z yvad fmk  (Millipore)


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    Structured Review

    Millipore z yvad fmk
    oA β induces IL-1 β secretion/release via caspase-1 activation. LPS-primed microglia were treated with <t>Z-VAD-FMK</t> ( a ) or <t>Z-YVAD-FMK</t> ( b ) for 30 min before oA β stimulation, and IL-1 β in the culture supernatant was measured at 48 h. Data indicate means±S.D. for four independent experiments. *** P
    Z Yvad Fmk, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1633 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/z yvad fmk/product/Millipore
    Average 99 stars, based on 1633 article reviews
    Price from $9.99 to $1999.99
    z yvad fmk - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Oligomeric amyloid β induces IL-1β processing via production of ROS: implication in Alzheimer's disease"

    Article Title: Oligomeric amyloid β induces IL-1β processing via production of ROS: implication in Alzheimer's disease

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.503

    oA β induces IL-1 β secretion/release via caspase-1 activation. LPS-primed microglia were treated with Z-VAD-FMK ( a ) or Z-YVAD-FMK ( b ) for 30 min before oA β stimulation, and IL-1 β in the culture supernatant was measured at 48 h. Data indicate means±S.D. for four independent experiments. *** P
    Figure Legend Snippet: oA β induces IL-1 β secretion/release via caspase-1 activation. LPS-primed microglia were treated with Z-VAD-FMK ( a ) or Z-YVAD-FMK ( b ) for 30 min before oA β stimulation, and IL-1 β in the culture supernatant was measured at 48 h. Data indicate means±S.D. for four independent experiments. *** P

    Techniques Used: Activation Assay

    LPS-primed microglia treated with oA β induce neuronal cell death via caspase-1, and IL-1 β . LPS-primed microglia were cocultured with primary cortical neurons. The cells were then treated with Z-YVAD-FMK or IL-1ra before oA β treatment. Neurons were stained with anti-MAP2 antibodies (green), microglia were stained with Cy-5-conjugated anti-CD11b antibodies (blue), and oA β was stained with anti-4G8 antibodies (red). Neuronal viability was assessed by MAP2 staining ( a and b ). Data indicate means±S.D. for three independent experiments. * P
    Figure Legend Snippet: LPS-primed microglia treated with oA β induce neuronal cell death via caspase-1, and IL-1 β . LPS-primed microglia were cocultured with primary cortical neurons. The cells were then treated with Z-YVAD-FMK or IL-1ra before oA β treatment. Neurons were stained with anti-MAP2 antibodies (green), microglia were stained with Cy-5-conjugated anti-CD11b antibodies (blue), and oA β was stained with anti-4G8 antibodies (red). Neuronal viability was assessed by MAP2 staining ( a and b ). Data indicate means±S.D. for three independent experiments. * P

    Techniques Used: Staining

    2) Product Images from "Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth"

    Article Title: Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth

    Journal: Molecular Systems Biology

    doi: 10.1038/msb.2011.56

    Relative metabolites concentrations in N, T, and R cell lines and effect of aminooxyacetate (AOA) and epigallocatechin gallate (EGCG) treatments on their proliferation. Evaluation of Cit ( A ), Mal ( B ), Glu ( C ), and Asp ( D ) intracellular concentrations was carried out after 54 h of grown in normal medium by using enzymatic assays. Analysis of AOA ( E ) and EGCG ( F ) treatments on N, T, and R cell lines proliferation. Cells, seeded at density of 3000 cells/cm 2 , after 18 h and following a complete medium replacement, were treated with 100 μM AOA, or 1 mM dimethyl aspartate (DMD), or 2 mM dimethyl α-ketoglutarate (DMK) or 20 μM EGCG and counted at indicated time points. Error bars indicate s.e.m. ( n =3).
    Figure Legend Snippet: Relative metabolites concentrations in N, T, and R cell lines and effect of aminooxyacetate (AOA) and epigallocatechin gallate (EGCG) treatments on their proliferation. Evaluation of Cit ( A ), Mal ( B ), Glu ( C ), and Asp ( D ) intracellular concentrations was carried out after 54 h of grown in normal medium by using enzymatic assays. Analysis of AOA ( E ) and EGCG ( F ) treatments on N, T, and R cell lines proliferation. Cells, seeded at density of 3000 cells/cm 2 , after 18 h and following a complete medium replacement, were treated with 100 μM AOA, or 1 mM dimethyl aspartate (DMD), or 2 mM dimethyl α-ketoglutarate (DMK) or 20 μM EGCG and counted at indicated time points. Error bars indicate s.e.m. ( n =3).

    Techniques Used:

    3) Product Images from "Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose"

    Article Title: Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4443-1

    Cell death and morphology in GOT1-null 143B and siRNA knock-down A549 cells upon glucose deprivation. a Cells grown in 1 g/L glucose, 0 g/L glucose or treated with 2 mM H 2 O 2. for 24 h Arrows in black indicate ischemic-like cell death in GOT1-null 143B cells. Arrows in white indicate apoptotic cell death. Bars represent 25 μm. b A549 non-template control (NTC) and GOT1-siRNA A549 cells grown in 1 g/L glucose or 0 g/L glucose for 24 h. Arrows in black indicate ischemic-like cell death. Bars represent 25 μm. c Prevention of ischemic-like cell morphological changes by OAA and PEP in medium without glucose. Cells grown in 4.5 g/L glucose for 4 days, and then the old medium was replaced with glucose-free-medium containing 10 Mm Asp, 5 mM OAA or 2.5 mM PEP. The cell morphological changes 5 min after replacing nutrient-depleted-medium. d Prevention of ischemic-like cell morphological changes by OAA and PEP in medium with 4.5 g/L glucose. Cells grown in 4.5 g/L glucose for 4 days, and then the old medium was replaced with 4.5 g/L glucose-medium containing 10 Mm Asp, 5 mM OAA or 2.5 mM PEP. The cell morphological changes 5 min after replacing nutrient-depleted-medium. Bars indicate 25 μm
    Figure Legend Snippet: Cell death and morphology in GOT1-null 143B and siRNA knock-down A549 cells upon glucose deprivation. a Cells grown in 1 g/L glucose, 0 g/L glucose or treated with 2 mM H 2 O 2. for 24 h Arrows in black indicate ischemic-like cell death in GOT1-null 143B cells. Arrows in white indicate apoptotic cell death. Bars represent 25 μm. b A549 non-template control (NTC) and GOT1-siRNA A549 cells grown in 1 g/L glucose or 0 g/L glucose for 24 h. Arrows in black indicate ischemic-like cell death. Bars represent 25 μm. c Prevention of ischemic-like cell morphological changes by OAA and PEP in medium without glucose. Cells grown in 4.5 g/L glucose for 4 days, and then the old medium was replaced with glucose-free-medium containing 10 Mm Asp, 5 mM OAA or 2.5 mM PEP. The cell morphological changes 5 min after replacing nutrient-depleted-medium. d Prevention of ischemic-like cell morphological changes by OAA and PEP in medium with 4.5 g/L glucose. Cells grown in 4.5 g/L glucose for 4 days, and then the old medium was replaced with 4.5 g/L glucose-medium containing 10 Mm Asp, 5 mM OAA or 2.5 mM PEP. The cell morphological changes 5 min after replacing nutrient-depleted-medium. Bars indicate 25 μm

    Techniques Used:

    Gene expression profile and rescue of GOT1-null 143B cells with different metabolites. a Gene expression profile before glucose deprivation b , Gene expression profile 4 h after glucose deprivation. c Changes of LC3-II levels before and after glucose deprivation for 4 h. d Cell viability in different concentrations of glucose for 24 h. e Cell viability upon glutamine deprivation for 24 h. f Relative viabilities of wild type 143B and A549 cells in medium with glucose concentration at 4.5 g/L or 0 g/L in the presence of AOA at concentration of 5 mM. g Relative cell viability in medium supplemented with different metabolites (Glc: glucose; Gly: glycine; Ser: serine; Gal: galactose). h Illustration of intermediates in gluconeogenesis pathway. i Wild type and GOT1-null 143B cells grown in glucose free medium supplemented with 10 mM aspartate (Asp), 5 mM OAA, or 2.5 mM PEP for 4 h, j , 16 h and k, 24 h. All the experiments have been repeated three times, and data are represented as mean ± s.d. One-way ANOVA test was performed for d , g , i , j and k . Unpaired student’s t-test was performed for a , b , e and f . *** p
    Figure Legend Snippet: Gene expression profile and rescue of GOT1-null 143B cells with different metabolites. a Gene expression profile before glucose deprivation b , Gene expression profile 4 h after glucose deprivation. c Changes of LC3-II levels before and after glucose deprivation for 4 h. d Cell viability in different concentrations of glucose for 24 h. e Cell viability upon glutamine deprivation for 24 h. f Relative viabilities of wild type 143B and A549 cells in medium with glucose concentration at 4.5 g/L or 0 g/L in the presence of AOA at concentration of 5 mM. g Relative cell viability in medium supplemented with different metabolites (Glc: glucose; Gly: glycine; Ser: serine; Gal: galactose). h Illustration of intermediates in gluconeogenesis pathway. i Wild type and GOT1-null 143B cells grown in glucose free medium supplemented with 10 mM aspartate (Asp), 5 mM OAA, or 2.5 mM PEP for 4 h, j , 16 h and k, 24 h. All the experiments have been repeated three times, and data are represented as mean ± s.d. One-way ANOVA test was performed for d , g , i , j and k . Unpaired student’s t-test was performed for a , b , e and f . *** p

    Techniques Used: Expressing, Concentration Assay, Gas Chromatography

    4) Product Images from "TBP-like protein (TLP) interferes with Taspase1-mediated processing of TFIIA and represses TATA box gene expression"

    Article Title: TBP-like protein (TLP) interferes with Taspase1-mediated processing of TFIIA and represses TATA box gene expression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv576

    Inhibition of Taspae1-mediated processing of the TFIIAαβ precursor by TLP. ( A ) Effect of MG132 on TFIIA proteins. HCT116 cells were treated with MG132 and CHX for the indicated time, and the amount of TFIIA protein was determined. ( B ) Effect of overexpressed Taspase1 on the endogenous TFIIAαβ precursor. HCT116 cells were transfected with expression plasmids of FH-Taspase1, and the amount of the TFIIAαβ precursor was determined. ( C ) Taspase1-mediated processing of the TFIIAαβ precursor in vitro . Five nanograms of purified recombinant TFIIAαβ was mixed with 20 ng of purified recombinant FH-Taspase1 and incubated at 37°C for 1.5 h, and proteins were detected by Western blotting. Effect of TLP on Taspase1-mediated processing of the TFIIAαβ precursor in vitro . Twelve nanograms of TFIIAαβ and 6 ng of TFIIAγ were incubated with 50 to 200 ng of TLP or ΔIIA mutant protein at 37°C for 1 h. Fifty nanograms of FH-Taspase1 was then added to the mixture and incubated for 1.5 h.
    Figure Legend Snippet: Inhibition of Taspae1-mediated processing of the TFIIAαβ precursor by TLP. ( A ) Effect of MG132 on TFIIA proteins. HCT116 cells were treated with MG132 and CHX for the indicated time, and the amount of TFIIA protein was determined. ( B ) Effect of overexpressed Taspase1 on the endogenous TFIIAαβ precursor. HCT116 cells were transfected with expression plasmids of FH-Taspase1, and the amount of the TFIIAαβ precursor was determined. ( C ) Taspase1-mediated processing of the TFIIAαβ precursor in vitro . Five nanograms of purified recombinant TFIIAαβ was mixed with 20 ng of purified recombinant FH-Taspase1 and incubated at 37°C for 1.5 h, and proteins were detected by Western blotting. Effect of TLP on Taspase1-mediated processing of the TFIIAαβ precursor in vitro . Twelve nanograms of TFIIAαβ and 6 ng of TFIIAγ were incubated with 50 to 200 ng of TLP or ΔIIA mutant protein at 37°C for 1 h. Fifty nanograms of FH-Taspase1 was then added to the mixture and incubated for 1.5 h.

    Techniques Used: Inhibition, Transfection, Expressing, In Vitro, Purification, Recombinant, Incubation, Western Blot, Mutagenesis

    EMSA to detect the association of TFIIA with TATA the box. ( A ) in vitro processing of recombinant TFIIAαβ precursor protein. Twenty nanograms of purified recombinant TFIIAαβ and DGAA expressed in E. coli were mixed with 70 ng of purified recombinant FH-Taspase1 and incubated at 37°C for 1 h. Proteins were separated by SDS-PAGE and detected by silver staining. ( B – D ) EMSA of TFIIA and TBP to detect TATA box binding of the GAPDH promoter. Purified TFIIAαβ, His-TFIIAγ and FH-TBP were used. Panel B: (a) processed TFIIAαβ (indicated as Processing +, same with αβ of panel b) and unprocessed TFIIAαβ (indicated as Processing -, same with α/β of panel b) were used for EMSA. Processing: purified recombinant TFIIAαβ and DGAA were incubated with FH-Taspase1, and TFIIA proteins were purified. (b) Indicated combinations of purified proteins were used for EMSA. Panel C: cold probe DNA (WT) and its mutant (mut) were used as competitors in EMSA binding reactions. Sequences of wild-type and mutant competitors were 5′-CGGTTTCTATAAATTGAGCC and 5′-CGGTTTCCAGTAACTGAGCC, respectively. Panel D: specific antibodies against TBP, TFIIAαβ and control IgG were included in the EMSA. αβ and α/β indicate unprocessed and processed TFIIAαβ, respectively.
    Figure Legend Snippet: EMSA to detect the association of TFIIA with TATA the box. ( A ) in vitro processing of recombinant TFIIAαβ precursor protein. Twenty nanograms of purified recombinant TFIIAαβ and DGAA expressed in E. coli were mixed with 70 ng of purified recombinant FH-Taspase1 and incubated at 37°C for 1 h. Proteins were separated by SDS-PAGE and detected by silver staining. ( B – D ) EMSA of TFIIA and TBP to detect TATA box binding of the GAPDH promoter. Purified TFIIAαβ, His-TFIIAγ and FH-TBP were used. Panel B: (a) processed TFIIAαβ (indicated as Processing +, same with αβ of panel b) and unprocessed TFIIAαβ (indicated as Processing -, same with α/β of panel b) were used for EMSA. Processing: purified recombinant TFIIAαβ and DGAA were incubated with FH-Taspase1, and TFIIA proteins were purified. (b) Indicated combinations of purified proteins were used for EMSA. Panel C: cold probe DNA (WT) and its mutant (mut) were used as competitors in EMSA binding reactions. Sequences of wild-type and mutant competitors were 5′-CGGTTTCTATAAATTGAGCC and 5′-CGGTTTCCAGTAACTGAGCC, respectively. Panel D: specific antibodies against TBP, TFIIAαβ and control IgG were included in the EMSA. αβ and α/β indicate unprocessed and processed TFIIAαβ, respectively.

    Techniques Used: In Vitro, Recombinant, Purification, Incubation, SDS Page, Silver Staining, Binding Assay, Mutagenesis

    In vivo function of mature TFIIAα and TFIIAβ. ( A ) Chromatin binding of TFIIAαβ. Wild-type FH-TFIIAαβ (WT) and FH-DGAA (DGAA) were introduced into HeLa cells. Processing of wild-type TFIIAαβ was checked by Western blotting (a). Amounts of chromatin-bound exogenous FH-TFIIAαβ and FH-DGAA were determined by ChIP using M2 Agarose beads. ChIP enrichment at the TATA box-containing GAPDH promoter (GAPDH pro) and control DNA region (p21FUR) was determined by qPCR (b). Ctrl indicates control. ( B ) Activation of TATA promoter. Wild-type FH-TFIIAαβ and FH-DGAA were introduced into HeLa cells together with indicated p21 reporter constructs, and the luciferase activity was determined. ( C ) Affinity of TFIIAαβ to its interacting proteins. Exogenously expressed FH-TFIIAαβ and FH-DGAA were immunoprecipitated with M2 Agarose beads, and co-precipitated TLP and TFIIAγ were detected. Inp: input. ( D ) A dose-responsive effect of TFIIAγ on TATA promoter activation. Indicated combinations of TFIIAs were introduced into HCT116 cells together with p21-65/GL4 reporter plasmid, and luciferase activity was determined. For dose-dependent analysis, 100 ng (+) or 200 ng (++) of TFIIAγ expression plasmid was used.
    Figure Legend Snippet: In vivo function of mature TFIIAα and TFIIAβ. ( A ) Chromatin binding of TFIIAαβ. Wild-type FH-TFIIAαβ (WT) and FH-DGAA (DGAA) were introduced into HeLa cells. Processing of wild-type TFIIAαβ was checked by Western blotting (a). Amounts of chromatin-bound exogenous FH-TFIIAαβ and FH-DGAA were determined by ChIP using M2 Agarose beads. ChIP enrichment at the TATA box-containing GAPDH promoter (GAPDH pro) and control DNA region (p21FUR) was determined by qPCR (b). Ctrl indicates control. ( B ) Activation of TATA promoter. Wild-type FH-TFIIAαβ and FH-DGAA were introduced into HeLa cells together with indicated p21 reporter constructs, and the luciferase activity was determined. ( C ) Affinity of TFIIAαβ to its interacting proteins. Exogenously expressed FH-TFIIAαβ and FH-DGAA were immunoprecipitated with M2 Agarose beads, and co-precipitated TLP and TFIIAγ were detected. Inp: input. ( D ) A dose-responsive effect of TFIIAγ on TATA promoter activation. Indicated combinations of TFIIAs were introduced into HCT116 cells together with p21-65/GL4 reporter plasmid, and luciferase activity was determined. For dose-dependent analysis, 100 ng (+) or 200 ng (++) of TFIIAγ expression plasmid was used.

    Techniques Used: In Vivo, Binding Assay, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activation Assay, Construct, Luciferase, Activity Assay, Immunoprecipitation, Plasmid Preparation, Expressing

    5) Product Images from "Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SAICAR synthase from Streptococcus suis serotype 2"

    Article Title: Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SAICAR synthase from Streptococcus suis serotype 2

    Journal: Acta Crystallographica Section F: Structural Biology and Crystallization Communications

    doi: 10.1107/S1744309110020518

    A typical X-ray diffraction image collected from a crystal of SAICAR synthase. The crystal diffracted to 2.8 Å resolution.
    Figure Legend Snippet: A typical X-ray diffraction image collected from a crystal of SAICAR synthase. The crystal diffracted to 2.8 Å resolution.

    Techniques Used:

    Representative crystals of SAICAR synthase. The size of the crystal was measured as approximately 1.0–1.5 mm.
    Figure Legend Snippet: Representative crystals of SAICAR synthase. The size of the crystal was measured as approximately 1.0–1.5 mm.

    Techniques Used:

    6) Product Images from "Rescue from galactose-induced death of Leigh Syndrome patient cells by pyruvate and NAD+"

    Article Title: Rescue from galactose-induced death of Leigh Syndrome patient cells by pyruvate and NAD+

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1179-4

    The viability of LS fibroblasts is reduced in galactose medium in a cell density-dependent manner and rescued by specific metabolites. a Effect of cell seeding density on the galactose-induced reduction in viability of LS cells (S7, S8, V1; measured at 96 h; N = 1, n = 8). b Specificity of the galactose-induced reduction in viability (2500 cells seeded; measured at 96 h; N = 3, n ≥ 12) for LS cells relative to CT cells (CT1, CT2, CT3). c Experimental protocol used in this study to quantify the effects of galactose and other treatments on control and LS cells. d Dose-dependent rescue of the galactose-induced reduction in viability of LS cells (S7, S8, V1) at 96 h by pyruvate in the presence of a constant glutamine concentration ( N = 3, n ≥ 9). The EC50 value was determined by fitting a Hill equation to the data ( R 2 = 0.97). e Same as d , but now for the dose-dependent effect of glutamine in the presence of a constant pyruvate concentration ( N = 3, n ≥ 18). The EC50 value was determined by fitting a Hill equation to the data ( R 2 = 0.98). f Rescuing effect of specific metabolites on the galactose-induced reduction in LS cell viability (96 h; Calcein MSK images for patient S7 are shown as a typical example): sodium pyruvate (10 mM), sodium l -lactate (10 mM), sodium succinate dibasic hexahydrate (10 mM), l -(-)-malic acid (10 mM), oxaloacetic acid (10 mM), dimethyl α-ketoglutarate (10 mM), 2-ketobutyric acid (10 mM), l -aspartic acid (15 mM), and uridine (1 mM). The “ + ” conditions reflect combined galactose + metabolite treatment. g Quantification of the rescuing effect of the metabolites ( N = 3, n ≥ 18) in LS patient cells (S7, S8, V1) at 96 h ( N = 3, n ≥ 18). Statistics: * P
    Figure Legend Snippet: The viability of LS fibroblasts is reduced in galactose medium in a cell density-dependent manner and rescued by specific metabolites. a Effect of cell seeding density on the galactose-induced reduction in viability of LS cells (S7, S8, V1; measured at 96 h; N = 1, n = 8). b Specificity of the galactose-induced reduction in viability (2500 cells seeded; measured at 96 h; N = 3, n ≥ 12) for LS cells relative to CT cells (CT1, CT2, CT3). c Experimental protocol used in this study to quantify the effects of galactose and other treatments on control and LS cells. d Dose-dependent rescue of the galactose-induced reduction in viability of LS cells (S7, S8, V1) at 96 h by pyruvate in the presence of a constant glutamine concentration ( N = 3, n ≥ 9). The EC50 value was determined by fitting a Hill equation to the data ( R 2 = 0.97). e Same as d , but now for the dose-dependent effect of glutamine in the presence of a constant pyruvate concentration ( N = 3, n ≥ 18). The EC50 value was determined by fitting a Hill equation to the data ( R 2 = 0.98). f Rescuing effect of specific metabolites on the galactose-induced reduction in LS cell viability (96 h; Calcein MSK images for patient S7 are shown as a typical example): sodium pyruvate (10 mM), sodium l -lactate (10 mM), sodium succinate dibasic hexahydrate (10 mM), l -(-)-malic acid (10 mM), oxaloacetic acid (10 mM), dimethyl α-ketoglutarate (10 mM), 2-ketobutyric acid (10 mM), l -aspartic acid (15 mM), and uridine (1 mM). The “ + ” conditions reflect combined galactose + metabolite treatment. g Quantification of the rescuing effect of the metabolites ( N = 3, n ≥ 18) in LS patient cells (S7, S8, V1) at 96 h ( N = 3, n ≥ 18). Statistics: * P

    Techniques Used: Concentration Assay

    Exogenous NAD + (eNAD) rescues the galactose-induced reduction in cell viability and intracellular NAD + (iNAD) content in LS fibroblasts. a Dose-dependent effect of extracellular sodium pyruvate (10 mM), oxaloacetic acid (10 mM), dimethyl α-ketoglutarate (10 mM), 2-ketobutyric acid (10 mM), and l -aspartic acid (15 mM) on intracellular NAD (iNAD) levels in LS cells (24 h; N = 1, n = 3). b Dose-dependent effect of extracellular NAD (eNAD; β-nicotinamide adenine dinucleotide hydrate) on intracellular NAD (iNAD) levels (24 h; N = 1, n ≥ 6). c Dose-dependent rescue of the galactose-induced reduction in viability of LS cells (Calcein MSK images) by eNAD (β-nicotinamide adenine dinucleotide hydrate; S7 shown as a typical example; 96 h). d Quantification of the rescuing effect of eNAD in LS patient cells (96 h; N = 2 n ≥ 9). e Dose-dependent inhibition of the rescuing effect of eNAD by oleamide (OLE) in LS patient cells (96 h; N = 3, n = ≥ 6). Statistics: * P
    Figure Legend Snippet: Exogenous NAD + (eNAD) rescues the galactose-induced reduction in cell viability and intracellular NAD + (iNAD) content in LS fibroblasts. a Dose-dependent effect of extracellular sodium pyruvate (10 mM), oxaloacetic acid (10 mM), dimethyl α-ketoglutarate (10 mM), 2-ketobutyric acid (10 mM), and l -aspartic acid (15 mM) on intracellular NAD (iNAD) levels in LS cells (24 h; N = 1, n = 3). b Dose-dependent effect of extracellular NAD (eNAD; β-nicotinamide adenine dinucleotide hydrate) on intracellular NAD (iNAD) levels (24 h; N = 1, n ≥ 6). c Dose-dependent rescue of the galactose-induced reduction in viability of LS cells (Calcein MSK images) by eNAD (β-nicotinamide adenine dinucleotide hydrate; S7 shown as a typical example; 96 h). d Quantification of the rescuing effect of eNAD in LS patient cells (96 h; N = 2 n ≥ 9). e Dose-dependent inhibition of the rescuing effect of eNAD by oleamide (OLE) in LS patient cells (96 h; N = 3, n = ≥ 6). Statistics: * P

    Techniques Used: Inhibition

    7) Product Images from "Mitochondrial Division Inhibitor 1 (mdivi-1) Protects Neurons against Excitotoxicity through the Modulation of Mitochondrial Function and Intracellular Ca2+ Signaling"

    Article Title: Mitochondrial Division Inhibitor 1 (mdivi-1) Protects Neurons against Excitotoxicity through the Modulation of Mitochondrial Function and Intracellular Ca2+ Signaling

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00003

    Preincubation of neurons with mdivi-1 depletes ER Ca 2+ store. (A,B) Neurons were incubated with Fluo-4 in a Ca 2+ -free medium and exposed to (A) thapsigargin and (B) ionomycin (2 μM) plus FCCP (2 μM) in the presence or absence of mdivi-1 (50 μM). Resulting cytosolic Ca 2+ increase was measured to determine ER Ca 2+ content. (C) Ionomycin (2 μM) plus FCCP (2 μM) was added in the presence ( n = 60 cells) or absence ( n = 59 cells) of acute mdivi-1 (50 μM, 5 min). Resulting cytosolic Ca 2+ increase was measured to determine ER Ca 2+ content. (D) Fluo-4-loaded neurons were exposed to NMDA (30 μM) as indicated in the absence ( n = 83) or presence ( n = 90) of mdivi-1 (50 μM, 5 min) and cytosolic Ca 2+ load quantified. Traces represent normalized means ± SEM of cells from at least three independent cultures/experiments. *** p
    Figure Legend Snippet: Preincubation of neurons with mdivi-1 depletes ER Ca 2+ store. (A,B) Neurons were incubated with Fluo-4 in a Ca 2+ -free medium and exposed to (A) thapsigargin and (B) ionomycin (2 μM) plus FCCP (2 μM) in the presence or absence of mdivi-1 (50 μM). Resulting cytosolic Ca 2+ increase was measured to determine ER Ca 2+ content. (C) Ionomycin (2 μM) plus FCCP (2 μM) was added in the presence ( n = 60 cells) or absence ( n = 59 cells) of acute mdivi-1 (50 μM, 5 min). Resulting cytosolic Ca 2+ increase was measured to determine ER Ca 2+ content. (D) Fluo-4-loaded neurons were exposed to NMDA (30 μM) as indicated in the absence ( n = 83) or presence ( n = 90) of mdivi-1 (50 μM, 5 min) and cytosolic Ca 2+ load quantified. Traces represent normalized means ± SEM of cells from at least three independent cultures/experiments. *** p

    Techniques Used: Incubation

    8) Product Images from "Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity"

    Article Title: Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004365

    Analysis of recombinant Li AS-A. A) Coomassie blue stained 12% SDS-PAGE gel of 10 μg of recombinant Li AS-A post affinity chromatography purification. B) Western-blot analysis of 1 μg of purified recombinant Li AS-A using a rabbit anti-HisTag monoclonal antibody (1:1000). MW, molecular weight marker. C) Analytic size exclusion chromatogram of recombinant Li AS-A after purification by affinity, size exclusion and ion exchange chromatographies. D and E) Calibration curve for Li ASA Stokes’ radius and MW determination, respectively. K av was determined considering the elution volume of the proteins used as standards, the total volume of the column and the exclusion volume given by the elution of blue dextran. The used standards were as follows: ribonuclease (R), chymotrypsinogen A (CtA), ovalbumin (OA), albumin (A), aldolase (Ald), catalase (C). Data is representative of two independent experiments. F and G) Differential scanning fluorimetry analysis of recombinant Li AS-A in the presence of several ligands, expressed in T m variation (∆T m - °C) determined as T m (protein + ligand)–T m (protein without ligand). F) Single ligand effect at 1 mM concentration: ATP, AMP, pyrophosphate (Pyro), ammonium chloride (NH 4 Cl), magnesium chloride (MgCl 2 ), asparagine (Asn), aspartate (Asp), glutamate (Glu), glutamine (Gln). G) Concentration dependent effect of AMP in Li AS-A stabilization. These results represent the mean values of two independent experiments plus the standard deviation.
    Figure Legend Snippet: Analysis of recombinant Li AS-A. A) Coomassie blue stained 12% SDS-PAGE gel of 10 μg of recombinant Li AS-A post affinity chromatography purification. B) Western-blot analysis of 1 μg of purified recombinant Li AS-A using a rabbit anti-HisTag monoclonal antibody (1:1000). MW, molecular weight marker. C) Analytic size exclusion chromatogram of recombinant Li AS-A after purification by affinity, size exclusion and ion exchange chromatographies. D and E) Calibration curve for Li ASA Stokes’ radius and MW determination, respectively. K av was determined considering the elution volume of the proteins used as standards, the total volume of the column and the exclusion volume given by the elution of blue dextran. The used standards were as follows: ribonuclease (R), chymotrypsinogen A (CtA), ovalbumin (OA), albumin (A), aldolase (Ald), catalase (C). Data is representative of two independent experiments. F and G) Differential scanning fluorimetry analysis of recombinant Li AS-A in the presence of several ligands, expressed in T m variation (∆T m - °C) determined as T m (protein + ligand)–T m (protein without ligand). F) Single ligand effect at 1 mM concentration: ATP, AMP, pyrophosphate (Pyro), ammonium chloride (NH 4 Cl), magnesium chloride (MgCl 2 ), asparagine (Asn), aspartate (Asp), glutamate (Glu), glutamine (Gln). G) Concentration dependent effect of AMP in Li AS-A stabilization. These results represent the mean values of two independent experiments plus the standard deviation.

    Techniques Used: Recombinant, Staining, SDS Page, Affinity Chromatography, Purification, Western Blot, Molecular Weight, Marker, Concentration Assay, Standard Deviation

    Multiple-sequence alignment of prokaryote and eukaryote AS-A proteins and 3D homology models of Li AS-A and Lm AS-A. A) Alignment of Li AS-A (NCBI-Gene ID: 5069795/ LinJ.26.0790), Lm AS-A (NCBI-Gene ID: 5652811/LmjF.26.0830), Tb AS-A (NCBI-GeneID:3658321/ Tb927.7.1110), Tc AS-A (NCBI-GeneID:3534325/Tc00.1047053503625.10) and Ec AS-A (NCBI-GeneID:948258/pdb:12AS). A pre-established colour pattern was used, according to ALSCRIPT Calcons (Aline version 011208): red, identical residues; orange to blue, scale of conservation of amino acid properties in each column alignment; white, dissimilar residues). Secondary structure components of Ec AS-A crystal structure (black) are represented above the alignment. In all sequences, binding residues for several ligands were represented: AMP (circles), asparagine (squares), ATP (triangle) and aspartate (inverted triangle). B) Superposition of Ec AS-A structure (green) (PDB accession code 12AS), with Li AS-A (blue) and Lm AS-A (purple) homology models (obtained from the SWISS-MODEL server, using PDB 12AS as a template). The dashed box points a structurally divergent region.
    Figure Legend Snippet: Multiple-sequence alignment of prokaryote and eukaryote AS-A proteins and 3D homology models of Li AS-A and Lm AS-A. A) Alignment of Li AS-A (NCBI-Gene ID: 5069795/ LinJ.26.0790), Lm AS-A (NCBI-Gene ID: 5652811/LmjF.26.0830), Tb AS-A (NCBI-GeneID:3658321/ Tb927.7.1110), Tc AS-A (NCBI-GeneID:3534325/Tc00.1047053503625.10) and Ec AS-A (NCBI-GeneID:948258/pdb:12AS). A pre-established colour pattern was used, according to ALSCRIPT Calcons (Aline version 011208): red, identical residues; orange to blue, scale of conservation of amino acid properties in each column alignment; white, dissimilar residues). Secondary structure components of Ec AS-A crystal structure (black) are represented above the alignment. In all sequences, binding residues for several ligands were represented: AMP (circles), asparagine (squares), ATP (triangle) and aspartate (inverted triangle). B) Superposition of Ec AS-A structure (green) (PDB accession code 12AS), with Li AS-A (blue) and Lm AS-A (purple) homology models (obtained from the SWISS-MODEL server, using PDB 12AS as a template). The dashed box points a structurally divergent region.

    Techniques Used: Sequencing, Binding Assay

    9) Product Images from "Endothelin‐1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention) Endothelin‐1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention"

    Article Title: Endothelin‐1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention) Endothelin‐1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13719

    Low‐resolution autoradiographs of [ 125 I]‐ET‐1 binding in haemorrhoid sections; (A) total [ 125 I]‐ET‐1 binding (TB), (B) NSB in the presence of 1 μM unlabelled ET‐1, (C) in the presence of 1 μM selective ET B receptor antagonist BQ788 and (D) in the presence of 1 μM selective ET A receptor antagonist BQ123: identifying ET A and ET B receptor binding sites respectively.
    Figure Legend Snippet: Low‐resolution autoradiographs of [ 125 I]‐ET‐1 binding in haemorrhoid sections; (A) total [ 125 I]‐ET‐1 binding (TB), (B) NSB in the presence of 1 μM unlabelled ET‐1, (C) in the presence of 1 μM selective ET B receptor antagonist BQ788 and (D) in the presence of 1 μM selective ET A receptor antagonist BQ123: identifying ET A and ET B receptor binding sites respectively.

    Techniques Used: Binding Assay

    10) Product Images from "PM2.5 exposure aggravates oligomeric amyloid beta-induced neuronal injury and promotes NLRP3 inflammasome activation in an in vitro model of Alzheimer’s disease"

    Article Title: PM2.5 exposure aggravates oligomeric amyloid beta-induced neuronal injury and promotes NLRP3 inflammasome activation in an in vitro model of Alzheimer’s disease

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1178-5

    ROS and NLRP3 inflammasome activation is required for PM2.5-induced neuronal injury in neurons-microglia co-cultures. In a transwell co-culture system, neurons-microglia co-cultures were stimulated with oAβ for 12 h and treated with ROS inhibitors and caspase-1 inhibitors for 30 min before PM2.5 exposure. Z-VAD-FMK, Z-YVAD-FMK, NAC, DPI, and MitoQ were included. After PM2.5 exposure for 4 h, cell apoptosis was assessed by annexin V/PI method, and a representative experiment is shown
    Figure Legend Snippet: ROS and NLRP3 inflammasome activation is required for PM2.5-induced neuronal injury in neurons-microglia co-cultures. In a transwell co-culture system, neurons-microglia co-cultures were stimulated with oAβ for 12 h and treated with ROS inhibitors and caspase-1 inhibitors for 30 min before PM2.5 exposure. Z-VAD-FMK, Z-YVAD-FMK, NAC, DPI, and MitoQ were included. After PM2.5 exposure for 4 h, cell apoptosis was assessed by annexin V/PI method, and a representative experiment is shown

    Techniques Used: Activation Assay, Co-Culture Assay

    ROS and NLRP3 inflammasome activation is required for PM2.5-induced neuronal injury in neurons-microglia co-cultures. In a transwell co-culture system, neurons-microglia co-cultures were stimulated with oAβ for 12 h. Then, the co-cultures were treated with ROS inhibitors and caspase-1 inhibitors for 30 min before PM2.5 exposure for 4 h. Z-VAD-FMK, Z-YVAD-FMK, NAC, DPI, and MitoQ were included. a Apoptosis of co-cultured neurons were evaluated by flow cytometry with annexin V/PI staining. b Cell viability of co-cultured neurons were assessed via MTT assay. All figures are representative of three independent experiments, performed in triplicate. * P
    Figure Legend Snippet: ROS and NLRP3 inflammasome activation is required for PM2.5-induced neuronal injury in neurons-microglia co-cultures. In a transwell co-culture system, neurons-microglia co-cultures were stimulated with oAβ for 12 h. Then, the co-cultures were treated with ROS inhibitors and caspase-1 inhibitors for 30 min before PM2.5 exposure for 4 h. Z-VAD-FMK, Z-YVAD-FMK, NAC, DPI, and MitoQ were included. a Apoptosis of co-cultured neurons were evaluated by flow cytometry with annexin V/PI staining. b Cell viability of co-cultured neurons were assessed via MTT assay. All figures are representative of three independent experiments, performed in triplicate. * P

    Techniques Used: Activation Assay, Co-Culture Assay, Cell Culture, Flow Cytometry, Cytometry, Staining, MTT Assay

    PM2.5-induced IL-1β production in oAβ-stimulated microglia is possibly dependent on NLRP3 inflammasome activation. Microglia were stimulated by LPS or oAβ alone for varying time, and the concentration of IL-1β in the culture supernatant was measured by ELISA ( a ). Microglia primed with LPS for 3 h were washed with fresh serum-free DMEM. LPS-primed microglia were stimulated with oAβ for 12 h and treated with pan-caspase inhibitor Z-VAD-FMK ( b ) or caspase-1 inhibitor Z-YVAD-FMK ( c ) for 30 min before PM2.5 exposure. After PM2.5 exposure for 4 h, IL-1β concentration in the culture supernatant was measured by ELISA. All figures are representative of three independent experiments, performed in triplicate. * P
    Figure Legend Snippet: PM2.5-induced IL-1β production in oAβ-stimulated microglia is possibly dependent on NLRP3 inflammasome activation. Microglia were stimulated by LPS or oAβ alone for varying time, and the concentration of IL-1β in the culture supernatant was measured by ELISA ( a ). Microglia primed with LPS for 3 h were washed with fresh serum-free DMEM. LPS-primed microglia were stimulated with oAβ for 12 h and treated with pan-caspase inhibitor Z-VAD-FMK ( b ) or caspase-1 inhibitor Z-YVAD-FMK ( c ) for 30 min before PM2.5 exposure. After PM2.5 exposure for 4 h, IL-1β concentration in the culture supernatant was measured by ELISA. All figures are representative of three independent experiments, performed in triplicate. * P

    Techniques Used: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Jaridonin, a Novel Ent-Kaurene Diterpenoid from Isodon rubescens, Inducing Apoptosis via Production of Reactive Oxygen Species in Esophageal Cancer Cells"

    Article Title: Jaridonin, a Novel Ent-Kaurene Diterpenoid from Isodon rubescens, Inducing Apoptosis via Production of Reactive Oxygen Species in Esophageal Cancer Cells

    Journal: Current cancer drug targets

    doi:

    Jaridonin induces release of cytochrome c and the cleavage of Caspase-9/3
    Figure Legend Snippet: Jaridonin induces release of cytochrome c and the cleavage of Caspase-9/3

    Techniques Used:

    12) Product Images from "Comparison of the Drug-Drug Interaction Potential of Daptomycin in Combination with Rifampin in Healthy Adult Volunteers"

    Article Title: Comparison of the Drug-Drug Interaction Potential of Daptomycin in Combination with Rifampin in Healthy Adult Volunteers

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01525-18

    Daptomycin clearance based on MDR1 genotype and study visit. Observed daptomycin CL values at each study visit according to MDR1 genotype are presented. Open circle, observations from visit 1 (daptomycin alone); open triangle, observations from visit 2 (rifampin coadministered with daptomycin); **, subject with undeterminable genotype. Horizontal lines denote mean CL values observed for each of the categories.
    Figure Legend Snippet: Daptomycin clearance based on MDR1 genotype and study visit. Observed daptomycin CL values at each study visit according to MDR1 genotype are presented. Open circle, observations from visit 1 (daptomycin alone); open triangle, observations from visit 2 (rifampin coadministered with daptomycin); **, subject with undeterminable genotype. Horizontal lines denote mean CL values observed for each of the categories.

    Techniques Used:

    13) Product Images from "Stability of daptomycin in peritoneal dialysis solutions packaged in dual-compartment infusion bags"

    Article Title: Stability of daptomycin in peritoneal dialysis solutions packaged in dual-compartment infusion bags

    Journal: European Journal of Hospital Pharmacy

    doi: 10.1136/ejhpharm-2015-000651

    Stability of daptomycin
    Figure Legend Snippet: Stability of daptomycin

    Techniques Used:

    The percentage of daptomycin (20 µg/mL) remaining in the Balance peritoneal dialysis (PD) solution before and after storage at 4°C, 25°C or 37°C for various time intervals. Results are presented as mean±SD.
    Figure Legend Snippet: The percentage of daptomycin (20 µg/mL) remaining in the Balance peritoneal dialysis (PD) solution before and after storage at 4°C, 25°C or 37°C for various time intervals. Results are presented as mean±SD.

    Techniques Used:

    14) Product Images from "Stability of daptomycin in peritoneal dialysis solutions packaged in dual-compartment infusion bags"

    Article Title: Stability of daptomycin in peritoneal dialysis solutions packaged in dual-compartment infusion bags

    Journal: European Journal of Hospital Pharmacy

    doi: 10.1136/ejhpharm-2015-000651

    Stability of daptomycin
    Figure Legend Snippet: Stability of daptomycin

    Techniques Used:

    The percentage of daptomycin (20 µg/mL) remaining in the Balance peritoneal dialysis (PD) solution before and after storage at 4°C, 25°C or 37°C for various time intervals. Results are presented as mean±SD.
    Figure Legend Snippet: The percentage of daptomycin (20 µg/mL) remaining in the Balance peritoneal dialysis (PD) solution before and after storage at 4°C, 25°C or 37°C for various time intervals. Results are presented as mean±SD.

    Techniques Used:

    15) Product Images from "Drug Combinations against Borrelia burgdorferi Persisters In Vitro: Eradication Achieved by Using Daptomycin, Cefoperazone and Doxycycline"

    Article Title: Drug Combinations against Borrelia burgdorferi Persisters In Vitro: Eradication Achieved by Using Daptomycin, Cefoperazone and Doxycycline

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0117207

    Effect of antibiotics alone and in combinations on aggregated microcolony form and planktonic forms of B. burgdorferi . Stationary phase B. burgdorferi culture (10 day old) was treated with 10 μg/ml drugs (labeled on the image) for 7 days followed by staining by SYBR Green I/PI assay. Green cells indicate live cells whereas red cells dead cells. (A) B. burgdorferi aggregated microcolony (MC) form was more resistant to different antibiotics or their combinations than planktonic form (round body and spirochetal form) (PT) as observed by fluorescence microscopy at 400 × magnification. (B) Susceptibility of B. burgdorferi microcolony form to antibiotics and antibiotic combinations was assessed by fluorescence microscopy at 200 × magnification. The luminance of individual RB is much weaker than that of microcolony, which makes the individual cells hard to be observed when the microcolonies were being examined. Abbreviation: Dox, doxycycline; CefP, cefoperazone; Cfz, clofazimine; Dap, daptomycin; Smx, sulfamethoxazole; Cab, carbencillin; Car, carbomycin.
    Figure Legend Snippet: Effect of antibiotics alone and in combinations on aggregated microcolony form and planktonic forms of B. burgdorferi . Stationary phase B. burgdorferi culture (10 day old) was treated with 10 μg/ml drugs (labeled on the image) for 7 days followed by staining by SYBR Green I/PI assay. Green cells indicate live cells whereas red cells dead cells. (A) B. burgdorferi aggregated microcolony (MC) form was more resistant to different antibiotics or their combinations than planktonic form (round body and spirochetal form) (PT) as observed by fluorescence microscopy at 400 × magnification. (B) Susceptibility of B. burgdorferi microcolony form to antibiotics and antibiotic combinations was assessed by fluorescence microscopy at 200 × magnification. The luminance of individual RB is much weaker than that of microcolony, which makes the individual cells hard to be observed when the microcolonies were being examined. Abbreviation: Dox, doxycycline; CefP, cefoperazone; Cfz, clofazimine; Dap, daptomycin; Smx, sulfamethoxazole; Cab, carbencillin; Car, carbomycin.

    Techniques Used: Labeling, Staining, SYBR Green Assay, Fluorescence, Microscopy

    Subculture (15 days) of 10 day old B. burgdorferi stationary phase culture treated with different antibiotics alone or in combinations. Representative images were taken with fluorescence microscopy (400 × magnification) using SYBR Green I/PI staining. Only Dox+Dap+CefP completely killed all forms including the microcolony form of B. burgdorferi persisters as shown by lack of any viable green spirochetal form after 15 day subculture. Abbreviation: Dox, doxycycline; CefP, cefoperazone; Cfz, clofazimine; Dap, daptomycin; Smx, sulfamethoxazole.
    Figure Legend Snippet: Subculture (15 days) of 10 day old B. burgdorferi stationary phase culture treated with different antibiotics alone or in combinations. Representative images were taken with fluorescence microscopy (400 × magnification) using SYBR Green I/PI staining. Only Dox+Dap+CefP completely killed all forms including the microcolony form of B. burgdorferi persisters as shown by lack of any viable green spirochetal form after 15 day subculture. Abbreviation: Dox, doxycycline; CefP, cefoperazone; Cfz, clofazimine; Dap, daptomycin; Smx, sulfamethoxazole.

    Techniques Used: Fluorescence, Microscopy, SYBR Green Assay, Staining

    16) Product Images from "Carboxylated molecules regulate magnesium content of amorphous calcium carbonates during calcification"

    Article Title: Carboxylated molecules regulate magnesium content of amorphous calcium carbonates during calcification

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

    doi: 10.1073/pnas.0906741106

    Mg/Ca ratio in ACC vs. log( K Mg-ligand / K Ca-ligand ) at 0.025 M oxydiacetic, d -tartaric, citric, glutamic, malonic, and aspartic acids. ( A ) Solution Mg/Ca ratio of 2.0. ( B ) Solution Mg/Ca ratio of 4.0. ( C ) Solution Mg/Ca ratio of 5.0 (modern seawater).
    Figure Legend Snippet: Mg/Ca ratio in ACC vs. log( K Mg-ligand / K Ca-ligand ) at 0.025 M oxydiacetic, d -tartaric, citric, glutamic, malonic, and aspartic acids. ( A ) Solution Mg/Ca ratio of 2.0. ( B ) Solution Mg/Ca ratio of 4.0. ( C ) Solution Mg/Ca ratio of 5.0 (modern seawater).

    Techniques Used:

    Mg/Ca ratio in solution vs. Mg/Ca in ACC for the inorganic control experiments ( A ) for aspartic acid at 0.1, 0.05, and 0.025 M, ( B ) for glutamic acid at 0.1, 0.05, and 0.025 M, ( C ) four carboxylic acids at 0.025 M: oxydiacetic, d -tartaric, citric, and
    Figure Legend Snippet: Mg/Ca ratio in solution vs. Mg/Ca in ACC for the inorganic control experiments ( A ) for aspartic acid at 0.1, 0.05, and 0.025 M, ( B ) for glutamic acid at 0.1, 0.05, and 0.025 M, ( C ) four carboxylic acids at 0.025 M: oxydiacetic, d -tartaric, citric, and

    Techniques Used:

    Mg/Ca ratio in the solid vs. log( K Mg-ligand / K Ca-ligand ) for 0.025 M oxydiacetic, d -tartaric, citric, glutamic, malonic, and aspartic acids. Shaded boxes show compositional ranges of high magnesium calcite and dolomite.
    Figure Legend Snippet: Mg/Ca ratio in the solid vs. log( K Mg-ligand / K Ca-ligand ) for 0.025 M oxydiacetic, d -tartaric, citric, glutamic, malonic, and aspartic acids. Shaded boxes show compositional ranges of high magnesium calcite and dolomite.

    Techniques Used:

    17) Product Images from "Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity"

    Article Title: Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004365

    Li AS-A expression and localization in  L .  infantum . A)  AS-A expression in different stages of  L .  infantum  life cycle. Promastigote forms: logarithmic phase (Log), early stationary phase (ES), late stationary phase (LS); axenic amastigote forms (Am). Twenty μg of total extract were analysed by Western-blot and probed with rabbit polyclonal anti- Li AS-A. α-tubulin (mouse monoclonal antibody) was used as loading control. These results are representative of 3 independent experiments.  B)  Immunofluorescence analysis showing AS-A (red upper panel; green lower panel) localization in  L .  infantum  promastigote form. Nucleus and kinetoplast DNA, cytosol and mitochondria were stained with DAPI (blue), sheep anti- Li TDR1 (thiol-dependent reductase in green) and Mitotracker Orange CMTMROS (red), respectively. Images were acquired with a 100x objective, using a Zeiss AxioImager Z1. The scale bar corresponds to 5 μm. Data is representative of 4 independent experiments.  C)  Digitonin fractionation of mid-log  L .  infantum  promastigotes. Pellet (P) and supernatant (S) fractions obtained using increasing concentrations of digitonin or positive control with 1% of Triton X-100 (TR–Total Release) were subjected to Western-blot analysis and probed with antibodies against  Li Enolase (cytosolic marker) and hypoxanthine guanine phosphoribosyltransferase  Li HGPRT (glycosomal marker). Data is representative of 5 independent experiments.
    Figure Legend Snippet: Li AS-A expression and localization in L . infantum . A) AS-A expression in different stages of L . infantum life cycle. Promastigote forms: logarithmic phase (Log), early stationary phase (ES), late stationary phase (LS); axenic amastigote forms (Am). Twenty μg of total extract were analysed by Western-blot and probed with rabbit polyclonal anti- Li AS-A. α-tubulin (mouse monoclonal antibody) was used as loading control. These results are representative of 3 independent experiments. B) Immunofluorescence analysis showing AS-A (red upper panel; green lower panel) localization in L . infantum promastigote form. Nucleus and kinetoplast DNA, cytosol and mitochondria were stained with DAPI (blue), sheep anti- Li TDR1 (thiol-dependent reductase in green) and Mitotracker Orange CMTMROS (red), respectively. Images were acquired with a 100x objective, using a Zeiss AxioImager Z1. The scale bar corresponds to 5 μm. Data is representative of 4 independent experiments. C) Digitonin fractionation of mid-log L . infantum promastigotes. Pellet (P) and supernatant (S) fractions obtained using increasing concentrations of digitonin or positive control with 1% of Triton X-100 (TR–Total Release) were subjected to Western-blot analysis and probed with antibodies against Li Enolase (cytosolic marker) and hypoxanthine guanine phosphoribosyltransferase Li HGPRT (glycosomal marker). Data is representative of 5 independent experiments.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining, Fractionation, Positive Control, Marker

    18) Product Images from "Proteases Inhibition Assessment on PC12 and NGF Treated Cells after Oxygen and Glucose Deprivation Reveals a Distinct Role for Aspartyl Proteases"

    Article Title: Proteases Inhibition Assessment on PC12 and NGF Treated Cells after Oxygen and Glucose Deprivation Reveals a Distinct Role for Aspartyl Proteases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025950

    Effect of 16 hours Oxygen-Glucose Deprivation (OGD) on % survival of PC12 cells in the absence or presence of different concentrations of the aspartyl protease inhibitor pepstatin A (1 µM and 5 µM). The values are presented as mean ± SEM, *p
    Figure Legend Snippet: Effect of 16 hours Oxygen-Glucose Deprivation (OGD) on % survival of PC12 cells in the absence or presence of different concentrations of the aspartyl protease inhibitor pepstatin A (1 µM and 5 µM). The values are presented as mean ± SEM, *p

    Techniques Used: Protease Inhibitor

    19) Product Images from "Two sources of endogenous H2O2 in Escherichia coli"

    Article Title: Two sources of endogenous H2O2 in Escherichia coli

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2010.07059.x

    Pathways of NAD + biosynthesis. Top: NadB-dependent formation of NAD + from aspartate. Bottom: Proposed conversion of β-alanine supplements to NAD + . The requirement for PuuE and PncB was demonstrated; the involvement of NadA is speculative (see
    Figure Legend Snippet: Pathways of NAD + biosynthesis. Top: NadB-dependent formation of NAD + from aspartate. Bottom: Proposed conversion of β-alanine supplements to NAD + . The requirement for PuuE and PncB was demonstrated; the involvement of NadA is speculative (see

    Techniques Used:

    Suppression of H 2 O 2 formation by β-alanine occurs through catabolism rather than pantothenate synthesis. A. In vivo H 2 O 2 production by the catalase/peroxidase-deficient parent strain (LC106, triangles) and a panC mutant (SSK62, diamonds). Open
    Figure Legend Snippet: Suppression of H 2 O 2 formation by β-alanine occurs through catabolism rather than pantothenate synthesis. A. In vivo H 2 O 2 production by the catalase/peroxidase-deficient parent strain (LC106, triangles) and a panC mutant (SSK62, diamonds). Open

    Techniques Used: In Vivo, Mutagenesis

    20) Product Images from "Developmental and neurochemical features of cholinergic neurons in the murine cerebral cortex"

    Article Title: Developmental and neurochemical features of cholinergic neurons in the murine cerebral cortex

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-10-18

    Confocal microscopical analysis of ChAT+ neurons labelled with calretinin (CR) and different GABAergic markers . A, B, C, D, E : during postnatal development at P11 ( A, B ), P16 ( C, D ) and P22 ( E ), ChAT+ cells (red) sometimes also contain (white arrows) GABAergic markers (green). GABA was revealed with either a polyclonal (GABAp in A ) or a monoclonal (GABAm in D ) antibody. Panels C and E show labelling of GAD67; in particular the large white arrowhead in C indicates a neuron that presented triple immunoreaction for ChAT/GAD67/CR (blue). No double labelling was found using GAD65 as GABAergic marker ( B ). In the adult (Ad), ChAT+ cells (red) only showed occasional immunoreactivity for GABAergic markers (green), as shown in F (GAD65), G (GABAm) and H (GABAp). In G , the large white arrowhead indicates again a cell labelled also for CR (blue). I, L : in the adult transgenic (Tg) GAD67-GFP mouse, no ChAT+ neurons (red) expressed GFP (green), but two of them contained CR (blue signal in L , small white arrowheads). Small red arrowheads: single ChAT+ neurons. Small green arrowheads: single GABAergic cells. Small white arrowheads: double ChAT+/CR+ neurons. Scale bar: 23 μm in A , B and E ; 20 μm in C , F and G ; 33 μm in D and I ; 11 μm in L .
    Figure Legend Snippet: Confocal microscopical analysis of ChAT+ neurons labelled with calretinin (CR) and different GABAergic markers . A, B, C, D, E : during postnatal development at P11 ( A, B ), P16 ( C, D ) and P22 ( E ), ChAT+ cells (red) sometimes also contain (white arrows) GABAergic markers (green). GABA was revealed with either a polyclonal (GABAp in A ) or a monoclonal (GABAm in D ) antibody. Panels C and E show labelling of GAD67; in particular the large white arrowhead in C indicates a neuron that presented triple immunoreaction for ChAT/GAD67/CR (blue). No double labelling was found using GAD65 as GABAergic marker ( B ). In the adult (Ad), ChAT+ cells (red) only showed occasional immunoreactivity for GABAergic markers (green), as shown in F (GAD65), G (GABAm) and H (GABAp). In G , the large white arrowhead indicates again a cell labelled also for CR (blue). I, L : in the adult transgenic (Tg) GAD67-GFP mouse, no ChAT+ neurons (red) expressed GFP (green), but two of them contained CR (blue signal in L , small white arrowheads). Small red arrowheads: single ChAT+ neurons. Small green arrowheads: single GABAergic cells. Small white arrowheads: double ChAT+/CR+ neurons. Scale bar: 23 μm in A , B and E ; 20 μm in C , F and G ; 33 μm in D and I ; 11 μm in L .

    Techniques Used: Marker, Transgenic Assay

    21) Product Images from "Striatal neurons directly converted from Huntington’s disease patient fibroblasts recapitulate age-associated disease phenotypes"

    Article Title: Striatal neurons directly converted from Huntington’s disease patient fibroblasts recapitulate age-associated disease phenotypes

    Journal: Nature neuroscience

    doi: 10.1038/s41593-018-0075-7

    HD patient fibroblasts can be directly reprogrammed into MSNs. Fibroblasts from three HD patients (with mHTT expansions of 40, 43 and 44 CAGs) and their respective age- and sex-matched controls (CAG sizes of 19, 17 and 18) reprogrammed into MSNs with miR-9/9*-124+CDM. (a), Reprogrammed HD.40 at post-induction day (PID) 30 immunostained with TUBB3, and HD.44 with TUBB3, NeuN, MAP2, DARPP-32 and GABA. (b), Images of all three pairs of cell lines immunostained with GABA and DARPP-32. (c), Quantification of TUBB3, GABA and DARPP-32-positive cells at PID 30; n=averages of 1,000 cells from 3 independent HD and control lines. Unpaired t-test corrected for multiple comparisons using the Holm-Sidak method; (from left, P =0.98, 0.97, 0.98; df=4) n.s.=not significant. Scale bar: 50 μm. Mean ± s.e.m.
    Figure Legend Snippet: HD patient fibroblasts can be directly reprogrammed into MSNs. Fibroblasts from three HD patients (with mHTT expansions of 40, 43 and 44 CAGs) and their respective age- and sex-matched controls (CAG sizes of 19, 17 and 18) reprogrammed into MSNs with miR-9/9*-124+CDM. (a), Reprogrammed HD.40 at post-induction day (PID) 30 immunostained with TUBB3, and HD.44 with TUBB3, NeuN, MAP2, DARPP-32 and GABA. (b), Images of all three pairs of cell lines immunostained with GABA and DARPP-32. (c), Quantification of TUBB3, GABA and DARPP-32-positive cells at PID 30; n=averages of 1,000 cells from 3 independent HD and control lines. Unpaired t-test corrected for multiple comparisons using the Holm-Sidak method; (from left, P =0.98, 0.97, 0.98; df=4) n.s.=not significant. Scale bar: 50 μm. Mean ± s.e.m.

    Techniques Used:

    22) Product Images from "Rescue from galactose-induced death of Leigh Syndrome patient cells by pyruvate and NAD+"

    Article Title: Rescue from galactose-induced death of Leigh Syndrome patient cells by pyruvate and NAD+

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1179-4

    The viability of LS fibroblasts is reduced in galactose medium in a cell density-dependent manner and rescued by specific metabolites. a Effect of cell seeding density on the galactose-induced reduction in viability of LS cells (S7, S8, V1; measured at 96 h; N = 1, n = 8). b Specificity of the galactose-induced reduction in viability (2500 cells seeded; measured at 96 h; N = 3, n ≥ 12) for LS cells relative to CT cells (CT1, CT2, CT3). c Experimental protocol used in this study to quantify the effects of galactose and other treatments on control and LS cells. d Dose-dependent rescue of the galactose-induced reduction in viability of LS cells (S7, S8, V1) at 96 h by pyruvate in the presence of a constant glutamine concentration ( N = 3, n ≥ 9). The EC50 value was determined by fitting a Hill equation to the data ( R 2 = 0.97). e Same as d , but now for the dose-dependent effect of glutamine in the presence of a constant pyruvate concentration ( N = 3, n ≥ 18). The EC50 value was determined by fitting a Hill equation to the data ( R 2 = 0.98). f Rescuing effect of specific metabolites on the galactose-induced reduction in LS cell viability (96 h; Calcein MSK images for patient S7 are shown as a typical example): sodium pyruvate (10 mM), sodium l -lactate (10 mM), sodium succinate dibasic hexahydrate (10 mM), l -(-)-malic acid (10 mM), oxaloacetic acid (10 mM), dimethyl α-ketoglutarate (10 mM), 2-ketobutyric acid (10 mM), l -aspartic acid (15 mM), and uridine (1 mM). The “ + ” conditions reflect combined galactose + metabolite treatment. g Quantification of the rescuing effect of the metabolites ( N = 3, n ≥ 18) in LS patient cells (S7, S8, V1) at 96 h ( N = 3, n ≥ 18). Statistics: * P
    Figure Legend Snippet: The viability of LS fibroblasts is reduced in galactose medium in a cell density-dependent manner and rescued by specific metabolites. a Effect of cell seeding density on the galactose-induced reduction in viability of LS cells (S7, S8, V1; measured at 96 h; N = 1, n = 8). b Specificity of the galactose-induced reduction in viability (2500 cells seeded; measured at 96 h; N = 3, n ≥ 12) for LS cells relative to CT cells (CT1, CT2, CT3). c Experimental protocol used in this study to quantify the effects of galactose and other treatments on control and LS cells. d Dose-dependent rescue of the galactose-induced reduction in viability of LS cells (S7, S8, V1) at 96 h by pyruvate in the presence of a constant glutamine concentration ( N = 3, n ≥ 9). The EC50 value was determined by fitting a Hill equation to the data ( R 2 = 0.97). e Same as d , but now for the dose-dependent effect of glutamine in the presence of a constant pyruvate concentration ( N = 3, n ≥ 18). The EC50 value was determined by fitting a Hill equation to the data ( R 2 = 0.98). f Rescuing effect of specific metabolites on the galactose-induced reduction in LS cell viability (96 h; Calcein MSK images for patient S7 are shown as a typical example): sodium pyruvate (10 mM), sodium l -lactate (10 mM), sodium succinate dibasic hexahydrate (10 mM), l -(-)-malic acid (10 mM), oxaloacetic acid (10 mM), dimethyl α-ketoglutarate (10 mM), 2-ketobutyric acid (10 mM), l -aspartic acid (15 mM), and uridine (1 mM). The “ + ” conditions reflect combined galactose + metabolite treatment. g Quantification of the rescuing effect of the metabolites ( N = 3, n ≥ 18) in LS patient cells (S7, S8, V1) at 96 h ( N = 3, n ≥ 18). Statistics: * P

    Techniques Used: Concentration Assay

    Exogenous NAD + (eNAD) rescues the galactose-induced reduction in cell viability and intracellular NAD + (iNAD) content in LS fibroblasts. a Dose-dependent effect of extracellular sodium pyruvate (10 mM), oxaloacetic acid (10 mM), dimethyl α-ketoglutarate (10 mM), 2-ketobutyric acid (10 mM), and l -aspartic acid (15 mM) on intracellular NAD (iNAD) levels in LS cells (24 h; N = 1, n = 3). b Dose-dependent effect of extracellular NAD (eNAD; β-nicotinamide adenine dinucleotide hydrate) on intracellular NAD (iNAD) levels (24 h; N = 1, n ≥ 6). c Dose-dependent rescue of the galactose-induced reduction in viability of LS cells (Calcein MSK images) by eNAD (β-nicotinamide adenine dinucleotide hydrate; S7 shown as a typical example; 96 h). d Quantification of the rescuing effect of eNAD in LS patient cells (96 h; N = 2 n ≥ 9). e Dose-dependent inhibition of the rescuing effect of eNAD by oleamide (OLE) in LS patient cells (96 h; N = 3, n = ≥ 6). Statistics: * P
    Figure Legend Snippet: Exogenous NAD + (eNAD) rescues the galactose-induced reduction in cell viability and intracellular NAD + (iNAD) content in LS fibroblasts. a Dose-dependent effect of extracellular sodium pyruvate (10 mM), oxaloacetic acid (10 mM), dimethyl α-ketoglutarate (10 mM), 2-ketobutyric acid (10 mM), and l -aspartic acid (15 mM) on intracellular NAD (iNAD) levels in LS cells (24 h; N = 1, n = 3). b Dose-dependent effect of extracellular NAD (eNAD; β-nicotinamide adenine dinucleotide hydrate) on intracellular NAD (iNAD) levels (24 h; N = 1, n ≥ 6). c Dose-dependent rescue of the galactose-induced reduction in viability of LS cells (Calcein MSK images) by eNAD (β-nicotinamide adenine dinucleotide hydrate; S7 shown as a typical example; 96 h). d Quantification of the rescuing effect of eNAD in LS patient cells (96 h; N = 2 n ≥ 9). e Dose-dependent inhibition of the rescuing effect of eNAD by oleamide (OLE) in LS patient cells (96 h; N = 3, n = ≥ 6). Statistics: * P

    Techniques Used: Inhibition

    23) Product Images from "Activation of apoptosis in NAF-1-deficient human epithelial breast cancer cells"

    Article Title: Activation of apoptosis in NAF-1-deficient human epithelial breast cancer cells

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.178293

    Activation of apoptosis in xenograft tumors and cancer cells with suppressed levels of NAF-1. (A) Left, immunohistochemistry (IHC) analysis using an antibody against activated caspase-3 showing a higher number of active-caspase-3-positive cells in NAF-1(−) tumors. Active-caspase-3-positive cells are marked by white arrowheads. Right, quantification of staining of active caspase-3. (B) Left, IHC analysis using an antibody against γH2AX showing a higher number of γH2AX-positive cells in NAF-1(−) tumors. Right, quantification of γH2AX staining. For A,B, cells were counted in ten high-power fields (×40) for each section obtained from five mice in each group; *** P
    Figure Legend Snippet: Activation of apoptosis in xenograft tumors and cancer cells with suppressed levels of NAF-1. (A) Left, immunohistochemistry (IHC) analysis using an antibody against activated caspase-3 showing a higher number of active-caspase-3-positive cells in NAF-1(−) tumors. Active-caspase-3-positive cells are marked by white arrowheads. Right, quantification of staining of active caspase-3. (B) Left, IHC analysis using an antibody against γH2AX showing a higher number of γH2AX-positive cells in NAF-1(−) tumors. Right, quantification of γH2AX staining. For A,B, cells were counted in ten high-power fields (×40) for each section obtained from five mice in each group; *** P

    Techniques Used: Activation Assay, Immunohistochemistry, Staining, Mouse Assay

    24) Product Images from "Probing the Dynamics of Doxorubicin-DNA Intercalation during the Initial Activation of Apoptosis by Fluorescence Lifetime Imaging Microscopy (FLIM)"

    Article Title: Probing the Dynamics of Doxorubicin-DNA Intercalation during the Initial Activation of Apoptosis by Fluorescence Lifetime Imaging Microscopy (FLIM)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044947

    Doxorubicin induces caspase-3 activation and H2AX phosphorylation. HeLa cells were exposed to 5 µg/ml doxorubicin for different periods, after which cell lysates of each time point were extracted for either measurement of caspase-3 activity or fixed for the γH2AX staining. Doxorubicin fluorescence lifetimes following treatment (black) vs. (A) caspase-3 activity (blue); and (B) γH2AX activation (red). γH2AX activation was quantified from confocal microscopic images, as shown in (C), using MetaMorph imaging processing software. As illustrated, caspase-3 activation is preceded by the H2AX phosphorylation. (Scale bars in C: 20 µm).
    Figure Legend Snippet: Doxorubicin induces caspase-3 activation and H2AX phosphorylation. HeLa cells were exposed to 5 µg/ml doxorubicin for different periods, after which cell lysates of each time point were extracted for either measurement of caspase-3 activity or fixed for the γH2AX staining. Doxorubicin fluorescence lifetimes following treatment (black) vs. (A) caspase-3 activity (blue); and (B) γH2AX activation (red). γH2AX activation was quantified from confocal microscopic images, as shown in (C), using MetaMorph imaging processing software. As illustrated, caspase-3 activation is preceded by the H2AX phosphorylation. (Scale bars in C: 20 µm).

    Techniques Used: Activation Assay, Activity Assay, Staining, Fluorescence, Imaging, Software

    25) Product Images from "Modulation of redox signaling promotes apoptosis in epithelial ovarian cancer cells"

    Article Title: Modulation of redox signaling promotes apoptosis in epithelial ovarian cancer cells

    Journal: Gynecologic oncology

    doi: 10.1016/j.ygyno.2011.04.051

    (A) Caspase-3 activity and (B) apoptosis in EOC cells. (A) Caspase-3 activity was measured in cell lysates from SKOV-3 and MDAH-2774 before and after DPI treatment (10 μM) at various time points. (B) The amount of DNA fragmentation (apoptosis) was assessed by TUNEL assay in MDAH-2774 and SKOV-3, before and after DPI treatment (10 μM, 12 and 24 hrs) as compared to control cells. Nuclei were stained with DAPI (blue) and apoptotic cells were visualized (60x) with fluorescein-12-dUTP (green). Results are representative of the mean of three independent experiments. (* All p
    Figure Legend Snippet: (A) Caspase-3 activity and (B) apoptosis in EOC cells. (A) Caspase-3 activity was measured in cell lysates from SKOV-3 and MDAH-2774 before and after DPI treatment (10 μM) at various time points. (B) The amount of DNA fragmentation (apoptosis) was assessed by TUNEL assay in MDAH-2774 and SKOV-3, before and after DPI treatment (10 μM, 12 and 24 hrs) as compared to control cells. Nuclei were stained with DAPI (blue) and apoptotic cells were visualized (60x) with fluorescein-12-dUTP (green). Results are representative of the mean of three independent experiments. (* All p

    Techniques Used: Activity Assay, TUNEL Assay, Staining

    26) Product Images from "The Protective Effect of Different Extracts of Three Artemisia Species against H2O2-Induced Oxidative Stress and Apoptosis in PC12 Neuronal Cells"

    Article Title: The Protective Effect of Different Extracts of Three Artemisia Species against H2O2-Induced Oxidative Stress and Apoptosis in PC12 Neuronal Cells

    Journal: Pharmacognosy Research

    doi: 10.4103/pr.pr_98_17

    The effect of selected Artemisia turanica (6.25 μg/mL) (a) and Artemisia turcomanica extracts (6.25 μg/mL) (b) extracts on the caspase-3 activity. Caspase-3 activity was measured by colorimetric detection and expressed as percent of control. Data are expressed as the mean ± standard error of the mean of three separate experiments. # P
    Figure Legend Snippet: The effect of selected Artemisia turanica (6.25 μg/mL) (a) and Artemisia turcomanica extracts (6.25 μg/mL) (b) extracts on the caspase-3 activity. Caspase-3 activity was measured by colorimetric detection and expressed as percent of control. Data are expressed as the mean ± standard error of the mean of three separate experiments. # P

    Techniques Used: Activity Assay

    27) Product Images from "Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis"

    Article Title: Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145460

    Ceramide induces GSK-3β-, caspase-2-, and proteasome-regulated Mcl-1 degradation followed by lysosomal-mitochondrial apoptosis. (A) We used Western blotting to determine the expression of Mcl-1 in 10I cells treated with different doses of C 2 -ceramide. C 2 -dihydroceramide was used as a negative control. (B) 10I cells were treated with 25 μM C 2 -ceramide in the presence or absence of 10 mM LiCl for the indicated time periods. We used Western blotting to determine the expression of Mcl-1. (C, D, and E) We used Western blotting to determine Mcl-1 expression and the activation of caspase-2, caspase-8, Bid, and caspase-3 in 25 μM C 2 -ceramide-treated 10I cells with or without z-VAD-fmk (10 μM), z-VDVAD-fmk (10 μM), z-IETD-fmk (10 μM), LiCl (10 mM), or the proteasome inhibitor MG132 (25 μM) for 6 h. 10I cells were transfected with shRNAs against GSK-3β (shGSK-3β) or caspase-2 (shCasp-2) or a negative-control shRNA (shLuc). The expression of Mcl-1 in ceramide-treated transfected cells was detected using Western blot analysis. β-actin served as an internal control. (F) A549 cells were treated with 25 μM C 2 -ceramide for 24 h in the presence or absence of 25 μM MG132. The translocation of cathepsin D was determined using a cathepsin D-specific antibody followed by an Alexa Fluor 488-labeled secondary antibody and DAPI nuclear staining. The scale bar is 10 μm. (G) 10I cells were treated with 25 μM C 2 -ceramide for 6 h in the presence or absence of 25 μM MG132. Using AO (top), rhodamine 123 (middle), and PI (bottom) staining, respectively, followed by flow cytometric analysis, the percentages of cells with LMP, MTP reduction, and apoptosis are shown (means ± S.D. of three individual experiments). A representative histogram is shown. *, P
    Figure Legend Snippet: Ceramide induces GSK-3β-, caspase-2-, and proteasome-regulated Mcl-1 degradation followed by lysosomal-mitochondrial apoptosis. (A) We used Western blotting to determine the expression of Mcl-1 in 10I cells treated with different doses of C 2 -ceramide. C 2 -dihydroceramide was used as a negative control. (B) 10I cells were treated with 25 μM C 2 -ceramide in the presence or absence of 10 mM LiCl for the indicated time periods. We used Western blotting to determine the expression of Mcl-1. (C, D, and E) We used Western blotting to determine Mcl-1 expression and the activation of caspase-2, caspase-8, Bid, and caspase-3 in 25 μM C 2 -ceramide-treated 10I cells with or without z-VAD-fmk (10 μM), z-VDVAD-fmk (10 μM), z-IETD-fmk (10 μM), LiCl (10 mM), or the proteasome inhibitor MG132 (25 μM) for 6 h. 10I cells were transfected with shRNAs against GSK-3β (shGSK-3β) or caspase-2 (shCasp-2) or a negative-control shRNA (shLuc). The expression of Mcl-1 in ceramide-treated transfected cells was detected using Western blot analysis. β-actin served as an internal control. (F) A549 cells were treated with 25 μM C 2 -ceramide for 24 h in the presence or absence of 25 μM MG132. The translocation of cathepsin D was determined using a cathepsin D-specific antibody followed by an Alexa Fluor 488-labeled secondary antibody and DAPI nuclear staining. The scale bar is 10 μm. (G) 10I cells were treated with 25 μM C 2 -ceramide for 6 h in the presence or absence of 25 μM MG132. Using AO (top), rhodamine 123 (middle), and PI (bottom) staining, respectively, followed by flow cytometric analysis, the percentages of cells with LMP, MTP reduction, and apoptosis are shown (means ± S.D. of three individual experiments). A representative histogram is shown. *, P

    Techniques Used: Western Blot, Expressing, Negative Control, Activation Assay, Transfection, shRNA, Translocation Assay, Labeling, Staining, Flow Cytometry

    Inhibiting cathepsin D inhibits the ceramide- or etoposide-induced activation of caspase-8, but not caspase-2, and mitochondrial apoptosis. (A) 10I cells were treated with 25 μM C 2 -ceramide or 50 μM etoposide for 6 h in the presence or absence of the cathepsin D inhibitor pepstatin A (25 μM). (B) A549 cells were treated with 25 μM C 2 -ceramide for 24 h with or without cathepsin D siRNA (50 nM) pretreatment. (C) To further determine whether cathepsin D affects caspase-8 activation, recombinant human cathepsin D was incubated together with lysates from untreated A549 cells for 0.5 h at 37°C with or without 25 μM pepstatin A or 10 μM of the caspase-8 inhibitor z-IETD-fmk. We used enzymatic cleavage of the specific substrates benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-pNA and benzyloxycarbonyl-Ile-Glu(-OMe)-Thr-Asp(-OMe)-pNA to determine the activities of caspase-2 and caspase-8, respectively. OD, optical density. Data are given as the average of three individual experiments (means ± S.D.). *, P
    Figure Legend Snippet: Inhibiting cathepsin D inhibits the ceramide- or etoposide-induced activation of caspase-8, but not caspase-2, and mitochondrial apoptosis. (A) 10I cells were treated with 25 μM C 2 -ceramide or 50 μM etoposide for 6 h in the presence or absence of the cathepsin D inhibitor pepstatin A (25 μM). (B) A549 cells were treated with 25 μM C 2 -ceramide for 24 h with or without cathepsin D siRNA (50 nM) pretreatment. (C) To further determine whether cathepsin D affects caspase-8 activation, recombinant human cathepsin D was incubated together with lysates from untreated A549 cells for 0.5 h at 37°C with or without 25 μM pepstatin A or 10 μM of the caspase-8 inhibitor z-IETD-fmk. We used enzymatic cleavage of the specific substrates benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-pNA and benzyloxycarbonyl-Ile-Glu(-OMe)-Thr-Asp(-OMe)-pNA to determine the activities of caspase-2 and caspase-8, respectively. OD, optical density. Data are given as the average of three individual experiments (means ± S.D.). *, P

    Techniques Used: Activation Assay, Recombinant, Incubation

    28) Product Images from "Achyranthes bidentata Polypeptides Reduces Oxidative Stress and Exerts Protective Effects against Myocardial Ischemic/Reperfusion Injury in Rats"

    Article Title: Achyranthes bidentata Polypeptides Reduces Oxidative Stress and Exerts Protective Effects against Myocardial Ischemic/Reperfusion Injury in Rats

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms141019792

    Achyranthes bidentata polypeptides (ABPP) preconditioning reduced myocardial apoptotic index and caspase-3 activity in rats subjected to myocardial ischemia/reperfusion. ( A ) Top: representative photomicrographs of in situ detection of apoptotic myocytes by terminal deoxynucleotidyl nickend labeling (TUNEL) staining in ischemic heart tissue from rats subjected to 30 min of ischemia and 4 h of reperfusion. Green fluorescence shows TUNEL-positive nuclei; Blue fluorescence shows nuclei of total cardiomyocytes. Bottom: percentage of TUNEL-positive nuclei in heart tissue sections; ( B ) Myocardial caspase-3 activity. MI/R, myocardial ischaemia/reperfusion (30 min/4 h); Sham, sham-operated; ABPP, Achyranthes bidentata polypeptides. Values presented are means ± SEM. n = 8/group. ** p
    Figure Legend Snippet: Achyranthes bidentata polypeptides (ABPP) preconditioning reduced myocardial apoptotic index and caspase-3 activity in rats subjected to myocardial ischemia/reperfusion. ( A ) Top: representative photomicrographs of in situ detection of apoptotic myocytes by terminal deoxynucleotidyl nickend labeling (TUNEL) staining in ischemic heart tissue from rats subjected to 30 min of ischemia and 4 h of reperfusion. Green fluorescence shows TUNEL-positive nuclei; Blue fluorescence shows nuclei of total cardiomyocytes. Bottom: percentage of TUNEL-positive nuclei in heart tissue sections; ( B ) Myocardial caspase-3 activity. MI/R, myocardial ischaemia/reperfusion (30 min/4 h); Sham, sham-operated; ABPP, Achyranthes bidentata polypeptides. Values presented are means ± SEM. n = 8/group. ** p

    Techniques Used: Activity Assay, In Situ, Labeling, TUNEL Assay, Staining, Fluorescence

    29) Product Images from "Enhanced NMDA receptor NR1 phosphorylation and neuronal activity in the arcuate nucleus of hypothalamus following peripheral inflammation"

    Article Title: Enhanced NMDA receptor NR1 phosphorylation and neuronal activity in the arcuate nucleus of hypothalamus following peripheral inflammation

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2010.190

    MK-801 (NMDA receptor antagonist) and CNQX (non-NMDA receptor antagonist) inhibit spontaneous discharge of ARC neurons in inflamed rats. (A) A significant decrease in neuronal discharge frequency was observed following MK-801 application (300 μmol/L) in inflamed rats ( n =11; P
    Figure Legend Snippet: MK-801 (NMDA receptor antagonist) and CNQX (non-NMDA receptor antagonist) inhibit spontaneous discharge of ARC neurons in inflamed rats. (A) A significant decrease in neuronal discharge frequency was observed following MK-801 application (300 μmol/L) in inflamed rats ( n =11; P

    Techniques Used:

    30) Product Images from "Long-term depression-inducing stimuli promote cleavage of the synaptic adhesion molecule NGL-3 through NMDA receptors, matrix metalloproteinases and presenilin/?-secretase"

    Article Title: Long-term depression-inducing stimuli promote cleavage of the synaptic adhesion molecule NGL-3 through NMDA receptors, matrix metalloproteinases and presenilin/?-secretase

    Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

    doi: 10.1098/rstb.2013.0158

    NGL-3 cleavage is blocked by inhibition of MMPs and presenilin/γ-secretase. ( a ) MMP inhibition blocks NMDA-induced NGL-3 cleavage. Rat hippocampal neurons at DIV 18–21 were incubated with GM6001 (2.5 and 25 µM, 30 min) before and during NMDA stimulation (20 µM, 3 min), followed by immunoblotting. The bar graphs represent mean ± s.e.m; n = 4; ** p
    Figure Legend Snippet: NGL-3 cleavage is blocked by inhibition of MMPs and presenilin/γ-secretase. ( a ) MMP inhibition blocks NMDA-induced NGL-3 cleavage. Rat hippocampal neurons at DIV 18–21 were incubated with GM6001 (2.5 and 25 µM, 30 min) before and during NMDA stimulation (20 µM, 3 min), followed by immunoblotting. The bar graphs represent mean ± s.e.m; n = 4; ** p

    Techniques Used: Inhibition, Incubation

    31) Product Images from "Selective cytotoxicity of intracellular amyloid ? peptide1–42 through p53 and Bax in cultured primary human neurons"

    Article Title: Selective cytotoxicity of intracellular amyloid ? peptide1–42 through p53 and Bax in cultured primary human neurons

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200110119

    Inhibition of Aβ 1–42 - mediated neuronal cell death with caspase inhibitors. Neurons were preincubated for 1 h in the presence of 5 μM of each inhibitor, microinjected with Aβ 1–42 peptide and incubated for 24 h in the presence of the inhibitors before revealing cell death of injected cells with TUNEL. The data represent the mean ± SEM of three independent experiments. One-way ANOVA (df = 11) followed by Sheffé's test showed a statistically significant difference between Aβ 1–42 and Aβ 1–42 + Z-VAD-fmk, Z-VEID-fmk, and Z-IETD-fmk. *, P
    Figure Legend Snippet: Inhibition of Aβ 1–42 - mediated neuronal cell death with caspase inhibitors. Neurons were preincubated for 1 h in the presence of 5 μM of each inhibitor, microinjected with Aβ 1–42 peptide and incubated for 24 h in the presence of the inhibitors before revealing cell death of injected cells with TUNEL. The data represent the mean ± SEM of three independent experiments. One-way ANOVA (df = 11) followed by Sheffé's test showed a statistically significant difference between Aβ 1–42 and Aβ 1–42 + Z-VAD-fmk, Z-VEID-fmk, and Z-IETD-fmk. *, P

    Techniques Used: Inhibition, Incubation, Injection, TUNEL Assay

    32) Product Images from "Aspartame and Phe-Containing Degradation Products in Soft Drinks across Europe"

    Article Title: Aspartame and Phe-Containing Degradation Products in Soft Drinks across Europe

    Journal: Nutrients

    doi: 10.3390/nu12061887

    Aspartame and total phenylalanine contents of all 111 soft drinks. Specifics regarding the soft drinks and their countries of origin can be found in Supplementary Table S2 , where the first letter indicates the group, the second letter indicates the cluster, and the number indicates the number of the soft drink. APM, aspartame, Phe, phenylalanine.
    Figure Legend Snippet: Aspartame and total phenylalanine contents of all 111 soft drinks. Specifics regarding the soft drinks and their countries of origin can be found in Supplementary Table S2 , where the first letter indicates the group, the second letter indicates the cluster, and the number indicates the number of the soft drink. APM, aspartame, Phe, phenylalanine.

    Techniques Used:

    33) Product Images from "Positive/Negative Allosteric Modulation Switching in an Umami Taste Receptor (T1R1/T1R3) by a Natural Flavor Compound, Methional"

    Article Title: Positive/Negative Allosteric Modulation Switching in an Umami Taste Receptor (T1R1/T1R3) by a Natural Flavor Compound, Methional

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30315-x

    Methional and 3-(methylthio)butanal (8) enhanced l -Glu responses in the presence of IMP. ( a ) HEK293T cells coexpressing hT1R1/hT1R3 together with hG16gi3 were stimulated with 2 mM l -Glu in the absence or presence of 120 μM methional (1) , 120 μM IMP, or both. ( b ) HEK293T cells coexpressing hT1R1/hT1R3 together with hG16gi3 were stimulated with 2 mM l -Glu in the absence or presence of 120 μM 3-(methylthio)butanal (8) , 120 μM IMP, or both. Values represent the mean ± SE of the RLU(AUC) of 6 recorded wells. Means followed by a different letter are significantly different, as analyzed by Tukey’s test (p
    Figure Legend Snippet: Methional and 3-(methylthio)butanal (8) enhanced l -Glu responses in the presence of IMP. ( a ) HEK293T cells coexpressing hT1R1/hT1R3 together with hG16gi3 were stimulated with 2 mM l -Glu in the absence or presence of 120 μM methional (1) , 120 μM IMP, or both. ( b ) HEK293T cells coexpressing hT1R1/hT1R3 together with hG16gi3 were stimulated with 2 mM l -Glu in the absence or presence of 120 μM 3-(methylthio)butanal (8) , 120 μM IMP, or both. Values represent the mean ± SE of the RLU(AUC) of 6 recorded wells. Means followed by a different letter are significantly different, as analyzed by Tukey’s test (p

    Techniques Used:

    Widespread areas engendering the PAM/NAM activities of methional. ( a , b ) Two distinct putative binding sites for methional. A methional molecule docked into the homology model of the transmembrane domain of mouse-type hT1R1. The residues that confer PAM activity are represented in magenta, those that confer NAM activity are represented in blue, and those that contribute to PAM/NAM mode-switching are represented in yellow. Two residues in which an alanine substitution changed the activity of methional from that of a PAM to that of a NAM are represented in green. The residues are also numbered according to the Ballesteros-Weinstein numbering scheme 29 . A representative docking pose with the highest Glide docking score is shown ( a ). Methional is shown as a space-filling model. All of the top 50 poses, ranked by the Glide docking score, are superimposed and displayed, and methional molecules are shown in black ( b ). All of the 40 top poses showed that methional binds to the lower region of the allosteric pocket, while 8 of the top poses ranked from 41 to 50 suggested a binding site in the upper region of the allosteric pocket. The dotted red circles represent the upper and lower putative binding regions of methional. ( c – f ) Methional was the strongest NAM for the mouse-type T1R1 receptor, while 3-(methylthio)butanal (8) was the strongest PAM for the human-type receptor. Dose-dependent responses to l -Glu ( c , f ) or l -Ala ( d , e ) were obtained in the presence and absence of methional and each of its analogs (120 μM). Values represent the mean ± SE of the RLU (AUC) of 6 recorded wells. The EC 50 and E max values of the receptors described in c and f are shown in Supplementary Table S3 .
    Figure Legend Snippet: Widespread areas engendering the PAM/NAM activities of methional. ( a , b ) Two distinct putative binding sites for methional. A methional molecule docked into the homology model of the transmembrane domain of mouse-type hT1R1. The residues that confer PAM activity are represented in magenta, those that confer NAM activity are represented in blue, and those that contribute to PAM/NAM mode-switching are represented in yellow. Two residues in which an alanine substitution changed the activity of methional from that of a PAM to that of a NAM are represented in green. The residues are also numbered according to the Ballesteros-Weinstein numbering scheme 29 . A representative docking pose with the highest Glide docking score is shown ( a ). Methional is shown as a space-filling model. All of the top 50 poses, ranked by the Glide docking score, are superimposed and displayed, and methional molecules are shown in black ( b ). All of the 40 top poses showed that methional binds to the lower region of the allosteric pocket, while 8 of the top poses ranked from 41 to 50 suggested a binding site in the upper region of the allosteric pocket. The dotted red circles represent the upper and lower putative binding regions of methional. ( c – f ) Methional was the strongest NAM for the mouse-type T1R1 receptor, while 3-(methylthio)butanal (8) was the strongest PAM for the human-type receptor. Dose-dependent responses to l -Glu ( c , f ) or l -Ala ( d , e ) were obtained in the presence and absence of methional and each of its analogs (120 μM). Values represent the mean ± SE of the RLU (AUC) of 6 recorded wells. The EC 50 and E max values of the receptors described in c and f are shown in Supplementary Table S3 .

    Techniques Used: Binding Assay, Activity Assay

    Cartoon representing a summary of the receptor expression experiments described in this paper. The PAM/NAM modes of methional primarily depended upon whether the residues at the bottom of the binding site were from the human or mouse amino acid sequences. PAM and NAM activities were conferred by distinct sites that were located at the upper or the lower regions, respectively, of the allosteric pocket across the microswitch responsible for receptor activation.
    Figure Legend Snippet: Cartoon representing a summary of the receptor expression experiments described in this paper. The PAM/NAM modes of methional primarily depended upon whether the residues at the bottom of the binding site were from the human or mouse amino acid sequences. PAM and NAM activities were conferred by distinct sites that were located at the upper or the lower regions, respectively, of the allosteric pocket across the microswitch responsible for receptor activation.

    Techniques Used: Expressing, Binding Assay, Activation Assay

    Identification of residues that engendered a switch in the PAM/NAM mode of methional activity. ( a ) T1R1 chimeras were designed to contain human (gray) and mouse amino acid sequences (white). ( b–d ) Dose-dependent responses to l -Ala ( b-1 , c-1 , d-1 ) or l -Glu ( b-2 , c-2 , d-2 ) were obtained for each T1R1 paired with mouse T1R3. Significant differences between amino acid responses with and without 120 μM methional were analyzed using Student’s t test (* p
    Figure Legend Snippet: Identification of residues that engendered a switch in the PAM/NAM mode of methional activity. ( a ) T1R1 chimeras were designed to contain human (gray) and mouse amino acid sequences (white). ( b–d ) Dose-dependent responses to l -Ala ( b-1 , c-1 , d-1 ) or l -Glu ( b-2 , c-2 , d-2 ) were obtained for each T1R1 paired with mouse T1R3. Significant differences between amino acid responses with and without 120 μM methional were analyzed using Student’s t test (* p

    Techniques Used: Activity Assay

    Residues that conferred the PAM and NAM activities of methional in T1R1. Dose-response curves were obtained to l -Glu for each human T1R1 mutant paired with mouse T1R3. ( a ) Of the 32 residues examined (Fig. 4 ), an alanine substitution in each of the six residues in hT1R1 caused either an abolition or a decrease in the PAM activity of methional. ( b ) An alanine substitution in mouse-type hT1R1 caused a decrease in the NAM activity of methional. ( c ) An alanine mutation in each of two residues in hT1R1 swapped the PAM and NAM activity of methional. Values represent the mean ± SE of the RLU (AUC) of 6 recorded wells. Significant differences between l -Glu responses with and without 120 μM methional were analyzed using Student’s t test (* p
    Figure Legend Snippet: Residues that conferred the PAM and NAM activities of methional in T1R1. Dose-response curves were obtained to l -Glu for each human T1R1 mutant paired with mouse T1R3. ( a ) Of the 32 residues examined (Fig. 4 ), an alanine substitution in each of the six residues in hT1R1 caused either an abolition or a decrease in the PAM activity of methional. ( b ) An alanine substitution in mouse-type hT1R1 caused a decrease in the NAM activity of methional. ( c ) An alanine mutation in each of two residues in hT1R1 swapped the PAM and NAM activity of methional. Values represent the mean ± SE of the RLU (AUC) of 6 recorded wells. Significant differences between l -Glu responses with and without 120 μM methional were analyzed using Student’s t test (* p

    Techniques Used: Mutagenesis, Activity Assay

    Methional and its structural analogs enhanced the responses of human T1R1/T1R3 to amino acids. ( a ) Methional enhanced the responses of hT1R1/hT1R3 to various amino acids. HEK293T cells coexpressing hT1R1/hT1R3 together with hG16gi3 were separately stimulated with 10 mM l -Glu or 50 mM concentrations of each amino acid except for l -Glu in the absence or presence of 120 μM methional. Significant differences between amino acid responses with and without methional were analyzed using Student’s t test (* p
    Figure Legend Snippet: Methional and its structural analogs enhanced the responses of human T1R1/T1R3 to amino acids. ( a ) Methional enhanced the responses of hT1R1/hT1R3 to various amino acids. HEK293T cells coexpressing hT1R1/hT1R3 together with hG16gi3 were separately stimulated with 10 mM l -Glu or 50 mM concentrations of each amino acid except for l -Glu in the absence or presence of 120 μM methional. Significant differences between amino acid responses with and without methional were analyzed using Student’s t test (* p

    Techniques Used:

    Distinct sets of residues conferred either the PAM or NAM activity of methional. ( a , b ) The residues described in Fig. 5a did not affect the activity of methional as a NAM. Dose-dependent responses were obtained to l -Ala for each mouse T1R1 mutant ( a ) and to l -Glu for each mouse-type human T1R1 mutant ( b ) paired with mouse T1R3. ( c , d ) The residue described in Fig. 5b did not affect the activity of methional as a PAM. Dose-dependent responses were obtained to l -Glu for the human T1R1 mutant ( c ) and to l -Ala for the human-type mouse T1R1 mutant ( d ) paired with mouse T1R3. Significant differences between l -amino acid responses with and without 120 μM methional were analyzed using Student’s t test (* p
    Figure Legend Snippet: Distinct sets of residues conferred either the PAM or NAM activity of methional. ( a , b ) The residues described in Fig. 5a did not affect the activity of methional as a NAM. Dose-dependent responses were obtained to l -Ala for each mouse T1R1 mutant ( a ) and to l -Glu for each mouse-type human T1R1 mutant ( b ) paired with mouse T1R3. ( c , d ) The residue described in Fig. 5b did not affect the activity of methional as a PAM. Dose-dependent responses were obtained to l -Glu for the human T1R1 mutant ( c ) and to l -Ala for the human-type mouse T1R1 mutant ( d ) paired with mouse T1R3. Significant differences between l -amino acid responses with and without 120 μM methional were analyzed using Student’s t test (* p

    Techniques Used: Activity Assay, Mutagenesis

    34) Product Images from "Functional connectivity of the main intercalated nucleus of the mouse amygdala"

    Article Title: Functional connectivity of the main intercalated nucleus of the mouse amygdala

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2010.201475

    AMPA/NMDA receptors mediated eEPSCs recorded in the Im
    Figure Legend Snippet: AMPA/NMDA receptors mediated eEPSCs recorded in the Im

    Techniques Used:

    35) Product Images from "Piper Essential Oils Inhibit Rhizopus oryzae Growth, Biofilm Formation, and Rhizopuspepsin Activity"

    Article Title: Piper Essential Oils Inhibit Rhizopus oryzae Growth, Biofilm Formation, and Rhizopuspepsin Activity

    Journal: The Canadian Journal of Infectious Diseases & Medical Microbiology = Journal Canadien des Maladies Infectieuses et de la Microbiologie Médicale

    doi: 10.1155/2018/5295619

    Proteolytic activity of rhizopuspepsin after overnight treatment with 48 µ g/ml of Piper essential oils. The plates were incubated at 37°C.
    Figure Legend Snippet: Proteolytic activity of rhizopuspepsin after overnight treatment with 48 µ g/ml of Piper essential oils. The plates were incubated at 37°C.

    Techniques Used: Activity Assay, Incubation

    36) Product Images from "Beyond the heterodimer model for mineralocorticoid and glucocorticoid receptor interactions in nuclei and at DNA"

    Article Title: Beyond the heterodimer model for mineralocorticoid and glucocorticoid receptor interactions in nuclei and at DNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0227520

    Co-immunoprecipitation supports MR-GR interaction in a cell line and whole rat hippocampus. (A) Anti-GR immunoprecipitation additionally pulls down mCherry-MR when 3617ChMR cells are treated with vehicle or corticosterone. Negative controls include a non-immune IgG as the immunoprecipitating antibody and a MOCK immunoprecipitation containing antibody and buffer but no lysate. Parent 3617 cells do not express mCherry-MR but contain GFP-GR and endogenous mouse GR. mCherry-MR expected at 136 kDa. (B) Immunoprecipitation of rat GR from whole hippocampus additionally captures MR supporting interaction following stress exposure. Mean plasma corticosterone for these animals was 366 ng/ml (range 326–434 ng/ml). Endogenous MR expected at 107 kDa. (C) MR does not co-immunoprecipitate with GR when animals are bilaterally adrenalectomized (ADX, mean corticosterone 17 ng/ml, range 6–36 ng/ml). Compare signal to positive control (i.p. CORT) animal. (D) Intraperitoneal corticosterone injection (i.p., 3 mg/kg) of ADX rats restores MR co-immunoprecipitation indicating hormone dependence. Mean corticosterone was 617 ng/ml (range 516–717 ng/ml) when killed 30 min after injection at the corticosterone peak [ 8 ]. Animals for i.p. CORT controls were adrenally intact and injected with 3 mg/kg corticosterone 30 min before death (mean plasma corticosterone 586 ng/ml, range 375–948 ng/ml). Smeared MR bands with the anti-MR 1D5 antibody may suggest MR post-translational modifications. Faint bands around 50 kDa represent the non-specific labelling of the immunoprecipitating IgG common to many co-IPs.
    Figure Legend Snippet: Co-immunoprecipitation supports MR-GR interaction in a cell line and whole rat hippocampus. (A) Anti-GR immunoprecipitation additionally pulls down mCherry-MR when 3617ChMR cells are treated with vehicle or corticosterone. Negative controls include a non-immune IgG as the immunoprecipitating antibody and a MOCK immunoprecipitation containing antibody and buffer but no lysate. Parent 3617 cells do not express mCherry-MR but contain GFP-GR and endogenous mouse GR. mCherry-MR expected at 136 kDa. (B) Immunoprecipitation of rat GR from whole hippocampus additionally captures MR supporting interaction following stress exposure. Mean plasma corticosterone for these animals was 366 ng/ml (range 326–434 ng/ml). Endogenous MR expected at 107 kDa. (C) MR does not co-immunoprecipitate with GR when animals are bilaterally adrenalectomized (ADX, mean corticosterone 17 ng/ml, range 6–36 ng/ml). Compare signal to positive control (i.p. CORT) animal. (D) Intraperitoneal corticosterone injection (i.p., 3 mg/kg) of ADX rats restores MR co-immunoprecipitation indicating hormone dependence. Mean corticosterone was 617 ng/ml (range 516–717 ng/ml) when killed 30 min after injection at the corticosterone peak [ 8 ]. Animals for i.p. CORT controls were adrenally intact and injected with 3 mg/kg corticosterone 30 min before death (mean plasma corticosterone 586 ng/ml, range 375–948 ng/ml). Smeared MR bands with the anti-MR 1D5 antibody may suggest MR post-translational modifications. Faint bands around 50 kDa represent the non-specific labelling of the immunoprecipitating IgG common to many co-IPs.

    Techniques Used: Immunoprecipitation, Positive Control, Injection

    MR and GR binding during simulated ultradian corticosterone pulsatility. (A) Representative 3617ChMR cells following 20 min, 100 nM corticosterone pulse (p) + washout (w). MMTV array formation (arrows) provides measurable loading of fluorescently tagged GR and MR at chromatinised DNA. (B) Percentage total cells over multiple fields of view forming arrays loading GFP-GR C656G , mCherry-MR, or both receptors on the same array. Mean ± SEM, n = 6 from two independent experiments, ≥130 cells per replicate. Counting 0 min samples was not routinely performed (cytoplasmic receptors) but a baseline of 2–3% cells formed weak intranuclear arrays in the absence of corticosterone. (C) ChIP targeting fluorescent receptor tags. Transient EGFP-GR C656G and prolonged mCherry-MR loading were observed at the MMTV array structure and at GRE-containing endogenous gene promoters Sgk1 and Per1 . Mean ± SEM, n = 3. (D) Percentage of total cells forming arrays loading GFP-GR C656G and/or mCherry-MR. An ultradian pulse in the physiological range was simulated with 10 nM corticosterone. Mean ± SEM from two independent experiments. (E) Three successive 20 min pulses of 30 nM were applied 1 hr apart to mimic the ultradian rhythm (arrows). Each pulse produced a transient increase in GFP-GR C656G array loading, while mCherry-MR array loading was largely unresponsive to ultradian stimulation. The downward drift in occupancy over the time course likely relates to accumulated media changes. Mean ± SEM, n = 6 from two independent experiments.
    Figure Legend Snippet: MR and GR binding during simulated ultradian corticosterone pulsatility. (A) Representative 3617ChMR cells following 20 min, 100 nM corticosterone pulse (p) + washout (w). MMTV array formation (arrows) provides measurable loading of fluorescently tagged GR and MR at chromatinised DNA. (B) Percentage total cells over multiple fields of view forming arrays loading GFP-GR C656G , mCherry-MR, or both receptors on the same array. Mean ± SEM, n = 6 from two independent experiments, ≥130 cells per replicate. Counting 0 min samples was not routinely performed (cytoplasmic receptors) but a baseline of 2–3% cells formed weak intranuclear arrays in the absence of corticosterone. (C) ChIP targeting fluorescent receptor tags. Transient EGFP-GR C656G and prolonged mCherry-MR loading were observed at the MMTV array structure and at GRE-containing endogenous gene promoters Sgk1 and Per1 . Mean ± SEM, n = 3. (D) Percentage of total cells forming arrays loading GFP-GR C656G and/or mCherry-MR. An ultradian pulse in the physiological range was simulated with 10 nM corticosterone. Mean ± SEM from two independent experiments. (E) Three successive 20 min pulses of 30 nM were applied 1 hr apart to mimic the ultradian rhythm (arrows). Each pulse produced a transient increase in GFP-GR C656G array loading, while mCherry-MR array loading was largely unresponsive to ultradian stimulation. The downward drift in occupancy over the time course likely relates to accumulated media changes. Mean ± SEM, n = 6 from two independent experiments.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Produced

    A cell line model for MR and GR co-expression. (A) The repeating unit in the MMTV array comprises the MMTV LTR driving expression of viral Harvey Ras (HaRas) cDNA. Mammary adenocarcinoma line 3617 contains approximately 200 copies at one location in chromosome 4. (B) 3617ChMR cells express fluorescently tagged receptors that localize to the cytoplasm in the absence of hormone but undergo nuclear translocation and bind the array structure (arrows) in the presence of corticosterone. (C) Dexamethasone (5 nM) induces array loading of both receptors, complete GFP-GR C656G translocation, but partial mCherry-MR translocation. Aldosterone (5 nM) induces mCherry-MR array loading, complete mCherry-MR translocation, but partial GFP-GR C656G translocation. 1 nM corticosterone favours mCherry-MR translocation, likely due MR’s higher affinity for corticosterone relative to GR. Treatments were 30 min. (D) The GR C656G mutation has little effect on the corticosterone sensitivity of EGFP-GR. COS-1 cells containing no endogenous GR or MR were transfected with equivalent amounts of wildtype (wt) or C656G mutant rat EGFP-GR prior to treatment with corticosterone for 24 hrs at the doses indicated. Co-transfected pFC31-Luc provided a MMTV-driven firefly luciferase reporter and pRL-CMV a Renilla transfection control. Two-way ANOVA of Renilla-corrected luminescence showed a significant effect of dose (F(5,24) = 190.4, p
    Figure Legend Snippet: A cell line model for MR and GR co-expression. (A) The repeating unit in the MMTV array comprises the MMTV LTR driving expression of viral Harvey Ras (HaRas) cDNA. Mammary adenocarcinoma line 3617 contains approximately 200 copies at one location in chromosome 4. (B) 3617ChMR cells express fluorescently tagged receptors that localize to the cytoplasm in the absence of hormone but undergo nuclear translocation and bind the array structure (arrows) in the presence of corticosterone. (C) Dexamethasone (5 nM) induces array loading of both receptors, complete GFP-GR C656G translocation, but partial mCherry-MR translocation. Aldosterone (5 nM) induces mCherry-MR array loading, complete mCherry-MR translocation, but partial GFP-GR C656G translocation. 1 nM corticosterone favours mCherry-MR translocation, likely due MR’s higher affinity for corticosterone relative to GR. Treatments were 30 min. (D) The GR C656G mutation has little effect on the corticosterone sensitivity of EGFP-GR. COS-1 cells containing no endogenous GR or MR were transfected with equivalent amounts of wildtype (wt) or C656G mutant rat EGFP-GR prior to treatment with corticosterone for 24 hrs at the doses indicated. Co-transfected pFC31-Luc provided a MMTV-driven firefly luciferase reporter and pRL-CMV a Renilla transfection control. Two-way ANOVA of Renilla-corrected luminescence showed a significant effect of dose (F(5,24) = 190.4, p

    Techniques Used: Expressing, Translocation Assay, Mutagenesis, Transfection, Luciferase

    37) Product Images from "Rapid HPLC-ESI-MS/MS Analysis of Neurotransmitters in the Brain Tissue of Alzheimer’s Disease Rats before and after Oral Administration of Xanthoceras sorbifolia Bunge"

    Article Title: Rapid HPLC-ESI-MS/MS Analysis of Neurotransmitters in the Brain Tissue of Alzheimer’s Disease Rats before and after Oral Administration of Xanthoceras sorbifolia Bunge

    Journal: Molecules

    doi: 10.3390/molecules23123111

    Typical multiple reaction monitoring chromatograms of eight standards and internal standard ( A ), the brain samples of the AD rats ( B ) and a blank sample spiked analytes and IS ( C ). DA, Dopamine; NE, norepinephrine; 5-HT, 5-hydroxytryptamine; Ach, acetyl choline; Trp, l -tryptophan; GABA, γ-aminobutyric acid; Glu, glutamic acid; Asp, aspartic acid.
    Figure Legend Snippet: Typical multiple reaction monitoring chromatograms of eight standards and internal standard ( A ), the brain samples of the AD rats ( B ) and a blank sample spiked analytes and IS ( C ). DA, Dopamine; NE, norepinephrine; 5-HT, 5-hydroxytryptamine; Ach, acetyl choline; Trp, l -tryptophan; GABA, γ-aminobutyric acid; Glu, glutamic acid; Asp, aspartic acid.

    Techniques Used:

    38) Product Images from "Glutamate receptor antagonist infusions into the basolateral and medial amygdala reveal differential contributions to olfactory vs. context fear conditioning and expression"

    Article Title: Glutamate receptor antagonist infusions into the basolateral and medial amygdala reveal differential contributions to olfactory vs. context fear conditioning and expression

    Journal:

    doi: 10.1101/lm.87105

    Effect on fear conditioning and fear-potentiated startle of glutamate receptor antagonist infusions into the basolateral amygdala. The NMDA receptor antagonist AP5 disrupted fear conditioning to an olfactory CS when infused prior to training ( leftmost
    Figure Legend Snippet: Effect on fear conditioning and fear-potentiated startle of glutamate receptor antagonist infusions into the basolateral amygdala. The NMDA receptor antagonist AP5 disrupted fear conditioning to an olfactory CS when infused prior to training ( leftmost

    Techniques Used:

    39) Product Images from "Humanin rescues cultured rat cortical neurons from NMDA-induced toxicity through the alleviation of mitochondrial dysfunction"

    Article Title: Humanin rescues cultured rat cortical neurons from NMDA-induced toxicity through the alleviation of mitochondrial dysfunction

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S133042

    Effect of HN on NMDA-induced ROS production. Notes: ( A ) ROS production was visualized under confocal microscope (×400). ( B ) Changes in cellular ROS production in cortical neurons treated with NMDA with or without HN. A total of 1×10 6 cells were quantified. Data are the mean ± SD of 5 independent observations by flow cytometry. Data were analyzed by 1-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. # Control group versus NMDA group, P =0.000, *NMDA group versus NMDA + MK-801 group, P =0.000; **NMDA group versus NMDA + HN group, P =0.001, ## control versus NMDA + S7A-HN, P =0.019; ***NMDA versus NMDA + AGA-HNG, P =0.022. NMDA: 100 μmol/L; MK-801: 10 μmol/L; HN, S7A-HN, and AGA-HNG: 1 μmol/L each. NMDA is an excitotoxin that induces the overactivation of the NMDA receptor, causing excitotoxicity. MK-801 is a known uncompetitive antagonist of NMDA receptor, which could block the binding of NMDA. Abbreviations: AGA-HNG, 100× more active form of HN; ANOVA, analysis of variance; HN, humanin; NMDA, N -methyl-D-aspartate; ROS, reactive oxygen species; S7A-HN, inactive form of HN; SD, standard deviation.
    Figure Legend Snippet: Effect of HN on NMDA-induced ROS production. Notes: ( A ) ROS production was visualized under confocal microscope (×400). ( B ) Changes in cellular ROS production in cortical neurons treated with NMDA with or without HN. A total of 1×10 6 cells were quantified. Data are the mean ± SD of 5 independent observations by flow cytometry. Data were analyzed by 1-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. # Control group versus NMDA group, P =0.000, *NMDA group versus NMDA + MK-801 group, P =0.000; **NMDA group versus NMDA + HN group, P =0.001, ## control versus NMDA + S7A-HN, P =0.019; ***NMDA versus NMDA + AGA-HNG, P =0.022. NMDA: 100 μmol/L; MK-801: 10 μmol/L; HN, S7A-HN, and AGA-HNG: 1 μmol/L each. NMDA is an excitotoxin that induces the overactivation of the NMDA receptor, causing excitotoxicity. MK-801 is a known uncompetitive antagonist of NMDA receptor, which could block the binding of NMDA. Abbreviations: AGA-HNG, 100× more active form of HN; ANOVA, analysis of variance; HN, humanin; NMDA, N -methyl-D-aspartate; ROS, reactive oxygen species; S7A-HN, inactive form of HN; SD, standard deviation.

    Techniques Used: Microscopy, Flow Cytometry, Cytometry, Blocking Assay, Binding Assay, Standard Deviation

    Effect of HN on NMDA-induced mitochondrial membrane potential reduction. Notes: ( A ) Visualization of mitochondrial membrane potential in cortical neurons under a laser confocal scanning microscope (×400). Primarily cultured cortical neurons (1×10 6 ) were stained by JC-1 on 9th day in vitro. ( B ) Changes of mitochondrial membrane potential in cortical neurons treated with NMDA with or without HN. Data are the mean ± SD of 5 independent experiments. Data were analyzed by 1-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. # Control group versus NMDA group, P =0.002; *NMDA group versus NMDA + MK-801 group, P =0.026; **NMDA group versus NMDA + HN group, P =0.000. NMDA: 100 μmol/L; MK-801: 10 μmol/L and HN: 1 μmol/L. NMDA is an excitotoxin that induces the overactivation of the NMDA receptor, causing excitotoxicity. MK-801 is a known uncompetitive antagonist of NMDA receptor, which could block the binding of NMDA. Abbreviations: ANOVA, analysis of variance; HN, humanin; NMDA, N -methyl-D-aspartate; SD, standard deviation.
    Figure Legend Snippet: Effect of HN on NMDA-induced mitochondrial membrane potential reduction. Notes: ( A ) Visualization of mitochondrial membrane potential in cortical neurons under a laser confocal scanning microscope (×400). Primarily cultured cortical neurons (1×10 6 ) were stained by JC-1 on 9th day in vitro. ( B ) Changes of mitochondrial membrane potential in cortical neurons treated with NMDA with or without HN. Data are the mean ± SD of 5 independent experiments. Data were analyzed by 1-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. # Control group versus NMDA group, P =0.002; *NMDA group versus NMDA + MK-801 group, P =0.026; **NMDA group versus NMDA + HN group, P =0.000. NMDA: 100 μmol/L; MK-801: 10 μmol/L and HN: 1 μmol/L. NMDA is an excitotoxin that induces the overactivation of the NMDA receptor, causing excitotoxicity. MK-801 is a known uncompetitive antagonist of NMDA receptor, which could block the binding of NMDA. Abbreviations: ANOVA, analysis of variance; HN, humanin; NMDA, N -methyl-D-aspartate; SD, standard deviation.

    Techniques Used: Microscopy, Cell Culture, Staining, In Vitro, Blocking Assay, Binding Assay, Standard Deviation

    Effect of HN on NMDA-induced nitric oxide production. Notes: Data are shown as mean ± SD (n=5). Data were analyzed by 1-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. # Control group versus NMDA group, P =0.000; *NMDA versus NMDA + MK-801, P =0.003; **NMDA versus NMDA + HN, P =0.002; ## control versus NMDA + S7A-HN, P =0.004; ***NMDA versus NMDA + AGA-HNG, P =0.005. NMDA: 100 μmol/L; MK-801: 10 μmol/L; MK-801: 10 μmol/L; HN, S7A-HN, and AGA-HNG: 1 μmol/L each. NMDA is an excitotoxin that induces the overactivation of NMDA receptor, causing excitotoxicity. MK-801 is a known uncompetitive antagonist of NMDA receptor, which could block the binding of NMDA. Abbreviations: AGA-HNG, 100× more active form of HN; ANOVA, analysis of variance; conc, concentration; HN, humanin; NMDA, N -methyl-D-aspartate; S7A-HN, inactive form of HN; SD, standard deviation.
    Figure Legend Snippet: Effect of HN on NMDA-induced nitric oxide production. Notes: Data are shown as mean ± SD (n=5). Data were analyzed by 1-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. # Control group versus NMDA group, P =0.000; *NMDA versus NMDA + MK-801, P =0.003; **NMDA versus NMDA + HN, P =0.002; ## control versus NMDA + S7A-HN, P =0.004; ***NMDA versus NMDA + AGA-HNG, P =0.005. NMDA: 100 μmol/L; MK-801: 10 μmol/L; MK-801: 10 μmol/L; HN, S7A-HN, and AGA-HNG: 1 μmol/L each. NMDA is an excitotoxin that induces the overactivation of NMDA receptor, causing excitotoxicity. MK-801 is a known uncompetitive antagonist of NMDA receptor, which could block the binding of NMDA. Abbreviations: AGA-HNG, 100× more active form of HN; ANOVA, analysis of variance; conc, concentration; HN, humanin; NMDA, N -methyl-D-aspartate; S7A-HN, inactive form of HN; SD, standard deviation.

    Techniques Used: Blocking Assay, Binding Assay, Concentration Assay, Standard Deviation

    40) Product Images from "Marine omega-3 polyunsaturated fatty acids induce sex-specific changes in reinforcer-controlled behaviour and neurotransmitter metabolism in a spontaneously hypertensive rat model of ADHD"

    Article Title: Marine omega-3 polyunsaturated fatty acids induce sex-specific changes in reinforcer-controlled behaviour and neurotransmitter metabolism in a spontaneously hypertensive rat model of ADHD

    Journal: Behavioral and Brain Functions : BBF

    doi: 10.1186/1744-9081-8-56

    Chromatographic profiles of rat neostriatal extracts. ( a ) Amino acids in a WKY control-fed female, and ( b ) monoamines in a control-fed SHR male. Panel a ) shows the peaks of aspartic acid (asp), glutamate (Glu), serine (Ser), glutamine (Gln), α-amino adipic acid (AAA), glycine (Gly), taurine (Tau) and γ-amino butyric acid (GABA). Panel b ) shows the peaks of 5-hydroxyindol-3-acetic acid (5-HIAA), homovanillic acid (HVA), 3,4-hydroxybenzylamine (DHBA), dopamine (DA) and serotonin (5-HT).
    Figure Legend Snippet: Chromatographic profiles of rat neostriatal extracts. ( a ) Amino acids in a WKY control-fed female, and ( b ) monoamines in a control-fed SHR male. Panel a ) shows the peaks of aspartic acid (asp), glutamate (Glu), serine (Ser), glutamine (Gln), α-amino adipic acid (AAA), glycine (Gly), taurine (Tau) and γ-amino butyric acid (GABA). Panel b ) shows the peaks of 5-hydroxyindol-3-acetic acid (5-HIAA), homovanillic acid (HVA), 3,4-hydroxybenzylamine (DHBA), dopamine (DA) and serotonin (5-HT).

    Techniques Used:

    Related Articles

    Functional Assay:

    Article Title: Probing Excited States and Activation Energy for the Integral Membrane Protein Phospholamban by NMR CPMG Relaxation Dispersion Experiments
    Article Snippet: .. A fully functional monomeric mutant of PLN (C36A, C41F, C46A, AFA-PLN) was expressed [U-15 N] and [13 Cδ1 -Ile] using 15 NH4 Cl and 2-ketobutyric acid-4-13 C,3,3-d2 (Isotec-Sigma/Aldrich), and purified as previously described [ ; ]. .. After HPLC purification, the protein was lyophilized with the subsequent powder stored at −20 °C until sample preparation.

    Mutagenesis:

    Article Title: Probing Excited States and Activation Energy for the Integral Membrane Protein Phospholamban by NMR CPMG Relaxation Dispersion Experiments
    Article Snippet: .. A fully functional monomeric mutant of PLN (C36A, C41F, C46A, AFA-PLN) was expressed [U-15 N] and [13 Cδ1 -Ile] using 15 NH4 Cl and 2-ketobutyric acid-4-13 C,3,3-d2 (Isotec-Sigma/Aldrich), and purified as previously described [ ; ]. .. After HPLC purification, the protein was lyophilized with the subsequent powder stored at −20 °C until sample preparation.

    Labeling:

    Article Title: Structural model for the protein-translocating element of the twin-arginine transport system
    Article Snippet: .. A methyl-protonated sample labeled with 1 H and 13 C at valine γ-methyls and leucine and isoleucine δ-methyls, and deuterated at all other nonexchangeable proton positions, was produced by expression in M9 medium containing 70 mg/L 2-Ketobutyric acid-4-13 C,3,3-d2 and 120 mg/L 2-Keto-3-methyl-13 C-butyric-4-13 C, 3-d (Sigma-Aldrich) ( ). .. When the cultures reached an A 600 nm of 0.6, expression of the tatA allele was induced with 1-mM final concentration of isopropyl β- D -thiogalactoside, and the growth continued for 7 h at 30 °C before harvesting.

    Purification:

    Article Title: Probing Excited States and Activation Energy for the Integral Membrane Protein Phospholamban by NMR CPMG Relaxation Dispersion Experiments
    Article Snippet: .. A fully functional monomeric mutant of PLN (C36A, C41F, C46A, AFA-PLN) was expressed [U-15 N] and [13 Cδ1 -Ile] using 15 NH4 Cl and 2-ketobutyric acid-4-13 C,3,3-d2 (Isotec-Sigma/Aldrich), and purified as previously described [ ; ]. .. After HPLC purification, the protein was lyophilized with the subsequent powder stored at −20 °C until sample preparation.

    Produced:

    Article Title: Structural model for the protein-translocating element of the twin-arginine transport system
    Article Snippet: .. A methyl-protonated sample labeled with 1 H and 13 C at valine γ-methyls and leucine and isoleucine δ-methyls, and deuterated at all other nonexchangeable proton positions, was produced by expression in M9 medium containing 70 mg/L 2-Ketobutyric acid-4-13 C,3,3-d2 and 120 mg/L 2-Keto-3-methyl-13 C-butyric-4-13 C, 3-d (Sigma-Aldrich) ( ). .. When the cultures reached an A 600 nm of 0.6, expression of the tatA allele was induced with 1-mM final concentration of isopropyl β- D -thiogalactoside, and the growth continued for 7 h at 30 °C before harvesting.

    Gas Chromatography-Mass Spectrometry:

    Article Title: Biochemical Characterization of the Fusarium graminearum Candidate ACC-Deaminases and Virulence Testing of Knockout Mutant Strains
    Article Snippet: .. GC-MS Measurements of ACC and KBA The standards for 1-aminocyclopropane-carboxylic acid (ACC; EMD 149101-1G) and 2-ketobutyric acid (KBA; K401, Sigma-Aldrich, Vienna, Austria) were purchased from Sigma-Aldrich. .. The solvents methanol LC-MS grade (Honeywell 34966) and pyridine p.A. (Merck 1.09728.0500) were obtained from Merck.

    other:

    Article Title: Rescue from galactose-induced death of Leigh Syndrome patient cells by pyruvate and NAD+
    Article Snippet: Medium additions In certain experiments (one of) the following compounds was/were added to the medium: sodium pyruvate (#11360070, Life Technologies, Carlsbad, CA, USA), l -aspartic acid (#A7219, Sigma-Aldrich), 2-ketobutyric acid (#K401, Sigma-Aldrich), oxaloacetic acid (#O4126, Sigma-Aldrich), sodium l -lactate (#L7022, Sigma-Aldrich), l -(-)-malic acid (#M1000, Sigma-Aldrich), sodium succinate dibasic hexahydrate (#S9637, Sigma-Aldrich), dimethyl α-ketoglutarate (#349631, Sigma-Aldrich), uridine (#U3003, Sigma-Aldrich), β-nicotinamide adenine dinucleotide hydrate (#43410, Sigma-Aldrich), Oleamide (#O2136, Sigma-Aldrich) These compounds were dissolved directly in the medium after which its pH was adjusted to 7.2 with NaOH.

    Article Title: Rescue from galactose-induced death of Leigh Syndrome patient cells by pyruvate and NAD+
    Article Snippet: In certain experiments (one of) the following compounds was/were added to the medium: sodium pyruvate (#11360070, Life Technologies, Carlsbad, CA, USA), l -aspartic acid (#A7219, Sigma-Aldrich), 2-ketobutyric acid (#K401, Sigma-Aldrich), oxaloacetic acid (#O4126, Sigma-Aldrich), sodium l -lactate (#L7022, Sigma-Aldrich), l -(-)-malic acid (#M1000, Sigma-Aldrich), sodium succinate dibasic hexahydrate (#S9637, Sigma-Aldrich), dimethyl α-ketoglutarate (#349631, Sigma-Aldrich), uridine (#U3003, Sigma-Aldrich), β-nicotinamide adenine dinucleotide hydrate (#43410, Sigma-Aldrich), Oleamide (#O2136, Sigma-Aldrich) These compounds were dissolved directly in the medium after which its pH was adjusted to 7.2 with NaOH.

    Expressing:

    Article Title: Structural model for the protein-translocating element of the twin-arginine transport system
    Article Snippet: .. A methyl-protonated sample labeled with 1 H and 13 C at valine γ-methyls and leucine and isoleucine δ-methyls, and deuterated at all other nonexchangeable proton positions, was produced by expression in M9 medium containing 70 mg/L 2-Ketobutyric acid-4-13 C,3,3-d2 and 120 mg/L 2-Keto-3-methyl-13 C-butyric-4-13 C, 3-d (Sigma-Aldrich) ( ). .. When the cultures reached an A 600 nm of 0.6, expression of the tatA allele was induced with 1-mM final concentration of isopropyl β- D -thiogalactoside, and the growth continued for 7 h at 30 °C before harvesting.

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  • 93
    Millipore anti sftpb
    Altered protein expression in distal airways of Foxn4 lacZ/lacZ mice. A-L: wild-type and Foxn4 lacZ/lacZ lung sections from the indicated stages were immunostained with antibodies against <t>PDGFA</t> (A,B), PDGFRα (C,D), <t>SFTPB</t> (E,F), SFTPC (G,H), α-SMA
    Anti Sftpb, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore aspartate α ketoglutarate transamination activity
    The active site and N-terminal arm of MbnN are modified to accommodate a substrate with a different shape and charge from histidinol phosphate ( A ) A model of MbnN from Ms. trichosporium OB3b (red) generated by I-Tasser and aligned against HisC from My. tuberculosis (blue, PDB: 4R8D). ( B ) Stereoview of the aligned active sites of MbnN (white) against PLP-loaded HisC (grey (chain B), dark grey (chain A), yellow (PLP), PDB: 4R8D) from My. tuberculosis . Residue labels followed by a single ’ correspond to residues that are present on chain A (and are not modeled for MbnN). Underlined residues are altered in MbnN: F29 and F124 are tyrosines in My. tuberculosis HisC, while T100 is an aspartate residue. These residues form a more hydrophobic pocket but do not affect interactions with the PLP/PMP cofactor or an <t>α-ketoglutarate-like</t> cosubstrate. ( C ) Stereoview of the aligned active sites of the MbnN model (white) against PLP- and histidinol-phosphate loaded HisC from E. coli (gray (chain B), dark gray (chain A), yellow (PLP), teal (Hsp), PDB: 1FG3). Residue labels that are followed by a single ’ are present on chain A (and are not modeled in for MbnN). Underlined residues are altered in MbnN: F29 and F124 are tyrosines in 1FG3, while S99 is an alanine, T100 is an aspartate residue, and T222 is a leucine while S224 is a threonine. None of the H-bonding interactions with the histidinol group present in the E. coli enzyme are possible in the hydrophobic MbnN active site.
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    85
    Millipore aspartase activity
    TGF-β inhibits expression of components of the NK cell cytotoxic apparatus. (A) Expression of granzymes B and H at the protein and mRNA level. For granzyme B, NK cells were stimulated 20 ng/ml IL-15 for 48 hrs and increasing amounts of TGF-β (ranging from 0 to 5 ng/ml). The immunoblot shows granzyme B expression (GZMB) and actin as a loading control. For granzyme H, expression was determined in unstimulated NK cells, NK cells treated with IL-15 and IL-15 plus TGF-β (for 48 hrs). The blots show granzyme H (GZMH) and actin levels. For gene expression, GZMB and GZMH steady state mRNA levels were quantitated from unstimulated (U), IL-15 stimulated (15) and IL-15 plus TGF-β (15+β) treated NK cells (using qRT-PCR) and expressed as arbitrary units, with expression in unstimulated NK cells defined as 1 unit (note the different scales). (B) Expression of the cathepsin C ( CTSC ) and perforin ( PRF1 ) gene in unstimulated (U), IL-15 stimulated (15) and IL-15 plus TGF-β (15+β) treated NK cells, determined as in (A). (C) Protease activity in cytokine treated NK cells. <t>Aspartase</t> (Aspase) activity (a measure of granzyme B activity) assayed by hydrolysis of AcIEPD-pNA (left panel) and cathepsin C activity assayed by hydrolysis of GF-pNA (right panel). Activity was measured in lysates derived from unstimulated NK cells (U), NK cells stimulated for 48 hrs with 20 ng/ml IL-15 (15) or IL-15 plus 5 ng/ml TGF-β (15+β). Each reaction contained lysate from 6×10 5 cells and was performed in triplicate, with the mean and SD calculated. Activity is expressed as arbitrary units. (D) NK cells were stimulated with either 20 ng/ml IL-15 (15) or IL-15 plus 5 ng/ml TGF-β (15+β) for 48 hrs and used in standard killing assays against K562, OVCA433 and SKOV3 tumour cell lines at an E∶T ratio of 3∶1 (with standard deviation shown).
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    Image Search Results


    Altered protein expression in distal airways of Foxn4 lacZ/lacZ mice. A-L: wild-type and Foxn4 lacZ/lacZ lung sections from the indicated stages were immunostained with antibodies against PDGFA (A,B), PDGFRα (C,D), SFTPB (E,F), SFTPC (G,H), α-SMA

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    Article Title: Foxn4 Influences Alveologenesis during Lung Development

    doi: 10.1002/dvdy.22610

    Figure Lengend Snippet: Altered protein expression in distal airways of Foxn4 lacZ/lacZ mice. A-L: wild-type and Foxn4 lacZ/lacZ lung sections from the indicated stages were immunostained with antibodies against PDGFA (A,B), PDGFRα (C,D), SFTPB (E,F), SFTPC (G,H), α-SMA

    Article Snippet: Antibodies used in this work are: anti-Foxn4 (rabbit, 1:50) ( ); anti-β-tubulin IV (mouse, 1:50, Sigma); anti-Trp63 (p63) (mouse, 1:500, Santa Cruz); anti-Scgb1a1/CC-10 (goat, 1:1000, Santa Cruz); anti-BrdU (mouse, 1:50, BD Pharmingen); anti-CFTR (rabbit, 1:500, Alomone); anti-β-gal (rabbit, 1:5000, Cappel); anti-PDGFA (rabbit, 1:500, Santa Cruz); anti-PDGFRα (rabbit, 1:500, Santa Cruz); anti-SFTPB (sheep, 1:1250, Millipore); anti-SFTPC (rabbit, 1:500, Santa Cruz); anti-elastin (goat, 1:500, Santa Cruz); and anti-α-SMA (mouse, 1:500, Sigma).

    Techniques: Expressing, Mouse Assay

    The active site and N-terminal arm of MbnN are modified to accommodate a substrate with a different shape and charge from histidinol phosphate ( A ) A model of MbnN from Ms. trichosporium OB3b (red) generated by I-Tasser and aligned against HisC from My. tuberculosis (blue, PDB: 4R8D). ( B ) Stereoview of the aligned active sites of MbnN (white) against PLP-loaded HisC (grey (chain B), dark grey (chain A), yellow (PLP), PDB: 4R8D) from My. tuberculosis . Residue labels followed by a single ’ correspond to residues that are present on chain A (and are not modeled for MbnN). Underlined residues are altered in MbnN: F29 and F124 are tyrosines in My. tuberculosis HisC, while T100 is an aspartate residue. These residues form a more hydrophobic pocket but do not affect interactions with the PLP/PMP cofactor or an α-ketoglutarate-like cosubstrate. ( C ) Stereoview of the aligned active sites of the MbnN model (white) against PLP- and histidinol-phosphate loaded HisC from E. coli (gray (chain B), dark gray (chain A), yellow (PLP), teal (Hsp), PDB: 1FG3). Residue labels that are followed by a single ’ are present on chain A (and are not modeled in for MbnN). Underlined residues are altered in MbnN: F29 and F124 are tyrosines in 1FG3, while S99 is an alanine, T100 is an aspartate residue, and T222 is a leucine while S224 is a threonine. None of the H-bonding interactions with the histidinol group present in the E. coli enzyme are possible in the hydrophobic MbnN active site.

    Journal: Biochemistry

    Article Title: Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins

    doi: 10.1021/acs.biochem.8b00296

    Figure Lengend Snippet: The active site and N-terminal arm of MbnN are modified to accommodate a substrate with a different shape and charge from histidinol phosphate ( A ) A model of MbnN from Ms. trichosporium OB3b (red) generated by I-Tasser and aligned against HisC from My. tuberculosis (blue, PDB: 4R8D). ( B ) Stereoview of the aligned active sites of MbnN (white) against PLP-loaded HisC (grey (chain B), dark grey (chain A), yellow (PLP), PDB: 4R8D) from My. tuberculosis . Residue labels followed by a single ’ correspond to residues that are present on chain A (and are not modeled for MbnN). Underlined residues are altered in MbnN: F29 and F124 are tyrosines in My. tuberculosis HisC, while T100 is an aspartate residue. These residues form a more hydrophobic pocket but do not affect interactions with the PLP/PMP cofactor or an α-ketoglutarate-like cosubstrate. ( C ) Stereoview of the aligned active sites of the MbnN model (white) against PLP- and histidinol-phosphate loaded HisC from E. coli (gray (chain B), dark gray (chain A), yellow (PLP), teal (Hsp), PDB: 1FG3). Residue labels that are followed by a single ’ are present on chain A (and are not modeled in for MbnN). Underlined residues are altered in MbnN: F29 and F124 are tyrosines in 1FG3, while S99 is an alanine, T100 is an aspartate residue, and T222 is a leucine while S224 is a threonine. None of the H-bonding interactions with the histidinol group present in the E. coli enzyme are possible in the hydrophobic MbnN active site.

    Article Snippet: Purified MbnN (at a final concentration of 25 μM) was assayed for aspartate/α-ketoglutarate transamination activity using an AST Activity Assay kit (MAK055, MilliporeSigma), following the instructions provided by the manufacturer.

    Techniques: Modification, Mass Spectrometry, Generated, Plasmid Purification

    TGF-β inhibits expression of components of the NK cell cytotoxic apparatus. (A) Expression of granzymes B and H at the protein and mRNA level. For granzyme B, NK cells were stimulated 20 ng/ml IL-15 for 48 hrs and increasing amounts of TGF-β (ranging from 0 to 5 ng/ml). The immunoblot shows granzyme B expression (GZMB) and actin as a loading control. For granzyme H, expression was determined in unstimulated NK cells, NK cells treated with IL-15 and IL-15 plus TGF-β (for 48 hrs). The blots show granzyme H (GZMH) and actin levels. For gene expression, GZMB and GZMH steady state mRNA levels were quantitated from unstimulated (U), IL-15 stimulated (15) and IL-15 plus TGF-β (15+β) treated NK cells (using qRT-PCR) and expressed as arbitrary units, with expression in unstimulated NK cells defined as 1 unit (note the different scales). (B) Expression of the cathepsin C ( CTSC ) and perforin ( PRF1 ) gene in unstimulated (U), IL-15 stimulated (15) and IL-15 plus TGF-β (15+β) treated NK cells, determined as in (A). (C) Protease activity in cytokine treated NK cells. Aspartase (Aspase) activity (a measure of granzyme B activity) assayed by hydrolysis of AcIEPD-pNA (left panel) and cathepsin C activity assayed by hydrolysis of GF-pNA (right panel). Activity was measured in lysates derived from unstimulated NK cells (U), NK cells stimulated for 48 hrs with 20 ng/ml IL-15 (15) or IL-15 plus 5 ng/ml TGF-β (15+β). Each reaction contained lysate from 6×10 5 cells and was performed in triplicate, with the mean and SD calculated. Activity is expressed as arbitrary units. (D) NK cells were stimulated with either 20 ng/ml IL-15 (15) or IL-15 plus 5 ng/ml TGF-β (15+β) for 48 hrs and used in standard killing assays against K562, OVCA433 and SKOV3 tumour cell lines at an E∶T ratio of 3∶1 (with standard deviation shown).

    Journal: PLoS ONE

    Article Title: Human Tumour Immune Evasion via TGF-? Blocks NK Cell Activation but Not Survival Allowing Therapeutic Restoration of Anti-Tumour Activity

    doi: 10.1371/journal.pone.0022842

    Figure Lengend Snippet: TGF-β inhibits expression of components of the NK cell cytotoxic apparatus. (A) Expression of granzymes B and H at the protein and mRNA level. For granzyme B, NK cells were stimulated 20 ng/ml IL-15 for 48 hrs and increasing amounts of TGF-β (ranging from 0 to 5 ng/ml). The immunoblot shows granzyme B expression (GZMB) and actin as a loading control. For granzyme H, expression was determined in unstimulated NK cells, NK cells treated with IL-15 and IL-15 plus TGF-β (for 48 hrs). The blots show granzyme H (GZMH) and actin levels. For gene expression, GZMB and GZMH steady state mRNA levels were quantitated from unstimulated (U), IL-15 stimulated (15) and IL-15 plus TGF-β (15+β) treated NK cells (using qRT-PCR) and expressed as arbitrary units, with expression in unstimulated NK cells defined as 1 unit (note the different scales). (B) Expression of the cathepsin C ( CTSC ) and perforin ( PRF1 ) gene in unstimulated (U), IL-15 stimulated (15) and IL-15 plus TGF-β (15+β) treated NK cells, determined as in (A). (C) Protease activity in cytokine treated NK cells. Aspartase (Aspase) activity (a measure of granzyme B activity) assayed by hydrolysis of AcIEPD-pNA (left panel) and cathepsin C activity assayed by hydrolysis of GF-pNA (right panel). Activity was measured in lysates derived from unstimulated NK cells (U), NK cells stimulated for 48 hrs with 20 ng/ml IL-15 (15) or IL-15 plus 5 ng/ml TGF-β (15+β). Each reaction contained lysate from 6×10 5 cells and was performed in triplicate, with the mean and SD calculated. Activity is expressed as arbitrary units. (D) NK cells were stimulated with either 20 ng/ml IL-15 (15) or IL-15 plus 5 ng/ml TGF-β (15+β) for 48 hrs and used in standard killing assays against K562, OVCA433 and SKOV3 tumour cell lines at an E∶T ratio of 3∶1 (with standard deviation shown).

    Article Snippet: For enzymatic assays, granzyme B activity was analysed as aspartase activity by hydrolysis of the substrate AcIEPD-pNA (Calbiochem) and cathepsin C activity by hydrolysis of GF-pNA (Sigma Aldrich) as previously described .

    Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Derivative Assay, Standard Deviation