification

    Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1
    Figure Legend Snippet: Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

    Techniques Used: In Vitro, Activity Assay, Fluorescence, Purification

    33) Product Images from "Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice"

    Article Title: Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156974

    Knockout of Cacna1f significantly prevented excess activation of calpain in rd1 mt/mt photoreceptors. (A-C) Representative vertical retinal cryo-sections of (A) wild type, (B) rd1 mt/mt and (C) rd1 mt/mt x Cacna1f ko/ko retinas at p11, p13, p15, and p18. Photoreceptor nuclei with increased calpain activity appear as grey dots or circles. The horizontal bars mark the outer nuclear layer (ONL) borders. (D) Summary graph showing the quantification of calpain-positive photoreceptors in the different genotypes at p11, p13, p15, and 18. Each data point represents the mean percentage of cells with increased calpain activity in the ONL (n = 3–8 per genotype and time point) ± SEM; ***p
    Figure Legend Snippet: Knockout of Cacna1f significantly prevented excess activation of calpain in rd1 mt/mt photoreceptors. (A-C) Representative vertical retinal cryo-sections of (A) wild type, (B) rd1 mt/mt and (C) rd1 mt/mt x Cacna1f ko/ko retinas at p11, p13, p15, and p18. Photoreceptor nuclei with increased calpain activity appear as grey dots or circles. The horizontal bars mark the outer nuclear layer (ONL) borders. (D) Summary graph showing the quantification of calpain-positive photoreceptors in the different genotypes at p11, p13, p15, and 18. Each data point represents the mean percentage of cells with increased calpain activity in the ONL (n = 3–8 per genotype and time point) ± SEM; ***p

    Techniques Used: Knock-Out, Activation Assay, Activity Assay

    34) Product Images from "Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease"

    Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165463

    2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.
    Figure Legend Snippet: 2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.

    Techniques Used: Thermal Shift Assay, Incubation

    35) Product Images from "Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *"

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.141580

    Schematic representation of the Hsp90 occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.
    Figure Legend Snippet: Schematic representation of the Hsp90 occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.

    Techniques Used: Filtration, Binding Assay

    Hsp90 open binding sites can be titrated with [ 3 H]17-AAG. Recombinant human Hsp90β protein (100 n m ) was incubated with increasing concentrations of unlabeled 17-AAG overnight at 4 °C followed by removal of unbound 17-AAG with prechilled size exclusion columns. Free Hsp90 sites were titrated with [ 3 H]17-AAG at 4 °C as described under “Experimental Procedures.” The data from triplicate binding experiments were fit to a four parameter logistic equation.
    Figure Legend Snippet: Hsp90 open binding sites can be titrated with [ 3 H]17-AAG. Recombinant human Hsp90β protein (100 n m ) was incubated with increasing concentrations of unlabeled 17-AAG overnight at 4 °C followed by removal of unbound 17-AAG with prechilled size exclusion columns. Free Hsp90 sites were titrated with [ 3 H]17-AAG at 4 °C as described under “Experimental Procedures.” The data from triplicate binding experiments were fit to a four parameter logistic equation.

    Techniques Used: Binding Assay, Recombinant, Incubation

    Determination of Hsp90 occupancy in living cells. H1650 cells were incubated with increasing concentrations of IPI-504 for 6 h at 37 °C. Total Hsp90 protein levels were determined by quantitative immunoblotting using separate anti Hsp90α and Hsp90β antibodies and recombinant proteins as internal standards ( A ). Percent Hsp90 occupancy was determined by titration of open binding sites at 4 °C and total Hsp90 ( B and C ). Data are from a representative experiment with n = 2.
    Figure Legend Snippet: Determination of Hsp90 occupancy in living cells. H1650 cells were incubated with increasing concentrations of IPI-504 for 6 h at 37 °C. Total Hsp90 protein levels were determined by quantitative immunoblotting using separate anti Hsp90α and Hsp90β antibodies and recombinant proteins as internal standards ( A ). Percent Hsp90 occupancy was determined by titration of open binding sites at 4 °C and total Hsp90 ( B and C ). Data are from a representative experiment with n = 2.

    Techniques Used: Incubation, Recombinant, Titration, Binding Assay

    Dissociation of [ 3 H]17-AAG from purified Hsp90 and SK-BR-3 lysates is highly temperature-dependent. Purified Hela Hsp90 (100 n m ) or lysate Hsp90·[ 3 H]17-AAG complexes were formed as described under “Experimental Procedures” and passed over size exclusion spin columns. Column eluates were incubated with 10 μ m cold 17-AAG, and samples were removed at different time points. A loss of bound radioactive 17-AAG counts from Hsp90 was measured at both 4 and 37 °C ( A ) and Hsp90 in SK-BR-3 cancer cell lysate at 4 °C ( B ). The data were fit to a monoexponential decay equation.
    Figure Legend Snippet: Dissociation of [ 3 H]17-AAG from purified Hsp90 and SK-BR-3 lysates is highly temperature-dependent. Purified Hela Hsp90 (100 n m ) or lysate Hsp90·[ 3 H]17-AAG complexes were formed as described under “Experimental Procedures” and passed over size exclusion spin columns. Column eluates were incubated with 10 μ m cold 17-AAG, and samples were removed at different time points. A loss of bound radioactive 17-AAG counts from Hsp90 was measured at both 4 and 37 °C ( A ) and Hsp90 in SK-BR-3 cancer cell lysate at 4 °C ( B ). The data were fit to a monoexponential decay equation.

    Techniques Used: Purification, Incubation

    Hsp90 occupancy is a better predictor of in vivo pharmacodynamic effects by IPI-504 than tumor or plasma PK. H1650 tumor-bearing mice were treated with a single dose of 100 mg/kg intravenous IPI-504. Tumors and blood plasma were harvested at designated time points post dose. Drug levels of Hsp90 active species (IPI-504, 17-AAG, and 17-AG) were quantified by LC-MS/MS in plasma and in tumor ( A ). EGFR protein levels ( B ) and Hsp90 occupancy ( C ) were measured in tumor tissue. Data are expressed as averages ± S.D. (vehicle ( veh ), n = 2; and 1–48 h, n = 3).
    Figure Legend Snippet: Hsp90 occupancy is a better predictor of in vivo pharmacodynamic effects by IPI-504 than tumor or plasma PK. H1650 tumor-bearing mice were treated with a single dose of 100 mg/kg intravenous IPI-504. Tumors and blood plasma were harvested at designated time points post dose. Drug levels of Hsp90 active species (IPI-504, 17-AAG, and 17-AG) were quantified by LC-MS/MS in plasma and in tumor ( A ). EGFR protein levels ( B ) and Hsp90 occupancy ( C ) were measured in tumor tissue. Data are expressed as averages ± S.D. (vehicle ( veh ), n = 2; and 1–48 h, n = 3).

    Techniques Used: In Vivo, Mouse Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Hsp90 occupancy correlates with antitumor activity of IPI-504 in a xenograft model of NSCLC. H1650 tumor bearing mice were treated with vehicle (●), 25 mg/kg (□), 50 mg/kg (▾), or 100 mg/kg (○) of IPI-504 (IP, twice weekly) and tumor growth was assessed by caliper measurement ( A ). Data are expressed as means and standard error ( n = 10 per arm). A separate group of H1650 tumor bearing mice was treated with a single dose of 25, 50, or 100 mg/kg of IPI-504 and sacrificed 2 h post dose. Hsp90 occupancy was determined as described and plotted against tumor size (mm 3 ) measured on day 50 of drug treatment ( B ). Data are expressed as averages with standard deviation ( n = 2 ( x -axis)) and as means with standard error ( n = 10 ( y -axis)). The points were fit to a linear least squares regression equation with a calculated R 2 = 0.98.
    Figure Legend Snippet: Hsp90 occupancy correlates with antitumor activity of IPI-504 in a xenograft model of NSCLC. H1650 tumor bearing mice were treated with vehicle (●), 25 mg/kg (□), 50 mg/kg (▾), or 100 mg/kg (○) of IPI-504 (IP, twice weekly) and tumor growth was assessed by caliper measurement ( A ). Data are expressed as means and standard error ( n = 10 per arm). A separate group of H1650 tumor bearing mice was treated with a single dose of 25, 50, or 100 mg/kg of IPI-504 and sacrificed 2 h post dose. Hsp90 occupancy was determined as described and plotted against tumor size (mm 3 ) measured on day 50 of drug treatment ( B ). Data are expressed as averages with standard deviation ( n = 2 ( x -axis)) and as means with standard error ( n = 10 ( y -axis)). The points were fit to a linear least squares regression equation with a calculated R 2 = 0.98.

    Techniques Used: Activity Assay, Mouse Assay, Standard Deviation

    Effect of Hsp90 occupancy on client protein abundance. H1650 cells were incubated for 6 or 24 h with increasing concentrations of IPI-504. HER2, mEGFR, Akt, and cRaf protein levels were assessed by immunoblotting ( A and C ). The fraction of degraded proteins was assessed by densitometry compared with untreated samples and plotted together with the occupancy curves ( B and D ).
    Figure Legend Snippet: Effect of Hsp90 occupancy on client protein abundance. H1650 cells were incubated for 6 or 24 h with increasing concentrations of IPI-504. HER2, mEGFR, Akt, and cRaf protein levels were assessed by immunoblotting ( A and C ). The fraction of degraded proteins was assessed by densitometry compared with untreated samples and plotted together with the occupancy curves ( B and D ).

    Techniques Used: Incubation

    Binding of [ 3 H]17-AAG to purified Hela Hsp90 and SK-BR-3 lysates at 4 °C. The binding reaction was initiated by adding 10 μ m [ 3 H]17-AAG to 100 n m purified Hsp90 or to SK-BR-3 lysate containing ∼100 n m Hsp90. Drug association was measured at 4 °C by a time-dependent increase in protein bound counts for purified Hsp90 ( A ) or Hsp90 in SK-BR-3 cell lysate ( B ). Data were fitted by nonlinear regression to a single exponential equation to obtain a ( k obs ) value. Half-life was calculated using the equation t ½ = 0.693/ k obs .
    Figure Legend Snippet: Binding of [ 3 H]17-AAG to purified Hela Hsp90 and SK-BR-3 lysates at 4 °C. The binding reaction was initiated by adding 10 μ m [ 3 H]17-AAG to 100 n m purified Hsp90 or to SK-BR-3 lysate containing ∼100 n m Hsp90. Drug association was measured at 4 °C by a time-dependent increase in protein bound counts for purified Hsp90 ( A ) or Hsp90 in SK-BR-3 cell lysate ( B ). Data were fitted by nonlinear regression to a single exponential equation to obtain a ( k obs ) value. Half-life was calculated using the equation t ½ = 0.693/ k obs .

    Techniques Used: Binding Assay, Purification

    36) Product Images from "Short-Term Storage of Rat Sperm in the Presence of Various Extenders"

    Article Title: Short-Term Storage of Rat Sperm in the Presence of Various Extenders

    Journal: Journal of the American Association for Laboratory Animal Science : JAALAS

    doi:

    Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 4 °C for 6, 24, 32, 48, and 72 h. Data are presented as mean ± SEM ( n = 6).
    Figure Legend Snippet: Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 4 °C for 6, 24, 32, 48, and 72 h. Data are presented as mean ± SEM ( n = 6).

    Techniques Used:

    Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 22 °C for 6, 24, 32, 48, and 72 h. Data are presented as mean ± SEM ( n = 6). *, Values within
    Figure Legend Snippet: Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 22 °C for 6, 24, 32, 48, and 72 h. Data are presented as mean ± SEM ( n = 6). *, Values within

    Techniques Used:

    Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 37 °C for 24, 48, and 72 h. Data are presented as mean ± SEM ( n = 6). *, Values within the same
    Figure Legend Snippet: Percentage motility of Sprague–Dawley epididymal sperm after dilution in TL-HEPES, Tris, mKRB, LEY, and PBS extenders and storage at 37 °C for 24, 48, and 72 h. Data are presented as mean ± SEM ( n = 6). *, Values within the same

    Techniques Used:

    37) Product Images from "The Roles of Integrins in Function of Human Neutrophils after Their Migration through Endothelium into Interstitial Matrix"

    Article Title: The Roles of Integrins in Function of Human Neutrophils after Their Migration through Endothelium into Interstitial Matrix

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118593

    Effects of migration and culture on phagocytosis by neutrophils. Neutrophil phagocytosis was analysed by measuring intensity of fluorescence of cells incubated with pHrodo E. coli BioParticles. Freshly isolated neutrophils (without or with collagenase treatment) were compared to neutrophils recovered from a gel 1h or 24 h after migrating through EC treated with 100U/ml TNF. A. Samples were incubated with bioparticles for 10, 30 or 60min before being fixed for flow cytometry. B. Confocal microscopic images of neutrophils recovered after 1h and incubated with bioparticles for 60min, imaged in the same plane for (i) bisbenzamide-stained nuclei or (ii) fluorescent intracellular bioparticles. Data are mean ± SEM from 3 experiments. ** = p
    Figure Legend Snippet: Effects of migration and culture on phagocytosis by neutrophils. Neutrophil phagocytosis was analysed by measuring intensity of fluorescence of cells incubated with pHrodo E. coli BioParticles. Freshly isolated neutrophils (without or with collagenase treatment) were compared to neutrophils recovered from a gel 1h or 24 h after migrating through EC treated with 100U/ml TNF. A. Samples were incubated with bioparticles for 10, 30 or 60min before being fixed for flow cytometry. B. Confocal microscopic images of neutrophils recovered after 1h and incubated with bioparticles for 60min, imaged in the same plane for (i) bisbenzamide-stained nuclei or (ii) fluorescent intracellular bioparticles. Data are mean ± SEM from 3 experiments. ** = p

    Techniques Used: Migration, Fluorescence, Incubation, Isolation, Flow Cytometry, Cytometry, Staining

    38) Product Images from "Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors †"

    Article Title: Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors †

    Journal: Journal of Virology

    doi:

    Comparison of RNA and protein syntheses from the RNA replication-competent and RNA replication-defective KUN replicon DNA vectors. (A) Northern blot hybridization analysis of total RNA isolated from BHK21 cells 36 h after transfection with equal amounts of pKUNrep2 (lane 1) or pKUNrep2(dGDD) (lane 2) plasmid DNAs, or untransfected cells (lane 3). The probe was a [ 32 ). Relative phosphorimager counts of the radiolabeled NS3 bands in corresponding RIP samples are shown with the background level deducted. DNA transfection, Northern blot hybridization, protein labeling, ACD treatment, and RIP procedures were performed as described in Materials and Methods.
    Figure Legend Snippet: Comparison of RNA and protein syntheses from the RNA replication-competent and RNA replication-defective KUN replicon DNA vectors. (A) Northern blot hybridization analysis of total RNA isolated from BHK21 cells 36 h after transfection with equal amounts of pKUNrep2 (lane 1) or pKUNrep2(dGDD) (lane 2) plasmid DNAs, or untransfected cells (lane 3). The probe was a [ 32 ). Relative phosphorimager counts of the radiolabeled NS3 bands in corresponding RIP samples are shown with the background level deducted. DNA transfection, Northern blot hybridization, protein labeling, ACD treatment, and RIP procedures were performed as described in Materials and Methods.

    Techniques Used: Northern Blot, Hybridization, Isolation, Transfection, Plasmid Preparation, Labeling

    Comparative analyses of transient β-Gal expression from the KUN replicon [pKUNβrep2, pKUNβrep2(dGDD); see Materials and Methods], pSCAβ (SFV replicon-based; 14), and pCMVβ (conventional plasmid; Clontech) DNA constructs. BHK21 cell monolayers (∼1.3 × 10 5 cells in 16-mm-diameter wells of 24-well plates) were transfected with ∼0.8 μg of corresponding DNAs and incubated until reaching confluency (∼2 days posttransfection). The cells were then trypsinized and transferred into larger plates to allow continuous cell division. Forty percent of the trypsinized cells were transferred into 35-mm-diameter plates, allowed to propagate, and assayed for β-Gal activity at day 3 after initial transfection. Fifty percent of the trypsinized cells were transferred into 60-mm-diameter plates, allowed to propagate, and assayed for β-Gal activity at day 5 after initial transfection. U*, the amounts of biochemically active β-Gal protein, in units of the enzymatic activity, produced in the total volume of each cell lysate collected from 16-mm-diameter wells (days 1 and 2), 35-mm-diameter plates (day 3), and 60-mm-diameter plates (day 5). The values are the means ± standard deviation for triplicate experiments.
    Figure Legend Snippet: Comparative analyses of transient β-Gal expression from the KUN replicon [pKUNβrep2, pKUNβrep2(dGDD); see Materials and Methods], pSCAβ (SFV replicon-based; 14), and pCMVβ (conventional plasmid; Clontech) DNA constructs. BHK21 cell monolayers (∼1.3 × 10 5 cells in 16-mm-diameter wells of 24-well plates) were transfected with ∼0.8 μg of corresponding DNAs and incubated until reaching confluency (∼2 days posttransfection). The cells were then trypsinized and transferred into larger plates to allow continuous cell division. Forty percent of the trypsinized cells were transferred into 35-mm-diameter plates, allowed to propagate, and assayed for β-Gal activity at day 3 after initial transfection. Fifty percent of the trypsinized cells were transferred into 60-mm-diameter plates, allowed to propagate, and assayed for β-Gal activity at day 5 after initial transfection. U*, the amounts of biochemically active β-Gal protein, in units of the enzymatic activity, produced in the total volume of each cell lysate collected from 16-mm-diameter wells (days 1 and 2), 35-mm-diameter plates (day 3), and 60-mm-diameter plates (day 5). The values are the means ± standard deviation for triplicate experiments.

    Techniques Used: Expressing, Plasmid Preparation, Construct, Transfection, Incubation, Activity Assay, Produced, Standard Deviation

    39) Product Images from "Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20"

    Article Title: Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20

    Journal: EMBO Reports

    doi: 10.15252/embr.201846433

    Characterization of pSer335 XErp1 antibody CSF extract was treated with Myc‐XErp1 IVT carrying the indicated combinations of the mutations DSG − (S33N S38N), DSA − (S284N S288N), ZBR − (C583A) and CaMKII − (T195A). Calcium was added, samples were taken at the indicated time points and as indicated treated with λ‐phosphatase. Samples were immunoblotted for the Myc‐tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α‐tubulin. Asterisk indicates unspecific bands. Several lanes were removed at the dashed line. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT at the indicated dilutions. An empty IVT not expressing XErp1 and an untreated condition were used as controls. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, pSer335 XErp1, and α‐tubulin. The XErp1 membrane was stripped and reprobed for the Myc‐tag. Asterisks indicate unspecific bands. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT that was either wild‐type or mutated to alanine at Ser335. An empty IVT reaction not expressing Myc‐XErp1 served as control. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, the Myc‐tag, pSer335 XErp1, and α‐tubulin. Asterisks indicate unspecific bands. Source data are available online for this figure.
    Figure Legend Snippet: Characterization of pSer335 XErp1 antibody CSF extract was treated with Myc‐XErp1 IVT carrying the indicated combinations of the mutations DSG − (S33N S38N), DSA − (S284N S288N), ZBR − (C583A) and CaMKII − (T195A). Calcium was added, samples were taken at the indicated time points and as indicated treated with λ‐phosphatase. Samples were immunoblotted for the Myc‐tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α‐tubulin. Asterisk indicates unspecific bands. Several lanes were removed at the dashed line. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT at the indicated dilutions. An empty IVT not expressing XErp1 and an untreated condition were used as controls. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, pSer335 XErp1, and α‐tubulin. The XErp1 membrane was stripped and reprobed for the Myc‐tag. Asterisks indicate unspecific bands. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT that was either wild‐type or mutated to alanine at Ser335. An empty IVT reaction not expressing Myc‐XErp1 served as control. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, the Myc‐tag, pSer335 XErp1, and α‐tubulin. Asterisks indicate unspecific bands. Source data are available online for this figure.

    Techniques Used: Expressing

    40) Product Images from "Multiplex live single-cell transcriptional analysis demarcates cellular functional heterogeneity"

    Article Title: Multiplex live single-cell transcriptional analysis demarcates cellular functional heterogeneity

    Journal: eLife

    doi: 10.7554/eLife.49599

    Optimization of fluorescence labeling of MAGIC Factor and MAGIC Probes. ( A ) Labeling strategy of MAGIC Factor. MAGIC Factor was fluorescently labeled with increasing amounts of Alexa Fluor 488 SDP Ester and run on a 12% SDS-PAGE gel after purification by size-exclusion chromatography. The ratios refer to dye:protein molar ratios during fluorescent labeling. The control reaction was performed with DMSO instead of the dye. The same gel was imaged for native Alexa Fluor 488 fluorescence (top gel) and after staining all protein with Sypro Orange (bottom gel). ( B ) Electrophoretic Mobility Shift Assay (EMSA) of fluorescently labeled MAGIC Factor. Proteins from A were reacted with Alexa Fluor 647-labeled dsRNA and run on a native gel to visualize the binding affinities of the proteins. The gel was visualized in the RNA channel, protein channel and FRET channel. Note that the appearance of MAGIC Factor as multiple bands is likely due to the use of an NHS-ester dye to attach Alexa Fluor 488 to the protein. Dependent on the exact location of the fluorophore molecule, each single protein molecule likely runs differently on the native polyacrylamide gel. ( C ) Quantitative assessment of the EMSA with respect to the relative shift of the dsRNA, the corrected FRET (cFRET) intensity of shifted dsRNA and the cFRET/shift ratio. ( D ) Unlabeled and fluorescently labeled MAGIC Factor were affinity purified using dsRNA-coupled agarose beads. The proteins were reacted with the beads for 1 hr, washed three times with binding buffer and then gradually eluted with increasing concentrations of KCl. At the end, remaining proteins were eluted with 1x SDS-PAGE sample buffer and all samples run on a 12% SDS-PAGE gel. As a control experiment, fluorescently labeled MAGIC Factor was reacted with agarose beads in the absence of dsRNA. Gels were imaged for native Alexa Fluor 488 fluorescence (right gel) and after staining all protein (left gel). ( E ) Effect of degree of RNA fluorescent labeling on probe hybridization kinetics. Fluorescently labeled RNA probes were gel purified to obtain one to four labeled RNA probes. They were then reacted with unlabeled sense probes to generate dsRNA in TEN 100 buffer (tris, EDTA, sodium chloride) at 95°C (positive control) or physiologic buffer resembling cytoplasmic ionic concentrations at 37°C for 30 min or 2 hr. ssRNA are shown as controls. Representative native 20% polyacrylamide gels are shown. ( F ) Quantification of RNA fluorescence intensities from ( E ). Quantified data are shown as mean ± s.e.m. *p
    Figure Legend Snippet: Optimization of fluorescence labeling of MAGIC Factor and MAGIC Probes. ( A ) Labeling strategy of MAGIC Factor. MAGIC Factor was fluorescently labeled with increasing amounts of Alexa Fluor 488 SDP Ester and run on a 12% SDS-PAGE gel after purification by size-exclusion chromatography. The ratios refer to dye:protein molar ratios during fluorescent labeling. The control reaction was performed with DMSO instead of the dye. The same gel was imaged for native Alexa Fluor 488 fluorescence (top gel) and after staining all protein with Sypro Orange (bottom gel). ( B ) Electrophoretic Mobility Shift Assay (EMSA) of fluorescently labeled MAGIC Factor. Proteins from A were reacted with Alexa Fluor 647-labeled dsRNA and run on a native gel to visualize the binding affinities of the proteins. The gel was visualized in the RNA channel, protein channel and FRET channel. Note that the appearance of MAGIC Factor as multiple bands is likely due to the use of an NHS-ester dye to attach Alexa Fluor 488 to the protein. Dependent on the exact location of the fluorophore molecule, each single protein molecule likely runs differently on the native polyacrylamide gel. ( C ) Quantitative assessment of the EMSA with respect to the relative shift of the dsRNA, the corrected FRET (cFRET) intensity of shifted dsRNA and the cFRET/shift ratio. ( D ) Unlabeled and fluorescently labeled MAGIC Factor were affinity purified using dsRNA-coupled agarose beads. The proteins were reacted with the beads for 1 hr, washed three times with binding buffer and then gradually eluted with increasing concentrations of KCl. At the end, remaining proteins were eluted with 1x SDS-PAGE sample buffer and all samples run on a 12% SDS-PAGE gel. As a control experiment, fluorescently labeled MAGIC Factor was reacted with agarose beads in the absence of dsRNA. Gels were imaged for native Alexa Fluor 488 fluorescence (right gel) and after staining all protein (left gel). ( E ) Effect of degree of RNA fluorescent labeling on probe hybridization kinetics. Fluorescently labeled RNA probes were gel purified to obtain one to four labeled RNA probes. They were then reacted with unlabeled sense probes to generate dsRNA in TEN 100 buffer (tris, EDTA, sodium chloride) at 95°C (positive control) or physiologic buffer resembling cytoplasmic ionic concentrations at 37°C for 30 min or 2 hr. ssRNA are shown as controls. Representative native 20% polyacrylamide gels are shown. ( F ) Quantification of RNA fluorescence intensities from ( E ). Quantified data are shown as mean ± s.e.m. *p

    Techniques Used: Fluorescence, Labeling, SDS Page, Purification, Size-exclusion Chromatography, Staining, Electrophoretic Mobility Shift Assay, Binding Assay, Affinity Purification, Hybridization, Positive Control

    41) Product Images from "Suppression by L-Methionine of Cell Cycle Progression in LNCaP and MCF-7 Cells but not Benign Cells"

    Article Title: Suppression by L-Methionine of Cell Cycle Progression in LNCaP and MCF-7 Cells but not Benign Cells

    Journal: Anticancer research

    doi:

    Flow cytometric analysis of propidium iodide-stained cells showing the effects of L-methionine treatment (5 mg/ml for 72 h) on cell cycle progression of MCF-7 breast cancer cells and non-tumorigenic MCF-10A breast epithelial cells. Data are expressed
    Figure Legend Snippet: Flow cytometric analysis of propidium iodide-stained cells showing the effects of L-methionine treatment (5 mg/ml for 72 h) on cell cycle progression of MCF-7 breast cancer cells and non-tumorigenic MCF-10A breast epithelial cells. Data are expressed

    Techniques Used: Flow Cytometry, Staining

    42) Product Images from "Cell-surface C-type lectin-like receptor CLEC-1 dampens dendritic cell activation and downstream Th17 responses"

    Article Title: Cell-surface C-type lectin-like receptor CLEC-1 dampens dendritic cell activation and downstream Th17 responses

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2016002360

    Human DCs express cell-surface CLEC-1. (A) Western blot analysis of CLEC-1 expression in human moDCs, HAECs, HUVECs, and HEKs. Cell extracts were immunoprecipitated with anti-human CLEC-1 mAb (D6 clone) and then analyzed by western blot with a second in-house anti-human CLEC-1 mAb (IgG1). Arrows indicate human CLEC-1 and IgG HC and LC at the expected size of 32, 50, and 25 kDa, respectively. M line represents molecular-weight size markers. (B) Representative dot plots and histograms of IgG1 isotype or CLEC-1 (IgG1) staining in non-permeabilized (non-perm) and permeabilized (perm) conditions, evaluated by flow cytometry for human blood: (i) CD16 + and CD16 − subpopulation of CD45 + CD14 − CD11c + HLA-DR high DCs; (ii) CD45 + CD14 + CD16 + monocytes; (iii) SSC high CD16 + neutrophils; and (iv) HAECs. Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (C) Representative dot plots or histograms of IgG1 isotype or CLEC-1 (IgG1) staining vs DC-SIGN or CD16 staining for human moDCs in non-perm or perm conditions and evaluated by flow cytometry (i-ii). Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (iii) Cell-surface expression of CLEC-1 vs HLA-DR on unstimulated (US), TLR 4-L (LPS), TLR 3-L (Poly I:C), TLR 7-L (R848), and TGF-β–stimulated moDCs. (iv) Histogram represents MFI ± standard error of the mean (SEM) of CLEC-1 staining of 6 independent experiments. Statistical analysis of CLEC-1 MFI staining was performed between US and each stimuli. Panel Di shows representative confocal microscopy images, and (ii) quantitation of CLEC-1 protein in non-perm and perm conditions for human HUVECs, moDCs, CD16 + monocytes and neutrophils. Panels exhibiting DAPI (blue) and CLEC-1 (green) staining revealed by anti-human CLEC-1 mAb (D6 clone) followed by secondary anti-mouse Alexa-488 antibody. Original magnification ×600. Images are representative of 4 independent experiments. CLEC-1 protein quantitation was performed by velocity software and expressed as histogram of mean ± SEM of numbers of fluorescent spots per cell (n ≥7). * P
    Figure Legend Snippet: Human DCs express cell-surface CLEC-1. (A) Western blot analysis of CLEC-1 expression in human moDCs, HAECs, HUVECs, and HEKs. Cell extracts were immunoprecipitated with anti-human CLEC-1 mAb (D6 clone) and then analyzed by western blot with a second in-house anti-human CLEC-1 mAb (IgG1). Arrows indicate human CLEC-1 and IgG HC and LC at the expected size of 32, 50, and 25 kDa, respectively. M line represents molecular-weight size markers. (B) Representative dot plots and histograms of IgG1 isotype or CLEC-1 (IgG1) staining in non-permeabilized (non-perm) and permeabilized (perm) conditions, evaluated by flow cytometry for human blood: (i) CD16 + and CD16 − subpopulation of CD45 + CD14 − CD11c + HLA-DR high DCs; (ii) CD45 + CD14 + CD16 + monocytes; (iii) SSC high CD16 + neutrophils; and (iv) HAECs. Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (C) Representative dot plots or histograms of IgG1 isotype or CLEC-1 (IgG1) staining vs DC-SIGN or CD16 staining for human moDCs in non-perm or perm conditions and evaluated by flow cytometry (i-ii). Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (iii) Cell-surface expression of CLEC-1 vs HLA-DR on unstimulated (US), TLR 4-L (LPS), TLR 3-L (Poly I:C), TLR 7-L (R848), and TGF-β–stimulated moDCs. (iv) Histogram represents MFI ± standard error of the mean (SEM) of CLEC-1 staining of 6 independent experiments. Statistical analysis of CLEC-1 MFI staining was performed between US and each stimuli. Panel Di shows representative confocal microscopy images, and (ii) quantitation of CLEC-1 protein in non-perm and perm conditions for human HUVECs, moDCs, CD16 + monocytes and neutrophils. Panels exhibiting DAPI (blue) and CLEC-1 (green) staining revealed by anti-human CLEC-1 mAb (D6 clone) followed by secondary anti-mouse Alexa-488 antibody. Original magnification ×600. Images are representative of 4 independent experiments. CLEC-1 protein quantitation was performed by velocity software and expressed as histogram of mean ± SEM of numbers of fluorescent spots per cell (n ≥7). * P

    Techniques Used: Western Blot, Expressing, Immunoprecipitation, Molecular Weight, Staining, Flow Cytometry, Cytometry, Confocal Microscopy, Quantitation Assay, Protein Quantitation, Software

    43) Product Images from "Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts"

    Article Title: Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.507525

    Wortmannin and GS-9820 induce osteoclast retraction. Rat osteoclasts were bathed in HEPES-buffered M199 medium with 15% FBS and antibiotics and imaged using time-lapse phase-contrast microscopy. A–E illustrates 5 different osteoclasts, each shown
    Figure Legend Snippet: Wortmannin and GS-9820 induce osteoclast retraction. Rat osteoclasts were bathed in HEPES-buffered M199 medium with 15% FBS and antibiotics and imaged using time-lapse phase-contrast microscopy. A–E illustrates 5 different osteoclasts, each shown

    Techniques Used: Microscopy

    44) Product Images from "mTORC1 Inhibition Induces Resistance to Methotrexate and 6-Mercaptopurine in Ph+ and Ph-like B-ALL"

    Article Title: mTORC1 Inhibition Induces Resistance to Methotrexate and 6-Mercaptopurine in Ph+ and Ph-like B-ALL

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-17-0024

    mTOR inhibition protects from methotrexate and 6-mercaptopurine (A) BV173 and (B) SUP-B15 Ph + B-ALL cells are treated with MTX and 6MP for 48 hours in presence of 100 nM MLN0128. Viability is measured by Annexin-V and PI staining. Unpaired t-tests of were performed, n=3, mean±SD, *p
    Figure Legend Snippet: mTOR inhibition protects from methotrexate and 6-mercaptopurine (A) BV173 and (B) SUP-B15 Ph + B-ALL cells are treated with MTX and 6MP for 48 hours in presence of 100 nM MLN0128. Viability is measured by Annexin-V and PI staining. Unpaired t-tests of were performed, n=3, mean±SD, *p

    Techniques Used: Inhibition, Staining

    45) Product Images from "The proline rich domain of p53 is dispensable for MGMT-dependent DNA repair and cell survival following alkylation damage"

    Article Title: The proline rich domain of p53 is dispensable for MGMT-dependent DNA repair and cell survival following alkylation damage

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2017.116

    Loss of MGMT in cells does not change their apoptotic response to death inducing stimuli regardless of p53 status. ( a ) MGMT and p53 protein levels in p53 WT , p53 ΔP and p53 −/− MEF cells, deficient or sufficient for MGMT, 18 h after IR (5 Gy) or Diazald (75 μ M) treatment. ( b ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells with or without MGMT expression treated with different doses of UV, IR or Diazald for 18 h
    Figure Legend Snippet: Loss of MGMT in cells does not change their apoptotic response to death inducing stimuli regardless of p53 status. ( a ) MGMT and p53 protein levels in p53 WT , p53 ΔP and p53 −/− MEF cells, deficient or sufficient for MGMT, 18 h after IR (5 Gy) or Diazald (75 μ M) treatment. ( b ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells with or without MGMT expression treated with different doses of UV, IR or Diazald for 18 h

    Techniques Used: Flow Cytometry, Staining, Expressing

    MGMT overexpression facilitates repair and some survival advantage in p53 −/− cells. ( a ) Cell death at 18 h as measured by flow cytometric analysis of Annexin V staining in MEF cells overexpressing MGMT and treated with 100 μ M Diazald. ( b ) Intracellular pH2AX levels measured by flow cytometry in MEF cells overexpressing MGMT and treated with 100 μ M Diazald for 3 h to assess damage, or at 24 h to assess repair. ( c ) Colony-forming assays examining survival in p53 WT , p53 ΔP or p53 −/− MEF cells with or without MGMT overexpression after 250 μ M Diazald for increasing time intervals. ( d ) Quantification of colony-forming assay in p53 WT , p53 ΔP , or p53 −/− MEF cells
    Figure Legend Snippet: MGMT overexpression facilitates repair and some survival advantage in p53 −/− cells. ( a ) Cell death at 18 h as measured by flow cytometric analysis of Annexin V staining in MEF cells overexpressing MGMT and treated with 100 μ M Diazald. ( b ) Intracellular pH2AX levels measured by flow cytometry in MEF cells overexpressing MGMT and treated with 100 μ M Diazald for 3 h to assess damage, or at 24 h to assess repair. ( c ) Colony-forming assays examining survival in p53 WT , p53 ΔP or p53 −/− MEF cells with or without MGMT overexpression after 250 μ M Diazald for increasing time intervals. ( d ) Quantification of colony-forming assay in p53 WT , p53 ΔP , or p53 −/− MEF cells

    Techniques Used: Over Expression, Flow Cytometry, Staining, Cytometry

    P53 ΔP cells do not express p21 or PUMA and are resistant to apoptosis, cell cycle arrest, and senescence. p21 and PUMA gene expression levels in ( a ) H1299 cells expressing p53 WT ERTam or p53 ΔP ERTam constructs 5 h after 4OHT treatment, and ( b ) primary B cells 6 h post irradiation. ( c ) p21 and PUMA protein levels in H1299 cells expressing p53 WT ERTam or p53 ΔP ERTam constructs 8 h post 4OHT. ( d ) Cell death at 18 h as measured by flow cytometric analysis of Annexin V staining in p53 WT , p53 ΔP , and p53 −/− MEF cells treated with a variety of apoptotic stimuli. ( e ) Brdu incorporation into MEFs 18 h after 4 Gy IR. Growth of ( f ) E1A/RAS transformed MEF cells over 4 days and ( g ) primary MEF cells over time. ( h ) Colony-forming assay looking at survival of primary MEF cells transduced with RAS oncogene
    Figure Legend Snippet: P53 ΔP cells do not express p21 or PUMA and are resistant to apoptosis, cell cycle arrest, and senescence. p21 and PUMA gene expression levels in ( a ) H1299 cells expressing p53 WT ERTam or p53 ΔP ERTam constructs 5 h after 4OHT treatment, and ( b ) primary B cells 6 h post irradiation. ( c ) p21 and PUMA protein levels in H1299 cells expressing p53 WT ERTam or p53 ΔP ERTam constructs 8 h post 4OHT. ( d ) Cell death at 18 h as measured by flow cytometric analysis of Annexin V staining in p53 WT , p53 ΔP , and p53 −/− MEF cells treated with a variety of apoptotic stimuli. ( e ) Brdu incorporation into MEFs 18 h after 4 Gy IR. Growth of ( f ) E1A/RAS transformed MEF cells over 4 days and ( g ) primary MEF cells over time. ( h ) Colony-forming assay looking at survival of primary MEF cells transduced with RAS oncogene

    Techniques Used: Expressing, Construct, Irradiation, Flow Cytometry, Staining, BrdU Incorporation Assay, Transformation Assay, Transduction

    Loss of MGMT in cells inhibits repair and survival of p53 ΔP cells. Intracellular pH2AX levels measured by flow cytometry in p53 WT , p53 ΔP and p53 −/− MEF cells ( a ) sufficient or deficient for MGMT or, ( d ) expressing BCLxL, treated with 100 μ M Diazald for 3 h to assess damage or 24 h to assess repair. ( b ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells treated for 3 h with 100 μ M Diazald, washed and assessed for Annexin V staining 24 h later. ( c ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells expressing BCLxL and treated with 100 μ M Diazald for 24 h. ( e ) Colony-forming assays examining survival in Left : p53 WT , p53 ΔP and p53 −/− MEF cells sufficient or deficient for MGMT; Right : MEF cells from p53 ΔP , MGMT −/− mice after 250 μ M Diazald treatment for increasing time intervals
    Figure Legend Snippet: Loss of MGMT in cells inhibits repair and survival of p53 ΔP cells. Intracellular pH2AX levels measured by flow cytometry in p53 WT , p53 ΔP and p53 −/− MEF cells ( a ) sufficient or deficient for MGMT or, ( d ) expressing BCLxL, treated with 100 μ M Diazald for 3 h to assess damage or 24 h to assess repair. ( b ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells treated for 3 h with 100 μ M Diazald, washed and assessed for Annexin V staining 24 h later. ( c ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells expressing BCLxL and treated with 100 μ M Diazald for 24 h. ( e ) Colony-forming assays examining survival in Left : p53 WT , p53 ΔP and p53 −/− MEF cells sufficient or deficient for MGMT; Right : MEF cells from p53 ΔP , MGMT −/− mice after 250 μ M Diazald treatment for increasing time intervals

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Staining, Mouse Assay

    46) Product Images from "Human Rhinovirus 3C protease cleaves RIPK1, concurrent with caspase 8 activation"

    Article Title: Human Rhinovirus 3C protease cleaves RIPK1, concurrent with caspase 8 activation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19839-4

    ( a ) HRV16 replication is down regulated with inhibition of caspase 8. O-HeLa cells were infected with HRV16 at an M.O.I of 3. At 3 h.p.i., ActoD (5 µg/mL) was added to selected samples. Caspase 8 was inhibited as indicated through addition caspase 8 inhibitor (4 µM) at 4 h.p.i. Cultures were frozen at 24 h.p.i. Once thawed, virus was clarified and titrated. Results are expressed as mean of 3 independent experiments. Error bars are +/− standard error of the mean. *p ≤ 0.05, ns – nonsignificant. ( b ) HRV16 restricts apoptotic pathways. O-HeLa cells were infected and treated with ActoD as in a) above. Cells were collected at 12 h.p.i and the effect of treatment and/or infection analysed by flow cytometry after staining with annexin V-FITC (An) and propidium iodide (Pi). Representative dual-parameter fluorescence density blots were acquired. ( c ) Bars are representative of percentage of viable (An−/Pi−), early apoptotic (An+/Pi−), late apoptotic (An+/Pi+) or necrotic (An−/Pi+) cells of total number of cells within each sample.
    Figure Legend Snippet: ( a ) HRV16 replication is down regulated with inhibition of caspase 8. O-HeLa cells were infected with HRV16 at an M.O.I of 3. At 3 h.p.i., ActoD (5 µg/mL) was added to selected samples. Caspase 8 was inhibited as indicated through addition caspase 8 inhibitor (4 µM) at 4 h.p.i. Cultures were frozen at 24 h.p.i. Once thawed, virus was clarified and titrated. Results are expressed as mean of 3 independent experiments. Error bars are +/− standard error of the mean. *p ≤ 0.05, ns – nonsignificant. ( b ) HRV16 restricts apoptotic pathways. O-HeLa cells were infected and treated with ActoD as in a) above. Cells were collected at 12 h.p.i and the effect of treatment and/or infection analysed by flow cytometry after staining with annexin V-FITC (An) and propidium iodide (Pi). Representative dual-parameter fluorescence density blots were acquired. ( c ) Bars are representative of percentage of viable (An−/Pi−), early apoptotic (An+/Pi−), late apoptotic (An+/Pi+) or necrotic (An−/Pi+) cells of total number of cells within each sample.

    Techniques Used: Inhibition, Infection, Flow Cytometry, Cytometry, Staining, Fluorescence

    47) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.220780

    Vpr from the incoming virion induces apoptosis independently of its G 2 arrest activity in Jurkat cells. A , Vpr wt and mutants are similarly incorporated into HIV-1 pseudoparticles. Pseudoparticles were produced in the absence or presence of HA-tagged wt or mutant Vpr proteins. The presence of Vpr and the p24 capsid in the viral particle was analyzed by Western blot ( left panel ). The signals obtained in the left panel were quantified and expressed as HA/p24 ratios ( right panel ). B and C , VprK27M and VprS79A, but not VprQ65R, induce apoptosis in Jurkat cells. Jurkat cells were incubated with empty VLP or VLP containing the indicated HA-Vpr proteins each day until harvesting. Cells were harvested 48, 72, and 96 h after the first incubation, and the number of apoptotic cells was determined by flow cytometry after labeling with annexin-FITC. Panel B shows the distribution of cells versus the staining intensity at 96h. Panel C shows the percentage of annexin-FITC-positive cells versus time.
    Figure Legend Snippet: Vpr from the incoming virion induces apoptosis independently of its G 2 arrest activity in Jurkat cells. A , Vpr wt and mutants are similarly incorporated into HIV-1 pseudoparticles. Pseudoparticles were produced in the absence or presence of HA-tagged wt or mutant Vpr proteins. The presence of Vpr and the p24 capsid in the viral particle was analyzed by Western blot ( left panel ). The signals obtained in the left panel were quantified and expressed as HA/p24 ratios ( right panel ). B and C , VprK27M and VprS79A, but not VprQ65R, induce apoptosis in Jurkat cells. Jurkat cells were incubated with empty VLP or VLP containing the indicated HA-Vpr proteins each day until harvesting. Cells were harvested 48, 72, and 96 h after the first incubation, and the number of apoptotic cells was determined by flow cytometry after labeling with annexin-FITC. Panel B shows the distribution of cells versus the staining intensity at 96h. Panel C shows the percentage of annexin-FITC-positive cells versus time.

    Techniques Used: Activity Assay, Produced, Mutagenesis, Western Blot, Incubation, Flow Cytometry, Cytometry, Labeling, Staining

    48) Product Images from "Redirecting Valvular Myofibroblasts into Dormant Fibroblasts through Light-mediated Reduction in Substrate Modulus"

    Article Title: Redirecting Valvular Myofibroblasts into Dormant Fibroblasts through Light-mediated Reduction in Substrate Modulus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039969

    Decreased number of myofibroblasts on stiff-to-soft gels was not due to apoptosis. VICs cultured on different PD-PEG gels were stained with Annexin V linked with Alexa Fluor 594 and DAPI to detect apoptosis. (A) Scatter plot of Annexin V and DAPI staining for VICs cultured on stiff gels. Red box: apoptotic cells with high fluorescence in the Annexin V channel and low fluorescence in the DAPI channel. (B) A representative confocal image of a cell stained positively for Annexin V (red) overlaid with transmitted light DIC on stiff gels. Positively-stained cells were observed to exhibit a rounded morphology. Blue: Nucleus. Scale bar: 20 µm. (C) Quantification of apoptosis based on flow cytometry as shown in (A). Low levels of apoptosis were detected for VICs cultured on either gels or plastic plates. VICs treated with camptothecin, an apoptosis-inducing reagent, showed a much higher level of apoptosis than any gel-based culture condition or plastic plate. * indicates p
    Figure Legend Snippet: Decreased number of myofibroblasts on stiff-to-soft gels was not due to apoptosis. VICs cultured on different PD-PEG gels were stained with Annexin V linked with Alexa Fluor 594 and DAPI to detect apoptosis. (A) Scatter plot of Annexin V and DAPI staining for VICs cultured on stiff gels. Red box: apoptotic cells with high fluorescence in the Annexin V channel and low fluorescence in the DAPI channel. (B) A representative confocal image of a cell stained positively for Annexin V (red) overlaid with transmitted light DIC on stiff gels. Positively-stained cells were observed to exhibit a rounded morphology. Blue: Nucleus. Scale bar: 20 µm. (C) Quantification of apoptosis based on flow cytometry as shown in (A). Low levels of apoptosis were detected for VICs cultured on either gels or plastic plates. VICs treated with camptothecin, an apoptosis-inducing reagent, showed a much higher level of apoptosis than any gel-based culture condition or plastic plate. * indicates p

    Techniques Used: Cell Culture, Staining, Fluorescence, Flow Cytometry, Cytometry

    49) Product Images from "The DNA damage response is developmentally regulated in the African trypanosome"

    Article Title: The DNA damage response is developmentally regulated in the African trypanosome

    Journal: DNA Repair

    doi: 10.1016/j.dnarep.2018.11.005

    Flow cytometry analysis of asynchronous log-phase populations in the presence and absence of DNA damage by FITC Annexin V and propidium iodide staining. A) Top row: BSF cells with no treatment and immediately after exposure to 0.5, 1.0 or 1.5 mM of MMS for 1 h. Bottom row: PCF cells 24 h after treatment with 0. 1.0 or 1.5 mM of MMS for 1 h. B) Quantification of A-). C)Top row: BSF cells with no treatment and 24 h after treatment with 100, 200 or -400 μM of cisplatin for 1 h. Bottom row: PCF cells treated and analyzed as described in the top row. D) Quantification of C-). FACS analysis was performed on 5000 cell counts. Channels FL2-H (y axis) and FL1-H (x axis) were used to detect, respectively, propidium iodide (PI) and FITC-Annexin V (Annexin-V) staining. Asterisks denote p values less than 0.05 calculated by chi-square test.
    Figure Legend Snippet: Flow cytometry analysis of asynchronous log-phase populations in the presence and absence of DNA damage by FITC Annexin V and propidium iodide staining. A) Top row: BSF cells with no treatment and immediately after exposure to 0.5, 1.0 or 1.5 mM of MMS for 1 h. Bottom row: PCF cells 24 h after treatment with 0. 1.0 or 1.5 mM of MMS for 1 h. B) Quantification of A-). C)Top row: BSF cells with no treatment and 24 h after treatment with 100, 200 or -400 μM of cisplatin for 1 h. Bottom row: PCF cells treated and analyzed as described in the top row. D) Quantification of C-). FACS analysis was performed on 5000 cell counts. Channels FL2-H (y axis) and FL1-H (x axis) were used to detect, respectively, propidium iodide (PI) and FITC-Annexin V (Annexin-V) staining. Asterisks denote p values less than 0.05 calculated by chi-square test.

    Techniques Used: Flow Cytometry, Cytometry, Staining, FACS

    50) Product Images from "Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes"

    Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02655-1

    In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1
    Figure Legend Snippet: In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

    Techniques Used: In Vitro, Activity Assay, Fluorescence, Purification

    Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1
    Figure Legend Snippet: Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

    Techniques Used: In Vitro, Activity Assay, Fluorescence, Purification

    51) Product Images from "Brief Exercise Increases Peripheral Blood NK Cell Counts without Immediate Functional Changes, but Impairs their Responses to ex vivo Stimulation"

    Article Title: Brief Exercise Increases Peripheral Blood NK Cell Counts without Immediate Functional Changes, but Impairs their Responses to ex vivo Stimulation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2013.00125

    Changes in NK cell activity following brief exercise . (A) Pre-exercise (white) and post-exercise (black) isolated NK cells were analyzed by flow cytometry to determine the percentage of IFNγ-secreting cells using an IFNγ capture assay, either immediately upon isolation or following culture in AIM-V medium supplemented with 2% HEPES without cytokines for 4 or 24 h. Data represent the mean% ± SD of seven different donors. (B) The frequency of CD107a expressing NK cells was assessed by flow cytometry immediately after isolation in response to incubation for 6 h with K562 cells without cytokine stimulation. Using a forward scatter/side scatter lymphocyte gate anti-CD107a and anti-CD56 Ab double positive NK cells were determined. Shown is the percentage of CD107a-positive NK cells of six different donors. Significant differences are indicated as follows: * p
    Figure Legend Snippet: Changes in NK cell activity following brief exercise . (A) Pre-exercise (white) and post-exercise (black) isolated NK cells were analyzed by flow cytometry to determine the percentage of IFNγ-secreting cells using an IFNγ capture assay, either immediately upon isolation or following culture in AIM-V medium supplemented with 2% HEPES without cytokines for 4 or 24 h. Data represent the mean% ± SD of seven different donors. (B) The frequency of CD107a expressing NK cells was assessed by flow cytometry immediately after isolation in response to incubation for 6 h with K562 cells without cytokine stimulation. Using a forward scatter/side scatter lymphocyte gate anti-CD107a and anti-CD56 Ab double positive NK cells were determined. Shown is the percentage of CD107a-positive NK cells of six different donors. Significant differences are indicated as follows: * p

    Techniques Used: Activity Assay, Isolation, Flow Cytometry, Cytometry, Expressing, Incubation

    52) Product Images from "A simple medium enables bovine embryos to be held for seven days at 4?C"

    Article Title: A simple medium enables bovine embryos to be held for seven days at 4?C

    Journal: Scientific Reports

    doi: 10.1038/srep01173

    (a) Bovine blastocyst just after collection from the uterus. (b) Chilled embryo stored for 168 h in medium 199 with 25 mM HEPES and 50% FBS.
    Figure Legend Snippet: (a) Bovine blastocyst just after collection from the uterus. (b) Chilled embryo stored for 168 h in medium 199 with 25 mM HEPES and 50% FBS.

    Techniques Used:

    53) Product Images from "Bioengineering functional smooth muscle with spontaneous rhythmic contraction in vitro"

    Article Title: Bioengineering functional smooth muscle with spontaneous rhythmic contraction in vitro

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31992-4

    Maintenance of intestinal smooth muscle cell mixture (ISMC Mix) and vascular aortic smooth muscle cell line (MOVAS cells) cultured on STO cells in vitro . Non-sorted cells from enzymatically digested intestinal muscle strips were used as ISMC Mix. All ISMC Mix and MOVAS cells were cultured separately in FBS medium for the first 4 days followed by FBS or F12 medium. ( a – c ) 100 k ISMC Mix were cultured on STO cells or Ge with FBS or F12 medium for up to 3 weeks. ( a ) Immunofluorescence of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), and neuronal marker β-tubulin (green) at day 7. Scale bar, 100 µm. ( b ) GFP + ISMC Mix were analyzed for mRNA expression ( Kit, Myh11, Tubb 3 , Kitl , and Acta2 ) and gfp DNA ( n = 3 ). ( c ) and frequency was measured ( n ≥ 5 ). ( d ) MOVAS cells were analyzed for mRNA expression ( Myh11 ) at day 7 ( n = 3 ; triplicate samples). FBS = 15% FBS in DMEM. F12 = advanced DMEM/F12. *Samples were normalized to de-epithelialized intestine. **Samples were normalized to 100 k cells from day 0. Error bars, s.d. ***P
    Figure Legend Snippet: Maintenance of intestinal smooth muscle cell mixture (ISMC Mix) and vascular aortic smooth muscle cell line (MOVAS cells) cultured on STO cells in vitro . Non-sorted cells from enzymatically digested intestinal muscle strips were used as ISMC Mix. All ISMC Mix and MOVAS cells were cultured separately in FBS medium for the first 4 days followed by FBS or F12 medium. ( a – c ) 100 k ISMC Mix were cultured on STO cells or Ge with FBS or F12 medium for up to 3 weeks. ( a ) Immunofluorescence of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), and neuronal marker β-tubulin (green) at day 7. Scale bar, 100 µm. ( b ) GFP + ISMC Mix were analyzed for mRNA expression ( Kit, Myh11, Tubb 3 , Kitl , and Acta2 ) and gfp DNA ( n = 3 ). ( c ) and frequency was measured ( n ≥ 5 ). ( d ) MOVAS cells were analyzed for mRNA expression ( Myh11 ) at day 7 ( n = 3 ; triplicate samples). FBS = 15% FBS in DMEM. F12 = advanced DMEM/F12. *Samples were normalized to de-epithelialized intestine. **Samples were normalized to 100 k cells from day 0. Error bars, s.d. ***P

    Techniques Used: Cell Culture, In Vitro, Immunofluorescence, Immunocytochemistry, Marker, Expressing

    Engineering aligned intestinal smooth muscle with periodic contraction over 10 weeks in vitro . 100 k ISMC Mix were cultured on STO cells seeded ePCL scaffolds in FBS medium for the first 4 days before changing to F12 medium. ( a ) Confocal images of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), neuronal marker β-tubulin (green), and GFP (green) at 10 weeks. Scale bar, 100 µm. ( b ) GFP + ISMC Mix were analyzed for mRNA expression ( Kit, Myh11, Tubb3, Kitl , and Acta2 ) ( n = 5 ) and gfp DNA ( n = 4 ). The dashed line indicates the seeding density. ( c – e ) Relaxed and contracted state comparison of engineered intestinal smooth muscle. ( c ). ( d) To show the degree of the periodic contraction, area of contracted ePCL scaffolds were normalized to that of relaxed ePCL scaffolds ( n = 10 ). ( e ) To show the directionality of the periodic constriction, height and width of contracted ePCL scaffolds were normalized to those of relaxed ePCL scaffolds ( n = 10 ). ( f ) Frequency of ePCL rhythmic contractions ( n = 4 ). The dark gray dashed line indicates the mean frequency over time and light gray dashed lines indicate its s.d. *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P
    Figure Legend Snippet: Engineering aligned intestinal smooth muscle with periodic contraction over 10 weeks in vitro . 100 k ISMC Mix were cultured on STO cells seeded ePCL scaffolds in FBS medium for the first 4 days before changing to F12 medium. ( a ) Confocal images of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), neuronal marker β-tubulin (green), and GFP (green) at 10 weeks. Scale bar, 100 µm. ( b ) GFP + ISMC Mix were analyzed for mRNA expression ( Kit, Myh11, Tubb3, Kitl , and Acta2 ) ( n = 5 ) and gfp DNA ( n = 4 ). The dashed line indicates the seeding density. ( c – e ) Relaxed and contracted state comparison of engineered intestinal smooth muscle. ( c ). ( d) To show the degree of the periodic contraction, area of contracted ePCL scaffolds were normalized to that of relaxed ePCL scaffolds ( n = 10 ). ( e ) To show the directionality of the periodic constriction, height and width of contracted ePCL scaffolds were normalized to those of relaxed ePCL scaffolds ( n = 10 ). ( f ) Frequency of ePCL rhythmic contractions ( n = 4 ). The dark gray dashed line indicates the mean frequency over time and light gray dashed lines indicate its s.d. *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P

    Techniques Used: In Vitro, Cell Culture, Immunocytochemistry, Marker, Expressing

    MACS+ cells’ role in cultured ISMC Mix with rhythmic contractions in vitro . ( a – c ) 100 k MACS0 (passaged ISMC Mix: mixture of MACS+ cells, SMCs and neuronal cells) or MACS− cells (ISMC Mix without MACS+ cells: mostly SMCs and neuronal cells) were cultured on STO cells for 3 weeks. Cells were seeded and cultured in FBS medium for the first 4 days before changing to F12 medium. ( d – f ) 100 k MACS− were seeded on STO cells for 5 weeks, with (MACS−/+) or without (MACS−) addition of 60 k MACS+ cells on day 5. Cells were cultured in FBS medium for the first 7 days before changing to F12 medium. ( a,d ) Confocal images of ICC markers, Kit (red), Ano1 (green), co-localization (yellow). Scale bar, 100 µm. ( b,e ) Frequency of cultured cells motility due to spontaneous contraction ( b : wk2 n = 2 , wk3 n = 4 ; ( e ) wk5 n = 4 ). ( c,f ) Cultured cells were analyzed for mRNA expression ( Kit, Myh11, Tubb3 ) at week 2 ( c ) or week 5 ( f ) ( n = 4 ). *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P
    Figure Legend Snippet: MACS+ cells’ role in cultured ISMC Mix with rhythmic contractions in vitro . ( a – c ) 100 k MACS0 (passaged ISMC Mix: mixture of MACS+ cells, SMCs and neuronal cells) or MACS− cells (ISMC Mix without MACS+ cells: mostly SMCs and neuronal cells) were cultured on STO cells for 3 weeks. Cells were seeded and cultured in FBS medium for the first 4 days before changing to F12 medium. ( d – f ) 100 k MACS− were seeded on STO cells for 5 weeks, with (MACS−/+) or without (MACS−) addition of 60 k MACS+ cells on day 5. Cells were cultured in FBS medium for the first 7 days before changing to F12 medium. ( a,d ) Confocal images of ICC markers, Kit (red), Ano1 (green), co-localization (yellow). Scale bar, 100 µm. ( b,e ) Frequency of cultured cells motility due to spontaneous contraction ( b : wk2 n = 2 , wk3 n = 4 ; ( e ) wk5 n = 4 ). ( c,f ) Cultured cells were analyzed for mRNA expression ( Kit, Myh11, Tubb3 ) at week 2 ( c ) or week 5 ( f ) ( n = 4 ). *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P

    Techniques Used: Magnetic Cell Separation, Cell Culture, In Vitro, Immunocytochemistry, Expressing

    54) Product Images from "UCP-3 uncoupling protein confers hypoxia resistance to renal epithelial cells and is upregulated in renal cell carcinoma"

    Article Title: UCP-3 uncoupling protein confers hypoxia resistance to renal epithelial cells and is upregulated in renal cell carcinoma

    Journal: Scientific Reports

    doi: 10.1038/srep13450

    H/R-adapted cultures exhibit after H/R stress less hyperpolarization of the inner mitochondrial membrane potential (ΔΨ m ) than control cultures. ( A,B ) Dot plots ( A ) and histograms ( B ) showing forward scatter and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) fluorescence as a measure of cell size and ΔΨ m , respectively. Depicted are a control (left) and a H/R-adapted PT culture (right) recorded by flow cytometry under normoxic conditions (black lines in ( B )) and after H/R stress (48 h hypoxia/24 h reoxygenation; ( A ) and red histograms in ( B )). Cell populations with dissipated ΔΨ m (low ΔΨ m ) are indicated by gate and marker in A and B, respectively. ( C,D ) Mean percentage of control (open bars) and H/R-adapted cells (closed bars) with broken-down ΔΨm ( C ) and ( D ) mean TMRE fluorescence intensity of the cell population with high ΔΨ m (±SE, n = 9 from 3 cultures each determined in triplicate) recorded as in ( B ) under normoxic conditions (left), after H/R stress (48 h hypoxia/24 h reoxygenation, middle), or after pharmacological break-down of ΔΨ m by the proton ionophore carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 1 μM). * and ** indicate p ≤ 0.05 and p ≤ 0.01, respectively (ANOVA).
    Figure Legend Snippet: H/R-adapted cultures exhibit after H/R stress less hyperpolarization of the inner mitochondrial membrane potential (ΔΨ m ) than control cultures. ( A,B ) Dot plots ( A ) and histograms ( B ) showing forward scatter and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) fluorescence as a measure of cell size and ΔΨ m , respectively. Depicted are a control (left) and a H/R-adapted PT culture (right) recorded by flow cytometry under normoxic conditions (black lines in ( B )) and after H/R stress (48 h hypoxia/24 h reoxygenation; ( A ) and red histograms in ( B )). Cell populations with dissipated ΔΨ m (low ΔΨ m ) are indicated by gate and marker in A and B, respectively. ( C,D ) Mean percentage of control (open bars) and H/R-adapted cells (closed bars) with broken-down ΔΨm ( C ) and ( D ) mean TMRE fluorescence intensity of the cell population with high ΔΨ m (±SE, n = 9 from 3 cultures each determined in triplicate) recorded as in ( B ) under normoxic conditions (left), after H/R stress (48 h hypoxia/24 h reoxygenation, middle), or after pharmacological break-down of ΔΨ m by the proton ionophore carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 1 μM). * and ** indicate p ≤ 0.05 and p ≤ 0.01, respectively (ANOVA).

    Techniques Used: Fluorescence, Flow Cytometry, Cytometry, Marker

    55) Product Images from "Green Tea component EGCG, Insulin and IGF-1 promote nuclear efflux of atrophy associated transcription factor Foxo1 in skeletal muscle fibers"

    Article Title: Green Tea component EGCG, Insulin and IGF-1 promote nuclear efflux of atrophy associated transcription factor Foxo1 in skeletal muscle fibers

    Journal: The Journal of nutritional biochemistry

    doi: 10.1016/j.jnutbio.2015.07.023

    IGF-1 Insulin dose response curve for inhibition of Foxo1-GFP nuclear localization A: Response of Foxo1-GFP N/C ratio following two hour treatment with various concentrations of IGF-1. Four chamber skeletal muscle fiber culture dish removed from incubator and replaced with serum free MEM with HEPES and 2mM L-Glutamine just before adding varying concentrations of IGF-1. B: Single binding sigmoidal curve fit for determination of EC50 dose response of IGF-1 (31pM) Foxo1 activation. C: Response of Foxo1-GFP N/C ratio following two hour treatment with various concentrations of insulin. Similar protocol as in figure A. D: Sigmoidal curve fit for determination of EC50 dose response of insulin (227pM) Foxo1 activation.
    Figure Legend Snippet: IGF-1 Insulin dose response curve for inhibition of Foxo1-GFP nuclear localization A: Response of Foxo1-GFP N/C ratio following two hour treatment with various concentrations of IGF-1. Four chamber skeletal muscle fiber culture dish removed from incubator and replaced with serum free MEM with HEPES and 2mM L-Glutamine just before adding varying concentrations of IGF-1. B: Single binding sigmoidal curve fit for determination of EC50 dose response of IGF-1 (31pM) Foxo1 activation. C: Response of Foxo1-GFP N/C ratio following two hour treatment with various concentrations of insulin. Similar protocol as in figure A. D: Sigmoidal curve fit for determination of EC50 dose response of insulin (227pM) Foxo1 activation.

    Techniques Used: Inhibition, Binding Assay, Activation Assay

    56) Product Images from "Unique Response Profile of Trabecular Meshwork Cells to the Novel Selective Glucocorticoid Receptor Agonist, GW870086X"

    Article Title: Unique Response Profile of Trabecular Meshwork Cells to the Novel Selective Glucocorticoid Receptor Agonist, GW870086X

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.12-11298

    Effect of glucocorticoids on hydraulic conductivity of human TM cell monolayers. Cells on permeable filters were treated for 5 days with DEX, GW870086X (GW87), or PRED. Cells on filters were then assayed for HC in Ussing-like chamber in HEPES-buffered DMEM (serum-free). Shown are mean results (±SEM) of five experiments using two different TM cell strains normalized to β-actin. Asterisks (*) indicate significant difference from untreated control where P
    Figure Legend Snippet: Effect of glucocorticoids on hydraulic conductivity of human TM cell monolayers. Cells on permeable filters were treated for 5 days with DEX, GW870086X (GW87), or PRED. Cells on filters were then assayed for HC in Ussing-like chamber in HEPES-buffered DMEM (serum-free). Shown are mean results (±SEM) of five experiments using two different TM cell strains normalized to β-actin. Asterisks (*) indicate significant difference from untreated control where P

    Techniques Used:

    57) Product Images from "Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme Disease tick vector Ixodes scapularis"

    Article Title: Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme Disease tick vector Ixodes scapularis

    Journal: Biochemical and biophysical research communications

    doi:

    Gelatinase activity of I. scapularis saliva. (A) Graph represents increase in fluorescence of fluoresceinated gelatin (0.1 mg/ml in Hepes buffer 25 mM NaCl 150 mM containing either 5 mM CaCl 2 or 5 mM EDTA) following addition of diluted tick saliva to give a total amount of 0.125 µl in a 50-µl reaction mixture. Similar results were obtained with four other pools of tick saliva. (B) Exponential loss of activity of tick gelatinase activity in the presence of 5 mM EDTA. Maximal fluorescence (MaxFluor) was estimated as the average of the last five fluorescence level determinations in the presence of EDTA. Each time point was plotted in the natural logarithmic scale as the ratio of the difference between MaxFluor minus the fluorescence at each time point (Fluor) divided by MaxFluor. Similar results were obtained with four other pools of saliva, each done at three different saliva dilutions.
    Figure Legend Snippet: Gelatinase activity of I. scapularis saliva. (A) Graph represents increase in fluorescence of fluoresceinated gelatin (0.1 mg/ml in Hepes buffer 25 mM NaCl 150 mM containing either 5 mM CaCl 2 or 5 mM EDTA) following addition of diluted tick saliva to give a total amount of 0.125 µl in a 50-µl reaction mixture. Similar results were obtained with four other pools of tick saliva. (B) Exponential loss of activity of tick gelatinase activity in the presence of 5 mM EDTA. Maximal fluorescence (MaxFluor) was estimated as the average of the last five fluorescence level determinations in the presence of EDTA. Each time point was plotted in the natural logarithmic scale as the ratio of the difference between MaxFluor minus the fluorescence at each time point (Fluor) divided by MaxFluor. Similar results were obtained with four other pools of saliva, each done at three different saliva dilutions.

    Techniques Used: Activity Assay, Fluorescence

    58) Product Images from "7SK-BAF axis controls pervasive transcription at enhancers"

    Article Title: 7SK-BAF axis controls pervasive transcription at enhancers

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.3176

    Two distinct 7SK RNA conformations in 7SK-BAF vs 7SK-Hexim complexes a , icSHAPE of Hexim1- or BAF-associated 7SK snRNA. The first 5 and last 15 nucleotides are not analyzed for structural reactivity and highly reactive bases are shown as blue. b , icSHAPE difference analysis between Hexim1- or BAF-associated 7SK from nucleotides 205 to 305. Positive values indicate more single stranded in Hexim1 and negative values are bases more reactive in BAF. c , Secondary structure prediction of the 7SK snRNA with nucleotides differentially reactive in Hexim1- or BAF-associated structures (above icSHAPE values of 0.5). Stem loops (SL) 1, 2, and 4 are cartooned as loops.
    Figure Legend Snippet: Two distinct 7SK RNA conformations in 7SK-BAF vs 7SK-Hexim complexes a , icSHAPE of Hexim1- or BAF-associated 7SK snRNA. The first 5 and last 15 nucleotides are not analyzed for structural reactivity and highly reactive bases are shown as blue. b , icSHAPE difference analysis between Hexim1- or BAF-associated 7SK from nucleotides 205 to 305. Positive values indicate more single stranded in Hexim1 and negative values are bases more reactive in BAF. c , Secondary structure prediction of the 7SK snRNA with nucleotides differentially reactive in Hexim1- or BAF-associated structures (above icSHAPE values of 0.5). Stem loops (SL) 1, 2, and 4 are cartooned as loops.

    Techniques Used:

    59) Product Images from "Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis"

    Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

    Journal: Scientific Reports

    doi: 10.1038/srep10132

    Role of the nuclear transcription factor RARB in altering the expression of genes involved in steroidogenesis. ( A ) H295R cells were transfected with the human −1.05 kb HSD3B2, −1.3 kb StAR and −3.7 kb CYP17A1, -2327 bp CYP11A1 promoter constructs along with an expression vector for the RARB transcription factor. Transfection medium was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA in serum-free medium for another 24 h. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Role of the nuclear transcription factor RARB in altering the expression of genes involved in steroidogenesis. ( A ) H295R cells were transfected with the human −1.05 kb HSD3B2, −1.3 kb StAR and −3.7 kb CYP17A1, -2327 bp CYP11A1 promoter constructs along with an expression vector for the RARB transcription factor. Transfection medium was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA in serum-free medium for another 24 h. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Transfection, Construct, Plasmid Preparation, Activation Assay, Luciferase

    RARB and Nur77 co-operation regulate HSD3B2 in H295R cells. H295R cells were transfected with the human −1.05 kb HSD3B2 promoter constructs along with an expression vector RARB, Nur77 and in combination of RARB/Nur77. Transfection medium was changed after 6 h and cells were cultivated in either serum free or in normal growth medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA for another 24 h. ( A ) RARB and Nur77 co-operation study under serum starvation condition. ( B ) RARB and Nur77 co-operation study under normal growth condition. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of four independent experiments. * p
    Figure Legend Snippet: RARB and Nur77 co-operation regulate HSD3B2 in H295R cells. H295R cells were transfected with the human −1.05 kb HSD3B2 promoter constructs along with an expression vector RARB, Nur77 and in combination of RARB/Nur77. Transfection medium was changed after 6 h and cells were cultivated in either serum free or in normal growth medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA for another 24 h. ( A ) RARB and Nur77 co-operation study under serum starvation condition. ( B ) RARB and Nur77 co-operation study under normal growth condition. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of four independent experiments. * p

    Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Activation Assay, Luciferase

    60) Product Images from "S100A9 protein is a novel ligand for the CD85j receptor and its interaction is implicated in the control of HIV-1 replication by NK cells"

    Article Title: S100A9 protein is a novel ligand for the CD85j receptor and its interaction is implicated in the control of HIV-1 replication by NK cells

    Journal: Retrovirology

    doi: 10.1186/1742-4690-10-122

    ELISA-based CD85j/S100A8 and CD85j/S100A9 binding assay. (A) Anti-GST mAbs well-coated microtiter plates were incubated with 4 μg/mL of recombinant GST-tagged human S100A8 or S100A9 proteins followed by incubation with increasing amounts of CD85j-Fc protein. (B) CD85j-Fc protein well-coated microtiter plates were incubated with increasing concentrations of recombinant GST-tagged human S100A8 or S100A9 proteins. (C) CD85j-Fc protein well-coated were incubated with 4 μg/mL of human S100A8 or S100A9 proteins in the presence of increasing amounts of soluble CD85j-Fc protein. (D) CD85j-Fc protein well-coated were incubated with 4 μg/mL of human S100A8 or S100A9 proteins in the presence of increasing concentrations of soluble NKG2D-Fc protein. Each point is the mean ± SE of 4 determinations.
    Figure Legend Snippet: ELISA-based CD85j/S100A8 and CD85j/S100A9 binding assay. (A) Anti-GST mAbs well-coated microtiter plates were incubated with 4 μg/mL of recombinant GST-tagged human S100A8 or S100A9 proteins followed by incubation with increasing amounts of CD85j-Fc protein. (B) CD85j-Fc protein well-coated microtiter plates were incubated with increasing concentrations of recombinant GST-tagged human S100A8 or S100A9 proteins. (C) CD85j-Fc protein well-coated were incubated with 4 μg/mL of human S100A8 or S100A9 proteins in the presence of increasing amounts of soluble CD85j-Fc protein. (D) CD85j-Fc protein well-coated were incubated with 4 μg/mL of human S100A8 or S100A9 proteins in the presence of increasing concentrations of soluble NKG2D-Fc protein. Each point is the mean ± SE of 4 determinations.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Recombinant

    Anti-HIV-1 activity of NK cell stimulated by S100A9 monomer or tetramer proteins. MDDC or CD4+ T cells were infected by HIV-1 and then cultured with unstimulated, S100A9 monomer- or S100A9 tetramer-stimulated NK cells (black, grey and white bars respectively) at the ratio E/T of 1:5. (A-B) HIV-1 replication was measured by intracellular p24 expression in MDDC and CD4+ T cells after 7 and 10 days of culture. (A) Graph represents cumulative results from 4 independent experiments showing the percentage of p24+ MDDC. (B) Graph represents cumulative results from 8 independent experiments showing the percentage of p24+ CD4+ T cells. (C) Graph represents cumulative results from 6 independent experiments showing the amount of p24 HIV-1 antigen in the supernatant of HIV-1-infected CD4+ T cells in culture with unstimulated, S100A9 monomer- or S100A9 tetramer-stimulated NK cells (black, grey and white bars respectively). For blocking experiments, NK cells were stimulated with an equimolar combination of CD85j-Fc + S100A9 tetramer. Results are expressed as mean ± SE and p values are shown.
    Figure Legend Snippet: Anti-HIV-1 activity of NK cell stimulated by S100A9 monomer or tetramer proteins. MDDC or CD4+ T cells were infected by HIV-1 and then cultured with unstimulated, S100A9 monomer- or S100A9 tetramer-stimulated NK cells (black, grey and white bars respectively) at the ratio E/T of 1:5. (A-B) HIV-1 replication was measured by intracellular p24 expression in MDDC and CD4+ T cells after 7 and 10 days of culture. (A) Graph represents cumulative results from 4 independent experiments showing the percentage of p24+ MDDC. (B) Graph represents cumulative results from 8 independent experiments showing the percentage of p24+ CD4+ T cells. (C) Graph represents cumulative results from 6 independent experiments showing the amount of p24 HIV-1 antigen in the supernatant of HIV-1-infected CD4+ T cells in culture with unstimulated, S100A9 monomer- or S100A9 tetramer-stimulated NK cells (black, grey and white bars respectively). For blocking experiments, NK cells were stimulated with an equimolar combination of CD85j-Fc + S100A9 tetramer. Results are expressed as mean ± SE and p values are shown.

    Techniques Used: Activity Assay, Infection, Cell Culture, Expressing, Blocking Assay

    Identification of CD85j ligand(s) expressed by HIV-1-infected MDDC. (A) MDDC were lysed at day 2 post HIV-1 infection. After blocking CD85j receptors and HLA class-I ligands as described in Materials and Methods, CD85j-Fc fusion protein was used to capture and purify proteins from uninfected or HIV-1-infected MDDC lysates onto the Protein chip array. Eluted proteins from the column were separated by electrophoresis in non-reducing conditions and revealed by silver staining. Lane 1, Seeblue® Plus2 Pre-Stained Standard, lanes 2–7, subsequent eluted fractions of HIV-1-infected MDDC lysates after pre-incubation with the CD85j-Fc-coupled gel from two representative donors (lanes 2–4, donor 1; lanes 5–7, donor 2). The proteins were identified as belonging to S100A8/A9 complex (S100A8, S100A9 and S100A8/A9) by protein sequencing. (B) Eluted fractions of HIV-1-infected MDDC lysates were analyzed by immunoblotting using anti-S100A9 (lane 1), anti-S100A8 (lane 2) and anti-CD85j (lane 3) mAbs. (C) CD85j-Fc fusion protein was used to capture and purify proteins from uninfected or HIV-1-infected MDDC lysates, where HLA-I molecules were blocked, onto the Protein chip Array.
    Figure Legend Snippet: Identification of CD85j ligand(s) expressed by HIV-1-infected MDDC. (A) MDDC were lysed at day 2 post HIV-1 infection. After blocking CD85j receptors and HLA class-I ligands as described in Materials and Methods, CD85j-Fc fusion protein was used to capture and purify proteins from uninfected or HIV-1-infected MDDC lysates onto the Protein chip array. Eluted proteins from the column were separated by electrophoresis in non-reducing conditions and revealed by silver staining. Lane 1, Seeblue® Plus2 Pre-Stained Standard, lanes 2–7, subsequent eluted fractions of HIV-1-infected MDDC lysates after pre-incubation with the CD85j-Fc-coupled gel from two representative donors (lanes 2–4, donor 1; lanes 5–7, donor 2). The proteins were identified as belonging to S100A8/A9 complex (S100A8, S100A9 and S100A8/A9) by protein sequencing. (B) Eluted fractions of HIV-1-infected MDDC lysates were analyzed by immunoblotting using anti-S100A9 (lane 1), anti-S100A8 (lane 2) and anti-CD85j (lane 3) mAbs. (C) CD85j-Fc fusion protein was used to capture and purify proteins from uninfected or HIV-1-infected MDDC lysates, where HLA-I molecules were blocked, onto the Protein chip Array.

    Techniques Used: Infection, Blocking Assay, Chromatin Immunoprecipitation, Electrophoresis, Silver Staining, Staining, Incubation, Sequencing

    Cytokine production and CD107a expression on NK cell after stimulation by S100A9 monomer and tetramer proteins. (A) Intracellular expression of TNF-α and IFN-γ and surface expression of CD107a on NK cells unstimulated, stimulated by S100A9 monomers or tetramers for 4 hours at 37°C (black, grey and white bars respectively). Graph shows cumulative results from 7 independent experiments. (B) NK cells were stimulated with S100A9 tetrater or with an equimolar combination of CD85j-Fc + S100A9 tetramer for 4 h at 37°C. For the later stimulation, CD85j-Fc and S100A9 tetramer were co-incubated for 2 hours at 37°C prior stimulation of NK cells. Graph represents the percentage of increase in TNF-α expression following stimulation compared to the unstimulated NK cells. Graph shows cumulative results from 4 independent experiments. (C) Expression of intracellular TNF-α and IFN-γ and surface CD107a by NK cells unstimulated, pre-stimulated by S100A9 monomer or tetramers (black, grey and white bars respectively) following a secondary stimulation by K562 target cells for 4 h at 37°C. For the blocking experiment regarding intracellular TNF-α expression, NK cells were stimulated with an equimolar combination of CD85j-Fc + S100A9 tetramer. Graph shows cumulative results from 8 independent experiments. Results are expressed as mean ± SE and p values are indicated.
    Figure Legend Snippet: Cytokine production and CD107a expression on NK cell after stimulation by S100A9 monomer and tetramer proteins. (A) Intracellular expression of TNF-α and IFN-γ and surface expression of CD107a on NK cells unstimulated, stimulated by S100A9 monomers or tetramers for 4 hours at 37°C (black, grey and white bars respectively). Graph shows cumulative results from 7 independent experiments. (B) NK cells were stimulated with S100A9 tetrater or with an equimolar combination of CD85j-Fc + S100A9 tetramer for 4 h at 37°C. For the later stimulation, CD85j-Fc and S100A9 tetramer were co-incubated for 2 hours at 37°C prior stimulation of NK cells. Graph represents the percentage of increase in TNF-α expression following stimulation compared to the unstimulated NK cells. Graph shows cumulative results from 4 independent experiments. (C) Expression of intracellular TNF-α and IFN-γ and surface CD107a by NK cells unstimulated, pre-stimulated by S100A9 monomer or tetramers (black, grey and white bars respectively) following a secondary stimulation by K562 target cells for 4 h at 37°C. For the blocking experiment regarding intracellular TNF-α expression, NK cells were stimulated with an equimolar combination of CD85j-Fc + S100A9 tetramer. Graph shows cumulative results from 8 independent experiments. Results are expressed as mean ± SE and p values are indicated.

    Techniques Used: Expressing, Incubation, Blocking Assay

    61) Product Images from "Activity of Trifluoperazine against Replicating, Non-Replicating and Drug Resistant M. tuberculosis"

    Article Title: Activity of Trifluoperazine against Replicating, Non-Replicating and Drug Resistant M. tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044245

    Viability of macrophages in presence of trifluoperazine. ( a )THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FCS at 37°C, 5% CO 2 for a period of 5 days. Dose dependent response of TFP on THP-1 cells viability was determined by culturing THP-1 cells in different concentration of TFP (0, 10 and 20 µg/ml). Viability of cells was determined by trypan blue exclusion method at days 1, 2, 3 and 5. ( b ) Monocyte derived macrophages were treated with different concentrations of TFP (0, 10, 20, 30 and 40 µg/ml). Cell viability was checked by MTT assays after 24, 48 and 72 hours of treatment. Values represent means ± standard errors of the means of duplicates.
    Figure Legend Snippet: Viability of macrophages in presence of trifluoperazine. ( a )THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FCS at 37°C, 5% CO 2 for a period of 5 days. Dose dependent response of TFP on THP-1 cells viability was determined by culturing THP-1 cells in different concentration of TFP (0, 10 and 20 µg/ml). Viability of cells was determined by trypan blue exclusion method at days 1, 2, 3 and 5. ( b ) Monocyte derived macrophages were treated with different concentrations of TFP (0, 10, 20, 30 and 40 µg/ml). Cell viability was checked by MTT assays after 24, 48 and 72 hours of treatment. Values represent means ± standard errors of the means of duplicates.

    Techniques Used: Cell Culture, Concentration Assay, Derivative Assay, MTT Assay

    62) Product Images from "Shiga toxin-induced apoptosis is more efficiently inhibited by dimeric recombinant hybrid-IgG/IgA immunoglobulins than by the parental IgG monoclonal antibodies"

    Article Title: Shiga toxin-induced apoptosis is more efficiently inhibited by dimeric recombinant hybrid-IgG/IgA immunoglobulins than by the parental IgG monoclonal antibodies

    Journal: Virulence

    doi: 10.4161/21505594.2014.973804

    Toxin neutralization by the dimeric hybrid IgG/IgA. Stx1 (5 pg/ml) and varying concentrations (abscissa) of the dimeric hybrid IgG/IgA were incubated for 1 h at 37°C. Each mixture was added to Vero cells (2 × 10 4 ), followed by
    Figure Legend Snippet: Toxin neutralization by the dimeric hybrid IgG/IgA. Stx1 (5 pg/ml) and varying concentrations (abscissa) of the dimeric hybrid IgG/IgA were incubated for 1 h at 37°C. Each mixture was added to Vero cells (2 × 10 4 ), followed by

    Techniques Used: Neutralization, Incubation

    Separation of dimeric and monomeric forms of a recombinant hybrid-IgG/IgA specific for Stx1B. A serum-free culture supernatant (60 ml) of CHO-K1 cells triple transfected with vector constructs for H, L and J chains of the hybrid-IgG/IgA was concentrated
    Figure Legend Snippet: Separation of dimeric and monomeric forms of a recombinant hybrid-IgG/IgA specific for Stx1B. A serum-free culture supernatant (60 ml) of CHO-K1 cells triple transfected with vector constructs for H, L and J chains of the hybrid-IgG/IgA was concentrated

    Techniques Used: Recombinant, Transfection, Plasmid Preparation, Construct

    Inhibiton of Stx1-induced caspase-3 activation in Vero cells by the hybrid IgG/IgA. Stx1 (5 pg/ml) was incubated with varying concentrations of the dimeric hybrid IgG/IgA or IgG mAb (D11C6) for 1 h at 37°C. Each mixture was added to Vero
    Figure Legend Snippet: Inhibiton of Stx1-induced caspase-3 activation in Vero cells by the hybrid IgG/IgA. Stx1 (5 pg/ml) was incubated with varying concentrations of the dimeric hybrid IgG/IgA or IgG mAb (D11C6) for 1 h at 37°C. Each mixture was added to Vero

    Techniques Used: Activation Assay, Incubation

    Inhibition of Stx1-induced phosphatidylserine exposure on Ramos cells by the hybrid IgG/IgA. Ramos cells (1 × 10 6 /ml) were incubated with Stx1 (5 pg/ml) in the presence of 10 ng/ml of IgA mAb, IgG1 mAb or the dimeric hybrid IgG/IgA for
    Figure Legend Snippet: Inhibition of Stx1-induced phosphatidylserine exposure on Ramos cells by the hybrid IgG/IgA. Ramos cells (1 × 10 6 /ml) were incubated with Stx1 (5 pg/ml) in the presence of 10 ng/ml of IgA mAb, IgG1 mAb or the dimeric hybrid IgG/IgA for

    Techniques Used: Inhibition, Incubation

    63) Product Images from "The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation"

    Article Title: The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation

    Journal: Redox Biology

    doi: 10.1016/j.redox.2013.12.023

    Collagen-dependent thrombus formation at arterial shear requires both Nox1 and Nox2. Hirudin-anticoagulated whole blood from wildtype (Wt) or Nox2 knockout (KO) mice was diluted 1:2 with modified HEPES-Tyrode's buffer, then pre-incubated with vehicle control (0.5% DMSO), Nox1 inhibitor (5 µM ML171) or aspirin (500 µM). Samples were perfused over a collagen-coated microfluidic channel at a constant shear of 1500 s −1 for 6 min, followed by washing in modified HEPES-Tyrode's buffer for 5 min with subsequent fixation in 3.7% formaldehyde. (A) Representative fluorescent, end point images of platelet thrombi from Wt (upper panel) and Nox2 KO (lower panel) mice following vehicle control/inhibitor treatment. (B) Platelet surface coverage (left panel) and thrombus volume (right panel) are expressed as mean±SEM, n =5 ( n =3 for aspirin), ⁎ P
    Figure Legend Snippet: Collagen-dependent thrombus formation at arterial shear requires both Nox1 and Nox2. Hirudin-anticoagulated whole blood from wildtype (Wt) or Nox2 knockout (KO) mice was diluted 1:2 with modified HEPES-Tyrode's buffer, then pre-incubated with vehicle control (0.5% DMSO), Nox1 inhibitor (5 µM ML171) or aspirin (500 µM). Samples were perfused over a collagen-coated microfluidic channel at a constant shear of 1500 s −1 for 6 min, followed by washing in modified HEPES-Tyrode's buffer for 5 min with subsequent fixation in 3.7% formaldehyde. (A) Representative fluorescent, end point images of platelet thrombi from Wt (upper panel) and Nox2 KO (lower panel) mice following vehicle control/inhibitor treatment. (B) Platelet surface coverage (left panel) and thrombus volume (right panel) are expressed as mean±SEM, n =5 ( n =3 for aspirin), ⁎ P

    Techniques Used: Knock-Out, Mouse Assay, Modification, Incubation

    64) Product Images from "Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures"

    Article Title: Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures

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

    doi: 10.1073/pnas.1834448100

    Stimulation of astrocytic Ca 2 + waves causes a decrease in the frequency of neuronal Ca 2 + oscillations. ( a ) Image of a field of hippocampal cultures that were fixed and stained with anti-MAP2 (green) and anti-GFAP (red) antibodies. (Scale bar, 50 μm.) ( b ) The graph shows individual traces of ratiometric fura 2 fluorescence in a neuron (cell 1) and astrocyte (cell 2) shown in a before fixation and staining. ATP (1 μM) was bath applied as indicated by the bar. ( c Left ) Ratiometric image of fura 2 fluorescence superimposed on a phase-contrast image of a field of hippocampal cultured cells. ( c Right ) Astrocytes (cells 1–3in Left ) are schematically shown as red and neurons (cells 4–6) are shown as blue. Astrocyte 1 was mechanically stimulated as indicated by the arrowhead. (Scale bar, 50 μm.) ( d ) The graph shows individual traces of ratiometric fura 2 fluorescence as a function of time in the astrocytes (traces 1–3) and neurons (4–6) indicated in c . Astrocyte 1 was mechanically stimulated twice, separated by 10 min (the arrowheads, MS1 and MS2). ( e ) The histogram shows the mean ± SEM frequency of Ca 2 + oscillations in neurons ( n = 73 neurons in four experiments) measured every 60 s before and after an astrocyte was mechanically stimulated (arrowheads). The Ca 2 + oscillation frequency was normalized to the prestimulation level. ( f ) Ratiometric and schematic images of a field of hippocampal cultured cells in which astrocytes are shown in red and neurons are shown in blue. (Scale bar, 50 μm.) ( g ) Individual traces of ratiometric fura 2 fluorescence in the astrocytes (1–3) and neurons (4–7) shown in f . Astrocyte 1 was mechanically stimulated (arrowheads) under normal conditions and 10 min later was mechanically stimulated in the presence of apyrase (20 units/ml; hatched bar). ( h ) The histogram shows the mean ± SEM frequency of Ca 2 + oscillations in neurons before stimulation (Pre; n = 48 neurons in five experiments), after the application of exogenous ATP (1 μM; n = 36 neurons in five experiments), mechanical stimulation of astrocytes (MS; n = 51 neurons in six experiments), and mechanical stimulation of astrocytes in the presence of apyrase (MS/apyrase; n = 38 neurons in five experiments). Ca 2 + oscillation frequency was normalized to the prestimulation level (Pre). Asterisks show significant difference from the response of Pre (**, P
    Figure Legend Snippet: Stimulation of astrocytic Ca 2 + waves causes a decrease in the frequency of neuronal Ca 2 + oscillations. ( a ) Image of a field of hippocampal cultures that were fixed and stained with anti-MAP2 (green) and anti-GFAP (red) antibodies. (Scale bar, 50 μm.) ( b ) The graph shows individual traces of ratiometric fura 2 fluorescence in a neuron (cell 1) and astrocyte (cell 2) shown in a before fixation and staining. ATP (1 μM) was bath applied as indicated by the bar. ( c Left ) Ratiometric image of fura 2 fluorescence superimposed on a phase-contrast image of a field of hippocampal cultured cells. ( c Right ) Astrocytes (cells 1–3in Left ) are schematically shown as red and neurons (cells 4–6) are shown as blue. Astrocyte 1 was mechanically stimulated as indicated by the arrowhead. (Scale bar, 50 μm.) ( d ) The graph shows individual traces of ratiometric fura 2 fluorescence as a function of time in the astrocytes (traces 1–3) and neurons (4–6) indicated in c . Astrocyte 1 was mechanically stimulated twice, separated by 10 min (the arrowheads, MS1 and MS2). ( e ) The histogram shows the mean ± SEM frequency of Ca 2 + oscillations in neurons ( n = 73 neurons in four experiments) measured every 60 s before and after an astrocyte was mechanically stimulated (arrowheads). The Ca 2 + oscillation frequency was normalized to the prestimulation level. ( f ) Ratiometric and schematic images of a field of hippocampal cultured cells in which astrocytes are shown in red and neurons are shown in blue. (Scale bar, 50 μm.) ( g ) Individual traces of ratiometric fura 2 fluorescence in the astrocytes (1–3) and neurons (4–7) shown in f . Astrocyte 1 was mechanically stimulated (arrowheads) under normal conditions and 10 min later was mechanically stimulated in the presence of apyrase (20 units/ml; hatched bar). ( h ) The histogram shows the mean ± SEM frequency of Ca 2 + oscillations in neurons before stimulation (Pre; n = 48 neurons in five experiments), after the application of exogenous ATP (1 μM; n = 36 neurons in five experiments), mechanical stimulation of astrocytes (MS; n = 51 neurons in six experiments), and mechanical stimulation of astrocytes in the presence of apyrase (MS/apyrase; n = 38 neurons in five experiments). Ca 2 + oscillation frequency was normalized to the prestimulation level (Pre). Asterisks show significant difference from the response of Pre (**, P

    Techniques Used: Staining, Fluorescence, Cell Culture, Mass Spectrometry

    65) Product Images from "Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts"

    Article Title: Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.507525

    Wortmannin and GS-9820 induce osteoclast retraction. Rat osteoclasts were bathed in HEPES-buffered M199 medium with 15% FBS and antibiotics and imaged using time-lapse phase-contrast microscopy. A–E illustrates 5 different osteoclasts, each shown
    Figure Legend Snippet: Wortmannin and GS-9820 induce osteoclast retraction. Rat osteoclasts were bathed in HEPES-buffered M199 medium with 15% FBS and antibiotics and imaged using time-lapse phase-contrast microscopy. A–E illustrates 5 different osteoclasts, each shown

    Techniques Used: Microscopy

    66) Product Images from "The DEXD/H-box RNA Helicase DDX19 Is Regulated by an ?-Helical Switch *D/H-box RNA Helicase DDX19 Is Regulated by an ?-Helical Switch * S⃞"

    Article Title: The DEXD/H-box RNA Helicase DDX19 Is Regulated by an ?-Helical Switch *D/H-box RNA Helicase DDX19 Is Regulated by an ?-Helical Switch * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.C900018200

    Structure of human DDX19. A , overview of DDX19 with ADP bound and the N-terminal flanking helix in the central cleft. The Arg 429 side chain that acts as an arginine finger is presented as sticks. B , schematic representation of the cleft-inserted helix with the two conserved domains of the protein, shown in the same view as in panel A . Residues that are conserved in DDX25 are shown in blue. C , overview of the DDX19-RNA complex, with Mg-ADPNP bound in the central cleft. The Arg 429 side chain is presented as sticks. D , detail of the RNA binding site of the DDX19-RNA complex. E , detail of the nucleotide binding site in the open conformation, with the electron density (2 F obs – F calc ) for ADP rendered at 1.5 σ. F , detail of the nucleotide binding site in the RNA complex, with the electron density (2 F obs – F calc ) for Mg-ADPNP rendered at 1.5 σ. In all panels, the conserved domain-1 ( yellow ), the conserved domain-2 ( red ), and the N-terminal flanking sequence ( green ) are indicated.
    Figure Legend Snippet: Structure of human DDX19. A , overview of DDX19 with ADP bound and the N-terminal flanking helix in the central cleft. The Arg 429 side chain that acts as an arginine finger is presented as sticks. B , schematic representation of the cleft-inserted helix with the two conserved domains of the protein, shown in the same view as in panel A . Residues that are conserved in DDX25 are shown in blue. C , overview of the DDX19-RNA complex, with Mg-ADPNP bound in the central cleft. The Arg 429 side chain is presented as sticks. D , detail of the RNA binding site of the DDX19-RNA complex. E , detail of the nucleotide binding site in the open conformation, with the electron density (2 F obs – F calc ) for ADP rendered at 1.5 σ. F , detail of the nucleotide binding site in the RNA complex, with the electron density (2 F obs – F calc ) for Mg-ADPNP rendered at 1.5 σ. In all panels, the conserved domain-1 ( yellow ), the conserved domain-2 ( red ), and the N-terminal flanking sequence ( green ) are indicated.

    Techniques Used: RNA Binding Assay, Binding Assay, Sequencing

    Role of the N-terminal flanking sequence in the regulation of DDX19 ATPase activity. A , schematic diagram of the DDX19 protein constructs used in this study (not drawn to scale). N-term represents the N terminus. B , relative ATPase activities of DDX19 protein constructs in the presence of between 0 and 0.5 mg/ml ssRNA.
    Figure Legend Snippet: Role of the N-terminal flanking sequence in the regulation of DDX19 ATPase activity. A , schematic diagram of the DDX19 protein constructs used in this study (not drawn to scale). N-term represents the N terminus. B , relative ATPase activities of DDX19 protein constructs in the presence of between 0 and 0.5 mg/ml ssRNA.

    Techniques Used: Sequencing, Activity Assay, Construct

    67) Product Images from "Synthesis and Evaluation of 1-(1-(Benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase"

    Article Title: Synthesis and Evaluation of 1-(1-(Benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase

    Journal: Chemmedchem

    doi: 10.1002/cmdc.200900098

    a) The structure of trypanothione (T[S] 2 ), the substrate of TryR. b) The principle of the DTNB-coupled assay for TryR.
    Figure Legend Snippet: a) The structure of trypanothione (T[S] 2 ), the substrate of TryR. b) The principle of the DTNB-coupled assay for TryR.

    Techniques Used:

    68) Product Images from "Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b"

    Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2014.00079

    The OX42-immunogene forms large aggregates and triggers an immune response in microglia. (A) Comparison of the size-distribution profiles between complete cell culture medium (DMEM + 10% FBS) and the OX42-immunoporter obtained by DLS shows that the non-viral vehicle did not form aggregates. After adding pDNA to form the OX42-immunogene (N/P = 4), aggregates formed over a large range (≈50–1300 nm). (B) Adding FBS to polyplexes after maturation in HBS and maturation of polyplexes in FBS-containing medium caused a shift in size-distribution to lower aggregate sizes, but it had no effect on the width of size-distributions. Values are plotted as Mean ± SEM (two experiments). FBS, fetal bovine serum; OX42-IP, OX42-immunoporter; HBS, HEPES-buffered saline; D (+/–), DMEM with/without 10% FBS. (C) ROS production in microglial cells was measured by quantifying ROS-indicator fluorescence after 60 min and reported as percentage (%) of the internal standard PDBu. The aggregated OX42-immunogene triggered the respiratory burst (38.8 ± 3.3%) which was inhibited by the respiratory burst inhibitor diphenyliodonium (–7.02 ± 4.49%). The positive control zymosan caused the release of ROS at similar levels (45.8 ± 4.7%) to the OX42-immunogene. However, PEI–PEG (–0.97 ± 3.74%), OX42 antibody (2.57 ± 1.68%) and the OX42-immunoporter (9.83 ± 2.84%) did not trigger significant ROS production. Values are plotted as Mean ± SEM. ** P
    Figure Legend Snippet: The OX42-immunogene forms large aggregates and triggers an immune response in microglia. (A) Comparison of the size-distribution profiles between complete cell culture medium (DMEM + 10% FBS) and the OX42-immunoporter obtained by DLS shows that the non-viral vehicle did not form aggregates. After adding pDNA to form the OX42-immunogene (N/P = 4), aggregates formed over a large range (≈50–1300 nm). (B) Adding FBS to polyplexes after maturation in HBS and maturation of polyplexes in FBS-containing medium caused a shift in size-distribution to lower aggregate sizes, but it had no effect on the width of size-distributions. Values are plotted as Mean ± SEM (two experiments). FBS, fetal bovine serum; OX42-IP, OX42-immunoporter; HBS, HEPES-buffered saline; D (+/–), DMEM with/without 10% FBS. (C) ROS production in microglial cells was measured by quantifying ROS-indicator fluorescence after 60 min and reported as percentage (%) of the internal standard PDBu. The aggregated OX42-immunogene triggered the respiratory burst (38.8 ± 3.3%) which was inhibited by the respiratory burst inhibitor diphenyliodonium (–7.02 ± 4.49%). The positive control zymosan caused the release of ROS at similar levels (45.8 ± 4.7%) to the OX42-immunogene. However, PEI–PEG (–0.97 ± 3.74%), OX42 antibody (2.57 ± 1.68%) and the OX42-immunoporter (9.83 ± 2.84%) did not trigger significant ROS production. Values are plotted as Mean ± SEM. ** P

    Techniques Used: Cell Culture, Fluorescence, Positive Control

    69) Product Images from "Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5? Capped 2?-O Unmethylated RNA"

    Article Title: Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5? Capped 2?-O Unmethylated RNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.00883-13

    Ifit1 selectively inhibits the translation of mRNA lacking 2′-O methylation. (A) The luciferase RNA amounts at 6 h after RNA transfection were determined by quantitative real-time RT-PCR. The relative luciferase mRNA amounts, calculated as the
    Figure Legend Snippet: Ifit1 selectively inhibits the translation of mRNA lacking 2′-O methylation. (A) The luciferase RNA amounts at 6 h after RNA transfection were determined by quantitative real-time RT-PCR. The relative luciferase mRNA amounts, calculated as the

    Techniques Used: Methylation, Luciferase, Transfection, Quantitative RT-PCR

    Ifit1 preferentially binds to virus RNA lacking 2′-O methylation. (A) Electrophoretic mobility shift of biotin-labeled RNA (JEV 5′-terminal 200 nucleotides) with recombinant Ifit1. The presence or absence of a 5′ cap and 2′-O
    Figure Legend Snippet: Ifit1 preferentially binds to virus RNA lacking 2′-O methylation. (A) Electrophoretic mobility shift of biotin-labeled RNA (JEV 5′-terminal 200 nucleotides) with recombinant Ifit1. The presence or absence of a 5′ cap and 2′-O

    Techniques Used: Methylation, Electrophoretic Mobility Shift Assay, Labeling, Recombinant

    70) Product Images from "The SMAC mimetic LCL-161 displays antitumor activity in preclinical models of rituximab-resistant B-cell lymphoma"

    Article Title: The SMAC mimetic LCL-161 displays antitumor activity in preclinical models of rituximab-resistant B-cell lymphoma

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2018018168

    LCL-161 has a synergistic antitumor effect with chemotherapy agents in RRCLs and sensitive parent cell lines. (A) The RRCL Raji 4RH and its sensitive parent cell line Raji were incubated with gemcitabine in combination with LCL-161 for 48 hours at the doses indicated. Viability measurements were performed with the CellTiter-Glo assay. LCL-161 synergistically enhanced the antitumor activity of 50-μM gemcitabine in the Raji 4RH cell line, but it was less effective in Raji cells. (B) A CI synergism calculation, performed with CalcuSyn software, indicated limited synergy in the Raji cell line; however, robust synergy (CI
    Figure Legend Snippet: LCL-161 has a synergistic antitumor effect with chemotherapy agents in RRCLs and sensitive parent cell lines. (A) The RRCL Raji 4RH and its sensitive parent cell line Raji were incubated with gemcitabine in combination with LCL-161 for 48 hours at the doses indicated. Viability measurements were performed with the CellTiter-Glo assay. LCL-161 synergistically enhanced the antitumor activity of 50-μM gemcitabine in the Raji 4RH cell line, but it was less effective in Raji cells. (B) A CI synergism calculation, performed with CalcuSyn software, indicated limited synergy in the Raji cell line; however, robust synergy (CI

    Techniques Used: Incubation, Glo Assay, Activity Assay, Software

    71) Product Images from "Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva"

    Article Title: Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

    Journal: Memórias do Instituto Oswaldo Cruz

    doi: 10.1590/0074-0276108062013002

    procoagulant activity of Leishmania amazonensis metacyclic promastigotes. A: analysis of phosphatidylserine exposure on metacyclic promastigotes. Parasites were analysed by flow cytometry in a dot plot of Alexa 488 conjugated-annexin V vs. propidium iodide (PI) double staining. Quadrant regions were defined from a negative control with no labelling. Data are representative of two independent experiments; B: recalcification time of human plasma was measured in the absence (CT) or in the presence of different numbers of metacyclic promastigotes. Results from 10 independent experiments are expressed as mean ± standard error of the means (SEM). Asterisks mean p
    Figure Legend Snippet: procoagulant activity of Leishmania amazonensis metacyclic promastigotes. A: analysis of phosphatidylserine exposure on metacyclic promastigotes. Parasites were analysed by flow cytometry in a dot plot of Alexa 488 conjugated-annexin V vs. propidium iodide (PI) double staining. Quadrant regions were defined from a negative control with no labelling. Data are representative of two independent experiments; B: recalcification time of human plasma was measured in the absence (CT) or in the presence of different numbers of metacyclic promastigotes. Results from 10 independent experiments are expressed as mean ± standard error of the means (SEM). Asterisks mean p

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Double Staining, Negative Control

    72) Product Images from "Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones"

    Article Title: Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones

    Journal: Scientific Reports

    doi: 10.1038/srep41105

    Electrophysiological properties and monovalent cation permeability of CpomOR channels. (a) CpomOrco + OR1 expressing HEK cells respond to the unspecific agonist VUAA3 200 μM generating inward currents in dose dependent manner (a1). Experimental conditions: whole-cell voltage clamp recording; holding potential: −50 mV; intracellular solution (mM): NaCl 140, EGTA 0.5, Hepes 10, pH 7.4; extracellular solution (mM): 140 NaCl, 2.0 CaCl 2 , 10 HEPES, pH 7.5. Stimulus intensity was changed by changing stimulus pulse duration. Basal current level was subtracted. VUAA3, 200 μM, applied repeatedly to the extracellular surface of membrane patch in outside-out configuration reversibly increased membrane current noise that can be associated with the activity of ion channels (a2). Experimental conditions: outside-out patch recording; holding potential +50 mV; intracellular solution (mM): KCl 140, EGTA 1, Hepes 10, pH 7.4; extracellular solution (mM): 140 NaCl, 2.0 CaCl 2 , 10 HEPES, pH 7.5; stimulus: VUAA3, 200 μM. Diagrams in a1 and a2 depict a time course of stimulus presentation. Note: the VUAA3 activated OR channels demonstrate little if any rundown. (b) To estimate the selectivity of the OR channels to monovalent cations we used whole-cell recordings. VUAA3 (200 μM) was added to all extracellular test solutions. Cells were first exposed to NaCl 140 mM solution. Then, the same whole-cell preparation was exposed to a solution in which the Na + ions were replaced by one of the following cations: Li + , K + , Cs + , Rb + . A series of ramps (50–100, black lines) were used for every solution tested and averaged (color lines). A potential at which current voltage characteristic of VUAA3 activated integral current intercepts current voltage characteristic of basal current was used as a Vr of the OR channel current in a given ion conditions (vertical colour lines). To determine the reversal potential shift (ΔVr), the Vr of the currents obtained in symmetrical Na + conditions (VrNa + ) was subtracted from the Vr obtained, then Na + was replaced by respective cations (VrX). The ΔVrX means were then used to estimate the permeability ratios (PX + /PNa + ). The permeability ratio sequence for some inorganic monovalent cations (PX + /PNa + ) was: Rb + (2 ± 0.12) > K + (1.37 ± 0.03) ≥ Cs + (1.36 ± 0.03) ~ Na + > Li + (0.93 ± 0.06). Experimental conditions: whole-cell voltage clamp recording; holding potential: −60 mV; intracellular solution (mM): NaCl 140, EGTA 0.5, Hepes 10, pH 7.4; extracellular solution: varied in accordance with the above description.
    Figure Legend Snippet: Electrophysiological properties and monovalent cation permeability of CpomOR channels. (a) CpomOrco + OR1 expressing HEK cells respond to the unspecific agonist VUAA3 200 μM generating inward currents in dose dependent manner (a1). Experimental conditions: whole-cell voltage clamp recording; holding potential: −50 mV; intracellular solution (mM): NaCl 140, EGTA 0.5, Hepes 10, pH 7.4; extracellular solution (mM): 140 NaCl, 2.0 CaCl 2 , 10 HEPES, pH 7.5. Stimulus intensity was changed by changing stimulus pulse duration. Basal current level was subtracted. VUAA3, 200 μM, applied repeatedly to the extracellular surface of membrane patch in outside-out configuration reversibly increased membrane current noise that can be associated with the activity of ion channels (a2). Experimental conditions: outside-out patch recording; holding potential +50 mV; intracellular solution (mM): KCl 140, EGTA 1, Hepes 10, pH 7.4; extracellular solution (mM): 140 NaCl, 2.0 CaCl 2 , 10 HEPES, pH 7.5; stimulus: VUAA3, 200 μM. Diagrams in a1 and a2 depict a time course of stimulus presentation. Note: the VUAA3 activated OR channels demonstrate little if any rundown. (b) To estimate the selectivity of the OR channels to monovalent cations we used whole-cell recordings. VUAA3 (200 μM) was added to all extracellular test solutions. Cells were first exposed to NaCl 140 mM solution. Then, the same whole-cell preparation was exposed to a solution in which the Na + ions were replaced by one of the following cations: Li + , K + , Cs + , Rb + . A series of ramps (50–100, black lines) were used for every solution tested and averaged (color lines). A potential at which current voltage characteristic of VUAA3 activated integral current intercepts current voltage characteristic of basal current was used as a Vr of the OR channel current in a given ion conditions (vertical colour lines). To determine the reversal potential shift (ΔVr), the Vr of the currents obtained in symmetrical Na + conditions (VrNa + ) was subtracted from the Vr obtained, then Na + was replaced by respective cations (VrX). The ΔVrX means were then used to estimate the permeability ratios (PX + /PNa + ). The permeability ratio sequence for some inorganic monovalent cations (PX + /PNa + ) was: Rb + (2 ± 0.12) > K + (1.37 ± 0.03) ≥ Cs + (1.36 ± 0.03) ~ Na + > Li + (0.93 ± 0.06). Experimental conditions: whole-cell voltage clamp recording; holding potential: −60 mV; intracellular solution (mM): NaCl 140, EGTA 0.5, Hepes 10, pH 7.4; extracellular solution: varied in accordance with the above description.

    Techniques Used: Permeability, Expressing, Activity Assay, Sequencing

    73) Product Images from "Insights into the Acquisition of Virulence of Avian Influenza Viruses during a Single Passage in Ferrets"

    Article Title: Insights into the Acquisition of Virulence of Avian Influenza Viruses during a Single Passage in Ferrets

    Journal: Viruses

    doi: 10.3390/v11100915

    The effect of PB2 627K and the PB2 489P, NP 408I combination on viral polymerase activity in mammalian cells. Different combinations of ( A ) the pHWLaos and pHWViet PB2, PB1, PA and NP genome segment plasmids (designated as LPB2, VPB2, LPB1, VPB1, LPA, VPA, LNP and VNP, respectively); as well as ( B ) each of the plasmids encoding the single AA changes, pHWLaos PB2 489P (LPB2 489P), pHWLaos PB2 627K (LPB2 627K), pHWLaos NP 408I (LNP 408I) and pHWViet PB2 627E (VPB2 627E), were co-transfected into 293T cells along with plasmids pPOL-NP-LUC and pRL-TK. Following incubation at 37 °C for 24 h in a humidified incubator the relative luciferase activity of each polymerase complex was assessed using the Dual-Luciferase Reporter Assay System (Promega). Values (mean of three replicates ± standard error) are shown relative to the polymerase activity of the luciferase control (pRL-TK + pPOL-NP-LUC only) which represents one unit. Luciferase activity of each polymerase complex genetic constellation was compared to the luciferase activity of the native A/Laos polymerase constellation (LPB2, LPB1, LPA and LNP) using a one-way analysis of variance test. * P ≤ 0.05.
    Figure Legend Snippet: The effect of PB2 627K and the PB2 489P, NP 408I combination on viral polymerase activity in mammalian cells. Different combinations of ( A ) the pHWLaos and pHWViet PB2, PB1, PA and NP genome segment plasmids (designated as LPB2, VPB2, LPB1, VPB1, LPA, VPA, LNP and VNP, respectively); as well as ( B ) each of the plasmids encoding the single AA changes, pHWLaos PB2 489P (LPB2 489P), pHWLaos PB2 627K (LPB2 627K), pHWLaos NP 408I (LNP 408I) and pHWViet PB2 627E (VPB2 627E), were co-transfected into 293T cells along with plasmids pPOL-NP-LUC and pRL-TK. Following incubation at 37 °C for 24 h in a humidified incubator the relative luciferase activity of each polymerase complex was assessed using the Dual-Luciferase Reporter Assay System (Promega). Values (mean of three replicates ± standard error) are shown relative to the polymerase activity of the luciferase control (pRL-TK + pPOL-NP-LUC only) which represents one unit. Luciferase activity of each polymerase complex genetic constellation was compared to the luciferase activity of the native A/Laos polymerase constellation (LPB2, LPB1, LPA and LNP) using a one-way analysis of variance test. * P ≤ 0.05.

    Techniques Used: Activity Assay, Transfection, Incubation, Luciferase, Reporter Assay

    74) Product Images from "Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis"

    Article Title: Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00211-17

    Visualization of BtubA/B polymerization and filament bundling at intermediate KCl concentration. (A) Time course of BtubA/B polymerization (3 μM BtubA/B, 30% TAMRA labeled) from GMPCPP-stabilized seeds (brighter labeling) in 50 mM HEPES (pH 7), 5 mM MgCl 2 , 100 mM KCl, and 1 mM GTP. (Right) Filament length versus time for eight individual filaments. (B) Two filaments growing in opposite directions (“1” and “2,” where + denotes the growing end of the filament) interact end to end, forming a bundle (B2). Two single BtubA/B filaments (“3+” and “4+”) and a bundle (B1) interact longitudinally to form a thicker bundle (B3). At 480 s and 580 s, bundles B2 and B3 zipper together (B4 and B5). (C) Dynamics of BtubA/B bundles formed by parallel and antiparallel filament annealing. A bundle (B1) formed by the interaction of two antiparallel growing BtubA/B filaments (“1+” and “2+”) interacts with a bundle (B2) formed by two parallel growing BtubA/B filaments (“3+” and “4+”), forming bundle B3. (D) Filament treadmilling. The rate of polymerization of the BtubA/B filament growing end (“+”) is similar to the depolymerization rate at the other end (“−”). Images were preprocessed as described in Materials and Methods. Scale bars, 10 μm (A) and 5 μm (B to D).
    Figure Legend Snippet: Visualization of BtubA/B polymerization and filament bundling at intermediate KCl concentration. (A) Time course of BtubA/B polymerization (3 μM BtubA/B, 30% TAMRA labeled) from GMPCPP-stabilized seeds (brighter labeling) in 50 mM HEPES (pH 7), 5 mM MgCl 2 , 100 mM KCl, and 1 mM GTP. (Right) Filament length versus time for eight individual filaments. (B) Two filaments growing in opposite directions (“1” and “2,” where + denotes the growing end of the filament) interact end to end, forming a bundle (B2). Two single BtubA/B filaments (“3+” and “4+”) and a bundle (B1) interact longitudinally to form a thicker bundle (B3). At 480 s and 580 s, bundles B2 and B3 zipper together (B4 and B5). (C) Dynamics of BtubA/B bundles formed by parallel and antiparallel filament annealing. A bundle (B1) formed by the interaction of two antiparallel growing BtubA/B filaments (“1+” and “2+”) interacts with a bundle (B2) formed by two parallel growing BtubA/B filaments (“3+” and “4+”), forming bundle B3. (D) Filament treadmilling. The rate of polymerization of the BtubA/B filament growing end (“+”) is similar to the depolymerization rate at the other end (“−”). Images were preprocessed as described in Materials and Methods. Scale bars, 10 μm (A) and 5 μm (B to D).

    Techniques Used: Concentration Assay, Labeling

    75) Product Images from "Inhibition of Mycobacterium tuberculosis Transaminase BioA by Aryl Hydrazines and Hydrazides"

    Article Title: Inhibition of Mycobacterium tuberculosis Transaminase BioA by Aryl Hydrazines and Hydrazides

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201300748

    Left: UV-Vis spectroscopy of 0.16 mM PLP bound BioA (Holo BioA) upon mixing with 0.4 mM Compound 2 at different time points. Right: Time-lapsed photos of a PLP-bound BioA crystal soaked in Compound 2 (15% PEG 8000, 100 mM HEPES pH 7.5, 100 mM MgCl 2 , and
    Figure Legend Snippet: Left: UV-Vis spectroscopy of 0.16 mM PLP bound BioA (Holo BioA) upon mixing with 0.4 mM Compound 2 at different time points. Right: Time-lapsed photos of a PLP-bound BioA crystal soaked in Compound 2 (15% PEG 8000, 100 mM HEPES pH 7.5, 100 mM MgCl 2 , and

    Techniques Used: UV-Vis Spectroscopy, Plasmid Purification

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    Article Snippet: .. To assess the externalization of phosphatidylserine in the plasma membrane as marker for early stage of apoptosis, ECs adherent to 25-mm-diameter glass coverslips were incubated with the conjugate Annexin V–Alexa Fluor 488 (Invitrogen, 1 μg/ml) and propidium iodide (PI; Invitrogen; 0.5 μg/ml) for 15 minutes in Annexin V binding buffer (Invitrogen). .. Annexin V– and PI-stained cells were visualized by a Zeiss LSM510 confocal imaging system using a ×40 oil objective and later quantified.

    Article Title: 1,4-dihydroxy-2-naphthoic Acid Induces Apoptosis in Human Keratinocyte: Potential Application for Psoriasis Treatment
    Article Snippet: .. Annexin V/Propidium Iodide Staining After DHNA treatment, HaCaT cells were resuspended in annexin V binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 , pH 7.4) and then incubated with FITC-conjugated annexin V (Biosource International, Inc., USA), and 0.5 μ g/mL propidium iodide (PI) for 15 min at room temperature in the dark. .. Cells were analyzed on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) immediately and then analyzed using WinMDI 2.9 (developed by Dr. J. Trotter, Scripps Institute, La Jolla, CA, USA).

    Article Title: Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y human neuroblastoma and T98G human glioblastoma cells
    Article Snippet: .. The cells were incubated at room temperature for 15 min. After that, 400 µl of 1 × annexin-binding buffer was added and the cells were analysed immediately using Attune™ Nxt Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Alexa Fluor® 488 annexin V and PI fluorescence emission was measured using the 530/30 nm (BL1) and 695/40 nm (BL3) emission filters, respectively, with excitation at 488 nm.

    Article Title: Microglial-derived microparticles mediate neuroinflammation after traumatic brain injury
    Article Snippet: .. Microglial staining was performed using anti-P2Y12 (1:1000, AnaSpec Inc., Fremont, CA) and anti-CD45-PerCP (1:10, Miltenyi Biotec, Auburn, CA) as follows: MP were incubated with anti-P2Y12 in annexin buffer for 1 h at RT, washed and incubated with Alexa Fluor 488 goat anti-rabbit secondary antibodies (1:500; Life Technologies) for 30 min, washed and further incubated with pre-conjugated anti-CD45 for 30 min at RT, and washed in annexin buffer prior to analysis by flow cytometry. .. FlowJo software (Vx; Tree Star, Inc., Ashland, OR) was used for analysis, and data are presented as percent of annexin V-positive MP in the MP gate as set by microbeads, unless otherwise specified.

    Infection:

    Article Title: Endogenous Human Papillomavirus E6 and E7 Proteins Differentially Regulate Proliferation, Senescence, and Apoptosis in HeLa Cervical Carcinoma Cells
    Article Snippet: .. Six days after infection, the cells were harvested by trypsinization, resuspended in fresh medium, washed once with cold phosphate-buffered saline, and resuspended in annexin binding buffer (Molecular Probes, Eugene, Oreg.). ..

    Apoptosis Assay:

    Article Title: Phosphatidylserine Is Not the Cell Surface Receptor for Vesicular Stomatitis Virus
    Article Snippet: .. Alexa Fluor 488-conjugated annexin V, propidium iodide (PI), and annexin binding buffer were obtained from the Vybrant Apoptosis Assay Kit #2 (Molecular Probes, Eugene, Oreg.). .. Annexin V staining was performed by using a slight variation of the manufacturer's protocol.

    Article Title: Endogenous Human Papillomavirus E6 and E7 Proteins Differentially Regulate Proliferation, Senescence, and Apoptosis in HeLa Cervical Carcinoma Cells
    Article Snippet: Six days after infection, the cells were harvested by trypsinization, resuspended in fresh medium, washed once with cold phosphate-buffered saline, and resuspended in annexin binding buffer (Molecular Probes, Eugene, Oreg.). .. For annexin V binding, the samples were then processed and analyzed by using a Vybrant apoptosis assay no. 2 kit (Molecular Probes) according to the manufacturer's instructions for flow cytometry.

    Expressing:

    Article Title: Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control
    Article Snippet: Cells and MPs were characterized for the expression of following antigens: CD19 and 20 for B-cells, CD3, 8, 5, and 27 for T-cells, CD16 and 56 for NK-cells, CD14 and 11c for monocytes, CD41 and 61 for platelets. .. The samples were previously diluted with 20 μl of PBS and 50 μl specific buffer for annexin-V binding (Invitrogen).

    Article Title: Microglial-derived microparticles mediate neuroinflammation after traumatic brain injury
    Article Snippet: The expression of phosphatidylserine (PS) on MP was detected using an anti-annexin V (FITC or APC) (1:50, BD Biosciences PharMingen, catalog no: 556419) in annexin buffer (5 mM KCl, 1 mM MgCl2 , 136 mM NaCl, 2 mM CaCl2 , 1% BSA; pH 7.4). .. Microglial staining was performed using anti-P2Y12 (1:1000, AnaSpec Inc., Fremont, CA) and anti-CD45-PerCP (1:10, Miltenyi Biotec, Auburn, CA) as follows: MP were incubated with anti-P2Y12 in annexin buffer for 1 h at RT, washed and incubated with Alexa Fluor 488 goat anti-rabbit secondary antibodies (1:500; Life Technologies) for 30 min, washed and further incubated with pre-conjugated anti-CD45 for 30 min at RT, and washed in annexin buffer prior to analysis by flow cytometry.

    Staining:

    Article Title: Short and Long Term, In Vitro and In Vivo Correlates of Cellular and Tissue Response to Mesoporous Silicon Nanovectors**
    Article Snippet: Paragraph title: Annexin V staining ... An annexin-binding buffer was prepared by combining HEPES (10 mM , Invitrogen), NaCl (140 mM, Sigma Aldrich), CaCl2 (2.5 mM, Sigma Aldrich), and adjusted to pH 7.4.

    Article Title: Phosphatidylserine Is Not the Cell Surface Receptor for Vesicular Stomatitis Virus
    Article Snippet: Paragraph title: Annexin V staining. ... Alexa Fluor 488-conjugated annexin V, propidium iodide (PI), and annexin binding buffer were obtained from the Vybrant Apoptosis Assay Kit #2 (Molecular Probes, Eugene, Oreg.).

    Article Title: Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation
    Article Snippet: Paragraph title: Annexin V binding and PI staining. ... To assess the externalization of phosphatidylserine in the plasma membrane as marker for early stage of apoptosis, ECs adherent to 25-mm-diameter glass coverslips were incubated with the conjugate Annexin V–Alexa Fluor 488 (Invitrogen, 1 μg/ml) and propidium iodide (PI; Invitrogen; 0.5 μg/ml) for 15 minutes in Annexin V binding buffer (Invitrogen).

    Article Title: Cymbopogon citratus and Camellia sinensis extracts selectively induce apoptosis in cancer cells and reduce growth of lymphoma xenografts in vivo
    Article Snippet: Analysis of cell death: annexin V binding assay and propidium iodide (PI) Annexin V binding assay and propidium iodide staining were performed to respectively monitor early apoptosis and cell permeabilization, a marker of necrotic or late apoptotic cell death. .. Cells were washed with phosphate buffer saline (PBS) and suspended in Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) with green fluorescent Annexin V AlexaFluor-488 (1:20) (Life Technologies Inc, Cat. No. A13201, Burlington, ON, Canada) and 0.01 mg/mL of red fluorescent PI (Life Technologies Inc, Cat. No. P3566, Burlington, ON, Canada) for 15 minutes at 37°C protected from light.

    Article Title: 1,4-dihydroxy-2-naphthoic Acid Induces Apoptosis in Human Keratinocyte: Potential Application for Psoriasis Treatment
    Article Snippet: .. Annexin V/Propidium Iodide Staining After DHNA treatment, HaCaT cells were resuspended in annexin V binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 , pH 7.4) and then incubated with FITC-conjugated annexin V (Biosource International, Inc., USA), and 0.5 μ g/mL propidium iodide (PI) for 15 min at room temperature in the dark. .. Cells were analyzed on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) immediately and then analyzed using WinMDI 2.9 (developed by Dr. J. Trotter, Scripps Institute, La Jolla, CA, USA).

    Article Title: Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y human neuroblastoma and T98G human glioblastoma cells
    Article Snippet: The cells were stained with annexin V and PI using Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Cat. No.: V13241; Thermo Fisher Scientific, Waltham, MA, USA). .. The cells were incubated at room temperature for 15 min. After that, 400 µl of 1 × annexin-binding buffer was added and the cells were analysed immediately using Attune™ Nxt Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control
    Article Snippet: A total of 10 μl of isolated MPs were stained for 15 minutes at room temperature in the dark with 10 μl of annexin-V-FITC and 10 μl of specific antibody. .. The samples were previously diluted with 20 μl of PBS and 50 μl specific buffer for annexin-V binding (Invitrogen).

    Article Title: Anergy Induction by Dimeric TCR Ligands
    Article Snippet: .. T cells were washed twice in RPMI, resuspended in 100 μ l annexin V buffer (10 mM HEPES, 140 mM l Alexa 594-labeled NaCl, 2.5 mM CaCl2 , pH 7.4), and stained with 5 μ annexin V (Molecular Probes) for 20 min at room temperature. .. Cells were then diluted by addition of 400 μ l of annexin V buffer and analyzed in an EPICS XL FACS (Coulter, Miami, FL).

    Article Title: Direct activation of PP2A for the treatment of tyrosine kinase inhibitor–resistant lung adenocarcinoma
    Article Snippet: .. Annexin V staining was performed using annexin V conjugate Alexa Fluor 488 from Invitrogen (Life Technologies) and annexin-binding buffer (catalog V13246) from Invitrogen (Life Technologies), according to the manufacturer’s protocol. .. For cell cycle analysis, cells were stained with 7-Aminoactinomycin D (Roche) to ascertain the DNA content and determine cell cycle distribution within the cell population ( ) Each experiment was performed in triplicate.

    Article Title: Microglial-derived microparticles mediate neuroinflammation after traumatic brain injury
    Article Snippet: .. Microglial staining was performed using anti-P2Y12 (1:1000, AnaSpec Inc., Fremont, CA) and anti-CD45-PerCP (1:10, Miltenyi Biotec, Auburn, CA) as follows: MP were incubated with anti-P2Y12 in annexin buffer for 1 h at RT, washed and incubated with Alexa Fluor 488 goat anti-rabbit secondary antibodies (1:500; Life Technologies) for 30 min, washed and further incubated with pre-conjugated anti-CD45 for 30 min at RT, and washed in annexin buffer prior to analysis by flow cytometry. .. FlowJo software (Vx; Tree Star, Inc., Ashland, OR) was used for analysis, and data are presented as percent of annexin V-positive MP in the MP gate as set by microbeads, unless otherwise specified.

    Article Title: Epigenetic Determinants of Erythropoiesis: Role of the Histone Methyltransferase SetD8 in Promoting Erythroid Cell Maturation and Survival
    Article Snippet: Cells were resuspended in 100 μl annexin binding buffer and labeled with 5 μl annexin V-Alexa Fluor 350 conjugate (Life Technologies) and propidium iodide (PI). .. As an additional method of analyzing apoptosis, fetal liver cells were stained for active caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP).

    Marker:

    Article Title: Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation
    Article Snippet: .. To assess the externalization of phosphatidylserine in the plasma membrane as marker for early stage of apoptosis, ECs adherent to 25-mm-diameter glass coverslips were incubated with the conjugate Annexin V–Alexa Fluor 488 (Invitrogen, 1 μg/ml) and propidium iodide (PI; Invitrogen; 0.5 μg/ml) for 15 minutes in Annexin V binding buffer (Invitrogen). .. Annexin V– and PI-stained cells were visualized by a Zeiss LSM510 confocal imaging system using a ×40 oil objective and later quantified.

    Article Title: Cymbopogon citratus and Camellia sinensis extracts selectively induce apoptosis in cancer cells and reduce growth of lymphoma xenografts in vivo
    Article Snippet: Analysis of cell death: annexin V binding assay and propidium iodide (PI) Annexin V binding assay and propidium iodide staining were performed to respectively monitor early apoptosis and cell permeabilization, a marker of necrotic or late apoptotic cell death. .. Cells were washed with phosphate buffer saline (PBS) and suspended in Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) with green fluorescent Annexin V AlexaFluor-488 (1:20) (Life Technologies Inc, Cat. No. A13201, Burlington, ON, Canada) and 0.01 mg/mL of red fluorescent PI (Life Technologies Inc, Cat. No. P3566, Burlington, ON, Canada) for 15 minutes at 37°C protected from light.

    Article Title: Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control
    Article Snippet: Annexin-V-FITC was used as a general marker for MPs [ ; ]. .. The samples were previously diluted with 20 μl of PBS and 50 μl specific buffer for annexin-V binding (Invitrogen).

    FACS:

    Article Title: Regulation of hypoxia responses by flavin adenine dinucleotide‐dependent modulation of HIF‐1α protein stability
    Article Snippet: Paragraph title: Fluorescent‐activating cell sorting (FACS) analysis ... Cells harvested with trypinase were washed with ice‐cold PBS containing 2% FBS twice, followed by re‐suspension in 1× Annexin‐binding buffer (Molecular Probes).

    Article Title: Anergy Induction by Dimeric TCR Ligands
    Article Snippet: T cells were washed twice in RPMI, resuspended in 100 μ l annexin V buffer (10 mM HEPES, 140 mM l Alexa 594-labeled NaCl, 2.5 mM CaCl2 , pH 7.4), and stained with 5 μ annexin V (Molecular Probes) for 20 min at room temperature. .. Cells were then diluted by addition of 400 μ l of annexin V buffer and analyzed in an EPICS XL FACS (Coulter, Miami, FL).

    Binding Assay:

    Article Title: Phosphatidylserine Is Not the Cell Surface Receptor for Vesicular Stomatitis Virus
    Article Snippet: .. Alexa Fluor 488-conjugated annexin V, propidium iodide (PI), and annexin binding buffer were obtained from the Vybrant Apoptosis Assay Kit #2 (Molecular Probes, Eugene, Oreg.). .. Annexin V staining was performed by using a slight variation of the manufacturer's protocol.

    Article Title: Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation
    Article Snippet: .. To assess the externalization of phosphatidylserine in the plasma membrane as marker for early stage of apoptosis, ECs adherent to 25-mm-diameter glass coverslips were incubated with the conjugate Annexin V–Alexa Fluor 488 (Invitrogen, 1 μg/ml) and propidium iodide (PI; Invitrogen; 0.5 μg/ml) for 15 minutes in Annexin V binding buffer (Invitrogen). .. Annexin V– and PI-stained cells were visualized by a Zeiss LSM510 confocal imaging system using a ×40 oil objective and later quantified.

    Article Title: Cymbopogon citratus and Camellia sinensis extracts selectively induce apoptosis in cancer cells and reduce growth of lymphoma xenografts in vivo
    Article Snippet: .. Cells were washed with phosphate buffer saline (PBS) and suspended in Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) with green fluorescent Annexin V AlexaFluor-488 (1:20) (Life Technologies Inc, Cat. No. A13201, Burlington, ON, Canada) and 0.01 mg/mL of red fluorescent PI (Life Technologies Inc, Cat. No. P3566, Burlington, ON, Canada) for 15 minutes at 37°C protected from light. .. Percentage of early (green), late apoptotic cells (green and red), and necrotic cells (red) were quantified with a Tali Image-Based Cytometer (Life Technologies Inc., Cat. No. T10796, Burlington, ON, Canada).

    Article Title: Dengue Virus Ensures Its Fusion in Late Endosomes Using Compartment-Specific Lipids
    Article Snippet: The cells were pre-incubated with annexin-binding buffer for 30 min at room temperature and fluorescent annexin was applied in the same buffer at concentration recommended by Invitrogen. .. BS-C-1 cells were incubated with DiD-labeled DEN at 10°C for 30 min to allow binding but not internalization of the virions.

    Article Title: 1,4-dihydroxy-2-naphthoic Acid Induces Apoptosis in Human Keratinocyte: Potential Application for Psoriasis Treatment
    Article Snippet: .. Annexin V/Propidium Iodide Staining After DHNA treatment, HaCaT cells were resuspended in annexin V binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 , pH 7.4) and then incubated with FITC-conjugated annexin V (Biosource International, Inc., USA), and 0.5 μ g/mL propidium iodide (PI) for 15 min at room temperature in the dark. .. Cells were analyzed on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) immediately and then analyzed using WinMDI 2.9 (developed by Dr. J. Trotter, Scripps Institute, La Jolla, CA, USA).

    Article Title: Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control
    Article Snippet: .. The samples were previously diluted with 20 μl of PBS and 50 μl specific buffer for annexin-V binding (Invitrogen). ..

    Article Title: Imiquimod Induces Apoptosis in Human Endometrial Cancer Cells In vitro and Prevents Tumor Progression In vivo
    Article Snippet: .. Annexin-V conjugated to allophycocyanin (APC), 7-Aminoactinomycin D (7-AAD), and Annexin Binding Buffer were purchased from Life Technologies (NY, USA). .. Immunoblotting detection was conducted using the following antibodies: Anti-beta actin (Sigma, A5541), Anti Caspase-3 (Cell Signaling, 9662S), Anti-PARP (Cell Signaling, 9542T), Anti-Bcl-2 (Cell Signaling, 2876S), Anti –Bcl-xL (Abcam, GR192701-3) and Anti-BAX (Abcam, GR151406-1).

    Article Title: Endogenous Human Papillomavirus E6 and E7 Proteins Differentially Regulate Proliferation, Senescence, and Apoptosis in HeLa Cervical Carcinoma Cells
    Article Snippet: .. Six days after infection, the cells were harvested by trypsinization, resuspended in fresh medium, washed once with cold phosphate-buffered saline, and resuspended in annexin binding buffer (Molecular Probes, Eugene, Oreg.). ..

    Article Title: Epigenetic Determinants of Erythropoiesis: Role of the Histone Methyltransferase SetD8 in Promoting Erythroid Cell Maturation and Survival
    Article Snippet: .. Cells were resuspended in 100 μl annexin binding buffer and labeled with 5 μl annexin V-Alexa Fluor 350 conjugate (Life Technologies) and propidium iodide (PI). .. Samples were analyzed using an LSRFortessa or LSRII cytometer (BD Biosciences).

    Derivative Assay:

    Article Title: Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control
    Article Snippet: The samples were previously diluted with 20 μl of PBS and 50 μl specific buffer for annexin-V binding (Invitrogen). .. The CD3 titration on T-cells and T-cell derived MPs and CD41 on platelet derived MPs are shown on and Figs. Events were acquired during 2 minutes of medium flow.

    Imaging:

    Article Title: Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation
    Article Snippet: To assess the externalization of phosphatidylserine in the plasma membrane as marker for early stage of apoptosis, ECs adherent to 25-mm-diameter glass coverslips were incubated with the conjugate Annexin V–Alexa Fluor 488 (Invitrogen, 1 μg/ml) and propidium iodide (PI; Invitrogen; 0.5 μg/ml) for 15 minutes in Annexin V binding buffer (Invitrogen). .. Annexin V– and PI-stained cells were visualized by a Zeiss LSM510 confocal imaging system using a ×40 oil objective and later quantified.

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  • 95
    Thermo Fisher rna processing buffer
    Dependence of <t>RNA</t> targeting on crRNA variants, temperature and point mutations a, <t>LbuC2c2</t> ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.
    Rna Processing Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hepes buffer
    In vitro activity of <t>mitoDPP-3.</t> a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in <t>HEPES</t> (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1
    Hepes Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher hsp90 binding buffer
    Schematic representation of the <t>Hsp90</t> occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.
    Hsp90 Binding Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher csf xb buffer
    Characterization of pSer335 <t>XErp1</t> antibody <t>CSF</t> extract was treated with Myc‐XErp1 IVT carrying the indicated combinations of the mutations DSG − (S33N S38N), DSA − (S284N S288N), ZBR − (C583A) and CaMKII − (T195A). Calcium was added, samples were taken at the indicated time points and as indicated treated with λ‐phosphatase. Samples were immunoblotted for the Myc‐tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α‐tubulin. Asterisk indicates unspecific bands. Several lanes were removed at the dashed line. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT at the indicated dilutions. An empty IVT not expressing XErp1 and an untreated condition were used as controls. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, pSer335 XErp1, and α‐tubulin. The XErp1 membrane was stripped and reprobed for the Myc‐tag. Asterisks indicate unspecific bands. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT that was either wild‐type or mutated to alanine at Ser335. An empty IVT reaction not expressing Myc‐XErp1 served as control. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, the Myc‐tag, pSer335 XErp1, and α‐tubulin. Asterisks indicate unspecific bands. Source data are available online for this figure.
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    Image Search Results


    Dependence of RNA targeting on crRNA variants, temperature and point mutations a, LbuC2c2 ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: Dependence of RNA targeting on crRNA variants, temperature and point mutations a, LbuC2c2 ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: Cleavage Assay, Activity Assay

    Binding data for LbuC2c2 to mature crRNA and target ssRNA a, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GG to LbuC2c2-WT, LbuC2c2-dHEPN1, LbuC2c2-dHEPN2, or LbuC2c2-dHEPN1/dHEPN2. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 27.1 ± 7.5 nM (LbuC2c2-WT), 15.2 ± 3.2 nM (LbuC2c2-dHEPN1), 11.5 ± 2.5 nM (LbuC2c2-dHEPN2), and 43.3 ± 11.5 nM (LbuC2c2- dHEPN1/dHEPN2). b, Representative electrophoretic mobility shift assay for binding reactions between LbuC2c2-dHEPN1/dHEPN2: crRNA-A_GG and either ‘on-target’ A ssRNA or ‘off-target’ B ssRNA, as indicated. Three independent experiments were conducted as described in the Methods. The gel was cropped for clarity. c, Quantified binding data from (b) were fitted to standard binding isoforms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 1.62 ± 0.43 nM for ssRNA A and N.D (≫10 nM) for ssRNA B. d, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GA to LbuC2c2-WT and LbuC2c2-R1079A. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 4.65 ± 0.6 nM (LbuC2c2-WT) and 2.52 ± 0.5 nM (LbuC2c2-R1079A). It is of note that these binding affinities differ from panel a. This difference is accounted for in a slight difference in the 5′ sequence of the guide with panel a guides beginning with a 5′-G G CCA… and panel d 5′-G A CCA. While the native sequence guide (5′-G A CCA) binds tighter to LbuC2c2, no difference is seen in the RNA targeting efficiencies of these guide variants (). Extended Data Fig. 6c

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: Binding data for LbuC2c2 to mature crRNA and target ssRNA a, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GG to LbuC2c2-WT, LbuC2c2-dHEPN1, LbuC2c2-dHEPN2, or LbuC2c2-dHEPN1/dHEPN2. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 27.1 ± 7.5 nM (LbuC2c2-WT), 15.2 ± 3.2 nM (LbuC2c2-dHEPN1), 11.5 ± 2.5 nM (LbuC2c2-dHEPN2), and 43.3 ± 11.5 nM (LbuC2c2- dHEPN1/dHEPN2). b, Representative electrophoretic mobility shift assay for binding reactions between LbuC2c2-dHEPN1/dHEPN2: crRNA-A_GG and either ‘on-target’ A ssRNA or ‘off-target’ B ssRNA, as indicated. Three independent experiments were conducted as described in the Methods. The gel was cropped for clarity. c, Quantified binding data from (b) were fitted to standard binding isoforms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 1.62 ± 0.43 nM for ssRNA A and N.D (≫10 nM) for ssRNA B. d, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GA to LbuC2c2-WT and LbuC2c2-R1079A. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 4.65 ± 0.6 nM (LbuC2c2-WT) and 2.52 ± 0.5 nM (LbuC2c2-R1079A). It is of note that these binding affinities differ from panel a. This difference is accounted for in a slight difference in the 5′ sequence of the guide with panel a guides beginning with a 5′-G G CCA… and panel d 5′-G A CCA. While the native sequence guide (5′-G A CCA) binds tighter to LbuC2c2, no difference is seen in the RNA targeting efficiencies of these guide variants (). Extended Data Fig. 6c

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: Binding Assay, Standard Deviation, Electrophoretic Mobility Shift Assay, Sequencing

    C2c2 provides sensitive detection of transcripts in complex mixtures a , Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b , Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c ,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β -actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β -actin null negative control). (mean ± s.d., n = 3) d , Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e , Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: C2c2 provides sensitive detection of transcripts in complex mixtures a , Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b , Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c ,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β -actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β -actin null negative control). (mean ± s.d., n = 3) d , Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e , Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: RNA Detection, Fluorescence, Generated, Negative Control, CRISPR

    In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

    Journal: Nature Communications

    Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

    doi: 10.1038/s41467-017-02655-1

    Figure Lengend Snippet: In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

    Article Snippet: In total 150 µL of 2 µM mitoDPP-2 in HEPES buffer (20 mM, 150 mM NaCl, pH = 8.0) were added to a 96-well optical bottom plate (Nunc 265301, Thermo Scientific) at room temperature.

    Techniques: In Vitro, Activity Assay, Fluorescence, Purification

    Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

    Journal: Nature Communications

    Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

    doi: 10.1038/s41467-017-02655-1

    Figure Lengend Snippet: Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

    Article Snippet: In total 150 µL of 2 µM mitoDPP-2 in HEPES buffer (20 mM, 150 mM NaCl, pH = 8.0) were added to a 96-well optical bottom plate (Nunc 265301, Thermo Scientific) at room temperature.

    Techniques: In Vitro, Activity Assay, Fluorescence, Purification

    Schematic representation of the Hsp90 occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Schematic representation of the Hsp90 occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Filtration, Binding Assay

    Hsp90 open binding sites can be titrated with [ 3 H]17-AAG. Recombinant human Hsp90β protein (100 n m ) was incubated with increasing concentrations of unlabeled 17-AAG overnight at 4 °C followed by removal of unbound 17-AAG with prechilled size exclusion columns. Free Hsp90 sites were titrated with [ 3 H]17-AAG at 4 °C as described under “Experimental Procedures.” The data from triplicate binding experiments were fit to a four parameter logistic equation.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Hsp90 open binding sites can be titrated with [ 3 H]17-AAG. Recombinant human Hsp90β protein (100 n m ) was incubated with increasing concentrations of unlabeled 17-AAG overnight at 4 °C followed by removal of unbound 17-AAG with prechilled size exclusion columns. Free Hsp90 sites were titrated with [ 3 H]17-AAG at 4 °C as described under “Experimental Procedures.” The data from triplicate binding experiments were fit to a four parameter logistic equation.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Binding Assay, Recombinant, Incubation

    Determination of Hsp90 occupancy in living cells. H1650 cells were incubated with increasing concentrations of IPI-504 for 6 h at 37 °C. Total Hsp90 protein levels were determined by quantitative immunoblotting using separate anti Hsp90α and Hsp90β antibodies and recombinant proteins as internal standards ( A ). Percent Hsp90 occupancy was determined by titration of open binding sites at 4 °C and total Hsp90 ( B and C ). Data are from a representative experiment with n = 2.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Determination of Hsp90 occupancy in living cells. H1650 cells were incubated with increasing concentrations of IPI-504 for 6 h at 37 °C. Total Hsp90 protein levels were determined by quantitative immunoblotting using separate anti Hsp90α and Hsp90β antibodies and recombinant proteins as internal standards ( A ). Percent Hsp90 occupancy was determined by titration of open binding sites at 4 °C and total Hsp90 ( B and C ). Data are from a representative experiment with n = 2.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Incubation, Recombinant, Titration, Binding Assay

    Dissociation of [ 3 H]17-AAG from purified Hsp90 and SK-BR-3 lysates is highly temperature-dependent. Purified Hela Hsp90 (100 n m ) or lysate Hsp90·[ 3 H]17-AAG complexes were formed as described under “Experimental Procedures” and passed over size exclusion spin columns. Column eluates were incubated with 10 μ m cold 17-AAG, and samples were removed at different time points. A loss of bound radioactive 17-AAG counts from Hsp90 was measured at both 4 and 37 °C ( A ) and Hsp90 in SK-BR-3 cancer cell lysate at 4 °C ( B ). The data were fit to a monoexponential decay equation.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Dissociation of [ 3 H]17-AAG from purified Hsp90 and SK-BR-3 lysates is highly temperature-dependent. Purified Hela Hsp90 (100 n m ) or lysate Hsp90·[ 3 H]17-AAG complexes were formed as described under “Experimental Procedures” and passed over size exclusion spin columns. Column eluates were incubated with 10 μ m cold 17-AAG, and samples were removed at different time points. A loss of bound radioactive 17-AAG counts from Hsp90 was measured at both 4 and 37 °C ( A ) and Hsp90 in SK-BR-3 cancer cell lysate at 4 °C ( B ). The data were fit to a monoexponential decay equation.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Purification, Incubation

    Hsp90 occupancy is a better predictor of in vivo pharmacodynamic effects by IPI-504 than tumor or plasma PK. H1650 tumor-bearing mice were treated with a single dose of 100 mg/kg intravenous IPI-504. Tumors and blood plasma were harvested at designated time points post dose. Drug levels of Hsp90 active species (IPI-504, 17-AAG, and 17-AG) were quantified by LC-MS/MS in plasma and in tumor ( A ). EGFR protein levels ( B ) and Hsp90 occupancy ( C ) were measured in tumor tissue. Data are expressed as averages ± S.D. (vehicle ( veh ), n = 2; and 1–48 h, n = 3).

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Hsp90 occupancy is a better predictor of in vivo pharmacodynamic effects by IPI-504 than tumor or plasma PK. H1650 tumor-bearing mice were treated with a single dose of 100 mg/kg intravenous IPI-504. Tumors and blood plasma were harvested at designated time points post dose. Drug levels of Hsp90 active species (IPI-504, 17-AAG, and 17-AG) were quantified by LC-MS/MS in plasma and in tumor ( A ). EGFR protein levels ( B ) and Hsp90 occupancy ( C ) were measured in tumor tissue. Data are expressed as averages ± S.D. (vehicle ( veh ), n = 2; and 1–48 h, n = 3).

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: In Vivo, Mouse Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Hsp90 occupancy correlates with antitumor activity of IPI-504 in a xenograft model of NSCLC. H1650 tumor bearing mice were treated with vehicle (●), 25 mg/kg (□), 50 mg/kg (▾), or 100 mg/kg (○) of IPI-504 (IP, twice weekly) and tumor growth was assessed by caliper measurement ( A ). Data are expressed as means and standard error ( n = 10 per arm). A separate group of H1650 tumor bearing mice was treated with a single dose of 25, 50, or 100 mg/kg of IPI-504 and sacrificed 2 h post dose. Hsp90 occupancy was determined as described and plotted against tumor size (mm 3 ) measured on day 50 of drug treatment ( B ). Data are expressed as averages with standard deviation ( n = 2 ( x -axis)) and as means with standard error ( n = 10 ( y -axis)). The points were fit to a linear least squares regression equation with a calculated R 2 = 0.98.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Hsp90 occupancy correlates with antitumor activity of IPI-504 in a xenograft model of NSCLC. H1650 tumor bearing mice were treated with vehicle (●), 25 mg/kg (□), 50 mg/kg (▾), or 100 mg/kg (○) of IPI-504 (IP, twice weekly) and tumor growth was assessed by caliper measurement ( A ). Data are expressed as means and standard error ( n = 10 per arm). A separate group of H1650 tumor bearing mice was treated with a single dose of 25, 50, or 100 mg/kg of IPI-504 and sacrificed 2 h post dose. Hsp90 occupancy was determined as described and plotted against tumor size (mm 3 ) measured on day 50 of drug treatment ( B ). Data are expressed as averages with standard deviation ( n = 2 ( x -axis)) and as means with standard error ( n = 10 ( y -axis)). The points were fit to a linear least squares regression equation with a calculated R 2 = 0.98.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Activity Assay, Mouse Assay, Standard Deviation

    Effect of Hsp90 occupancy on client protein abundance. H1650 cells were incubated for 6 or 24 h with increasing concentrations of IPI-504. HER2, mEGFR, Akt, and cRaf protein levels were assessed by immunoblotting ( A and C ). The fraction of degraded proteins was assessed by densitometry compared with untreated samples and plotted together with the occupancy curves ( B and D ).

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Effect of Hsp90 occupancy on client protein abundance. H1650 cells were incubated for 6 or 24 h with increasing concentrations of IPI-504. HER2, mEGFR, Akt, and cRaf protein levels were assessed by immunoblotting ( A and C ). The fraction of degraded proteins was assessed by densitometry compared with untreated samples and plotted together with the occupancy curves ( B and D ).

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Incubation

    Binding of [ 3 H]17-AAG to purified Hela Hsp90 and SK-BR-3 lysates at 4 °C. The binding reaction was initiated by adding 10 μ m [ 3 H]17-AAG to 100 n m purified Hsp90 or to SK-BR-3 lysate containing ∼100 n m Hsp90. Drug association was measured at 4 °C by a time-dependent increase in protein bound counts for purified Hsp90 ( A ) or Hsp90 in SK-BR-3 cell lysate ( B ). Data were fitted by nonlinear regression to a single exponential equation to obtain a ( k obs ) value. Half-life was calculated using the equation t ½ = 0.693/ k obs .

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Binding of [ 3 H]17-AAG to purified Hela Hsp90 and SK-BR-3 lysates at 4 °C. The binding reaction was initiated by adding 10 μ m [ 3 H]17-AAG to 100 n m purified Hsp90 or to SK-BR-3 lysate containing ∼100 n m Hsp90. Drug association was measured at 4 °C by a time-dependent increase in protein bound counts for purified Hsp90 ( A ) or Hsp90 in SK-BR-3 cell lysate ( B ). Data were fitted by nonlinear regression to a single exponential equation to obtain a ( k obs ) value. Half-life was calculated using the equation t ½ = 0.693/ k obs .

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Binding Assay, Purification

    Characterization of pSer335 XErp1 antibody CSF extract was treated with Myc‐XErp1 IVT carrying the indicated combinations of the mutations DSG − (S33N S38N), DSA − (S284N S288N), ZBR − (C583A) and CaMKII − (T195A). Calcium was added, samples were taken at the indicated time points and as indicated treated with λ‐phosphatase. Samples were immunoblotted for the Myc‐tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α‐tubulin. Asterisk indicates unspecific bands. Several lanes were removed at the dashed line. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT at the indicated dilutions. An empty IVT not expressing XErp1 and an untreated condition were used as controls. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, pSer335 XErp1, and α‐tubulin. The XErp1 membrane was stripped and reprobed for the Myc‐tag. Asterisks indicate unspecific bands. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT that was either wild‐type or mutated to alanine at Ser335. An empty IVT reaction not expressing Myc‐XErp1 served as control. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, the Myc‐tag, pSer335 XErp1, and α‐tubulin. Asterisks indicate unspecific bands. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20

    doi: 10.15252/embr.201846433

    Figure Lengend Snippet: Characterization of pSer335 XErp1 antibody CSF extract was treated with Myc‐XErp1 IVT carrying the indicated combinations of the mutations DSG − (S33N S38N), DSA − (S284N S288N), ZBR − (C583A) and CaMKII − (T195A). Calcium was added, samples were taken at the indicated time points and as indicated treated with λ‐phosphatase. Samples were immunoblotted for the Myc‐tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α‐tubulin. Asterisk indicates unspecific bands. Several lanes were removed at the dashed line. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT at the indicated dilutions. An empty IVT not expressing XErp1 and an untreated condition were used as controls. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, pSer335 XErp1, and α‐tubulin. The XErp1 membrane was stripped and reprobed for the Myc‐tag. Asterisks indicate unspecific bands. CSF extract was treated with Myc‐XErp1 CaMKII − ZBR − (T195A C583A) IVT that was either wild‐type or mutated to alanine at Ser335. An empty IVT reaction not expressing Myc‐XErp1 served as control. Samples were taken, treated as indicated with λ‐phosphatase and immunoblotted for XErp1, the Myc‐tag, pSer335 XErp1, and α‐tubulin. Asterisks indicate unspecific bands. Source data are available online for this figure.

    Article Snippet: XErp1: 2 μl Myc‐XErp1 IVT were diluted in 8 μl CSF‐XB Buffer (10 mM HEPES; 100 mM KCl; 2 mM MgCl2 ; 0.1 mM CaCl2 ; 50 mM sucrose; 5 mM EGTA; pH = 7.7) and added to 0.5 μg α‐Myc antibodies coupled to Dynabeads® Protein G (Thermo Fisher).

    Techniques: Expressing