synthetic aβ preparations  (Thermo Fisher)


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    Structured Review

    Thermo Fisher synthetic aβ preparations
    The β-amyloid-inducing activity is partly proteinase K-resistant Brain extracts from an aged APP23 tg mouse (Tg extract), an aged non-tg mouse (Wt extract) spiked with synthetic <t>Aβ</t> fibrils (syn. Aβ fibrils in Wt extract; 10µM Aβ), and synthetic Aβ fibrils in PBS (syn. Aβ fibrils in PBS; 10µM) were treated with 50µg/ml proteinase K (PK) for 0min, 30min, 1h, or 2h at 37°C. ( A ) Silver staining of a 12% Bis-Tris NuPage® gel for total protein reveals the digestion of the majority of proteins in the brain extracts and of synthetic Aβ fibrils already after 30 min PK treatment. ( B ) Immunoblot analysis with an antibody specific to human Aβ (6E10) shows that Aβ in the Tg extract is largely PK-resistant for up to 2h. Aβ in the spiked Wt extract revealed some, but significantly less PK-resistance, whereas synthetic Aβ fibrils in PBS were almost completely digested by the PK treatment. ( C ) Densitometric quantification of Aβ-immunoblots after PK treatment of 5 Tg extracts [each from a different animal] and 3 independent synthetic Aβ fibril preparations are shown. Each preparation was tested at least 3 times and the mean was taken. Aβ concentration at time point 0 was designated as 100%. Indicated is the mean ± SEM. Repeated Measures 2-way ANOVA revealed a significant difference between the groups (F (2,34) = 112.2). Multiple Bonferroni post hoc tests showed significances after PK digestion for all group comparisons, *** p
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    1) Product Images from "Soluble A? seeds are potent inducers of cerebral ?-amyloid deposition"

    Article Title: Soluble A? seeds are potent inducers of cerebral ?-amyloid deposition

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

    doi: 10.1523/JNEUROSCI.3088-11.2011

    The β-amyloid-inducing activity is partly proteinase K-resistant Brain extracts from an aged APP23 tg mouse (Tg extract), an aged non-tg mouse (Wt extract) spiked with synthetic Aβ fibrils (syn. Aβ fibrils in Wt extract; 10µM Aβ), and synthetic Aβ fibrils in PBS (syn. Aβ fibrils in PBS; 10µM) were treated with 50µg/ml proteinase K (PK) for 0min, 30min, 1h, or 2h at 37°C. ( A ) Silver staining of a 12% Bis-Tris NuPage® gel for total protein reveals the digestion of the majority of proteins in the brain extracts and of synthetic Aβ fibrils already after 30 min PK treatment. ( B ) Immunoblot analysis with an antibody specific to human Aβ (6E10) shows that Aβ in the Tg extract is largely PK-resistant for up to 2h. Aβ in the spiked Wt extract revealed some, but significantly less PK-resistance, whereas synthetic Aβ fibrils in PBS were almost completely digested by the PK treatment. ( C ) Densitometric quantification of Aβ-immunoblots after PK treatment of 5 Tg extracts [each from a different animal] and 3 independent synthetic Aβ fibril preparations are shown. Each preparation was tested at least 3 times and the mean was taken. Aβ concentration at time point 0 was designated as 100%. Indicated is the mean ± SEM. Repeated Measures 2-way ANOVA revealed a significant difference between the groups (F (2,34) = 112.2). Multiple Bonferroni post hoc tests showed significances after PK digestion for all group comparisons, *** p
    Figure Legend Snippet: The β-amyloid-inducing activity is partly proteinase K-resistant Brain extracts from an aged APP23 tg mouse (Tg extract), an aged non-tg mouse (Wt extract) spiked with synthetic Aβ fibrils (syn. Aβ fibrils in Wt extract; 10µM Aβ), and synthetic Aβ fibrils in PBS (syn. Aβ fibrils in PBS; 10µM) were treated with 50µg/ml proteinase K (PK) for 0min, 30min, 1h, or 2h at 37°C. ( A ) Silver staining of a 12% Bis-Tris NuPage® gel for total protein reveals the digestion of the majority of proteins in the brain extracts and of synthetic Aβ fibrils already after 30 min PK treatment. ( B ) Immunoblot analysis with an antibody specific to human Aβ (6E10) shows that Aβ in the Tg extract is largely PK-resistant for up to 2h. Aβ in the spiked Wt extract revealed some, but significantly less PK-resistance, whereas synthetic Aβ fibrils in PBS were almost completely digested by the PK treatment. ( C ) Densitometric quantification of Aβ-immunoblots after PK treatment of 5 Tg extracts [each from a different animal] and 3 independent synthetic Aβ fibril preparations are shown. Each preparation was tested at least 3 times and the mean was taken. Aβ concentration at time point 0 was designated as 100%. Indicated is the mean ± SEM. Repeated Measures 2-way ANOVA revealed a significant difference between the groups (F (2,34) = 112.2). Multiple Bonferroni post hoc tests showed significances after PK digestion for all group comparisons, *** p

    Techniques Used: Activity Assay, Silver Staining, Western Blot, Concentration Assay

    Extended sonication increases β-amyloid-inducing activity Brain extracts (10% [w/v]) from aged APP23 mice (Tg extract) were subjected to additional sonication (3 × 20sec). ( A ) Immunoblot analysis with an antibody specific to human Aβ reveals no change in total Aβ between the original Tg extract (−) and the extract with extended sonication (+). However, more Aβ was found in the supernatant (SN) of the extra-sonicated extract after ultracentrifugation (100,000 × g; 30 min) (long exposure). ( B–C ) In vivo seeding activity of the original and extra-sonicated extracts was tested by intrahippocampal injections into young, pre-depositing APP23 mice. Brains were immunohistochemically analyzed for Aβ deposition 4 months post-injection. Shown is the dentate gyrus of the hippocampus. Injection of original Tg extract ( B ) induced robust congophilic Aβ deposition with a filamentous and dense pattern, while Aβ induction with the extra-sonicated Tg extract generated more punctate and small deposits ( C ). The insert shows double labeling of Aβ immunoreactivity and Congo red binding of the induced β-amyloid. Scale bars: 200µm and 20µm. ( D ) Stereological quantification of Aβ load in the hippocampus revealed a significant increase in β-amyloid induction by extended sonication. (n = 5–6 mice per group; mean ± SEM, t[9]=3.188; * p
    Figure Legend Snippet: Extended sonication increases β-amyloid-inducing activity Brain extracts (10% [w/v]) from aged APP23 mice (Tg extract) were subjected to additional sonication (3 × 20sec). ( A ) Immunoblot analysis with an antibody specific to human Aβ reveals no change in total Aβ between the original Tg extract (−) and the extract with extended sonication (+). However, more Aβ was found in the supernatant (SN) of the extra-sonicated extract after ultracentrifugation (100,000 × g; 30 min) (long exposure). ( B–C ) In vivo seeding activity of the original and extra-sonicated extracts was tested by intrahippocampal injections into young, pre-depositing APP23 mice. Brains were immunohistochemically analyzed for Aβ deposition 4 months post-injection. Shown is the dentate gyrus of the hippocampus. Injection of original Tg extract ( B ) induced robust congophilic Aβ deposition with a filamentous and dense pattern, while Aβ induction with the extra-sonicated Tg extract generated more punctate and small deposits ( C ). The insert shows double labeling of Aβ immunoreactivity and Congo red binding of the induced β-amyloid. Scale bars: 200µm and 20µm. ( D ) Stereological quantification of Aβ load in the hippocampus revealed a significant increase in β-amyloid induction by extended sonication. (n = 5–6 mice per group; mean ± SEM, t[9]=3.188; * p

    Techniques Used: Sonication, Activity Assay, Mouse Assay, In Vivo, Injection, Generated, Labeling, Binding Assay

    2) Product Images from "Lack of MD2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness"

    Article Title: Lack of MD2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness

    Journal: Immunology and cell biology

    doi: 10.1038/icb.2008.75

    LPS induced IL-8 mRNA expression in THK, but not HUCL cells ( A ) HUCL were incubated with 1 µg/ml LPS for the indicated times; as a control, cells were also treated with 5% SMCM. ( B ) THK cells were treated with 1 µg/ml LPS for the indicated times. Total RNA was extracted, reverse transcripted, and amplified using IL-8 primers with GAPDH as control. PCR products were separated and stained as described in Materials and Methods . Results are representative of three independent experiments.
    Figure Legend Snippet: LPS induced IL-8 mRNA expression in THK, but not HUCL cells ( A ) HUCL were incubated with 1 µg/ml LPS for the indicated times; as a control, cells were also treated with 5% SMCM. ( B ) THK cells were treated with 1 µg/ml LPS for the indicated times. Total RNA was extracted, reverse transcripted, and amplified using IL-8 primers with GAPDH as control. PCR products were separated and stained as described in Materials and Methods . Results are representative of three independent experiments.

    Techniques Used: Expressing, Incubation, Amplification, Polymerase Chain Reaction, Staining

    LPS stimulated IκB-α phosphorylation and degradation in THK, but not HUCL cells HUCL ( A) or THK ( B ) cells were stimulated with phenol-extracted LPS (1 µg/ml) for the indicated times. As a positive control, HUCL cells were also treated with 5% supplemented P. aeruginosa -conditioned medium (PA-CM) in KBM or KBM alone (Con). Total protein was extracted, and 20 µg of protein were subjected to SDS-PAGE followed by phospho-IκB-α (p-IκB-α) and IκB-α immunoblotting using a chemiluminescence technique. The results are representative of two independent experiments.
    Figure Legend Snippet: LPS stimulated IκB-α phosphorylation and degradation in THK, but not HUCL cells HUCL ( A) or THK ( B ) cells were stimulated with phenol-extracted LPS (1 µg/ml) for the indicated times. As a positive control, HUCL cells were also treated with 5% supplemented P. aeruginosa -conditioned medium (PA-CM) in KBM or KBM alone (Con). Total protein was extracted, and 20 µg of protein were subjected to SDS-PAGE followed by phospho-IκB-α (p-IκB-α) and IκB-α immunoblotting using a chemiluminescence technique. The results are representative of two independent experiments.

    Techniques Used: Positive Control, SDS Page

    LPS failed to induce IL-6 and IL-8 secretion in HUCL cells ( A ) HUCL cells were stimulated with phenol-extracted LPS (1 µg/ml and 100 ng/ml) or medium alone (control) for 8 h; as a positive control, cells were also treated with 250 ng/ml purified flagellin for 8h. ( B ) HUCL cells were treated with 1 µg/ml phenol-extracted LPS with or without 0.1% human serum for 24 h. The effects of LPS and flagellin on IL-6 and -8 secretion were measured in cell culture supernatants by ELISA. Data are representative of triplicate experiments and are expressed as the mean ± SD. Statistically significant differences in secreted IL-6 and IL-8 in LPS-treated cells were determined by ANOVA with probabilities shown both for overall significance and pairwise comparison (* P
    Figure Legend Snippet: LPS failed to induce IL-6 and IL-8 secretion in HUCL cells ( A ) HUCL cells were stimulated with phenol-extracted LPS (1 µg/ml and 100 ng/ml) or medium alone (control) for 8 h; as a positive control, cells were also treated with 250 ng/ml purified flagellin for 8h. ( B ) HUCL cells were treated with 1 µg/ml phenol-extracted LPS with or without 0.1% human serum for 24 h. The effects of LPS and flagellin on IL-6 and -8 secretion were measured in cell culture supernatants by ELISA. Data are representative of triplicate experiments and are expressed as the mean ± SD. Statistically significant differences in secreted IL-6 and IL-8 in LPS-treated cells were determined by ANOVA with probabilities shown both for overall significance and pairwise comparison (* P

    Techniques Used: Positive Control, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

    3) Product Images from "WNT5A induces castration-resistant prostate cancer via CCL2 and tumour-infiltrating macrophages"

    Article Title: WNT5A induces castration-resistant prostate cancer via CCL2 and tumour-infiltrating macrophages

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2017.451

    Induction of CCL2 by WNT5A signalling. ( A ) LNCaP and 22RV1 cells expressed ROR2 mRNA (left) and protein (right). ( B ) When ROR2 was blocked with ROR2 shRNA, CCL2 expression was decreased. ( C ) When ROR2 was overexpressed with flag-ROR2 expression construct, CCL2 expression was significantly increased. ( D ) ERK inhibitor (U0126) significantly decreased CCL2 expression in LNCaP cells after treating WNT5A. ( E ) When WNT5A was treated on LNCaP cells, immunoblot of ERK demonstrated that ERK was phosphorylated. All study was repeated three times. * P
    Figure Legend Snippet: Induction of CCL2 by WNT5A signalling. ( A ) LNCaP and 22RV1 cells expressed ROR2 mRNA (left) and protein (right). ( B ) When ROR2 was blocked with ROR2 shRNA, CCL2 expression was decreased. ( C ) When ROR2 was overexpressed with flag-ROR2 expression construct, CCL2 expression was significantly increased. ( D ) ERK inhibitor (U0126) significantly decreased CCL2 expression in LNCaP cells after treating WNT5A. ( E ) When WNT5A was treated on LNCaP cells, immunoblot of ERK demonstrated that ERK was phosphorylated. All study was repeated three times. * P

    Techniques Used: shRNA, Expressing, Construct

    4) Product Images from "Type III secretion system of Pseudomonas aeruginosa affects mucin gene expression via NF-κB and AKT signaling in human carcinoma epithelial cells and a pneumonia mouse model"

    Article Title: Type III secretion system of Pseudomonas aeruginosa affects mucin gene expression via NF-κB and AKT signaling in human carcinoma epithelial cells and a pneumonia mouse model

    Journal: bioRxiv

    doi: 10.1101/442061

    ExoS and ExoT are required and each sufficient to increase NF-κB and AKT signaling in A549 cells. A549 cells were infected with the indicated strains for 1 h. (A) AKT and p-AKT expression as detected by western blotting. Quantification of p-AKT and SP1 expression using RAS-4000. (B) A549 cells were transfected with SP1 luciferase (Luc) reporter plasmid (0.1 μg). (C) NF-κB expression as detected by western blotting. Quantification of p-p65 and p-lκB-α expression using RAS-4000. β-actin was used as the internal control. (D) A549 cells were transfected with expression NF-κkB luciferase (Luc) reporter plasmid (0.1 μg). At 24 h after transfection, A549 cells were treated with the strains at MOI = 20 for 1 h, and then, luciferase activity was measured. The data were normalized to β-galactosidase activity. All data are representative of 3 independent experiments. Luciferase activities were measured 24 h after the transfection. “–”, control A549 cells treated with PBS only; PAK, A549 cells infected with P. aeruginosa; ExsA::Ω, A549 cells infected with ExsA::Ω (no T3SS); ΔST, PAK-ΔST mutant; Mock, PAKΔSTmt-pUCP18; S, PAKΔSTmt-pUCP18PAKexoS; T, PAKΔSTmt-pUCP18PAKexoT (MOI = 20).
    Figure Legend Snippet: ExoS and ExoT are required and each sufficient to increase NF-κB and AKT signaling in A549 cells. A549 cells were infected with the indicated strains for 1 h. (A) AKT and p-AKT expression as detected by western blotting. Quantification of p-AKT and SP1 expression using RAS-4000. (B) A549 cells were transfected with SP1 luciferase (Luc) reporter plasmid (0.1 μg). (C) NF-κB expression as detected by western blotting. Quantification of p-p65 and p-lκB-α expression using RAS-4000. β-actin was used as the internal control. (D) A549 cells were transfected with expression NF-κkB luciferase (Luc) reporter plasmid (0.1 μg). At 24 h after transfection, A549 cells were treated with the strains at MOI = 20 for 1 h, and then, luciferase activity was measured. The data were normalized to β-galactosidase activity. All data are representative of 3 independent experiments. Luciferase activities were measured 24 h after the transfection. “–”, control A549 cells treated with PBS only; PAK, A549 cells infected with P. aeruginosa; ExsA::Ω, A549 cells infected with ExsA::Ω (no T3SS); ΔST, PAK-ΔST mutant; Mock, PAKΔSTmt-pUCP18; S, PAKΔSTmt-pUCP18PAKexoS; T, PAKΔSTmt-pUCP18PAKexoT (MOI = 20).

    Techniques Used: Infection, Expressing, Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Mutagenesis

    5) Product Images from "Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry"

    Article Title: Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21093315

    AA disrupts mitochondrial membrane potential in MDA-MB-231 cells. MDA-MB-231 cells were grown in 6-well plate for 48 h in the presence of AA (8 μM). At the indicated times (0, 24, and 48 h), cells were incubated with 2 μM of JC-1 for 30 min. After incubation, images of the cells were obtained by exciting at a 488 nm wavelength and the fluorescence emitted was alternatively recorded at 530 and 580 nm wavelengths. Bars represent 30 μm. Percentages ± S.E.M. of the JC-1 fluorescence ratio (JC-1 aggregates: red, 580 nm)/(JC-1 monomers: green, 530 nm) at the different time points evaluated are represented in the bar graph. Images are representative of four fields from three independent experiments. ** p
    Figure Legend Snippet: AA disrupts mitochondrial membrane potential in MDA-MB-231 cells. MDA-MB-231 cells were grown in 6-well plate for 48 h in the presence of AA (8 μM). At the indicated times (0, 24, and 48 h), cells were incubated with 2 μM of JC-1 for 30 min. After incubation, images of the cells were obtained by exciting at a 488 nm wavelength and the fluorescence emitted was alternatively recorded at 530 and 580 nm wavelengths. Bars represent 30 μm. Percentages ± S.E.M. of the JC-1 fluorescence ratio (JC-1 aggregates: red, 580 nm)/(JC-1 monomers: green, 530 nm) at the different time points evaluated are represented in the bar graph. Images are representative of four fields from three independent experiments. ** p

    Techniques Used: Multiple Displacement Amplification, Incubation, Fluorescence

    6) Product Images from "Entinostat augments NK cell functions via epigenetic upregulation of IFIT1-STING-STAT4 pathway"

    Article Title: Entinostat augments NK cell functions via epigenetic upregulation of IFIT1-STING-STAT4 pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27546

    Transcriptomic alterations of IFIT1-associated genes and epigenetic upregulation of IFIT1 by entinostat. ( A ) Gene-expression heatmap of genes associated with IFIT1. Heat map showing genes that are altered in human NK cells following entinostat treatment. Differentially expressed gene-set shown was obtained by comparing RNA-Seq of human NK cells treated with or without entinostat with the previously published gene-set obtained from IFIT1 knockdown in THP1 cells [ 40 ]. ( B ) Venn diagram showing filtering of raw (6374) ATAC peaks by protein-coding genes (3671), association with transcription start site (389), and FDR
    Figure Legend Snippet: Transcriptomic alterations of IFIT1-associated genes and epigenetic upregulation of IFIT1 by entinostat. ( A ) Gene-expression heatmap of genes associated with IFIT1. Heat map showing genes that are altered in human NK cells following entinostat treatment. Differentially expressed gene-set shown was obtained by comparing RNA-Seq of human NK cells treated with or without entinostat with the previously published gene-set obtained from IFIT1 knockdown in THP1 cells [ 40 ]. ( B ) Venn diagram showing filtering of raw (6374) ATAC peaks by protein-coding genes (3671), association with transcription start site (389), and FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    7) Product Images from "MicroRNA-325-3p Facilitates Immune Escape of Mycobacterium tuberculosis through Targeting LNX1 via NEK6 Accumulation to Promote Anti-Apoptotic STAT3 Signaling"

    Article Title: MicroRNA-325-3p Facilitates Immune Escape of Mycobacterium tuberculosis through Targeting LNX1 via NEK6 Accumulation to Promote Anti-Apoptotic STAT3 Signaling

    Journal: mBio

    doi: 10.1128/mBio.00557-20

    NEK6 inhibits apoptosis through the activation of STAT3. (A) Immunoblot analysis of the phosphorylated STAT1 (p-STAT1), STAT1, phosphorylated STAT3 (p-STAT3), and STAT3 in RAW 264.7 cells stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis . (B) Immunoblot analysis of the p-STAT1, STAT1, p-STAT3, and STAT3 in RAW 264.7 cells and BMDMs transfected with Myc-NEK6. (C) BMDMs from WT and NEK6-deficient ( Nek6 −/− ) mice were stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis for 24 h. The expressions of p-STAT1, STAT1, p-STAT3, STAT3, and NEK6 were analyzed by Western blotting. (D) Immunoblot analysis of the p-STAT3 and STAT3 in lungs, spleens, and livers from WT and Nek6 −/− mice at 7 dpi. (E and G) The expression levels of BCL-2, BCL-X L , BCL-W, and MCL-1 in 10 μg/ml of gamma-irradiated M. tuberculosis -stimulated BMDMs from WT and Nek6 −/− mice were calculated by qRT-PCR at the indicated times (E) and by Western blotting (G). (F and H) BMDMs from Nek6 −/− mice were pretreated with 20 ng/ml IL-6 for 4 h h, then cells were stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis . The expression levels of BCL-2, BCL-X L , BCL-W, and MCL-1 were calculated by qRT-PCR (F) and Western blotting (H) at the indicated times. (I) BMDMs from WT and Nek6 −/− mice were stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis for 24 h, and the apoptosis rates were detected by flow cytometry. (J) BMDMs from Nek6 −/− mice were pretreated with 20 ng/ml of IL-6 for 4 h, and then cells were stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis for 24 h. The apoptosis rates were detected by flow cytometry. Statistical significance between groups was determined by two-tailed Student’s t test. All data are presented as the means ± SDs and were derived from three independent experiments. All blots are representative of three independent experiments. **, P
    Figure Legend Snippet: NEK6 inhibits apoptosis through the activation of STAT3. (A) Immunoblot analysis of the phosphorylated STAT1 (p-STAT1), STAT1, phosphorylated STAT3 (p-STAT3), and STAT3 in RAW 264.7 cells stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis . (B) Immunoblot analysis of the p-STAT1, STAT1, p-STAT3, and STAT3 in RAW 264.7 cells and BMDMs transfected with Myc-NEK6. (C) BMDMs from WT and NEK6-deficient ( Nek6 −/− ) mice were stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis for 24 h. The expressions of p-STAT1, STAT1, p-STAT3, STAT3, and NEK6 were analyzed by Western blotting. (D) Immunoblot analysis of the p-STAT3 and STAT3 in lungs, spleens, and livers from WT and Nek6 −/− mice at 7 dpi. (E and G) The expression levels of BCL-2, BCL-X L , BCL-W, and MCL-1 in 10 μg/ml of gamma-irradiated M. tuberculosis -stimulated BMDMs from WT and Nek6 −/− mice were calculated by qRT-PCR at the indicated times (E) and by Western blotting (G). (F and H) BMDMs from Nek6 −/− mice were pretreated with 20 ng/ml IL-6 for 4 h h, then cells were stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis . The expression levels of BCL-2, BCL-X L , BCL-W, and MCL-1 were calculated by qRT-PCR (F) and Western blotting (H) at the indicated times. (I) BMDMs from WT and Nek6 −/− mice were stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis for 24 h, and the apoptosis rates were detected by flow cytometry. (J) BMDMs from Nek6 −/− mice were pretreated with 20 ng/ml of IL-6 for 4 h, and then cells were stimulated with 10 μg/ml of gamma-irradiated M. tuberculosis for 24 h. The apoptosis rates were detected by flow cytometry. Statistical significance between groups was determined by two-tailed Student’s t test. All data are presented as the means ± SDs and were derived from three independent experiments. All blots are representative of three independent experiments. **, P

    Techniques Used: Activation Assay, Irradiation, Transfection, Mouse Assay, Western Blot, Expressing, Quantitative RT-PCR, Flow Cytometry, Two Tailed Test, Derivative Assay

    Suppression of NEK6/STAT3 pathway contributes to the defense against TB. (A) Survival of WT ( n = 10) and Nek6 −/− mice ( n = 10) after aerosol infection with approximately 400 CFU of M. tuberculosis . (B) M. tuberculosis bacterial loads in lungs, spleens, and livers of WT and Nek6 −/− mice at 21 dpi. (C) M. tuberculosis growth rates in BMDMs from WT and Nek6 −/− mice (MOI = 5). (D) Typical lung lesions of WT and Nek6 −/− mice at 21 dpi. Scale bar = 100 μm. (E) Survival of Lnx1 fl/fl Lyz2 -Cre mice ( n = 10) and Lnx1 fl/fl Lyz2 -Cre mice orally administered BP-1-102 (3 mg/kg) ( n = 10) after aerosol infection with approximately 400 CFU of M. tuberculosis . (F) M. tuberculosis bacterial loads in lungs, spleens, and livers of the Lnx1 fl/fl Lyz2 -Cre mice and Lnx1 fl/fl Lyz2 -Cre mice orally administered BP-1-102 (3 mg/kg) at 21 dpi. (G) M. tuberculosis growth rates in BMDMs from Lnx1 fl/fl Lyz2 -Cre mice and Lnx1 fl/fl Lyz2 -Cre mice treated with BP-1-102 (MOI = 5). (H) BMDMs from Lnx1 fl/fl Lyz2 -Cre mice were treated with BP-1-102 and infected with M. tuberculosis (MOI = 5) for 24 h; apoptosis rates were then detected by flow cytometry. (I) Typical lung lesions of the Lnx1 fl/fl Lyz2 -Cre mice and Lnx1 fl/fl Lyz2 -Cre mice orally administered BP-1-102 (3 mg/kg) at 21 dpi. Scale bar = 100 μm. Mouse survival data were plotted as Kaplan-Meier curves and compared by log rank (Mantel-Cox) test. Bacterial loads were analyzed using the Mann-Whitney U test. For other data, statistical significance between groups was determined by two-tailed Student’s t test. All data are presented as the means ± SDs and were derived from three independent experiments. *, P
    Figure Legend Snippet: Suppression of NEK6/STAT3 pathway contributes to the defense against TB. (A) Survival of WT ( n = 10) and Nek6 −/− mice ( n = 10) after aerosol infection with approximately 400 CFU of M. tuberculosis . (B) M. tuberculosis bacterial loads in lungs, spleens, and livers of WT and Nek6 −/− mice at 21 dpi. (C) M. tuberculosis growth rates in BMDMs from WT and Nek6 −/− mice (MOI = 5). (D) Typical lung lesions of WT and Nek6 −/− mice at 21 dpi. Scale bar = 100 μm. (E) Survival of Lnx1 fl/fl Lyz2 -Cre mice ( n = 10) and Lnx1 fl/fl Lyz2 -Cre mice orally administered BP-1-102 (3 mg/kg) ( n = 10) after aerosol infection with approximately 400 CFU of M. tuberculosis . (F) M. tuberculosis bacterial loads in lungs, spleens, and livers of the Lnx1 fl/fl Lyz2 -Cre mice and Lnx1 fl/fl Lyz2 -Cre mice orally administered BP-1-102 (3 mg/kg) at 21 dpi. (G) M. tuberculosis growth rates in BMDMs from Lnx1 fl/fl Lyz2 -Cre mice and Lnx1 fl/fl Lyz2 -Cre mice treated with BP-1-102 (MOI = 5). (H) BMDMs from Lnx1 fl/fl Lyz2 -Cre mice were treated with BP-1-102 and infected with M. tuberculosis (MOI = 5) for 24 h; apoptosis rates were then detected by flow cytometry. (I) Typical lung lesions of the Lnx1 fl/fl Lyz2 -Cre mice and Lnx1 fl/fl Lyz2 -Cre mice orally administered BP-1-102 (3 mg/kg) at 21 dpi. Scale bar = 100 μm. Mouse survival data were plotted as Kaplan-Meier curves and compared by log rank (Mantel-Cox) test. Bacterial loads were analyzed using the Mann-Whitney U test. For other data, statistical significance between groups was determined by two-tailed Student’s t test. All data are presented as the means ± SDs and were derived from three independent experiments. *, P

    Techniques Used: Mouse Assay, Infection, Flow Cytometry, MANN-WHITNEY, Two Tailed Test, Derivative Assay

    8) Product Images from "Two Novel CRX Mutant Proteins Causing Autosomal Dominant Leber Congenital Amaurosis Interact Differently With NRL"

    Article Title: Two Novel CRX Mutant Proteins Causing Autosomal Dominant Leber Congenital Amaurosis Interact Differently With NRL

    Journal: Human mutation

    doi: 10.1002/humu.21268

    Functional analysis of c.G264T (p.K88N) and c.413delT (p.I138fs48) mutant CRX genes compared to the wild-type gene, both in the presence and the absence of the retinal transcription factor, NRL. (A) Transactivation of wild-type and mutant CRX proteins in the presence and absence of NRL, as measured using a bovine rhodopsin promoter-luciferase construct. (B) Western blot of CRX protein compared to GAPDH, a housekeeping gene, in corresponding transactivation experiments. Presence of NRL is noted in brackets. (C) Western blot of NRL protein compared to GAPDH in corresponding transactivation experiments.
    Figure Legend Snippet: Functional analysis of c.G264T (p.K88N) and c.413delT (p.I138fs48) mutant CRX genes compared to the wild-type gene, both in the presence and the absence of the retinal transcription factor, NRL. (A) Transactivation of wild-type and mutant CRX proteins in the presence and absence of NRL, as measured using a bovine rhodopsin promoter-luciferase construct. (B) Western blot of CRX protein compared to GAPDH, a housekeeping gene, in corresponding transactivation experiments. Presence of NRL is noted in brackets. (C) Western blot of NRL protein compared to GAPDH in corresponding transactivation experiments.

    Techniques Used: Functional Assay, Mutagenesis, Luciferase, Construct, Western Blot

    9) Product Images from "Phosphorylation of BlaR1 in Manifestation of Antibiotic Resistance in Methicillin-Resistant Staphylococcus aureus and its Abrogation by Small Molecules"

    Article Title: Phosphorylation of BlaR1 in Manifestation of Antibiotic Resistance in Methicillin-Resistant Staphylococcus aureus and its Abrogation by Small Molecules

    Journal: ACS infectious diseases

    doi: 10.1021/acsinfecdis.5b00086

    The effect of compounds 10 , 11 , or 12 on BlaR1 tyrosine phosphorylation and β-lactamase activity. ( a ) Whole-cell extracts of NRS70 after induction with CBAP in the absence or presence of 0.7 or 7 μg/mL compounds 10 , 11 , or 12 were cleared of Protein A and other immunoglobin-binding proteins by incubation with IgG Sepharose and analyzed by western blot using antibodies against phosphotyrosine. ( b ) β-Lactamase activity of the culture media after induction with CBAP in the absence or presence of 7 μg/mL compounds 10 , 11 , or 12 .
    Figure Legend Snippet: The effect of compounds 10 , 11 , or 12 on BlaR1 tyrosine phosphorylation and β-lactamase activity. ( a ) Whole-cell extracts of NRS70 after induction with CBAP in the absence or presence of 0.7 or 7 μg/mL compounds 10 , 11 , or 12 were cleared of Protein A and other immunoglobin-binding proteins by incubation with IgG Sepharose and analyzed by western blot using antibodies against phosphotyrosine. ( b ) β-Lactamase activity of the culture media after induction with CBAP in the absence or presence of 7 μg/mL compounds 10 , 11 , or 12 .

    Techniques Used: Activity Assay, Binding Assay, Incubation, Western Blot

    10) Product Images from "Identification of Basic Fibroblast Growth Factor as the Dominant Protector of Laminar Shear Medium from the Modified Shear Device in Tumor Necrosis Factor-α Induced Endothelial Dysfunction"

    Article Title: Identification of Basic Fibroblast Growth Factor as the Dominant Protector of Laminar Shear Medium from the Modified Shear Device in Tumor Necrosis Factor-α Induced Endothelial Dysfunction

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2017.01095

    LSM and rbFGF suppresses TNF-α-induced ICAM-1 and PAI-1 protein and gene expression in vivo . (A) Immunohistochemical staining of ICAM-1 and PAI-1 proteins in thoracic aorta tissue. Representative images showing immunoreactivities of ICAM-1 and PAI-1 in the aortic endothelial layers (brown color as arrow) were decreased in the TNF-α+LSM and TNF-α+rbFGF groups compared to in the TNF-α group. The immunoreactivities of ICAM-1 and PAI-1 in the aortic endothelial layer were not decreased in the TNF-α+LSM+Ab group compared to in the TNF-α group ( n = 3). (B) Immunoreactivity signals in endothelial layers from 4 groups was quantified for all immunohistochemical images. ** p
    Figure Legend Snippet: LSM and rbFGF suppresses TNF-α-induced ICAM-1 and PAI-1 protein and gene expression in vivo . (A) Immunohistochemical staining of ICAM-1 and PAI-1 proteins in thoracic aorta tissue. Representative images showing immunoreactivities of ICAM-1 and PAI-1 in the aortic endothelial layers (brown color as arrow) were decreased in the TNF-α+LSM and TNF-α+rbFGF groups compared to in the TNF-α group. The immunoreactivities of ICAM-1 and PAI-1 in the aortic endothelial layer were not decreased in the TNF-α+LSM+Ab group compared to in the TNF-α group ( n = 3). (B) Immunoreactivity signals in endothelial layers from 4 groups was quantified for all immunohistochemical images. ** p

    Techniques Used: Expressing, In Vivo, Immunohistochemistry, Staining

    Determination of inflammation, ROS induction, and thrombosis-related gene and protein expression. Expression of (A) inflammation-related ICAM-1, VCAM-1 , and MCP-1 genes, (B) ROS induction–related HO-1, NQO-1 , and Keap-1 genes and (C) thrombosis-related TF, TM , and PAI-1 genes were all significantly increased in the TNF-α group compared to in the control group. Gene expression levels of ICAM-1, V-CAM-1, MCP-1, Keap-1, TF , and PAI-1 were attenuated significantly by LSM treatment compared to those in the TNF-α group, but similar trends were not observed in the TNF-α+SM group. However, gene expression of HO-1, NQO-1 , and TM were significantly enhanced by LSM treatment compared to those in the TNF-α group, but similar trends were not observed in the TNF-α+SM group. (A–C) Data are expressed as the mean ± S.E.M. ( n = 3). * p
    Figure Legend Snippet: Determination of inflammation, ROS induction, and thrombosis-related gene and protein expression. Expression of (A) inflammation-related ICAM-1, VCAM-1 , and MCP-1 genes, (B) ROS induction–related HO-1, NQO-1 , and Keap-1 genes and (C) thrombosis-related TF, TM , and PAI-1 genes were all significantly increased in the TNF-α group compared to in the control group. Gene expression levels of ICAM-1, V-CAM-1, MCP-1, Keap-1, TF , and PAI-1 were attenuated significantly by LSM treatment compared to those in the TNF-α group, but similar trends were not observed in the TNF-α+SM group. However, gene expression of HO-1, NQO-1 , and TM were significantly enhanced by LSM treatment compared to those in the TNF-α group, but similar trends were not observed in the TNF-α+SM group. (A–C) Data are expressed as the mean ± S.E.M. ( n = 3). * p

    Techniques Used: Expressing, Chick Chorioallantoic Membrane Assay

    Determination of inflammation, ROS induction, and thrombosis-related gene and protein expression. (A) Inflammation related ICAM-1, VCAM-1 , and MCP-1 genes, (B) ROS induction related Keap-1 genes, and (C) thrombosis-related TF and PAI-1 genes were attenuated significantly in the TNF-α+LSM, TNF-α+LSM+IgG, and TNF-α+rbFGF groups compared to in the TNF-α groups. Significant attenuation of ICAM-1, VCAM-1, MCP-1, TF , and Keap-1 were not observed in the TNF-α+LSM+Ab groups. Additionally, ROS induction-related antioxidant HO-1, NQO-1 and anti-thrombotic TM gene levels were enhanced in the TNF-α+LSM, TNF-α+LSM+IgG, and TNF-α+rbFGF groups compared to in the TNF-α groups. (A–C) Data are expressed as the mean ± S.E.M. ( n = 3). * p
    Figure Legend Snippet: Determination of inflammation, ROS induction, and thrombosis-related gene and protein expression. (A) Inflammation related ICAM-1, VCAM-1 , and MCP-1 genes, (B) ROS induction related Keap-1 genes, and (C) thrombosis-related TF and PAI-1 genes were attenuated significantly in the TNF-α+LSM, TNF-α+LSM+IgG, and TNF-α+rbFGF groups compared to in the TNF-α groups. Significant attenuation of ICAM-1, VCAM-1, MCP-1, TF , and Keap-1 were not observed in the TNF-α+LSM+Ab groups. Additionally, ROS induction-related antioxidant HO-1, NQO-1 and anti-thrombotic TM gene levels were enhanced in the TNF-α+LSM, TNF-α+LSM+IgG, and TNF-α+rbFGF groups compared to in the TNF-α groups. (A–C) Data are expressed as the mean ± S.E.M. ( n = 3). * p

    Techniques Used: Expressing

    11) Product Images from "The Y99C Mutation in Guanylyl Cyclase-Activating Protein 1 Increases Intracellular Ca2+ and Causes Photoreceptor Degeneration in Transgenic Mice"

    Article Title: The Y99C Mutation in Guanylyl Cyclase-Activating Protein 1 Increases Intracellular Ca2+ and Causes Photoreceptor Degeneration in Transgenic Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0963-04.2004

    Histological changes in the retinas of 6-month-old mice caused by Y99C GCAP1 expression. a , Light microscopy of retinal sections from a nontransgenic mouse (N/T), Y99C GCAP1 mice (L52, L52H, and L53), and a mouse that overexpressed normal GCAP1 (L300).
    Figure Legend Snippet: Histological changes in the retinas of 6-month-old mice caused by Y99C GCAP1 expression. a , Light microscopy of retinal sections from a nontransgenic mouse (N/T), Y99C GCAP1 mice (L52, L52H, and L53), and a mouse that overexpressed normal GCAP1 (L300).

    Techniques Used: Mouse Assay, Expressing, Light Microscopy

    Expression of the exogenous GCAP1 in transgenic mice. a , DNA construct for the Y99C GCAP1 expressed in transgenic mice (for details, see Materials and Methods). b , Electrophoretic mobility of purified mouse and bovine GCAP1. Shown is a Coomassie-stained
    Figure Legend Snippet: Expression of the exogenous GCAP1 in transgenic mice. a , DNA construct for the Y99C GCAP1 expressed in transgenic mice (for details, see Materials and Methods). b , Electrophoretic mobility of purified mouse and bovine GCAP1. Shown is a Coomassie-stained

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Construct, Purification, Staining

    Flash response kinetics and sensitivity in Y99C GCAP1 rods. a , Averaged responses recorded from a nontransgenic rod and a rod expressing Y99C GCAP1 (lineL53) to flashes of 4, 14.5, 35, 62, 227, 968, 3520, and 15,800 photons/μm -2 . Flash monitor
    Figure Legend Snippet: Flash response kinetics and sensitivity in Y99C GCAP1 rods. a , Averaged responses recorded from a nontransgenic rod and a rod expressing Y99C GCAP1 (lineL53) to flashes of 4, 14.5, 35, 62, 227, 968, 3520, and 15,800 photons/μm -2 . Flash monitor

    Techniques Used: Expressing

    12) Product Images from "Effects of cholesterol-tagged small interfering RNAs targeting 12/15-lipoxygenase on parameters of diabetic nephropathy in a mouse model of type 1 diabetes"

    Article Title: Effects of cholesterol-tagged small interfering RNAs targeting 12/15-lipoxygenase on parameters of diabetic nephropathy in a mouse model of type 1 diabetes

    Journal:

    doi: 10.1152/ajprenal.90268.2008

    MCP-1 expression and monocyte/macrophage infiltration in glomeruli. A : IHC staining of F4/80 in mouse renal sections. B : quantification of F4/80-positive macrophage staining in glomeruli. A significant increase in staining was observed in the diabetic
    Figure Legend Snippet: MCP-1 expression and monocyte/macrophage infiltration in glomeruli. A : IHC staining of F4/80 in mouse renal sections. B : quantification of F4/80-positive macrophage staining in glomeruli. A significant increase in staining was observed in the diabetic

    Techniques Used: Expressing, Immunohistochemistry, Staining

    13) Product Images from "Wnt/β-catenin signaling determines the vasculogenic fate of post-natal mesenchymal stem cells"

    Article Title: Wnt/β-catenin signaling determines the vasculogenic fate of post-natal mesenchymal stem cells

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.2334

    GSK-3β inhibition is sufficient to induce vasculogenic differentiation of dental pulp stem cells. (A): Western blots for Fzd-6, P-GSK-3β, and GSK-3β from DPSC treated with 10 µM CHIR99021 for up to 24 hours. Alternatively,
    Figure Legend Snippet: GSK-3β inhibition is sufficient to induce vasculogenic differentiation of dental pulp stem cells. (A): Western blots for Fzd-6, P-GSK-3β, and GSK-3β from DPSC treated with 10 µM CHIR99021 for up to 24 hours. Alternatively,

    Techniques Used: Inhibition, Western Blot

    14) Product Images from "Targeting Glycolysis with Epigallocatechin-3-Gallate Enhances the Efficacy of Chemotherapeutics in Pancreatic Cancer Cells and Xenografts"

    Article Title: Targeting Glycolysis with Epigallocatechin-3-Gallate Enhances the Efficacy of Chemotherapeutics in Pancreatic Cancer Cells and Xenografts

    Journal: Cancers

    doi: 10.3390/cancers11101496

    EGCG enhances the cell growth inhibitory effect of gemcitabine in pancreatic cancer cells. Combination index (CI) plots of EGCG (20, 40, 60 µM) in combination with gemcitabine (1, 10, 20 and 40 nM), abraxane (1, 10, 20 and 40 nM), 5-Fluorouracil (1, 10, 20 and 40 µM), irrinotecan (1, 10, 20 and 40 µM), and oxialiplatin (1, 10, 20 and 40 µM) in Panc-1 (left) and MIA PaCa-2 (right) cells. Drug interactions were quantitatively determined using the Chou–Talalay method, and CI
    Figure Legend Snippet: EGCG enhances the cell growth inhibitory effect of gemcitabine in pancreatic cancer cells. Combination index (CI) plots of EGCG (20, 40, 60 µM) in combination with gemcitabine (1, 10, 20 and 40 nM), abraxane (1, 10, 20 and 40 nM), 5-Fluorouracil (1, 10, 20 and 40 µM), irrinotecan (1, 10, 20 and 40 µM), and oxialiplatin (1, 10, 20 and 40 µM) in Panc-1 (left) and MIA PaCa-2 (right) cells. Drug interactions were quantitatively determined using the Chou–Talalay method, and CI

    Techniques Used:

    15) Product Images from "The Conserved PHD1-PHD2 Domain of ZFP-1/AF10 Is a Discrete Functional Module Essential for Viability in Caenorhabditis elegans"

    Article Title: The Conserved PHD1-PHD2 Domain of ZFP-1/AF10 Is a Discrete Functional Module Essential for Viability in Caenorhabditis elegans

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01462-12

    The long isoform of ZFP-1 is specifically expressed in the germ line. (A) Immunostaining of dissected gonads from adult worms by use of an anti-ZFP-1 N terminus-specific antibody (pink) for wild-type worms compared to  zfp-1 ( ok554 ) worms and worms with
    Figure Legend Snippet: The long isoform of ZFP-1 is specifically expressed in the germ line. (A) Immunostaining of dissected gonads from adult worms by use of an anti-ZFP-1 N terminus-specific antibody (pink) for wild-type worms compared to zfp-1 ( ok554 ) worms and worms with

    Techniques Used: Immunostaining

    16) Product Images from "Protein aggregation in Ehrlichia chaffeensis during infection of mammalian cells"

    Article Title: Protein aggregation in Ehrlichia chaffeensis during infection of mammalian cells

    Journal: FEMS Microbiology Letters

    doi: 10.1093/femsle/fnx059

    ClpB is targeted to protein aggregates in E. chaffeensis upon the treatment with GuHCl. Immunodetection of ClpB (∼95 kDa) in the soluble (S) and aggregated (A) fractions of E. chaffeensis cells isolated from the infected macrophages at the indicated times post infection. The infected macrophages were cultured in the absence or presence of 0.5 mM GuHCl. The samples loaded onto the gel contained equal protein amounts, as determined with the BCA method.
    Figure Legend Snippet: ClpB is targeted to protein aggregates in E. chaffeensis upon the treatment with GuHCl. Immunodetection of ClpB (∼95 kDa) in the soluble (S) and aggregated (A) fractions of E. chaffeensis cells isolated from the infected macrophages at the indicated times post infection. The infected macrophages were cultured in the absence or presence of 0.5 mM GuHCl. The samples loaded onto the gel contained equal protein amounts, as determined with the BCA method.

    Techniques Used: Immunodetection, Isolation, Infection, Cell Culture, BIA-KA

    Guanidinium chloride (GuHCl) inhibits the ClpB activity and E. chaffeensis growth, and increases protein aggregation in E. chaffeensis cells. ( A ) The ATPase activity of the purified E. chaffeensis ClpB was measured in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from two experiments are shown. ( B ) The growth of E. chaffeensis (right axis) in the infected DH82 cells and the host cell viability (left axis) were determined in cultures grown in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from three experiments are shown. ( C ) The amount of the aggregated proteins in E. chaffeensis expressed as the percentage of the total protein concentration after 24 and 48 h post infection of macrophages in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from three experiments are shown. ( D ) The amount of the aggregated proteins in E. chaffeensis expressed as the percentage of the total protein concentration as the function of time post infection of macrophages in the absence and presence of 0.5 mM GuHCl. The mean values and the standard deviations from three experiments are shown.
    Figure Legend Snippet: Guanidinium chloride (GuHCl) inhibits the ClpB activity and E. chaffeensis growth, and increases protein aggregation in E. chaffeensis cells. ( A ) The ATPase activity of the purified E. chaffeensis ClpB was measured in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from two experiments are shown. ( B ) The growth of E. chaffeensis (right axis) in the infected DH82 cells and the host cell viability (left axis) were determined in cultures grown in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from three experiments are shown. ( C ) The amount of the aggregated proteins in E. chaffeensis expressed as the percentage of the total protein concentration after 24 and 48 h post infection of macrophages in the presence of the indicated concentration of GuHCl. The mean values and the standard deviations from three experiments are shown. ( D ) The amount of the aggregated proteins in E. chaffeensis expressed as the percentage of the total protein concentration as the function of time post infection of macrophages in the absence and presence of 0.5 mM GuHCl. The mean values and the standard deviations from three experiments are shown.

    Techniques Used: Activity Assay, Purification, Concentration Assay, Infection, Protein Concentration

    17) Product Images from "Regulation of Apoptotic Endonucleases by EndoG"

    Article Title: Regulation of Apoptotic Endonucleases by EndoG

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2014.2772

    Induction of DNase I-like endonucleases in EndoG-overexpressing cells.  (A)  Western blotting for DNase I, DNase X (both at 8 h), DNase 1-like 2, DNase γ (both at 12 h), caspase-activated DNase (CAD) (at 8 h post-transfection),
    Figure Legend Snippet: Induction of DNase I-like endonucleases in EndoG-overexpressing cells. (A) Western blotting for DNase I, DNase X (both at 8 h), DNase 1-like 2, DNase γ (both at 12 h), caspase-activated DNase (CAD) (at 8 h post-transfection),

    Techniques Used: Western Blot, Transfection

    18) Product Images from "Neuregulin1 (NRG1) Signaling through Fyn Modulates NMDA Receptor Phosphorylation: Differential Synaptic Function in NRG1+/− Knock-Outs Compared with Wild-Type Mice"

    Article Title: Neuregulin1 (NRG1) Signaling through Fyn Modulates NMDA Receptor Phosphorylation: Differential Synaptic Function in NRG1+/− Knock-Outs Compared with Wild-Type Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4314-06.2007

    NMDAR is hypophosphorylated in NRG1( Δ TM) +/− and ErbB4 +/− mutant mice, and hypophosphorylation is reversed by clozapine. A , NR2B Y1472 was hypophosphorylated in hippocampal lysates from NRG1( Δ TM) +/− and ErbB4 +/− mutant mice (top, lanes 2 and 3, respectively) compared with age- and sex-matched wild-type (WT) C57BL/6 mice as shown by Western blot. Fyn/Src Y420 phosphorylation also was reduced in ErbB4 +/− and NRG1( Δ TM) +/− mutant mice (bottom, lanes 2 and 3, respectively). Hippocampal lysates from Fyn −/− mice served as a control for NR2B Y1472 hypophosphorylation, as well as for Fyn/Src Y420 phosphorylation (fourth lane, top and bottom panels, respectively). Data are representative of five to six animals in at least three independent experiments; 25 μg of total protein was loaded per lane. B , Clozapine reverses NR2B hypophosphorylation in NRG1( Δ TM) +/− mice. NR2B Y1472 phosphorylation (top) was increased 2.5- to 3-fold in NRG1( Δ TM) +/− mice when normalized against total loading of NR2B (bottom). The same dose of clozapine had no effect on NR2B Y1472 phosphorylation (top) when administered to age- and sex-matched wild-type mice. Data representative of two to three animals in at least two independent experiments; 20 μg of total protein was loaded per lane.
    Figure Legend Snippet: NMDAR is hypophosphorylated in NRG1( Δ TM) +/− and ErbB4 +/− mutant mice, and hypophosphorylation is reversed by clozapine. A , NR2B Y1472 was hypophosphorylated in hippocampal lysates from NRG1( Δ TM) +/− and ErbB4 +/− mutant mice (top, lanes 2 and 3, respectively) compared with age- and sex-matched wild-type (WT) C57BL/6 mice as shown by Western blot. Fyn/Src Y420 phosphorylation also was reduced in ErbB4 +/− and NRG1( Δ TM) +/− mutant mice (bottom, lanes 2 and 3, respectively). Hippocampal lysates from Fyn −/− mice served as a control for NR2B Y1472 hypophosphorylation, as well as for Fyn/Src Y420 phosphorylation (fourth lane, top and bottom panels, respectively). Data are representative of five to six animals in at least three independent experiments; 25 μg of total protein was loaded per lane. B , Clozapine reverses NR2B hypophosphorylation in NRG1( Δ TM) +/− mice. NR2B Y1472 phosphorylation (top) was increased 2.5- to 3-fold in NRG1( Δ TM) +/− mice when normalized against total loading of NR2B (bottom). The same dose of clozapine had no effect on NR2B Y1472 phosphorylation (top) when administered to age- and sex-matched wild-type mice. Data representative of two to three animals in at least two independent experiments; 20 μg of total protein was loaded per lane.

    Techniques Used: Mutagenesis, Mouse Assay, Western Blot

    19) Product Images from "Dual role of E-cadherin in the regulation of invasive collective migration of mammary carcinoma cells"

    Article Title: Dual role of E-cadherin in the regulation of invasive collective migration of mammary carcinoma cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22940-3

    E-cadherin is essential for tether formation. 4T1 cells, stably knocked down for E-cadherin (shEcad), and control cells (ShCon) were imaged by DIC ( A , D ) and double-labelled for E-cadherin ( B , E ) and β-catenin ( C , F ). Note that tether formation and E-cadherin-rich adhesions are markedly reduced in the knocked-down cells. ( G ) QRT–PCR analysis, pointing to an over 85% reduction in E-cadherin mRNA levels in shEcad compared to shCon 4T1cells, obtained in 3 independent experiments. ( H ) Western blotting revealed an ~83% decrease of E-cadherin protein levels in 4T1-shEcad, compared to 4T1-shCon. ( I ) Comparison of the percentage of tether-associated cells in shEcad and shCon 4T1cells, pointing to an ~68% reduction in the knocked-down cells, compared to controls. ***p
    Figure Legend Snippet: E-cadherin is essential for tether formation. 4T1 cells, stably knocked down for E-cadherin (shEcad), and control cells (ShCon) were imaged by DIC ( A , D ) and double-labelled for E-cadherin ( B , E ) and β-catenin ( C , F ). Note that tether formation and E-cadherin-rich adhesions are markedly reduced in the knocked-down cells. ( G ) QRT–PCR analysis, pointing to an over 85% reduction in E-cadherin mRNA levels in shEcad compared to shCon 4T1cells, obtained in 3 independent experiments. ( H ) Western blotting revealed an ~83% decrease of E-cadherin protein levels in 4T1-shEcad, compared to 4T1-shCon. ( I ) Comparison of the percentage of tether-associated cells in shEcad and shCon 4T1cells, pointing to an ~68% reduction in the knocked-down cells, compared to controls. ***p

    Techniques Used: Stable Transfection, Quantitative RT-PCR, Western Blot

    E-cadherin facilitates infiltration of 4T1 cell into stromal cells, ex vivo . GFP-expressing 4T1 cells (Con) or shEcad 4T1 cells (shEcad), and RFP expressing mouse embryo fibroblasts (MEF) were plated in two parallel compartments of a silicon insert (Ibidi®). When the cells within the two compartments reached confluence, the insert was removed, and the area between the two cell monolayers was imaged by live-cell video microscopy. ( A ) Snapshots taken at the indicated time points, showing the Con (left column, yellow) and shEcad (right column, yellow) and the MEF cells (magenta-colored). The vertical wavy line marks the position of the cancer cells at t = 0. Scale bar: 100 μm. ( B .
    Figure Legend Snippet: E-cadherin facilitates infiltration of 4T1 cell into stromal cells, ex vivo . GFP-expressing 4T1 cells (Con) or shEcad 4T1 cells (shEcad), and RFP expressing mouse embryo fibroblasts (MEF) were plated in two parallel compartments of a silicon insert (Ibidi®). When the cells within the two compartments reached confluence, the insert was removed, and the area between the two cell monolayers was imaged by live-cell video microscopy. ( A ) Snapshots taken at the indicated time points, showing the Con (left column, yellow) and shEcad (right column, yellow) and the MEF cells (magenta-colored). The vertical wavy line marks the position of the cancer cells at t = 0. Scale bar: 100 μm. ( B .

    Techniques Used: Ex Vivo, Expressing, Microscopy

    Tether-mediated cell-cell adhesions are anchored via adherens-type junctions. ( A , C ) Densely-plated 4T1 cells form E-cadherin- ( A ) and β-catenin-rich ( C ) adherens junctions, located either along the sub-apical region (arrows), or in multiple dorsal patches (arrowheads). ( B , D ) Sparsely plated 4T1 cells form structurally and molecularly polarized tethers that are attached to the neighboring cell via E-cadherin ( B ), co-localized actin (magenta) and β-catenin (yellow) ( D ). Arrowhead in Panel D denotes the tether adhesion area. Scale bar: 10 μm. (E,E’) Immunoblotting and corresponding histogram depicting E-cadherin levels in sparsely- (5.6*10 3 /cm 2 ) and densely-plated (8.9*10 4 /cm 2 ) 4T1 cells. The normalized levels of E-cadherin (relative to α-tubulin) are shown in E’. Blotting Lanes of Fig. 3E were extracted from the same gel (full blotting of the gel is shown in Supplementary). ( F ) Comparison of E-cadherin mRNA levels in densely- and sparsely-plated 4T1 cells normalized to HPRT1 and GUSB genes. The results are based on two independent experiments.
    Figure Legend Snippet: Tether-mediated cell-cell adhesions are anchored via adherens-type junctions. ( A , C ) Densely-plated 4T1 cells form E-cadherin- ( A ) and β-catenin-rich ( C ) adherens junctions, located either along the sub-apical region (arrows), or in multiple dorsal patches (arrowheads). ( B , D ) Sparsely plated 4T1 cells form structurally and molecularly polarized tethers that are attached to the neighboring cell via E-cadherin ( B ), co-localized actin (magenta) and β-catenin (yellow) ( D ). Arrowhead in Panel D denotes the tether adhesion area. Scale bar: 10 μm. (E,E’) Immunoblotting and corresponding histogram depicting E-cadherin levels in sparsely- (5.6*10 3 /cm 2 ) and densely-plated (8.9*10 4 /cm 2 ) 4T1 cells. The normalized levels of E-cadherin (relative to α-tubulin) are shown in E’. Blotting Lanes of Fig. 3E were extracted from the same gel (full blotting of the gel is shown in Supplementary). ( F ) Comparison of E-cadherin mRNA levels in densely- and sparsely-plated 4T1 cells normalized to HPRT1 and GUSB genes. The results are based on two independent experiments.

    Techniques Used:

    Effect of E-cadherin knockdown on the formation of lung metastases following injection of 4T1 cells into BALB/c mice. ( A ) 4T1-GFP cells (1*10 6 cells) were injected intravenously into the tail veins of BALB/c mice. Seven days later, animals were sacrificed, and their lungs imaged using 2-photon microscopy. Examination of the images revealed loosely packed metastatic nodules with multiple tethers running between the cells (arrowheads). Scale bar: Middle image − 10 mm; right image − 10 μm; insert − 5 μm. ( B ) Representative bioluminescence images (BLI), taken on days 5, 14, and 21, post-injection of 2.5*10 4 4T1-luciferase shCon or shEcad cells into the mammary fat pads of BALB/c mice (n = 4/5 mice in each group, respectively). ( C ,C’) BLI quantification of total flux in the lungs of shCon or shEcad mouse groups. Note that the accumulation of tumor cells in the lungs during the 14–21 day post-injection period was over twofold lower in the E-cadherin knocked-down cells, compared to the control 4T1 cells. ( D ) BLI quantification of total flux of 4T1-luciferase shCon or shEcad in the lungs of BALB/c mice, studied in two separate fat-pad injection experiments (n = 9/10 for each shCon or shEcad group, respectively). Data were collected on Day 14, and statistical significance of the differences between the groups was determined, using two-way ANOVA (**p = 0.009).
    Figure Legend Snippet: Effect of E-cadherin knockdown on the formation of lung metastases following injection of 4T1 cells into BALB/c mice. ( A ) 4T1-GFP cells (1*10 6 cells) were injected intravenously into the tail veins of BALB/c mice. Seven days later, animals were sacrificed, and their lungs imaged using 2-photon microscopy. Examination of the images revealed loosely packed metastatic nodules with multiple tethers running between the cells (arrowheads). Scale bar: Middle image − 10 mm; right image − 10 μm; insert − 5 μm. ( B ) Representative bioluminescence images (BLI), taken on days 5, 14, and 21, post-injection of 2.5*10 4 4T1-luciferase shCon or shEcad cells into the mammary fat pads of BALB/c mice (n = 4/5 mice in each group, respectively). ( C ,C’) BLI quantification of total flux in the lungs of shCon or shEcad mouse groups. Note that the accumulation of tumor cells in the lungs during the 14–21 day post-injection period was over twofold lower in the E-cadherin knocked-down cells, compared to the control 4T1 cells. ( D ) BLI quantification of total flux of 4T1-luciferase shCon or shEcad in the lungs of BALB/c mice, studied in two separate fat-pad injection experiments (n = 9/10 for each shCon or shEcad group, respectively). Data were collected on Day 14, and statistical significance of the differences between the groups was determined, using two-way ANOVA (**p = 0.009).

    Techniques Used: Injection, Mouse Assay, Microscopy, Luciferase

    Knockdown of E-cadherin significantly reduces lung metastases following intravenous injection of 4T1 cells into BALB/c mice. ( A ) BLI imaging at 1, 3, or 8 days post-injection of 1*10 5 4T1-luciferase shCon or shEcad cells into the tail vein of BALB/c mice. ( B ) Quantification of total flux in the lungs of mice injected with 4T1-luciferase shCon or shEcad cells. Histograms indicate a major reduction in the development of lung metastases in the E-cadherin knockded-down cells. Statistical significance of the differences between the total flux in the lungs of the mice injected with the knocked-down and control cells was determined using two-way ANOVA, based on three independent experiments, n = 11 in each group (**p = 0.001).
    Figure Legend Snippet: Knockdown of E-cadherin significantly reduces lung metastases following intravenous injection of 4T1 cells into BALB/c mice. ( A ) BLI imaging at 1, 3, or 8 days post-injection of 1*10 5 4T1-luciferase shCon or shEcad cells into the tail vein of BALB/c mice. ( B ) Quantification of total flux in the lungs of mice injected with 4T1-luciferase shCon or shEcad cells. Histograms indicate a major reduction in the development of lung metastases in the E-cadherin knockded-down cells. Statistical significance of the differences between the total flux in the lungs of the mice injected with the knocked-down and control cells was determined using two-way ANOVA, based on three independent experiments, n = 11 in each group (**p = 0.001).

    Techniques Used: Injection, Mouse Assay, Imaging, Luciferase

    20) Product Images from "Phytanic acid activates PPARα to promote beige adipogenic differentiation of preadipocytes"

    Article Title: Phytanic acid activates PPARα to promote beige adipogenic differentiation of preadipocytes

    Journal: The Journal of nutritional biochemistry

    doi: 10.1016/j.jnutbio.2019.02.013

    Effects of PA on the lipid accumulation and the expression of adipogenic markers in differentiated 3T3-L1 cells. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA for 8 days. (A) Confluent 3T3-L1 cells were cultured in the differentiation medium with or without PA for 2 days and then switched to the basic medium including insulin and T3 with or without PA for 6 days. (B) Oil-Red O staining of differentiated 3T3-L1 white adipocytes. Bar, 100 μm. (C) The absorbance of the stained Oil-Red O at 530 nm. (D) Western blotting analysis of adipogenic markers FABP4 and PPARγ in the differentiated 3T3-L1 white adipocytes. β-actin was used as the control. (E) Relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. **p≤0.01 versus control; ***p≤0.001 versus control.
    Figure Legend Snippet: Effects of PA on the lipid accumulation and the expression of adipogenic markers in differentiated 3T3-L1 cells. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA for 8 days. (A) Confluent 3T3-L1 cells were cultured in the differentiation medium with or without PA for 2 days and then switched to the basic medium including insulin and T3 with or without PA for 6 days. (B) Oil-Red O staining of differentiated 3T3-L1 white adipocytes. Bar, 100 μm. (C) The absorbance of the stained Oil-Red O at 530 nm. (D) Western blotting analysis of adipogenic markers FABP4 and PPARγ in the differentiated 3T3-L1 white adipocytes. β-actin was used as the control. (E) Relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. **p≤0.01 versus control; ***p≤0.001 versus control.

    Techniques Used: Expressing, Cell Culture, Staining, Western Blot

    PA has a positive effect on the phosphorylation of AMPK. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA for 8 days. (A) Western blotting analysis of p-AMPK and AMPK in beige adipocytes. β-actin was used as the control. (B) Relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. *p≤0.05 versus control.
    Figure Legend Snippet: PA has a positive effect on the phosphorylation of AMPK. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA for 8 days. (A) Western blotting analysis of p-AMPK and AMPK in beige adipocytes. β-actin was used as the control. (B) Relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. *p≤0.05 versus control.

    Techniques Used: Western Blot

    PA promotes beige adipocyte biogenesis. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA for 8 days. (A and B) Quantitative real-time PCR analysis of brown (A) and beige (B) adipocyte marker genes in beige adipocytes from 3T3-L1 white adipocytes. Data are shown as means ± SD of three independent experiments. (C) Western blotting analysis of brown adipogenic markers in beige adipocytes. β-actin was used as the control. (D) Relative quantitative analysis of protein bands. (E) Confluent 3T3-L1 cells were cultured in the differentiation medium for 2 days, switched to the basic medium including insulin and T3 for 4 days and then changed to the medium (DMEM and 10% FBS) with or without PA for 2 days and 5 days, respectively. (F and G) Western blotting analysis of PGC1α content and relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. *p≤0.05 versus control; **p≤0.01 versus control; ***p≤0.001 versus control; ns indicates no significant differences between PA treatment and the control group.
    Figure Legend Snippet: PA promotes beige adipocyte biogenesis. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA for 8 days. (A and B) Quantitative real-time PCR analysis of brown (A) and beige (B) adipocyte marker genes in beige adipocytes from 3T3-L1 white adipocytes. Data are shown as means ± SD of three independent experiments. (C) Western blotting analysis of brown adipogenic markers in beige adipocytes. β-actin was used as the control. (D) Relative quantitative analysis of protein bands. (E) Confluent 3T3-L1 cells were cultured in the differentiation medium for 2 days, switched to the basic medium including insulin and T3 for 4 days and then changed to the medium (DMEM and 10% FBS) with or without PA for 2 days and 5 days, respectively. (F and G) Western blotting analysis of PGC1α content and relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. *p≤0.05 versus control; **p≤0.01 versus control; ***p≤0.001 versus control; ns indicates no significant differences between PA treatment and the control group.

    Techniques Used: Real-time Polymerase Chain Reaction, Marker, Western Blot, Cell Culture

    PPARα antagonism inhibits PA-stimulated browning of white adipocytes. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA or GW6471 for 8 days. (A) The PPARα antagonist GW6471 inhibits PA- mediated browning effects. Oil-Red O staining of differentiated 3T3-L1 white adipocytes. Bar, 100 μm. (B) The absorbance of the stained Oil-Red O at 530 nm. (C and E) Western blotting analysis of PGC1α, UCP1, PPARα, PRDM16 (C) and p-AMPK/AMPK (E) in beige adipocytes. β-actin was used as the control. (D and F) Relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. *p≤0.05 versus control; **p≤0.01 versus control; ## p ≤0.01 versus PA treatment; ### p ≤0.001 versus PA treatment.
    Figure Legend Snippet: PPARα antagonism inhibits PA-stimulated browning of white adipocytes. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA or GW6471 for 8 days. (A) The PPARα antagonist GW6471 inhibits PA- mediated browning effects. Oil-Red O staining of differentiated 3T3-L1 white adipocytes. Bar, 100 μm. (B) The absorbance of the stained Oil-Red O at 530 nm. (C and E) Western blotting analysis of PGC1α, UCP1, PPARα, PRDM16 (C) and p-AMPK/AMPK (E) in beige adipocytes. β-actin was used as the control. (D and F) Relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. *p≤0.05 versus control; **p≤0.01 versus control; ## p ≤0.01 versus PA treatment; ### p ≤0.001 versus PA treatment.

    Techniques Used: Staining, Western Blot

    PA-stimulated browning of white adipocytes is independent on β3-AR. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA and SR59230A for 8 days. (A) Oil-Red O staining of differentiated 3T3-L1 white adipocytes. Bar, 100 μm. (B) The absorbance of the stained Oil-Red O at 530 nm. (C and E) Western blotting analysis of adipocyte markers (C) and brown adipocyte markers (E) in beige adipocytes. β-actin was used as the control. (D and F) Relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. (G and H) Western blotting analysis of PGC1α during differentiation of primary brown adipocytes (G) and relative quantitative analysis of protein bands (H). Data are shown as means ± SD of three independent experiments.*p≤0.05 versus control; **p≤0.01 versus control; ***p≤0.001 versus control; # p ≤0.05 versus PA treatment; ## p ≤0.01 versus PA treatment; ns indicates no significant differences.
    Figure Legend Snippet: PA-stimulated browning of white adipocytes is independent on β3-AR. 3T3-L1 preadipocytes were induced into beige adipocytes through the treatment of PA and SR59230A for 8 days. (A) Oil-Red O staining of differentiated 3T3-L1 white adipocytes. Bar, 100 μm. (B) The absorbance of the stained Oil-Red O at 530 nm. (C and E) Western blotting analysis of adipocyte markers (C) and brown adipocyte markers (E) in beige adipocytes. β-actin was used as the control. (D and F) Relative quantitative analysis of protein bands. Data are shown as means ± SD of three independent experiments. (G and H) Western blotting analysis of PGC1α during differentiation of primary brown adipocytes (G) and relative quantitative analysis of protein bands (H). Data are shown as means ± SD of three independent experiments.*p≤0.05 versus control; **p≤0.01 versus control; ***p≤0.001 versus control; # p ≤0.05 versus PA treatment; ## p ≤0.01 versus PA treatment; ns indicates no significant differences.

    Techniques Used: Staining, Western Blot

    PPARα is necessary for the effect of PA on beige adipocyte biogenesis. 3T3-L1 preadipocytes with knockdown of PPARα were induced into beige adipocytes through the treatment of PA for 8 days. (A) The target sequences used for shRNA-mediated experiments. (B) Quantitative real-time PCR analysis of Pparα (left) and western blot analysis of PPARα (right) in shRNA control and shRNA PPARα 3T3-L1 cells. (C) Oil-Red O staining of differentiated 3T3-L1 cells. Bar, 100 μm. (D) The absorbance of the stained Oil-Red O at 530 nm. (E, G and I) Western blotting analysis of brown adipogenic markers (E), PPARγ (G), AMPK and p-AMPK (I) contents in the indicated cells. β-actin was used as the control. (F, H and J) Relative quantitative analysis of protein bands. *p≤0.05 versus control; **p≤0.01 versus control; ns indicates no significant differences between PA treatment and the control group.
    Figure Legend Snippet: PPARα is necessary for the effect of PA on beige adipocyte biogenesis. 3T3-L1 preadipocytes with knockdown of PPARα were induced into beige adipocytes through the treatment of PA for 8 days. (A) The target sequences used for shRNA-mediated experiments. (B) Quantitative real-time PCR analysis of Pparα (left) and western blot analysis of PPARα (right) in shRNA control and shRNA PPARα 3T3-L1 cells. (C) Oil-Red O staining of differentiated 3T3-L1 cells. Bar, 100 μm. (D) The absorbance of the stained Oil-Red O at 530 nm. (E, G and I) Western blotting analysis of brown adipogenic markers (E), PPARγ (G), AMPK and p-AMPK (I) contents in the indicated cells. β-actin was used as the control. (F, H and J) Relative quantitative analysis of protein bands. *p≤0.05 versus control; **p≤0.01 versus control; ns indicates no significant differences between PA treatment and the control group.

    Techniques Used: shRNA, Real-time Polymerase Chain Reaction, Western Blot, Staining

    21) Product Images from "Transcriptome Profiling Reveals Novel Candidate Genes Related to Hippocampal Dysfunction in SREBP-1c Knockout Mice"

    Article Title: Transcriptome Profiling Reveals Novel Candidate Genes Related to Hippocampal Dysfunction in SREBP-1c Knockout Mice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21114131

    Validation of GLP2R, NDN, and ERBB4 protein expressions in the hippocampus. ( A ) Western blot analyses confirm that both GLP2R and NDN expressions are significantly decreased, while ERBB4 expression is significantly increased in the hippocampus of SREBP-1c KO mice compared to in those of WT mice (n = 3, * p
    Figure Legend Snippet: Validation of GLP2R, NDN, and ERBB4 protein expressions in the hippocampus. ( A ) Western blot analyses confirm that both GLP2R and NDN expressions are significantly decreased, while ERBB4 expression is significantly increased in the hippocampus of SREBP-1c KO mice compared to in those of WT mice (n = 3, * p

    Techniques Used: Western Blot, Expressing, Mouse Assay

    qRT-PCR validation of selected DEGs in the hippocampus of SREBP-1c KO mice. ( A ) Gip2r , ( B ) Ndn , ( C ) Il1r1 , ( D ) Gm16867 , ( E ) Erbb4 , ( F ) Aox4 (n = 8, * p
    Figure Legend Snippet: qRT-PCR validation of selected DEGs in the hippocampus of SREBP-1c KO mice. ( A ) Gip2r , ( B ) Ndn , ( C ) Il1r1 , ( D ) Gm16867 , ( E ) Erbb4 , ( F ) Aox4 (n = 8, * p

    Techniques Used: Quantitative RT-PCR, Mouse Assay

    22) Product Images from "Protective effect of P188 in the Model of Acute Trauma to Human Ankle Cartilage: the Mechanism of Action"

    Article Title: Protective effect of P188 in the Model of Acute Trauma to Human Ankle Cartilage: the Mechanism of Action

    Journal: Journal of orthopaedic trauma

    doi: 10.1097/BOT.0b013e3181ec4712

    Proposed mechanism of P188 effect. Impact to cartilage induces matrix disruption and cell death, which translates on the cellular level to the activation of IL-6, p38, and GSK3 signaling. In addition to sealing cell membrane, P188 inhibits necrosis, apoptosis, IL-6, p38, and GSK3 pathways.
    Figure Legend Snippet: Proposed mechanism of P188 effect. Impact to cartilage induces matrix disruption and cell death, which translates on the cellular level to the activation of IL-6, p38, and GSK3 signaling. In addition to sealing cell membrane, P188 inhibits necrosis, apoptosis, IL-6, p38, and GSK3 pathways.

    Techniques Used: Activation Assay

    23) Product Images from "Modulation of Cell Death Pathways by Hepatitis C Virus Proteins in Huh7.5 Hepatoma Cells"

    Article Title: Modulation of Cell Death Pathways by Hepatitis C Virus Proteins in Huh7.5 Hepatoma Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18112346

    HCV proteins activated autophagy in Huh7.5 cells, as revealed by the enhanced incorporation of monodansylcadaverine into autophagosomes and detection of LC3. ( a ) Huh7.5 cells harboring the HCV replicon, or transfected with the E1/E2-expressing plasmid or the empty pcDNA3.1(+) vector were stained 72 h posttransfection with the monodansylcadaverine (MDC) and with DAPI. Vertical panels from the left to the right are: MDC staining (green), nuclear staining with DAPI (blue), overlay of MDC and DAPI staining; ( b ) Percentages of MDC-positive cells. ( c ) immunofluorescent staining of the activated LC3 and NS5B protein in Huh7.5 cells (400× magnification). Vertical panels left to right: staining with rabbit anti-LC3 primary and anti-rabbit secondary antibodies conjugated to Cy3 (orange) or primary mouse anti-NS5B antibody and secondary anti-mouse antibodies conjugated to FITC (green), merge with nuclear staining with DAPI (blue) The arrows indicate cells with LC3 punctuate staining; ( d ) Percentages of LC3-positive cells. Values are means ± SEM of eight measurements done in three independent experiments, * p
    Figure Legend Snippet: HCV proteins activated autophagy in Huh7.5 cells, as revealed by the enhanced incorporation of monodansylcadaverine into autophagosomes and detection of LC3. ( a ) Huh7.5 cells harboring the HCV replicon, or transfected with the E1/E2-expressing plasmid or the empty pcDNA3.1(+) vector were stained 72 h posttransfection with the monodansylcadaverine (MDC) and with DAPI. Vertical panels from the left to the right are: MDC staining (green), nuclear staining with DAPI (blue), overlay of MDC and DAPI staining; ( b ) Percentages of MDC-positive cells. ( c ) immunofluorescent staining of the activated LC3 and NS5B protein in Huh7.5 cells (400× magnification). Vertical panels left to right: staining with rabbit anti-LC3 primary and anti-rabbit secondary antibodies conjugated to Cy3 (orange) or primary mouse anti-NS5B antibody and secondary anti-mouse antibodies conjugated to FITC (green), merge with nuclear staining with DAPI (blue) The arrows indicate cells with LC3 punctuate staining; ( d ) Percentages of LC3-positive cells. Values are means ± SEM of eight measurements done in three independent experiments, * p

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Staining

    24) Product Images from "Inhibition of miR-155 Limits Neuroinflammation and Improves Functional Recovery After Experimental Traumatic Brain Injury in Mice"

    Article Title: Inhibition of miR-155 Limits Neuroinflammation and Improves Functional Recovery After Experimental Traumatic Brain Injury in Mice

    Journal: Neurotherapeutics

    doi: 10.1007/s13311-018-0665-9

    Acute inhibition of miR-155 reduces TBI-induced pro-inflammatory gene expression in the cortex and alters expression of miR-155 direct targets. Acute ICV administration of miR-155 antagomir, beginning at 15 min post-injury, attenuated TBI-induced increases in miR-155, IL-1β, and TNFα expression in the injured cortex at 24 h post-injury (A). TBI decreased mRNA and protein levels of SOCS1, but not SHIP-1, in the injured cortex at 24 h post-injury. miR-155 antagomir treatment increased levels of SOCS1 mRNA similar to sham control levels (B). *** p
    Figure Legend Snippet: Acute inhibition of miR-155 reduces TBI-induced pro-inflammatory gene expression in the cortex and alters expression of miR-155 direct targets. Acute ICV administration of miR-155 antagomir, beginning at 15 min post-injury, attenuated TBI-induced increases in miR-155, IL-1β, and TNFα expression in the injured cortex at 24 h post-injury (A). TBI decreased mRNA and protein levels of SOCS1, but not SHIP-1, in the injured cortex at 24 h post-injury. miR-155 antagomir treatment increased levels of SOCS1 mRNA similar to sham control levels (B). *** p

    Techniques Used: Inhibition, Expressing

    25) Product Images from "Bro1 directly stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis"

    Article Title: Bro1 directly stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

    Journal: bioRxiv

    doi: 10.1101/2020.07.27.223255

    Bro1V M4 and M8 are not defective in their ability to stimulate Vps4-dependent ESCRT-III disassembly in vitro or in vivo. (A) Bro1 V domains [Bro1V (black squares), Bro1V(M4) (dark red triangles), Bro1V(M8) (light red diamonds)] (50-1000nM) were titrated into Vps4-dependent ESCRT-III disassembly assay 16 , containing 100nM Vps4. Representative blots of Snf7 distribution between the pellet (P) and soluble fractions (S) are shown (upper panels). Quantification of three experiments with reactions performed in duplicates is designated as “% Snf7 soluble” (lower graph). Error bars indicate mean ± S.E.M. (B) Subcellular fractionation was performed in bro1Δ (GOY65) cells transformed with an empty vector or plasmids expressing BRO1, bro1(M4), or bro1(M8). vps4Δ (MBY3) cells were used as a control highlighting the distribution upon complete loss of Vps4 function. Representative Western blots showing fractionation of Snf7, Bro1, and Vps4 are shown. Pep12 and Pgk1 were used as membrane and soluble markers respectively. Quantification represents six experiments, error bars indicate mean ± S.E.M. Statistical significance is indicated relative to bro1Δ (*, p
    Figure Legend Snippet: Bro1V M4 and M8 are not defective in their ability to stimulate Vps4-dependent ESCRT-III disassembly in vitro or in vivo. (A) Bro1 V domains [Bro1V (black squares), Bro1V(M4) (dark red triangles), Bro1V(M8) (light red diamonds)] (50-1000nM) were titrated into Vps4-dependent ESCRT-III disassembly assay 16 , containing 100nM Vps4. Representative blots of Snf7 distribution between the pellet (P) and soluble fractions (S) are shown (upper panels). Quantification of three experiments with reactions performed in duplicates is designated as “% Snf7 soluble” (lower graph). Error bars indicate mean ± S.E.M. (B) Subcellular fractionation was performed in bro1Δ (GOY65) cells transformed with an empty vector or plasmids expressing BRO1, bro1(M4), or bro1(M8). vps4Δ (MBY3) cells were used as a control highlighting the distribution upon complete loss of Vps4 function. Representative Western blots showing fractionation of Snf7, Bro1, and Vps4 are shown. Pep12 and Pgk1 were used as membrane and soluble markers respectively. Quantification represents six experiments, error bars indicate mean ± S.E.M. Statistical significance is indicated relative to bro1Δ (*, p

    Techniques Used: In Vitro, In Vivo, Fractionation, Transformation Assay, Plasmid Preparation, Expressing, Western Blot

    26) Product Images from "Modeling the effect of ascites-induced compression on ovarian cancer multicellular aggregates"

    Article Title: Modeling the effect of ascites-induced compression on ovarian cancer multicellular aggregates

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.034199

    Compression-induced changes in epithelial-mesenchymal-transition-associated genes. (A) DOV13 and (B) OvCa433 MCAs were compressed employing a FX Flexcell-4000C Compression Plus System, and RNA was extracted and processed for qPCR as described in the Materials and Methods. Data shown are fold change in the expression of the indicated genes at 6 h (filled circles) and 24 h (open boxes), with red bars depicting the mean for each gene (representative of n ≥3 experimental replicates, where each filled circle or open box is a separate replicate). Note that DOV13 cells do not express ROR2 or CDH1 (E-cadherin).
    Figure Legend Snippet: Compression-induced changes in epithelial-mesenchymal-transition-associated genes. (A) DOV13 and (B) OvCa433 MCAs were compressed employing a FX Flexcell-4000C Compression Plus System, and RNA was extracted and processed for qPCR as described in the Materials and Methods. Data shown are fold change in the expression of the indicated genes at 6 h (filled circles) and 24 h (open boxes), with red bars depicting the mean for each gene (representative of n ≥3 experimental replicates, where each filled circle or open box is a separate replicate). Note that DOV13 cells do not express ROR2 or CDH1 (E-cadherin).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    27) Product Images from "The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis"

    Article Title: The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-07-1196

    Gef3 binds to the Rho GTPase Rho4 in vitro. (A) Coomassie blue staining of SDS–PAGE showing purified 3FLAG-Gef3 from S. pombe . The asterisk marks the 3FLAG-Gef3 band with the expected size. (B, C) Gef3 binds to Rho4 in pull-down assays. Purified GST-Rho GTPases and GST control were bound to beads, depleted for nucleotides, and then incubated with purified 3FLAG-Gef3. The amount of pulled-down Gef3 was detected by Western blotting (B; representative of four independent assays) and quantified (C; mean ± 1 SD). The intensities of 3FLAG-Gef3 bands were normalized by the intensities of pulled-down Rho GTPases. The intensity of 3FLAG-Gef3 band in GST control was set as 1.
    Figure Legend Snippet: Gef3 binds to the Rho GTPase Rho4 in vitro. (A) Coomassie blue staining of SDS–PAGE showing purified 3FLAG-Gef3 from S. pombe . The asterisk marks the 3FLAG-Gef3 band with the expected size. (B, C) Gef3 binds to Rho4 in pull-down assays. Purified GST-Rho GTPases and GST control were bound to beads, depleted for nucleotides, and then incubated with purified 3FLAG-Gef3. The amount of pulled-down Gef3 was detected by Western blotting (B; representative of four independent assays) and quantified (C; mean ± 1 SD). The intensities of 3FLAG-Gef3 bands were normalized by the intensities of pulled-down Rho GTPases. The intensity of 3FLAG-Gef3 band in GST control was set as 1.

    Techniques Used: In Vitro, Staining, SDS Page, Purification, Incubation, Western Blot

    28) Product Images from "The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis"

    Article Title: The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-07-1196

    Gef3 regulates the localization of glucanases and Rho4 activity in vivo. (A, B) Eng1 localization is affected in gef3∆ cells. Micrographs (A) and quantification (B) showing that Eng1 localization at division site is affected in gef3∆ and rho4∆ cells. The arrowhead indicates a wt cell with Eng1 in the nonconstricting ring. Vertical views of the regions marked with red boxes are shown at the bottom corners. Arrows mark a gef3∆ cell without an intact Eng1 ring at the division site. (B) Only cells with complete septa were counted. For each strain, n > 40 cells. (C) RBD pulls down constitutively active Rho4-G23V more efficiently than wt and constitutively inactive Rho4-T28N. Yeast extracts from cells expressing 3HA tagged wt or mutant Rho4 were incubated with 200 μg of GST-RBD. The amount of Rho4 was detected by Western blotting. (D, E) Western blotting (D) and quantification (E) showing that gef3∆ cells have less active Rho4 than wt cells. (E) Mean ± 1 SD from three independent experiments. The level in wt is normalized to 1. (F, G) Overexpression of Rho4 or Eng1 rescues the septation defects in gef3∆ s cw1∆ cells. DIC images (F) and septation index (G) of gef3∆ s cw1∆ cells with plasmids grown under inducing conditions in liquid EMM5S – leucine for 24 h. (G) Mean ± 1 SD from three independent experiments, and n > 600 for each strain in each experiment. Bars, 5 μm.
    Figure Legend Snippet: Gef3 regulates the localization of glucanases and Rho4 activity in vivo. (A, B) Eng1 localization is affected in gef3∆ cells. Micrographs (A) and quantification (B) showing that Eng1 localization at division site is affected in gef3∆ and rho4∆ cells. The arrowhead indicates a wt cell with Eng1 in the nonconstricting ring. Vertical views of the regions marked with red boxes are shown at the bottom corners. Arrows mark a gef3∆ cell without an intact Eng1 ring at the division site. (B) Only cells with complete septa were counted. For each strain, n > 40 cells. (C) RBD pulls down constitutively active Rho4-G23V more efficiently than wt and constitutively inactive Rho4-T28N. Yeast extracts from cells expressing 3HA tagged wt or mutant Rho4 were incubated with 200 μg of GST-RBD. The amount of Rho4 was detected by Western blotting. (D, E) Western blotting (D) and quantification (E) showing that gef3∆ cells have less active Rho4 than wt cells. (E) Mean ± 1 SD from three independent experiments. The level in wt is normalized to 1. (F, G) Overexpression of Rho4 or Eng1 rescues the septation defects in gef3∆ s cw1∆ cells. DIC images (F) and septation index (G) of gef3∆ s cw1∆ cells with plasmids grown under inducing conditions in liquid EMM5S – leucine for 24 h. (G) Mean ± 1 SD from three independent experiments, and n > 600 for each strain in each experiment. Bars, 5 μm.

    Techniques Used: Activity Assay, In Vivo, Expressing, Mutagenesis, Incubation, Western Blot, Over Expression

    29) Product Images from "Growth Inhibitory Effects of Large Subunit Ribosomal Proteins in Melanoma"

    Article Title: Growth Inhibitory Effects of Large Subunit Ribosomal Proteins in Melanoma

    Journal: Pigment cell & melanoma research

    doi: 10.1111/pcmr.12259

    RPL5 and RPL11 mediated p53 and cell cycle effects following RPL13 silencing
    Figure Legend Snippet: RPL5 and RPL11 mediated p53 and cell cycle effects following RPL13 silencing

    Techniques Used:

    30) Product Images from "Post-translational allosteric activation of the P2X7 receptor through glycosaminoglycan chains of CD44 proteoglycans"

    Article Title: Post-translational allosteric activation of the P2X7 receptor through glycosaminoglycan chains of CD44 proteoglycans

    Journal: Cell Death Discovery

    doi: 10.1038/cddiscovery.2015.5

    ATP-gated P2X 7 receptor is expressed on the CHO cells surface. The P2X 7 receptor expression was determined through flow cytometry analysis. CHO cells were labeled with antibody anti-P2X 7 conjugated with Alexa Fluor 488, and the data were collected using a FACSCalibur flow cytometer (Becton–Dickinson) and analyzed using FlowJo software (Tree Star). The boundary between positive and negative cells labeled for the P2X 7 receptor was determined according to the fluorescence distribution of positive cells relative to unlabeled control samples. ( a ) The amount of P2X 7 receptor expressed in whole CHO cells. ( b ) P2X 7 receptor expressed at the surface of CHO cells. ( c ) Immunofluorescence labeling of P2X 7 in CHO-K1 and CHO-745 cells. Cells were stained with DAPI (blue) and immunolabelled with anti-P2X 7 (green) and Alexa Fluor 594 conjugated to WGA (red) at left column; ER-Tracker (red) at central column; and with CellLight Golgi Fluorescent Protein (red) at right column. The histograms and images are representative of the results of three experiments. Scale bars, 20 μ m.
    Figure Legend Snippet: ATP-gated P2X 7 receptor is expressed on the CHO cells surface. The P2X 7 receptor expression was determined through flow cytometry analysis. CHO cells were labeled with antibody anti-P2X 7 conjugated with Alexa Fluor 488, and the data were collected using a FACSCalibur flow cytometer (Becton–Dickinson) and analyzed using FlowJo software (Tree Star). The boundary between positive and negative cells labeled for the P2X 7 receptor was determined according to the fluorescence distribution of positive cells relative to unlabeled control samples. ( a ) The amount of P2X 7 receptor expressed in whole CHO cells. ( b ) P2X 7 receptor expressed at the surface of CHO cells. ( c ) Immunofluorescence labeling of P2X 7 in CHO-K1 and CHO-745 cells. Cells were stained with DAPI (blue) and immunolabelled with anti-P2X 7 (green) and Alexa Fluor 594 conjugated to WGA (red) at left column; ER-Tracker (red) at central column; and with CellLight Golgi Fluorescent Protein (red) at right column. The histograms and images are representative of the results of three experiments. Scale bars, 20 μ m.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Labeling, Software, Fluorescence, Immunofluorescence, Staining, Whole Genome Amplification

    31) Product Images from "d-serine levels in Alzheimer's disease: implications for novel biomarker development"

    Article Title: d-serine levels in Alzheimer's disease: implications for novel biomarker development

    Journal: Translational Psychiatry

    doi: 10.1038/tp.2015.52

    Amyloid-β oligomers (AβOs) increase d -serine and serine racemase (SR) levels in hippocampal cultures. Primary rat hippocampal neuronal cultures were exposed to 500 n M AβOs or vehicle (2% dimethyl sulfoxide in phosphate-buffered saline) for 24 h. ( a ) AβOs increased extracellular levels of d -serine. ( b and c ) AβOs increased total levels of SR messenger RNA (mRNA) ( b ) and protein ( c ). d -serine was measured by high-performance liquid chromatography and its values were corrected by total protein content in the analyzed samples. SR protein levels were detected by western blotting, using β-actin as a loading control. * P
    Figure Legend Snippet: Amyloid-β oligomers (AβOs) increase d -serine and serine racemase (SR) levels in hippocampal cultures. Primary rat hippocampal neuronal cultures were exposed to 500 n M AβOs or vehicle (2% dimethyl sulfoxide in phosphate-buffered saline) for 24 h. ( a ) AβOs increased extracellular levels of d -serine. ( b and c ) AβOs increased total levels of SR messenger RNA (mRNA) ( b ) and protein ( c ). d -serine was measured by high-performance liquid chromatography and its values were corrected by total protein content in the analyzed samples. SR protein levels were detected by western blotting, using β-actin as a loading control. * P

    Techniques Used: High Performance Liquid Chromatography, Western Blot

    32) Product Images from "Aberrant Regulation of Planar Cell Polarity in Polycystic Kidney Disease"

    Article Title: Aberrant Regulation of Planar Cell Polarity in Polycystic Kidney Disease

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2010010127

    CDC42 is activated in cystic kidneys. (A) Immunofluorescence micrographs represent the CDC42 expression pattern in Mx1 -Cre.Pkd1 F/null cystic and Pkd1 F/F control kidneys. Note that in Mx1 -Cre.Pkd1 F/null cystic kidneys, CDC42 localizes particularly to the apical membrane, in contrast to a diffused expression pattern in Pkd1 F/F control kidneys. Pictures are taken with the same exposure time. (B) Western blots demonstrate that total as well as activated CDC42 is increased in γGt -Cre.Pkd1 F/F cystic kidneys. Ponceau staining is used to show equal loading (bottom). Magnification, ×60.
    Figure Legend Snippet: CDC42 is activated in cystic kidneys. (A) Immunofluorescence micrographs represent the CDC42 expression pattern in Mx1 -Cre.Pkd1 F/null cystic and Pkd1 F/F control kidneys. Note that in Mx1 -Cre.Pkd1 F/null cystic kidneys, CDC42 localizes particularly to the apical membrane, in contrast to a diffused expression pattern in Pkd1 F/F control kidneys. Pictures are taken with the same exposure time. (B) Western blots demonstrate that total as well as activated CDC42 is increased in γGt -Cre.Pkd1 F/F cystic kidneys. Ponceau staining is used to show equal loading (bottom). Magnification, ×60.

    Techniques Used: Immunofluorescence, Expressing, Western Blot, Staining

    Fz3 is upregulated in cystic but not in precystic Pkd1 -inactivated mouse kidneys. (A and B) Western blots show that CDC42 but not Fz3 protein levels are upregulated in precystic Mx1 -Cre.Pkd1 F/F kidneys (A). Both Fz3 and CDC42 levels are greatly upregulated in 10-day-old Col2 -Cre.Pkd1 F/null and 3-week-old γGt -Cre.Pkd1 F/F cystic kidneys. GAPDH was used as loading control.
    Figure Legend Snippet: Fz3 is upregulated in cystic but not in precystic Pkd1 -inactivated mouse kidneys. (A and B) Western blots show that CDC42 but not Fz3 protein levels are upregulated in precystic Mx1 -Cre.Pkd1 F/F kidneys (A). Both Fz3 and CDC42 levels are greatly upregulated in 10-day-old Col2 -Cre.Pkd1 F/null and 3-week-old γGt -Cre.Pkd1 F/F cystic kidneys. GAPDH was used as loading control.

    Techniques Used: Western Blot

    Fz3 is upregulated in human ADPKD kidneys. (A) Western blots represent increased Fz3, Fz6, and CDC42 protein levels in human ADPKD kidneys (lanes 2 and 4) compared with normal kidneys (lanes 1 and 3). GAPDH is used as a loading control (bottom). (B and C) Immunofluorescence micrographs demonstrate Fz3 (B) and CDC42 (C) localization in normal (top) and ADPKD (bottom) kidneys. Note in DBA + tubules of normal kidneys, Fz3 localizes weakly at the apical membrane, whereas CDC42 localizes diffusely in the cytosol. In ADPKD kidneys, both Fz3 and CDC42 are strongly upregulated at the apical membrane of cyst-lining epithelial cells in distal cysts. Pictures are taken with the same exposure time. Magnification, ×20 in B; ×40 in C.
    Figure Legend Snippet: Fz3 is upregulated in human ADPKD kidneys. (A) Western blots represent increased Fz3, Fz6, and CDC42 protein levels in human ADPKD kidneys (lanes 2 and 4) compared with normal kidneys (lanes 1 and 3). GAPDH is used as a loading control (bottom). (B and C) Immunofluorescence micrographs demonstrate Fz3 (B) and CDC42 (C) localization in normal (top) and ADPKD (bottom) kidneys. Note in DBA + tubules of normal kidneys, Fz3 localizes weakly at the apical membrane, whereas CDC42 localizes diffusely in the cytosol. In ADPKD kidneys, both Fz3 and CDC42 are strongly upregulated at the apical membrane of cyst-lining epithelial cells in distal cysts. Pictures are taken with the same exposure time. Magnification, ×20 in B; ×40 in C.

    Techniques Used: Western Blot, Immunofluorescence

    Fz3 and PC1 antagonize each other in an inducible PC1-expressing cell line, and Axin2 expression is increased in precystic and cystic Pkd1 KO kidneys. (A) Western blots show that total CDC42 expression is not increased after PC1 induction (compare first and second lanes with GAPDH) but strongly after Fz3 overexpression (compare first and third lanes). Note that Fz3 and PC1 coexpression decreases total CDC42 expression levels (compare first and last lanes). GAPDH is used as loading control. (B) Chymographs demonstrate the distance traveled by YPC1m-HEK cells during a time-lapse videomicroscopy recording of wound-healing assays. Pictures are taken every 6 minutes during 20 hours. A white line is drawn next to the edge of the wound; a steeper white line indicates a slower closure. PC1 induction increases the speed, demonstrated by a gentle slope; Fz3 decreases the speed, showed by a steeper slope. Note that the slope in the first, control, and in the last, Fz3-transfected and PC1-induced chymograph, is the same. (C) Graph represents the quantification of the speed of migration of YPC1m-HEK cells in different fields in B. This confirms that cells transfected with Fz3 migrate slower, whereas cells transfected with Fz3 and induced with PC1 move with a similar speed as their counterpart controls. (D) Graph demonstrates the upregulation of Axin2 via quantitative reverse transcriptase–PCR in precystic Mx1- Cre.Pkd1 F/F and Mx1- Cre.Pkd1 F/null and in cystic γGt -Cre.Pkd1 F/F kidneys compared with their control littermates. All samples are normalized to the 2-week-old control kidney, which was set as 1. (E) Graph represents the relative luciferase activity in HEK control and YPC1m-HEK cells with or without Fz3 transfection and tetracycline induction. PC1 induction slightly reduces luciferase activity. All samples were normalized to the HEK control column, which was set as 1. Bars indicate SEs. All experiments were repeated at least three times. Magnification, ×10.
    Figure Legend Snippet: Fz3 and PC1 antagonize each other in an inducible PC1-expressing cell line, and Axin2 expression is increased in precystic and cystic Pkd1 KO kidneys. (A) Western blots show that total CDC42 expression is not increased after PC1 induction (compare first and second lanes with GAPDH) but strongly after Fz3 overexpression (compare first and third lanes). Note that Fz3 and PC1 coexpression decreases total CDC42 expression levels (compare first and last lanes). GAPDH is used as loading control. (B) Chymographs demonstrate the distance traveled by YPC1m-HEK cells during a time-lapse videomicroscopy recording of wound-healing assays. Pictures are taken every 6 minutes during 20 hours. A white line is drawn next to the edge of the wound; a steeper white line indicates a slower closure. PC1 induction increases the speed, demonstrated by a gentle slope; Fz3 decreases the speed, showed by a steeper slope. Note that the slope in the first, control, and in the last, Fz3-transfected and PC1-induced chymograph, is the same. (C) Graph represents the quantification of the speed of migration of YPC1m-HEK cells in different fields in B. This confirms that cells transfected with Fz3 migrate slower, whereas cells transfected with Fz3 and induced with PC1 move with a similar speed as their counterpart controls. (D) Graph demonstrates the upregulation of Axin2 via quantitative reverse transcriptase–PCR in precystic Mx1- Cre.Pkd1 F/F and Mx1- Cre.Pkd1 F/null and in cystic γGt -Cre.Pkd1 F/F kidneys compared with their control littermates. All samples are normalized to the 2-week-old control kidney, which was set as 1. (E) Graph represents the relative luciferase activity in HEK control and YPC1m-HEK cells with or without Fz3 transfection and tetracycline induction. PC1 induction slightly reduces luciferase activity. All samples were normalized to the HEK control column, which was set as 1. Bars indicate SEs. All experiments were repeated at least three times. Magnification, ×10.

    Techniques Used: Expressing, Western Blot, Over Expression, Transfection, Migration, Polymerase Chain Reaction, Luciferase, Activity Assay

    33) Product Images from "Distinct and Shared Roles of ?-Arrestin-1 and ?-Arrestin-2 on the Regulation of C3a Receptor Signaling in Human Mast Cells"

    Article Title: Distinct and Shared Roles of ?-Arrestin-1 and ?-Arrestin-2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019585

    C3a-induced ERK1/2 phosphorylation is enhanced in β-arrestin-1, β-arrestin-2 and double KD cells. shRNA control or β-arrestin KD HMC-1 cells (1×10 6 /ml) were exposed to C3a (100 nM) for 1, 5 and 10, 15 and 30 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody followed by anti-rabbit IgG-HRP. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A) Representative immunoblots from three similar experiments are shown. (B) ERK1/2 phosphorylation was quantified using Image J as shown in the line graph. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by two way ANOVA with Bonferroni's post test. ** indicates p
    Figure Legend Snippet: C3a-induced ERK1/2 phosphorylation is enhanced in β-arrestin-1, β-arrestin-2 and double KD cells. shRNA control or β-arrestin KD HMC-1 cells (1×10 6 /ml) were exposed to C3a (100 nM) for 1, 5 and 10, 15 and 30 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody followed by anti-rabbit IgG-HRP. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A) Representative immunoblots from three similar experiments are shown. (B) ERK1/2 phosphorylation was quantified using Image J as shown in the line graph. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by two way ANOVA with Bonferroni's post test. ** indicates p

    Techniques Used: shRNA, SDS Page, Western Blot

    34) Product Images from "Interleukin-21 Receptor Gene Induction in Human T Cells Is Mediated by T-Cell Receptor-Induced Sp1 Activity"

    Article Title: Interleukin-21 Receptor Gene Induction in Human T Cells Is Mediated by T-Cell Receptor-Induced Sp1 Activity

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.22.9741-9752.2005

    Sp1 and Sp3 bind to the GC motif in the IL-21R promoter in vitro. (A) Proteins purified by DNA affinity chromatography. Nuclear extracts from PI-stimulated (4 h) human PB T cells were subjected to DNA affinity chromatography. Eluates from WT or mutant
    Figure Legend Snippet: Sp1 and Sp3 bind to the GC motif in the IL-21R promoter in vitro. (A) Proteins purified by DNA affinity chromatography. Nuclear extracts from PI-stimulated (4 h) human PB T cells were subjected to DNA affinity chromatography. Eluates from WT or mutant

    Techniques Used: In Vitro, Purification, Affinity Chromatography, Mutagenesis

    Dephosphorylation of Sp1 is essential for TCR activation-induced IL-21R expression. Human PB T cells were not stimulated or stimulated with anti-CD3 and anti-CD28 for 2 h. Half of the cells were coincubated with 10 nM calyculin A or 0.8 μM okadaic
    Figure Legend Snippet: Dephosphorylation of Sp1 is essential for TCR activation-induced IL-21R expression. Human PB T cells were not stimulated or stimulated with anti-CD3 and anti-CD28 for 2 h. Half of the cells were coincubated with 10 nM calyculin A or 0.8 μM okadaic

    Techniques Used: De-Phosphorylation Assay, Activation Assay, Expressing

    Binding of Sp1 to the IL-21R promoter is essential for TCR-induced IL-21R expression. Suppression of IL-21R expression in human primary T cells by siRNA targeting of Sp1 is also shown. Human PB T cells were transfected with 100 nM siRNAs along with pEYFP-N1
    Figure Legend Snippet: Binding of Sp1 to the IL-21R promoter is essential for TCR-induced IL-21R expression. Suppression of IL-21R expression in human primary T cells by siRNA targeting of Sp1 is also shown. Human PB T cells were transfected with 100 nM siRNAs along with pEYFP-N1

    Techniques Used: Binding Assay, Expressing, Transfection

    35) Product Images from "TGF-β induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways"

    Article Title: TGF-β induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways

    Journal: American Journal of Clinical and Experimental Urology

    doi:

    Smad3 regulates FGF-2 expression in mouse embryo fibroblasts. A. TGF-β1-induced (100 pM) FGF-2 mRNA expression is equivalent in both Smad2 control MEFs and Smad2 null MEFs; B. A significant upregulation of FGF-2 mRNA by TGF-β1 (100 pM) is evident in Smad3 control MEFs with maximal expression at 9 hr, whereas FGF-2 message remains at near basal levels in TGF-β1 stimulated Smad3 null MEFs; C. TGF-β1 (100 pM) induces Smad2 and Smad3 phosphorylation in Smad3 wild type MEFs cells. In Smad3 null MEFs, Smad3 protein and phosphorylation is absent; however, Smad2 is phosphorylated by TGF-β1; D. A corresponding upregulation of total cellular FGF-2 protein levels by TGF-β1 (100 pM) at 24 hr is observed in Smad3 control MEFs; however, this effect is significantly attenuated in Smad3 null MEFs. All data were normalized to cell number and fold changes were determined relative to control. *P
    Figure Legend Snippet: Smad3 regulates FGF-2 expression in mouse embryo fibroblasts. A. TGF-β1-induced (100 pM) FGF-2 mRNA expression is equivalent in both Smad2 control MEFs and Smad2 null MEFs; B. A significant upregulation of FGF-2 mRNA by TGF-β1 (100 pM) is evident in Smad3 control MEFs with maximal expression at 9 hr, whereas FGF-2 message remains at near basal levels in TGF-β1 stimulated Smad3 null MEFs; C. TGF-β1 (100 pM) induces Smad2 and Smad3 phosphorylation in Smad3 wild type MEFs cells. In Smad3 null MEFs, Smad3 protein and phosphorylation is absent; however, Smad2 is phosphorylated by TGF-β1; D. A corresponding upregulation of total cellular FGF-2 protein levels by TGF-β1 (100 pM) at 24 hr is observed in Smad3 control MEFs; however, this effect is significantly attenuated in Smad3 null MEFs. All data were normalized to cell number and fold changes were determined relative to control. *P

    Techniques Used: Expressing

    Binding of Smad3/4 complex to the FGF-2 promoter. A. ChIP analysis after TGF-β1 (80 pM) treatment of Smad2 null MEFs showed Smad3 and Smad4 antibodies selectively immunoprecipitated the -2353 to -2168 region (arrow) of the FGF-2 promoter that contains a putative consensus Smad4 binding element; B. Chromatin shearing shows fragments of approximately 200-800 bp; C. Map of regions evaluated by PCR analysis (arrows). Primer sets were designed to scan 5 kb of the FGF-2 promoter, but only amplicons surrounding the potential binding site are shown. Sequence analysis in this region detected five potential SBEs. Subsequent analysis using MatInspector (Genomatix) predicted a gtct SBE motif at -1994 from the start codon as a putative Smad4 binding sequence in the FGF-2 promoter.
    Figure Legend Snippet: Binding of Smad3/4 complex to the FGF-2 promoter. A. ChIP analysis after TGF-β1 (80 pM) treatment of Smad2 null MEFs showed Smad3 and Smad4 antibodies selectively immunoprecipitated the -2353 to -2168 region (arrow) of the FGF-2 promoter that contains a putative consensus Smad4 binding element; B. Chromatin shearing shows fragments of approximately 200-800 bp; C. Map of regions evaluated by PCR analysis (arrows). Primer sets were designed to scan 5 kb of the FGF-2 promoter, but only amplicons surrounding the potential binding site are shown. Sequence analysis in this region detected five potential SBEs. Subsequent analysis using MatInspector (Genomatix) predicted a gtct SBE motif at -1994 from the start codon as a putative Smad4 binding sequence in the FGF-2 promoter.

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

    TGF-β1 induces phosphorylation of Smad3, p38, and ERK 1/2 in prostate gland fibroblasts. A. Western blots show induction of Smad3, p38 phosphorylation by TGF-β1 treatment in prostate gland fibroblasts; B. TGF-β1 induced ERK 1/2 phosphorylation in prostate gland fibroblasts. Basal phosphorylation was evident and TGF-β1 induced phosphorylation was most apparent at 4 hr, which was inhibited by pretreatment with MEK inhibitor U0126 (4 + U0126) as a control.
    Figure Legend Snippet: TGF-β1 induces phosphorylation of Smad3, p38, and ERK 1/2 in prostate gland fibroblasts. A. Western blots show induction of Smad3, p38 phosphorylation by TGF-β1 treatment in prostate gland fibroblasts; B. TGF-β1 induced ERK 1/2 phosphorylation in prostate gland fibroblasts. Basal phosphorylation was evident and TGF-β1 induced phosphorylation was most apparent at 4 hr, which was inhibited by pretreatment with MEK inhibitor U0126 (4 + U0126) as a control.

    Techniques Used: Western Blot

    36) Product Images from "Role of heparan sulfate in mediating CXCL8-induced endothelial cell migration"

    Article Title: Role of heparan sulfate in mediating CXCL8-induced endothelial cell migration

    Journal: PeerJ

    doi: 10.7717/peerj.1669

    Rho GTPases activity. After treatments, the Rho GTPase activity in HUVECs was detected by using the active Rho/Rac1 pulldown assay, and quantitative data were obtained. (A and B) CXCL8 increased the Rac1 activity at 12 h, and this was abolished by heparinase III; (C and D) Heparinase III significantly reduced the CXCL8-induced Rho activity at both 4 h and 12 h. ∗a P
    Figure Legend Snippet: Rho GTPases activity. After treatments, the Rho GTPase activity in HUVECs was detected by using the active Rho/Rac1 pulldown assay, and quantitative data were obtained. (A and B) CXCL8 increased the Rac1 activity at 12 h, and this was abolished by heparinase III; (C and D) Heparinase III significantly reduced the CXCL8-induced Rho activity at both 4 h and 12 h. ∗a P

    Techniques Used: Activity Assay

    Effect of heparinase III on the CXCL8-modulated expression of Rho GTPases. HUVECs were treated with 15 mU/ml heparinase III (HepIII), 100 ng/ml CXCL8, or both for indicated times (4 and 12 h), respectively. Normal cell without treatment was set as control (CT). The expression of Rho-GTPases was detected by western blot, and quantitative data were obtained using ImageJ: (A and B) Cdc42; (C and D) Rac1; (E and F) RhoA. ∗ P
    Figure Legend Snippet: Effect of heparinase III on the CXCL8-modulated expression of Rho GTPases. HUVECs were treated with 15 mU/ml heparinase III (HepIII), 100 ng/ml CXCL8, or both for indicated times (4 and 12 h), respectively. Normal cell without treatment was set as control (CT). The expression of Rho-GTPases was detected by western blot, and quantitative data were obtained using ImageJ: (A and B) Cdc42; (C and D) Rac1; (E and F) RhoA. ∗ P

    Techniques Used: Expressing, Western Blot

    37) Product Images from "Cytotoxicity of Selenium Immunoconjugates against Triple Negative Breast Cancer Cells"

    Article Title: Cytotoxicity of Selenium Immunoconjugates against Triple Negative Breast Cancer Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19113352

    SDS-PAGE of Native and Se-conjugated mAbs under reducing conditions followed by Coomassie Blue R-250 staining. Lane 1: Kadcyla ® 20 µg; Lane 2: Marker; Lane 3: BV 5 µg; Lane 4: BV 10 µg; Lane 5: BV 20 µg; Lane 6: Se-BV 5 µg; Lane 7: Se-BV 10 µg; Lane 8: Se-BV 20 µg; Lane 9: TZ 5 µg; Lane 10: TZ 10 µg; Lane 11: TZ 20 µg, Lane 12: Se-TZ 10 µg; Lane 13: Se-TZ 20 µg; Lane 14: Gamma globulin 20 µg. (BV: Bevacizumab, Se-BV: Selenobevacizumab, TZ: Trastuzumab, Se-TZ: Selenotrastuzumab).
    Figure Legend Snippet: SDS-PAGE of Native and Se-conjugated mAbs under reducing conditions followed by Coomassie Blue R-250 staining. Lane 1: Kadcyla ® 20 µg; Lane 2: Marker; Lane 3: BV 5 µg; Lane 4: BV 10 µg; Lane 5: BV 20 µg; Lane 6: Se-BV 5 µg; Lane 7: Se-BV 10 µg; Lane 8: Se-BV 20 µg; Lane 9: TZ 5 µg; Lane 10: TZ 10 µg; Lane 11: TZ 20 µg, Lane 12: Se-TZ 10 µg; Lane 13: Se-TZ 20 µg; Lane 14: Gamma globulin 20 µg. (BV: Bevacizumab, Se-BV: Selenobevacizumab, TZ: Trastuzumab, Se-TZ: Selenotrastuzumab).

    Techniques Used: SDS Page, Staining, Marker

    mAb migration on 4–20% Tris-Glycine PAGE gel under non-reducing conditions followed by Coomassie Blue R-250 stain. The image was taken using a Coomassie Blue filter. ( A ) Lane 1: molecular marker; Lane 2: purified human IgG 10 µg; Lane 3: bovine gamma globulin 5 µg; Lane 4: bovine gamma globulin 10 µg; Lane 5: T-DM1 5 µg; Lane 6: T-DM1 10 µg; Lane 7: BV 5 µg; Lane 8: BV 10 µg; Lane 9: Se-BV 5 µg; Lane 10: Se-BV 10 µg; Lane 11: TZ 5 µg; Lane 12: TZ 10 µg; Lane 13: Se-TZ 5 µg; Lane 14: Se-TZ 10 µg. ( B ) TZ migration on 4–20% Tris-Glycine PAGE gel under non-reducing conditions with electrophoretic poles reversed followed by Coomassie Blue R-250 stain. Lane 2: Ladder; Lane 3: TZ 5 µg; Lane 4: TZ 10 µg. (BV: Bevacizumab, Se-BV: Selenobevacizumab, TZ: Trastuzumab, Se-TZ: Selenotrastuzumab).
    Figure Legend Snippet: mAb migration on 4–20% Tris-Glycine PAGE gel under non-reducing conditions followed by Coomassie Blue R-250 stain. The image was taken using a Coomassie Blue filter. ( A ) Lane 1: molecular marker; Lane 2: purified human IgG 10 µg; Lane 3: bovine gamma globulin 5 µg; Lane 4: bovine gamma globulin 10 µg; Lane 5: T-DM1 5 µg; Lane 6: T-DM1 10 µg; Lane 7: BV 5 µg; Lane 8: BV 10 µg; Lane 9: Se-BV 5 µg; Lane 10: Se-BV 10 µg; Lane 11: TZ 5 µg; Lane 12: TZ 10 µg; Lane 13: Se-TZ 5 µg; Lane 14: Se-TZ 10 µg. ( B ) TZ migration on 4–20% Tris-Glycine PAGE gel under non-reducing conditions with electrophoretic poles reversed followed by Coomassie Blue R-250 stain. Lane 2: Ladder; Lane 3: TZ 5 µg; Lane 4: TZ 10 µg. (BV: Bevacizumab, Se-BV: Selenobevacizumab, TZ: Trastuzumab, Se-TZ: Selenotrastuzumab).

    Techniques Used: Migration, Polyacrylamide Gel Electrophoresis, Staining, Marker, Purification

    38) Product Images from "Targeting Glycolysis with Epigallocatechin-3-Gallate Enhances the Efficacy of Chemotherapeutics in Pancreatic Cancer Cells and Xenografts"

    Article Title: Targeting Glycolysis with Epigallocatechin-3-Gallate Enhances the Efficacy of Chemotherapeutics in Pancreatic Cancer Cells and Xenografts

    Journal: Cancers

    doi: 10.3390/cancers11101496

    EGCG and gemcitabine together further affected cell kinetic in pancreatic cancer cells. ( A ) EGCG (E) enhanced the effect of gemcitabine (G) on the cell cycle. Following treatment with 40 µM EGCG (E), 20 nM gemcitabine (G), or both (G + E) for 48 h, cells were stained with propidium iodide (PI) and the number of cells in each phase of the cell cycle was measured by flow cytometry. Results are expressed as a percentage of control. ( B ) EGCG (E) and gemcitabine (G) modulated S/G2 phase protein expression. Immunoblots for phosphorylated checkpoint kinases 1 (p-Chk1), phosphorylated and total tumor protein p53 (p53), cyclin-dependent kinase (cdk) inhibitor p21 Waf1/Cip1 (p21), cell division cycle 2 (cdc2) and Cyclin B1 in total cell protein extracts from Panc-1 and MIA PaCa-2 cells treated with EGCG (E), gemcitabine (G), or both (G + E), for 48 h. Loading control: β-Actin. Bands were quantified and results are expressed as a percentage of control. * p
    Figure Legend Snippet: EGCG and gemcitabine together further affected cell kinetic in pancreatic cancer cells. ( A ) EGCG (E) enhanced the effect of gemcitabine (G) on the cell cycle. Following treatment with 40 µM EGCG (E), 20 nM gemcitabine (G), or both (G + E) for 48 h, cells were stained with propidium iodide (PI) and the number of cells in each phase of the cell cycle was measured by flow cytometry. Results are expressed as a percentage of control. ( B ) EGCG (E) and gemcitabine (G) modulated S/G2 phase protein expression. Immunoblots for phosphorylated checkpoint kinases 1 (p-Chk1), phosphorylated and total tumor protein p53 (p53), cyclin-dependent kinase (cdk) inhibitor p21 Waf1/Cip1 (p21), cell division cycle 2 (cdc2) and Cyclin B1 in total cell protein extracts from Panc-1 and MIA PaCa-2 cells treated with EGCG (E), gemcitabine (G), or both (G + E), for 48 h. Loading control: β-Actin. Bands were quantified and results are expressed as a percentage of control. * p

    Techniques Used: Staining, Flow Cytometry, Cytometry, Expressing, Western Blot

    39) Product Images from "Development of a Cell-Based Hepatitis C Virus Infection Fluorescent Resonance Energy Transfer Assay for High-Throughput Antiviral Compound Screening "

    Article Title: Development of a Cell-Based Hepatitis C Virus Infection Fluorescent Resonance Energy Transfer Assay for High-Throughput Antiviral Compound Screening

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00495-09

    HCV NS3 protein accumulation parallels infection kinetics. (A) Huh7 cells were infected with JFH-1 HCVcc at an MOI of 0.01 FFU/cell. Culture supernatant, intracellular RNA, and cellular protein were collected at the indicated times p.i. Intracellular
    Figure Legend Snippet: HCV NS3 protein accumulation parallels infection kinetics. (A) Huh7 cells were infected with JFH-1 HCVcc at an MOI of 0.01 FFU/cell. Culture supernatant, intracellular RNA, and cellular protein were collected at the indicated times p.i. Intracellular

    Techniques Used: Infection, Cell Culture

    Reproducibility of HCV infection in nondividing Huh7 cell cultures. Huh7 cells were plated in a 96-well format, DMSO-treated for 20 days, and then infected with HCVcc JFH-1 at an MOI of 0.05 FFU/cell. (A) HCV RNA and NS3 protease activity were measured
    Figure Legend Snippet: Reproducibility of HCV infection in nondividing Huh7 cell cultures. Huh7 cells were plated in a 96-well format, DMSO-treated for 20 days, and then infected with HCVcc JFH-1 at an MOI of 0.05 FFU/cell. (A) HCV RNA and NS3 protease activity were measured

    Techniques Used: Infection, Activity Assay

    40) Product Images from "Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation reveals multisite regulation of surface expression and gating"

    Article Title: Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation reveals multisite regulation of surface expression and gating

    Journal: bioRxiv

    doi: 10.1101/2020.04.29.067835

    Phosphorylation at S671 regulates the cell surface expression of Na V 1.5. ( A ) Representative western blots of total (left panel) and cell surface (right panel) Na V 1.5 from HEK-293 cells transiently transfected with Na V 1.5-WT + Na V β1, Na V 1.5-S671A + Na V β1 or Na V 1.5-S671E + Na V β1. Samples were probed in parallel with the anti-transferrin receptor (TransR) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies. ( B ) Mean ± SEM total and cell surface Na V 1.5 protein expression in transiently transfected HEK-293 cells (n=12 in 6 different experiments). Expression of Na V 1.5 in each sample was first normalized to the TransR protein in the same blot and then expressed relative to Na V 1.5 protein expression (total or cell surface) in cells transfected with Na V 1.5-WT + Navβ1. Relative (mean ± SEM) Na V 1.5 cell surface expression is different (*** p
    Figure Legend Snippet: Phosphorylation at S671 regulates the cell surface expression of Na V 1.5. ( A ) Representative western blots of total (left panel) and cell surface (right panel) Na V 1.5 from HEK-293 cells transiently transfected with Na V 1.5-WT + Na V β1, Na V 1.5-S671A + Na V β1 or Na V 1.5-S671E + Na V β1. Samples were probed in parallel with the anti-transferrin receptor (TransR) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies. ( B ) Mean ± SEM total and cell surface Na V 1.5 protein expression in transiently transfected HEK-293 cells (n=12 in 6 different experiments). Expression of Na V 1.5 in each sample was first normalized to the TransR protein in the same blot and then expressed relative to Na V 1.5 protein expression (total or cell surface) in cells transfected with Na V 1.5-WT + Navβ1. Relative (mean ± SEM) Na V 1.5 cell surface expression is different (*** p

    Techniques Used: Expressing, Western Blot, Transfection

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    Article Snippet: .. For immunofluorescence analysis of E-cadherin, MECA32 and CD14, incubation with the primary antibody (overnight at 4°C) was followed by incubation (1 h at RT) with the secondary antibodies: Alexa fluor 488 Goat anti-rabbit (Life technologies, A11034) for E-cadherin and Alexa Fluor 488 Goat anti-rat (Life technologies, A11006) for CD14 and MECA 32. ..

    Article Title: FoxM1 overexpression promotes cell proliferation and migration and inhibits apoptosis in hypopharyngeal squamous cell carcinoma resulting in poor clinical prognosis
    Article Snippet: .. Cells were incubated with primary antibodies against FoxM1 (1:100; Bioworld Technology), cyclin A1 (#sc-751, 1:100; Santa Cruz Biotechnology), PCNA (#ab2426, 1:100; Abcam), E-cadherin (#RLT1453, 1:100; Suzhou Ruiying Biological Technology) vimentin (#RLT4879, 1:100; Suzhou Ruiying Biological Technologyl) and β-tubulin (#RLM3030, 1:100; Suzhou Ruiying Biological Technology) for 20 h, and then incubated with Alexa Fluor-conjugated secondary antibodies (1:1000; Invitrogen/Thermo Fisher Scientific) and Hoechst stain (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at room temperature in the dark. .. The images were viewed and recorded with a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

    HAT Assay:

    Article Title: Mechanical stress induces lung fibrosis by epithelial-mesenchymal transition (EMT)
    Article Snippet: .. The secondary antibodies were then used with 1:6000goat anti-mouse IgG-Horseradish Peroxidase (HRP) for cytokeratin-8, E-Cadherin, vimentin or 1:6000goat anti-rabbit IgG-HRP for α-SMA (Pierce, Thermo Scientific, Ottawa, ON, Canada) in blocking buffer for 1 hat room temperature. .. The bands were visualized using enhanced chemiluminescence (Amersham ECL Western Blotting Detection Reagents, GE Healthcare, Baie d’Urfe, QC, Canada).

    Confocal Microscopy:

    Article Title: ?-Catenin and Vinculin Cooperate to Promote High E-cadherin-based Adhesion Strength *
    Article Snippet: .. Samples were immunostained with vinculin, E-cadherin, and α18 epitope antibodies, mounted with Immumount medium (Thermo Shandon, Pittsburgh, PA), and analyzed by confocal microscopy. .. The ratio of intensities (expressed in arbitrary units) between vinculin and Ecad or α18 and Ecad was calculated using ImageJ software at the cell-cell contact area from compilation of the three-dimensional stack of confocal images.

    Staining:

    Article Title: LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis
    Article Snippet: .. E-cadherin was detected by staining with anti-E-cadherin antibodies (1 μg/ml) overnight at 4°C followed by incubating with Alexa 488-conjugated anti-mouse antibody (Molecular Probes) for 1 hr at room temperature. .. Cell nuclei were stained with Hoechst for 3 min.

    Article Title: FoxM1 overexpression promotes cell proliferation and migration and inhibits apoptosis in hypopharyngeal squamous cell carcinoma resulting in poor clinical prognosis
    Article Snippet: .. Cells were incubated with primary antibodies against FoxM1 (1:100; Bioworld Technology), cyclin A1 (#sc-751, 1:100; Santa Cruz Biotechnology), PCNA (#ab2426, 1:100; Abcam), E-cadherin (#RLT1453, 1:100; Suzhou Ruiying Biological Technology) vimentin (#RLT4879, 1:100; Suzhou Ruiying Biological Technologyl) and β-tubulin (#RLM3030, 1:100; Suzhou Ruiying Biological Technology) for 20 h, and then incubated with Alexa Fluor-conjugated secondary antibodies (1:1000; Invitrogen/Thermo Fisher Scientific) and Hoechst stain (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at room temperature in the dark. .. The images were viewed and recorded with a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

    Immunofluorescence:

    Article Title: C3G knock-down enhances migration and invasion by increasing Rap1-mediated p38α activation, while it impairs tumor growth through p38α-independent mechanisms
    Article Snippet: .. For immunofluorescence analysis of E-cadherin, MECA32 and CD14, incubation with the primary antibody (overnight at 4°C) was followed by incubation (1 h at RT) with the secondary antibodies: Alexa fluor 488 Goat anti-rabbit (Life technologies, A11034) for E-cadherin and Alexa Fluor 488 Goat anti-rat (Life technologies, A11006) for CD14 and MECA 32. ..

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  • 99
    Thermo Fisher supersignal elisa pico chemiluminescent substrate
    The protein expression level of PI3K p85. Cells were treated with samples, or control for 72 h. Total protein was extracted for the analysis using Western blot. The signal of target protein in MCF-7 cells ( a , b ) and MDA-MB-231 cells ( c , d ) was detected and quantified using <t>SuperSignal</t> <t>ELISA</t> Pico Chemiluminescent Substrate. Data are presented as mean ± SD. * indicates a significant difference compared to the control ( p
    Supersignal Elisa Pico Chemiluminescent Substrate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/supersignal elisa pico chemiluminescent substrate/product/Thermo Fisher
    Average 99 stars, based on 76 article reviews
    Price from $9.99 to $1999.99
    supersignal elisa pico chemiluminescent substrate - by Bioz Stars, 2020-09
    99/100 stars
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    99
    Thermo Fisher supersignal west pico chemiluminescent substrate
    Effects of pHK-PAS on HKII and cytochrome c localization. HeLa and HEK293 cells were treated with 50 µM pHK-PAS for 24–72 h. Thereafter, cells were harvested and mitochondria-enriched, and cytosolic fractions were isolated. Samples were normalized for protein content and electrophoresed on a 10–12% SDS polyacrylamide gel, then transferred to a nitrocellulose membrane; incubated overnight with mouse anti-human HK II, VDAC, or cytochrome c Abs, followed by 3 h with horseradish peroxidase–conjugated mouse IgG Ab; and finally visualized by using <t>SuperSignal</t> West Pico Chemiluminescent Substrate. A , C ) Immunoblots of HKII, VDAC1, and cytochrome c (Cyt-c) in the mitochondria-enriched (left) and cytosolic fractions (right) of control ( t = 0) and pHK-PAS-treated (24–72 h) HeLa ( A ) and HEK293 ( C ) cells. VDAC and β-actin were used as loading controls for the mitochondria-enriched and cytosolic fractions, respectively. B , D ) Changes in the HKII and cytochrome c content of the cytosolic fraction of control and pHK-PAS–treated HeLa ( B ) and HEK293 ( D ) cells determined by densitometric quantification of the band intensities in panels A and C , respectively. *** P
    Supersignal West Pico Chemiluminescent Substrate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5693 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/supersignal west pico chemiluminescent substrate/product/Thermo Fisher
    Average 99 stars, based on 5693 article reviews
    Price from $9.99 to $1999.99
    supersignal west pico chemiluminescent substrate - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    91
    Thermo Fisher supersignal chemiluminescence kit
    Effects of pHK-PAS on HKII and cytochrome c localization. HeLa and HEK293 cells were treated with 50 µM pHK-PAS for 24–72 h. Thereafter, cells were harvested and mitochondria-enriched, and cytosolic fractions were isolated. Samples were normalized for protein content and electrophoresed on a 10–12% SDS polyacrylamide gel, then transferred to a nitrocellulose membrane; incubated overnight with mouse anti-human HK II, VDAC, or cytochrome c Abs, followed by 3 h with horseradish peroxidase–conjugated mouse IgG Ab; and finally visualized by using <t>SuperSignal</t> West Pico Chemiluminescent Substrate. A , C ) Immunoblots of HKII, VDAC1, and cytochrome c (Cyt-c) in the mitochondria-enriched (left) and cytosolic fractions (right) of control ( t = 0) and pHK-PAS-treated (24–72 h) HeLa ( A ) and HEK293 ( C ) cells. VDAC and β-actin were used as loading controls for the mitochondria-enriched and cytosolic fractions, respectively. B , D ) Changes in the HKII and cytochrome c content of the cytosolic fraction of control and pHK-PAS–treated HeLa ( B ) and HEK293 ( D ) cells determined by densitometric quantification of the band intensities in panels A and C , respectively. *** P
    Supersignal Chemiluminescence Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/supersignal chemiluminescence kit/product/Thermo Fisher
    Average 91 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    supersignal chemiluminescence kit - by Bioz Stars, 2020-09
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    Image Search Results


    The protein expression level of PI3K p85. Cells were treated with samples, or control for 72 h. Total protein was extracted for the analysis using Western blot. The signal of target protein in MCF-7 cells ( a , b ) and MDA-MB-231 cells ( c , d ) was detected and quantified using SuperSignal ELISA Pico Chemiluminescent Substrate. Data are presented as mean ± SD. * indicates a significant difference compared to the control ( p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Synergistic Effect of Bioactive Anticarcinogens from Soybean on Anti-Proliferative Activity in MDA-MB-231 and MCF-7 Human Breast Cancer Cells In Vitro

    doi: 10.3390/molecules23071557

    Figure Lengend Snippet: The protein expression level of PI3K p85. Cells were treated with samples, or control for 72 h. Total protein was extracted for the analysis using Western blot. The signal of target protein in MCF-7 cells ( a , b ) and MDA-MB-231 cells ( c , d ) was detected and quantified using SuperSignal ELISA Pico Chemiluminescent Substrate. Data are presented as mean ± SD. * indicates a significant difference compared to the control ( p

    Article Snippet: Target protein were detected and quantified using SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Western Blot, Multiple Displacement Amplification, Enzyme-linked Immunosorbent Assay

    The protein expression level of PI3K p85. Cells were treated with samples, or control for 72 h. Total protein was extracted for the analysis using Western blot. The signal of target protein in MCF-7 cells ( a , b ) and MDA-MB-231 cells ( c , d ) was detected and quantified using SuperSignal ELISA Pico Chemiluminescent Substrate. Data are presented as mean ± SD. * indicates a significant difference compared to the control ( p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Synergistic Effect of Bioactive Anticarcinogens from Soybean on Anti-Proliferative Activity in MDA-MB-231 and MCF-7 Human Breast Cancer Cells In Vitro

    doi: 10.3390/molecules23071557

    Figure Lengend Snippet: The protein expression level of PI3K p85. Cells were treated with samples, or control for 72 h. Total protein was extracted for the analysis using Western blot. The signal of target protein in MCF-7 cells ( a , b ) and MDA-MB-231 cells ( c , d ) was detected and quantified using SuperSignal ELISA Pico Chemiluminescent Substrate. Data are presented as mean ± SD. * indicates a significant difference compared to the control ( p

    Article Snippet: Target protein were detected and quantified using SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Western Blot, Multiple Displacement Amplification, Enzyme-linked Immunosorbent Assay

    Effects of pHK-PAS on HKII and cytochrome c localization. HeLa and HEK293 cells were treated with 50 µM pHK-PAS for 24–72 h. Thereafter, cells were harvested and mitochondria-enriched, and cytosolic fractions were isolated. Samples were normalized for protein content and electrophoresed on a 10–12% SDS polyacrylamide gel, then transferred to a nitrocellulose membrane; incubated overnight with mouse anti-human HK II, VDAC, or cytochrome c Abs, followed by 3 h with horseradish peroxidase–conjugated mouse IgG Ab; and finally visualized by using SuperSignal West Pico Chemiluminescent Substrate. A , C ) Immunoblots of HKII, VDAC1, and cytochrome c (Cyt-c) in the mitochondria-enriched (left) and cytosolic fractions (right) of control ( t = 0) and pHK-PAS-treated (24–72 h) HeLa ( A ) and HEK293 ( C ) cells. VDAC and β-actin were used as loading controls for the mitochondria-enriched and cytosolic fractions, respectively. B , D ) Changes in the HKII and cytochrome c content of the cytosolic fraction of control and pHK-PAS–treated HeLa ( B ) and HEK293 ( D ) cells determined by densitometric quantification of the band intensities in panels A and C , respectively. *** P

    Journal: The FASEB Journal

    Article Title: Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells

    doi: 10.1096/fj.201601173R

    Figure Lengend Snippet: Effects of pHK-PAS on HKII and cytochrome c localization. HeLa and HEK293 cells were treated with 50 µM pHK-PAS for 24–72 h. Thereafter, cells were harvested and mitochondria-enriched, and cytosolic fractions were isolated. Samples were normalized for protein content and electrophoresed on a 10–12% SDS polyacrylamide gel, then transferred to a nitrocellulose membrane; incubated overnight with mouse anti-human HK II, VDAC, or cytochrome c Abs, followed by 3 h with horseradish peroxidase–conjugated mouse IgG Ab; and finally visualized by using SuperSignal West Pico Chemiluminescent Substrate. A , C ) Immunoblots of HKII, VDAC1, and cytochrome c (Cyt-c) in the mitochondria-enriched (left) and cytosolic fractions (right) of control ( t = 0) and pHK-PAS-treated (24–72 h) HeLa ( A ) and HEK293 ( C ) cells. VDAC and β-actin were used as loading controls for the mitochondria-enriched and cytosolic fractions, respectively. B , D ) Changes in the HKII and cytochrome c content of the cytosolic fraction of control and pHK-PAS–treated HeLa ( B ) and HEK293 ( D ) cells determined by densitometric quantification of the band intensities in panels A and C , respectively. *** P

    Article Snippet: Samples were then visualized by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Isolation, Incubation, Western Blot