sheep anti cathepsin h  (Millipore)


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    Structured Review

    Millipore sheep anti cathepsin h
    Expression of cathepsins C, H and L in TALL-104 cells and human CD8+ T cells and their co-localisation with cystatin F. (A-C) TALL-104 and pCTLs were analysed for cathepsins C, H and L expression by western blot. GAPDH or β-actin staining was used to show protein loading. (D, E) Co-localisation of cystatin F with cathepsins C, H and L was studied by immunofluorescence microscopy in TALL-104 (D) and pCTLs (E) . Cystatin F (green) and cathepsin C (red) co-localisation is shown in first row, cystatin F (red) and <t>cathepsin</t> H (green) in second row and cystatin F (red) and cathepsin L (green) in third row. Bars represent 10 μm. (F) TALL-104 cell lysates were immunoprecipitated with cystatin F antibody and analysed by western blot with anti-cathepsin C and H antibodies. (G) Proximity ligation experiment for cystatin F-cathepsin C interaction in TALL-104 cells and pCTLs. Signals were quantified in BlobFinder software. Bars represent 10 μm. ctrl = control; cysF = cystatin F; IP = immunoprecipitation; pCTL = primary human cytotoxic T cells
    Sheep Anti Cathepsin H, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 8466 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sheep anti cathepsin h - by Bioz Stars, 2020-04
    93/100 stars

    Images

    1) Product Images from "Increased Cystatin F Levels Correlate with Decreased Cytotoxicity of Cytotoxic T Cells"

    Article Title: Increased Cystatin F Levels Correlate with Decreased Cytotoxicity of Cytotoxic T Cells

    Journal: Radiology and Oncology

    doi: 10.2478/raon-2019-0007

    Expression of cathepsins C, H and L in TALL-104 cells and human CD8+ T cells and their co-localisation with cystatin F. (A-C) TALL-104 and pCTLs were analysed for cathepsins C, H and L expression by western blot. GAPDH or β-actin staining was used to show protein loading. (D, E) Co-localisation of cystatin F with cathepsins C, H and L was studied by immunofluorescence microscopy in TALL-104 (D) and pCTLs (E) . Cystatin F (green) and cathepsin C (red) co-localisation is shown in first row, cystatin F (red) and cathepsin H (green) in second row and cystatin F (red) and cathepsin L (green) in third row. Bars represent 10 μm. (F) TALL-104 cell lysates were immunoprecipitated with cystatin F antibody and analysed by western blot with anti-cathepsin C and H antibodies. (G) Proximity ligation experiment for cystatin F-cathepsin C interaction in TALL-104 cells and pCTLs. Signals were quantified in BlobFinder software. Bars represent 10 μm. ctrl = control; cysF = cystatin F; IP = immunoprecipitation; pCTL = primary human cytotoxic T cells
    Figure Legend Snippet: Expression of cathepsins C, H and L in TALL-104 cells and human CD8+ T cells and their co-localisation with cystatin F. (A-C) TALL-104 and pCTLs were analysed for cathepsins C, H and L expression by western blot. GAPDH or β-actin staining was used to show protein loading. (D, E) Co-localisation of cystatin F with cathepsins C, H and L was studied by immunofluorescence microscopy in TALL-104 (D) and pCTLs (E) . Cystatin F (green) and cathepsin C (red) co-localisation is shown in first row, cystatin F (red) and cathepsin H (green) in second row and cystatin F (red) and cathepsin L (green) in third row. Bars represent 10 μm. (F) TALL-104 cell lysates were immunoprecipitated with cystatin F antibody and analysed by western blot with anti-cathepsin C and H antibodies. (G) Proximity ligation experiment for cystatin F-cathepsin C interaction in TALL-104 cells and pCTLs. Signals were quantified in BlobFinder software. Bars represent 10 μm. ctrl = control; cysF = cystatin F; IP = immunoprecipitation; pCTL = primary human cytotoxic T cells

    Techniques Used: Expressing, Western Blot, Staining, Immunofluorescence, Microscopy, Immunoprecipitation, Ligation, Software

    2) Product Images from "3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: β-ketoamphetamine modulation of neurotoxicity by the dopamine transporter"

    Article Title: 3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: β-ketoamphetamine modulation of neurotoxicity by the dopamine transporter

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.13048

    Effects of mephedrone (4′ – 40 mg/kg), methylone (4′ –30 mg/kg), and MDPV (4′ – 30 mg/kg) alone or in combination on DA nerve endings of the striatum. Mice were treated with mephedrone (MEPH; 4′ – 40 mg/kg), MDPV (4′ – 30 mg/kg), or methylone (MTHY; 4′ – 30 mg/kg) singly or in the indicated two-drug combinations and the levels of DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH), (c) and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for the β-ketoamphetamines. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH, and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as relative band density by comparison to the respective control. Data are expressed as mean ± SEM for n = 4–5 mice per group.
    Figure Legend Snippet: Effects of mephedrone (4′ – 40 mg/kg), methylone (4′ –30 mg/kg), and MDPV (4′ – 30 mg/kg) alone or in combination on DA nerve endings of the striatum. Mice were treated with mephedrone (MEPH; 4′ – 40 mg/kg), MDPV (4′ – 30 mg/kg), or methylone (MTHY; 4′ – 30 mg/kg) singly or in the indicated two-drug combinations and the levels of DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH), (c) and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for the β-ketoamphetamines. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH, and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as relative band density by comparison to the respective control. Data are expressed as mean ± SEM for n = 4–5 mice per group.

    Techniques Used: Mouse Assay, Injection, High Performance Liquid Chromatography, Western Blot

    Effects of MDPV (4′ – 10, 20, or 30 mg/kg) on methamphetamine (2.5, 5, or 10 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with MDPV (4′ – 10, 20 or 30 mg/kg), methamphetamine (Meth; 2.5, 5 or 10 mg/kg), or their combination in the indicated doses and the levels of DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for MDPV and methamphetamine. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH, and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and expressed as relative band density by comparison with the respective control. Data are means for n = 4–8 mice per group. SEM bars (
    Figure Legend Snippet: Effects of MDPV (4′ – 10, 20, or 30 mg/kg) on methamphetamine (2.5, 5, or 10 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with MDPV (4′ – 10, 20 or 30 mg/kg), methamphetamine (Meth; 2.5, 5 or 10 mg/kg), or their combination in the indicated doses and the levels of DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for MDPV and methamphetamine. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH, and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and expressed as relative band density by comparison with the respective control. Data are means for n = 4–8 mice per group. SEM bars (

    Techniques Used: Mouse Assay, Injection, High Performance Liquid Chromatography, Western Blot

    Effects of MDPV (4′ – 30 mg/kg) on amphetamine (4′ – 5 mg/ kg)-, 3,4-methylenedioxymethamphetamine (MDMA) (4′ – 20 mg/kg)-, and MPTP (2′ – 20 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with MDPV (MV; 4′ – 30 mg/kg) in combination with amphetamine (AM; 4′ – 5 mg/kg), MDMA (MD; 4′ –20 mg/kg) or MPTP (MP; 2′ – 20 mg/kg) and the levels DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for all drugs. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as relative band density by comparison with the respective control. Data are mean ± SEM for n = 5–6 mice per group. * p
    Figure Legend Snippet: Effects of MDPV (4′ – 30 mg/kg) on amphetamine (4′ – 5 mg/ kg)-, 3,4-methylenedioxymethamphetamine (MDMA) (4′ – 20 mg/kg)-, and MPTP (2′ – 20 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with MDPV (MV; 4′ – 30 mg/kg) in combination with amphetamine (AM; 4′ – 5 mg/kg), MDMA (MD; 4′ –20 mg/kg) or MPTP (MP; 2′ – 20 mg/kg) and the levels DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for all drugs. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as relative band density by comparison with the respective control. Data are mean ± SEM for n = 5–6 mice per group. * p

    Techniques Used: Mouse Assay, Injection, High Performance Liquid Chromatography, Western Blot

    Effects of methylone (4′ – 10, 20 or 30 mg/kg) on methamphetamine (4′ – 2.5 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with methylone (4′ – 10, 20, or 30 mg/ kg) alone or in combination with methamphetamine (4′ – 2.5 mg/kg) and the levels DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for all drugs. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and expressed as relative band density by comparison with the respective control. Data are mean ± SEM for n = 4–6 mice per group. ** p
    Figure Legend Snippet: Effects of methylone (4′ – 10, 20 or 30 mg/kg) on methamphetamine (4′ – 2.5 mg/kg)-induced neurotoxicity to DA nerve endings. Mice were treated with methylone (4′ – 10, 20, or 30 mg/ kg) alone or in combination with methamphetamine (4′ – 2.5 mg/kg) and the levels DA (a), dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were determined 2 days after treatment. Controls were injected with physiological saline on the same binge schedule used for all drugs. DA levels were determined by HPLC and are reported as % control. Relative pixel densities for immunoblots of DAT, TH and GFAP were quantified using ImageJ, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and expressed as relative band density by comparison with the respective control. Data are mean ± SEM for n = 4–6 mice per group. ** p

    Techniques Used: Mouse Assay, Injection, High Performance Liquid Chromatography, Western Blot

    3) Product Images from "CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells"

    Article Title: CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038375

    EGFR/ERK signaling pathway is involved in the CsA-induced proliferation of human trophoblast cells. A : Primary human trophoblast cells and JEG-3 cells were treated with 1 μM CsA and neutralizing antibody against CXCR4 (20 µg/ml) or CXCL12 (40 µg/ml), or U0126 (20 μM), or LY294002 (20 μM), or AG1478 (200 nM) for 48 h, and then subjected to BrdU cell proliferation assay. Data are presented as mean ± SEM of three independent experiments. *P
    Figure Legend Snippet: EGFR/ERK signaling pathway is involved in the CsA-induced proliferation of human trophoblast cells. A : Primary human trophoblast cells and JEG-3 cells were treated with 1 μM CsA and neutralizing antibody against CXCR4 (20 µg/ml) or CXCL12 (40 µg/ml), or U0126 (20 μM), or LY294002 (20 μM), or AG1478 (200 nM) for 48 h, and then subjected to BrdU cell proliferation assay. Data are presented as mean ± SEM of three independent experiments. *P

    Techniques Used: BrdU Cell Proliferation Assay

    4) Product Images from "Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations *"

    Article Title: Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M110.003129

    Confirmation of NE residence for NETs by targeting tagged fusion proteins. A , NETs were tested as tagged fusions for NE targeting, which is determined by their enrichment in a rim at the limits of DNA staining. NET fusions are visualized in white in the left panels and in red in the right panels , whereas DNA is visualized in white in the right panels . NETs were sometimes expressed or targeted better in certain cell types: all panels shown are U2OS osteosarcoma cells except for TMEM38A in mouse C2C12 cells and TMEM70 and CKAP4 in HeLa cells. All micrographs are on the same scale except for CKAP4 with all scale bars at 10 μm. B and C , inner versus outer nuclear membrane targeting. Structured illumination microscopy can distinguish proteins in the INM from those in the ONM when co-stained with nuclear basket protein Nup153 and cytoplasmic filament protein Nup358. B , an ONM NET or ER/ONM protein ( red ) should be in the same plane with Nup358 ( green ) but should be external to Nup153 ( green ), and this is observed for the control ER protein Sec61β ( upper schematic and images ). Correspondingly, an INM NET should be in the same plane as Nup153 and internal to Nup358 as is observed for the control NET LAP2β ( lower schematic and images ). C , all new NETs tested appeared in the same plane of the INM with Nup153 and formed an internal ring to Nup358, indicating INM residence. Bars , 5 μm.
    Figure Legend Snippet: Confirmation of NE residence for NETs by targeting tagged fusion proteins. A , NETs were tested as tagged fusions for NE targeting, which is determined by their enrichment in a rim at the limits of DNA staining. NET fusions are visualized in white in the left panels and in red in the right panels , whereas DNA is visualized in white in the right panels . NETs were sometimes expressed or targeted better in certain cell types: all panels shown are U2OS osteosarcoma cells except for TMEM38A in mouse C2C12 cells and TMEM70 and CKAP4 in HeLa cells. All micrographs are on the same scale except for CKAP4 with all scale bars at 10 μm. B and C , inner versus outer nuclear membrane targeting. Structured illumination microscopy can distinguish proteins in the INM from those in the ONM when co-stained with nuclear basket protein Nup153 and cytoplasmic filament protein Nup358. B , an ONM NET or ER/ONM protein ( red ) should be in the same plane with Nup358 ( green ) but should be external to Nup153 ( green ), and this is observed for the control ER protein Sec61β ( upper schematic and images ). Correspondingly, an INM NET should be in the same plane as Nup153 and internal to Nup358 as is observed for the control NET LAP2β ( lower schematic and images ). C , all new NETs tested appeared in the same plane of the INM with Nup153 and formed an internal ring to Nup358, indicating INM residence. Bars , 5 μm.

    Techniques Used: Staining, Microscopy

    5) Product Images from "Experience-Driven Axon Retraction in the Pharmacologically Inactivated Visual Cortex Does Not Require Synaptic Transmission"

    Article Title: Experience-Driven Axon Retraction in the Pharmacologically Inactivated Visual Cortex Does Not Require Synaptic Transmission

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004193

    Example of labeled axons in BoNT/E-treated visual cortex. (A) Low-magnification image of coronal section across visual cortex of BoNT/E-injected kitten. An arrowhead indicates the BoNT/E injection site. (B, C) High-magnification view of two regions in A far from (dotted line, B) and close to (solid line, C) the injection site. Egr-1 signal is detectable in the area far from the injection site (B), whereas the signal is very low in the area near the injection site (C). The staining at the lower-left in C represents labeled axons. (D) Enlarged view of geniculocortical axons in BoNT/E inactivated area (box in C). Scale bar, A: 1 mm, B, C: 100 µm, D: 10 µm.
    Figure Legend Snippet: Example of labeled axons in BoNT/E-treated visual cortex. (A) Low-magnification image of coronal section across visual cortex of BoNT/E-injected kitten. An arrowhead indicates the BoNT/E injection site. (B, C) High-magnification view of two regions in A far from (dotted line, B) and close to (solid line, C) the injection site. Egr-1 signal is detectable in the area far from the injection site (B), whereas the signal is very low in the area near the injection site (C). The staining at the lower-left in C represents labeled axons. (D) Enlarged view of geniculocortical axons in BoNT/E inactivated area (box in C). Scale bar, A: 1 mm, B, C: 100 µm, D: 10 µm.

    Techniques Used: Labeling, Injection, Staining

    Effects of BoNT/E on cortical activity and Egr-1 expression level in visual cortex. (A) Examples of spike activity in rat visual cortex contralateral (upper trace, Control) and ipsilateral (lower trace, BoNT/E) to BoNT/E injection site recorded 1 week after injection. BoNT/E blocked neuronal activity only in the injected cortex. (B) Egr-1 expression level is significantly decreased in the BoNT/E injected cortex. The upper right panel shows examples of immunoblots for Egr-1 in the visual cortex of normal rats (Norm) and that 1 week after BoNT/E injection. Blot densities were normalized to those of GAPDH and expressed as the ratio to those of Egr-1 in normal animals. Error bars indicate SEM. *: P
    Figure Legend Snippet: Effects of BoNT/E on cortical activity and Egr-1 expression level in visual cortex. (A) Examples of spike activity in rat visual cortex contralateral (upper trace, Control) and ipsilateral (lower trace, BoNT/E) to BoNT/E injection site recorded 1 week after injection. BoNT/E blocked neuronal activity only in the injected cortex. (B) Egr-1 expression level is significantly decreased in the BoNT/E injected cortex. The upper right panel shows examples of immunoblots for Egr-1 in the visual cortex of normal rats (Norm) and that 1 week after BoNT/E injection. Blot densities were normalized to those of GAPDH and expressed as the ratio to those of Egr-1 in normal animals. Error bars indicate SEM. *: P

    Techniques Used: Activity Assay, Expressing, Injection, Western Blot

    6) Product Images from "Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells"

    Article Title: Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells

    Journal: Retrovirology

    doi: 10.1186/1742-4690-5-115

    RT isoforms are present in purified HIV virions . Viral particles from H3B cells were pelleted through 25% sucrose, solubilised in 2D gel electrophoresis buffer and an aliquot resolved by 1D SDS-PAGE (A). The remaining sample was spiked with 3 μg of GAPDH protein, focussed on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel and then resolved by SDS-PAGE followed by transfer to PVDF membranes (B). RT was detected by Western blot using an anti-RT antibody. RT isoforms (B) are designated by black arrows and the calculated pI and expected position of p66 and p51 indicated.
    Figure Legend Snippet: RT isoforms are present in purified HIV virions . Viral particles from H3B cells were pelleted through 25% sucrose, solubilised in 2D gel electrophoresis buffer and an aliquot resolved by 1D SDS-PAGE (A). The remaining sample was spiked with 3 μg of GAPDH protein, focussed on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel and then resolved by SDS-PAGE followed by transfer to PVDF membranes (B). RT was detected by Western blot using an anti-RT antibody. RT isoforms (B) are designated by black arrows and the calculated pI and expected position of p66 and p51 indicated.

    Techniques Used: Purification, Two-Dimensional Gel Electrophoresis, Electrophoresis, SDS Page, Western Blot

    2D gel electrophoresis analysis of recombinant RT identifies protein isoforms . Recombinant RT LAI + GAPDH protein (3 μg each) was solubilised in 2D gel electrophoresis buffer, focussed on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel then resolved on a 10% acrylamide SDS-PAGE gel followed by transfer to PVDF membranes. RT was detected by Western blot using an anti-RT antibody (upper panel) and GAPDH detected by Coomassie stain (lower panel). RT isoforms are designated by black arrows and calculated pI indicated. Position of triangles (Δ) denote the reference marks used for calculation of pI.
    Figure Legend Snippet: 2D gel electrophoresis analysis of recombinant RT identifies protein isoforms . Recombinant RT LAI + GAPDH protein (3 μg each) was solubilised in 2D gel electrophoresis buffer, focussed on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel then resolved on a 10% acrylamide SDS-PAGE gel followed by transfer to PVDF membranes. RT was detected by Western blot using an anti-RT antibody (upper panel) and GAPDH detected by Coomassie stain (lower panel). RT isoforms are designated by black arrows and calculated pI indicated. Position of triangles (Δ) denote the reference marks used for calculation of pI.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Recombinant, SDS Page, Western Blot, Staining

    The same major RT isoforms are present in virus producer cells, newly infected cells and HIV RTCs . H3B and Hut-78 cells were co-cultured for the indicated time period then lysed. For panels A and B, lysates were immunoprecipitated using heat-inactivated AIDS patient sera cross-linked to protein A sepharose beads and washed. In panels A, B and D, E samples were subjected to 2D gel electrophoresis on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel along with 3 μg of GAPDH protein. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. RT was detected by Western blot using an anti-RT antibody and RT isoforms are designated by a black arrow (n = 2 for each panel). Minor differences in the p66 and p51 profiles were observed between experiments and spots not routinely observed are indicated by a white arrow. (A) H3B virus producer cells. H3B and Hut-78 cells were co-cultured and lysed immediately. (B) Infected cell lysates. H3B and Hut-78 cells were co-cultured and lysed at 40 min post-cell mixing. (C-E) HIV RTC's. Lysates were subjected to 15–30% sucrose velocity gradient sedimentation. Fractions (1 ml) were collected from the top of the gradient and viral -ssDNA analysed by real time PCR (C). The remainder of two selected fractions; (D) from the top of the gradient (fraction 1) and (E) co-incident with the known sedimentation of RTCs (fraction 5), were TCA precipitated and subjected to 2D gel electrophoresis, as for panels A and B, above. Experiments were replicated, at least n = 2, for each presented biological situation.
    Figure Legend Snippet: The same major RT isoforms are present in virus producer cells, newly infected cells and HIV RTCs . H3B and Hut-78 cells were co-cultured for the indicated time period then lysed. For panels A and B, lysates were immunoprecipitated using heat-inactivated AIDS patient sera cross-linked to protein A sepharose beads and washed. In panels A, B and D, E samples were subjected to 2D gel electrophoresis on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel along with 3 μg of GAPDH protein. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. RT was detected by Western blot using an anti-RT antibody and RT isoforms are designated by a black arrow (n = 2 for each panel). Minor differences in the p66 and p51 profiles were observed between experiments and spots not routinely observed are indicated by a white arrow. (A) H3B virus producer cells. H3B and Hut-78 cells were co-cultured and lysed immediately. (B) Infected cell lysates. H3B and Hut-78 cells were co-cultured and lysed at 40 min post-cell mixing. (C-E) HIV RTC's. Lysates were subjected to 15–30% sucrose velocity gradient sedimentation. Fractions (1 ml) were collected from the top of the gradient and viral -ssDNA analysed by real time PCR (C). The remainder of two selected fractions; (D) from the top of the gradient (fraction 1) and (E) co-incident with the known sedimentation of RTCs (fraction 5), were TCA precipitated and subjected to 2D gel electrophoresis, as for panels A and B, above. Experiments were replicated, at least n = 2, for each presented biological situation.

    Techniques Used: Infection, Cell Culture, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, SDS Page, Western Blot, Sedimentation, Real-time Polymerase Chain Reaction

    Phosphatase treatment alters the RT isoforms detected . H3B and Hut-78 cells were mixed and incubated at 37°C for 40 mins, cells were then lysed and virus protein immunoprecipitated using heat-inactivated AIDS patient antibody cross-linked to protein A sepharose beads. Immunoprecipitates were incubated without (A) or with (B) calf intestinal alkaline phosphatase (CIAP), proteins pelleted, washed and subjected to 2D gel electrophoresis on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel along with 3 μg of GAPDH protein, and then resolved by SDS-PAGE. RT was detected by Western blot using an anti-RT antibody. RT isoforms are designated by a black arrow and spots not routinely observed are indicated by a white arrow. Experiments were replicated (n = 3).
    Figure Legend Snippet: Phosphatase treatment alters the RT isoforms detected . H3B and Hut-78 cells were mixed and incubated at 37°C for 40 mins, cells were then lysed and virus protein immunoprecipitated using heat-inactivated AIDS patient antibody cross-linked to protein A sepharose beads. Immunoprecipitates were incubated without (A) or with (B) calf intestinal alkaline phosphatase (CIAP), proteins pelleted, washed and subjected to 2D gel electrophoresis on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel along with 3 μg of GAPDH protein, and then resolved by SDS-PAGE. RT was detected by Western blot using an anti-RT antibody. RT isoforms are designated by a black arrow and spots not routinely observed are indicated by a white arrow. Experiments were replicated (n = 3).

    Techniques Used: Incubation, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, SDS Page, Western Blot

    7) Product Images from "Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton"

    Article Title: Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126214

    Distinct Tpm isoforms differentially impact on the elastic modulus of the cell. Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from n = 3 independent experiments. * P
    Figure Legend Snippet: Distinct Tpm isoforms differentially impact on the elastic modulus of the cell. Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from n = 3 independent experiments. * P

    Techniques Used: Clone Assay, Generated, Stable Transfection, Isolation, SDS Page, Western Blot, Whisker Assay

    8) Product Images from "BPIFB3 Regulates Autophagy and Coxsackievirus B Replication through a Noncanonical Pathway Independent of the Core Initiation Machinery"

    Article Title: BPIFB3 Regulates Autophagy and Coxsackievirus B Replication through a Noncanonical Pathway Independent of the Core Initiation Machinery

    Journal: mBio

    doi: 10.1128/mBio.02147-14

    Localization of BPIFB3 to the ER. (A, top) Schematic of BPIFB3. (Bottom) Confocal microscopy for Flag (green) and ER-mRFP (red) in U2OS cells transfected with BPIFB3-Flag and infected with CellLights ER-RFP baculovirus. (Left) Differential interference contrast (DIC) image. (Right) A 5× magnification of the area indicated by the white box in the merged image. (B) Confocal microscopy for Flag (green) and either p230/Golgi (red, top) or MTCO2 (to label mitochondria; red, bottom) in U2OS cells transfected with BPIFB3-Flag at ~48 h posttransfection. (C) Subcellular fractionation of BPIFB3 in U2OS cells stably expressing BPIFB3-Flag. Shown are immunoblots from collected fractions for BPIFB3-Flag, calnexin (CXN), p230/Golgin, and LAMP2. (D) Confocal microscopy of U2OS cells transiently transfected with BPIFB3-Flag. At 48 h posttransfection, cells were permeabilized with digitonin, fixed with PFA, and then incubated with anti-Flag-M2 (top row) or BPIFB3 (bottom row) antibodies. Cells were then permeabilized with Triton X-100 and stained with anti-BPIFB3 (top row) or anti-Flag M2 (bottom row) antibodies. Bar, 10 µm.
    Figure Legend Snippet: Localization of BPIFB3 to the ER. (A, top) Schematic of BPIFB3. (Bottom) Confocal microscopy for Flag (green) and ER-mRFP (red) in U2OS cells transfected with BPIFB3-Flag and infected with CellLights ER-RFP baculovirus. (Left) Differential interference contrast (DIC) image. (Right) A 5× magnification of the area indicated by the white box in the merged image. (B) Confocal microscopy for Flag (green) and either p230/Golgi (red, top) or MTCO2 (to label mitochondria; red, bottom) in U2OS cells transfected with BPIFB3-Flag at ~48 h posttransfection. (C) Subcellular fractionation of BPIFB3 in U2OS cells stably expressing BPIFB3-Flag. Shown are immunoblots from collected fractions for BPIFB3-Flag, calnexin (CXN), p230/Golgin, and LAMP2. (D) Confocal microscopy of U2OS cells transiently transfected with BPIFB3-Flag. At 48 h posttransfection, cells were permeabilized with digitonin, fixed with PFA, and then incubated with anti-Flag-M2 (top row) or BPIFB3 (bottom row) antibodies. Cells were then permeabilized with Triton X-100 and stained with anti-BPIFB3 (top row) or anti-Flag M2 (bottom row) antibodies. Bar, 10 µm.

    Techniques Used: Confocal Microscopy, Transfection, Infection, Fractionation, Stable Transfection, Expressing, Western Blot, Incubation, Staining

    9) Product Images from "Helicobacter Pylori Promotes the Expression of Kr?ppel-Like Factor 5, a Mediator of Carcinogenesis, In Vitro and In Vivo"

    Article Title: Helicobacter Pylori Promotes the Expression of Kr?ppel-Like Factor 5, a Mediator of Carcinogenesis, In Vitro and In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054344

    H. pylori induces expansion of a KLF5 + cell population in vivo . (A–G) KLF5 expression in murine gastric epithelial cells was assessed by flow cytometry analysis in uninfected and H. pylori -infected mice at acute time points (24, 48, 72 hours, and 1 week) and chronic time points (4 and 8 weeks) post-challenge. Percentage of KLF5 + cells at 4 weeks (A) and 8 weeks (C) and levels of KLF5 protein at 4 weeks (B) and 8 weeks (D), as determined by mean fluorescence units (MFU), were determined by flow cytometry. Data from 4 and 8 week time points were analyzed at separate times. H. pylori colonization density in mice infected for 24, 48, and 72 hours, and 1 week was assessed by quantitative culture (E). Percentage of KLF5 + cells (F) and levels of KLF5 protein (G) at 24, 48, or 72 hours, or 1 week were determined by flow cytometry. Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.
    Figure Legend Snippet: H. pylori induces expansion of a KLF5 + cell population in vivo . (A–G) KLF5 expression in murine gastric epithelial cells was assessed by flow cytometry analysis in uninfected and H. pylori -infected mice at acute time points (24, 48, 72 hours, and 1 week) and chronic time points (4 and 8 weeks) post-challenge. Percentage of KLF5 + cells at 4 weeks (A) and 8 weeks (C) and levels of KLF5 protein at 4 weeks (B) and 8 weeks (D), as determined by mean fluorescence units (MFU), were determined by flow cytometry. Data from 4 and 8 week time points were analyzed at separate times. H. pylori colonization density in mice infected for 24, 48, and 72 hours, and 1 week was assessed by quantitative culture (E). Percentage of KLF5 + cells (F) and levels of KLF5 protein (G) at 24, 48, or 72 hours, or 1 week were determined by flow cytometry. Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.

    Techniques Used: In Vivo, Expressing, Flow Cytometry, Cytometry, Infection, Mouse Assay, Fluorescence, MANN-WHITNEY

    KLF5 and Ki67 co-localize to the isthmal region. KLF5 and Ki67 immunohistochemistry staining was assessed on murine gastric tissue sections from uninfected mice (A and C) or H. pylori PMSS1-infected mice (B and D) at 400× magnification. Insets demonstrate regions of KLF5 and Ki67 co-localization (arrows) within the isthmal regions of the gastric epithelium (E and F). Nuclei are stained in blue.
    Figure Legend Snippet: KLF5 and Ki67 co-localize to the isthmal region. KLF5 and Ki67 immunohistochemistry staining was assessed on murine gastric tissue sections from uninfected mice (A and C) or H. pylori PMSS1-infected mice (B and D) at 400× magnification. Insets demonstrate regions of KLF5 and Ki67 co-localization (arrows) within the isthmal regions of the gastric epithelium (E and F). Nuclei are stained in blue.

    Techniques Used: Immunohistochemistry, Staining, Mouse Assay, Infection

    H. pylori -induced KLF5 upregulation is independent of the cag pathogenicity island, VacA, or LPS. AGS human gastric epithelial cells were co-cultured with wild-type cag + H. pylori strain 60190, or its isogenic cagE − , cagA − , slt − , or vacA − mutants at an MOI of 100∶1 for 2 hours. (A) Quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (B) Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. (C) Western blot analysis replicates were quantified using densitometry. (D) Gastric epithelial cells were co-cultured with the wild-type cag + H. pylori strain 60190, heat-killed (HK) H. pylori strain 60190, or with strain 60190 in a transwell (TW) system for 2 hours and quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (E) Gastric epithelial cells were treated with H. pylori LPS (10 ng/ml or 100 ng/ml) for 2 hours and quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. Data are represented as fold over uninfected (UI) control. Error bars indicate standard error of the mean from experiments performed on at least three independent occasions, and Mann-Whitney tests were used to determine statistical significance between groups.
    Figure Legend Snippet: H. pylori -induced KLF5 upregulation is independent of the cag pathogenicity island, VacA, or LPS. AGS human gastric epithelial cells were co-cultured with wild-type cag + H. pylori strain 60190, or its isogenic cagE − , cagA − , slt − , or vacA − mutants at an MOI of 100∶1 for 2 hours. (A) Quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (B) Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. (C) Western blot analysis replicates were quantified using densitometry. (D) Gastric epithelial cells were co-cultured with the wild-type cag + H. pylori strain 60190, heat-killed (HK) H. pylori strain 60190, or with strain 60190 in a transwell (TW) system for 2 hours and quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (E) Gastric epithelial cells were treated with H. pylori LPS (10 ng/ml or 100 ng/ml) for 2 hours and quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. Data are represented as fold over uninfected (UI) control. Error bars indicate standard error of the mean from experiments performed on at least three independent occasions, and Mann-Whitney tests were used to determine statistical significance between groups.

    Techniques Used: Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY

    H. pylori upregulates KLF5 in human gastric epithelial cells in vitro . AGS human gastric epithelial cells were co-cultured with wild-type cag + H. pylori strain 60190 at an MOI of 100∶1 for the indicated time points. (A) Quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (B) Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. (C) Western blot analysis replicates were quantified using densitometry. (D) Gastric epithelial cells were either left untreated or pretreated with actinomycin D for 1 hour prior to co-culture with H. pylori . Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. Data are represented as fold over uninfected control. − and + symbols indicate the absence or presence of H. pylori ( Hp ), respectively. Error bars indicate standard error of the mean from experiments performed on at least three independent occasions, and Mann-Whitney tests were used to determine statistical significance between groups.
    Figure Legend Snippet: H. pylori upregulates KLF5 in human gastric epithelial cells in vitro . AGS human gastric epithelial cells were co-cultured with wild-type cag + H. pylori strain 60190 at an MOI of 100∶1 for the indicated time points. (A) Quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (B) Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. (C) Western blot analysis replicates were quantified using densitometry. (D) Gastric epithelial cells were either left untreated or pretreated with actinomycin D for 1 hour prior to co-culture with H. pylori . Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. Data are represented as fold over uninfected control. − and + symbols indicate the absence or presence of H. pylori ( Hp ), respectively. Error bars indicate standard error of the mean from experiments performed on at least three independent occasions, and Mann-Whitney tests were used to determine statistical significance between groups.

    Techniques Used: In Vitro, Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, Co-Culture Assay, MANN-WHITNEY

    H. pylori induces expansion of a KLF5 + , Lrig1 + cell population in vivo . (A) Flow cytometry dot plots demonstrate Lrig1 and KLF5 immunostaining in representative gastric epithelial cells from uninfected and H. pylori -infected mice at 4 and 8 weeks. The percentage of Lrig1 + , KLF5 + cells was quantified in uninfected and H. pylori -infected mice at 4 weeks (B) and 8 weeks (C). Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.
    Figure Legend Snippet: H. pylori induces expansion of a KLF5 + , Lrig1 + cell population in vivo . (A) Flow cytometry dot plots demonstrate Lrig1 and KLF5 immunostaining in representative gastric epithelial cells from uninfected and H. pylori -infected mice at 4 and 8 weeks. The percentage of Lrig1 + , KLF5 + cells was quantified in uninfected and H. pylori -infected mice at 4 weeks (B) and 8 weeks (C). Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.

    Techniques Used: In Vivo, Flow Cytometry, Cytometry, Immunostaining, Infection, Mouse Assay, MANN-WHITNEY

    KLF5 expression parallels the severity of gastric premalignant lesions in H. pylori -infected humans. (A) KLF5 expression was evaluated by immunohistochemistry in a human population at high risk for gastric cancer. Gastric biopsies from uninfected patients with normal gastric mucosa and H. pylori -infected patients with non-atrophic gastritis, intestinal metaplasia (IM), and dysplasia were evaluated for KLF5 immunostaining at 200× magnification. (B and C) A single pathologist assessed the percentage of KLF5 + cells exhibiting cytoplasmic (B) or nuclear (C) staining. Each data point represents an individual biopsy and mean values are shown. The percentage and mean value of KLF5 + cells from biopsies from patients with normal gastric tissue (circles), gastritis (squares), intestinal metaplasia (IM, triangles), and dysplasia (inverted triangles) are shown. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.
    Figure Legend Snippet: KLF5 expression parallels the severity of gastric premalignant lesions in H. pylori -infected humans. (A) KLF5 expression was evaluated by immunohistochemistry in a human population at high risk for gastric cancer. Gastric biopsies from uninfected patients with normal gastric mucosa and H. pylori -infected patients with non-atrophic gastritis, intestinal metaplasia (IM), and dysplasia were evaluated for KLF5 immunostaining at 200× magnification. (B and C) A single pathologist assessed the percentage of KLF5 + cells exhibiting cytoplasmic (B) or nuclear (C) staining. Each data point represents an individual biopsy and mean values are shown. The percentage and mean value of KLF5 + cells from biopsies from patients with normal gastric tissue (circles), gastritis (squares), intestinal metaplasia (IM, triangles), and dysplasia (inverted triangles) are shown. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.

    Techniques Used: Expressing, Infection, Immunohistochemistry, Immunostaining, Staining, MANN-WHITNEY

    H. pylori upregulates KLF5 expression in vivo . (A–C) KLF5 expression in murine antral gastric tissue was assessed by KLF5 immunostaining in uninfected (A), H. pylori PMSS1-infected mice (B), and H. pylori PMSS1 cagE − -infected mice (C) at 400× magnification. (D and E) A single pathologist, blinded to treatment groups, assessed and scored KLF5 immunostaining. KLF5 immunohistochemistry (IHC) score was determined by assessing the percentage of KLF5 + epithelial cells multiplied by the intensity of epithelial KLF5 staining (1–3) in both the cytoplasm and nucleus of murine gastric epithelial cells (D and E). Each data point represents an individual animal and mean values are shown. Circles designate uninfected mice, squares represent H. pylori PMSS1-infected mice, and triangles represent H. pylori PMSS1 cagE − -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.
    Figure Legend Snippet: H. pylori upregulates KLF5 expression in vivo . (A–C) KLF5 expression in murine antral gastric tissue was assessed by KLF5 immunostaining in uninfected (A), H. pylori PMSS1-infected mice (B), and H. pylori PMSS1 cagE − -infected mice (C) at 400× magnification. (D and E) A single pathologist, blinded to treatment groups, assessed and scored KLF5 immunostaining. KLF5 immunohistochemistry (IHC) score was determined by assessing the percentage of KLF5 + epithelial cells multiplied by the intensity of epithelial KLF5 staining (1–3) in both the cytoplasm and nucleus of murine gastric epithelial cells (D and E). Each data point represents an individual animal and mean values are shown. Circles designate uninfected mice, squares represent H. pylori PMSS1-infected mice, and triangles represent H. pylori PMSS1 cagE − -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.

    Techniques Used: Expressing, In Vivo, Immunostaining, Infection, Mouse Assay, Immunohistochemistry, Staining, MANN-WHITNEY

    10) Product Images from "Comparative Analysis of Two Gene-Targeting Approaches Challenges the Tumor-Suppressive Role of the Protein Kinase MK5/PRAK"

    Article Title: Comparative Analysis of Two Gene-Targeting Approaches Challenges the Tumor-Suppressive Role of the Protein Kinase MK5/PRAK

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136138

    Kinase activity of wild type MK5/PRAK, MK5/PRAK-Δex6 and -Δex8. A) HEK293 cells were transfected with the constructs indicated. The total amount of DNA per well was kept constant (1,6μg DNA per well in 12-well plate) by adding pcDNA-flag expression vector. 24 h post transfection cells were lysed in the plates with 1x Laemmli buffer. Lysates were analyzed by Western blotting using the indicated antibodies. A significant increase in phosphorylation of the substrate Hsp27 was only detected for WT MK5/PRAK in the presence of overexpressed p38 MAPK, ERK3 or ERK4. B) HEK293 cells were transfected with the expression constructs indicated. 24 h post transfection the cells were lysed and GFP-tagged MK5/PRAK or its exon 6 (Δex6) or exon 8 (Δex8) deletion mutants were immuno-precipitated by GFP nanobodies coupled to M270 epoxy beads. The beads were used in a kinase reaction with recombinant Hsp27 as a substrate. The reaction mixture was resolved by SDS-PAGE and Hsp27 phosphorylating activity was detected by phospho-imaging (autoradiogram) and Western blot against pS82-Hsp27. Increased Hsp27-kinase activity is only detected for WT MK5/PRAK (asterisk). C) In vitro MK5/PRAK kinase assay with Hsp27 as substrates. GST–p38 was used to activate MK5/PRAK in vitro. While Hsp27-kinase activity is only detected for WT MK5/PRAK, auto-phosphorylating activity in the absence of p38 MAPK (left panel) is detected for both WT MK5/PRAK and Δex8 (asterisk).
    Figure Legend Snippet: Kinase activity of wild type MK5/PRAK, MK5/PRAK-Δex6 and -Δex8. A) HEK293 cells were transfected with the constructs indicated. The total amount of DNA per well was kept constant (1,6μg DNA per well in 12-well plate) by adding pcDNA-flag expression vector. 24 h post transfection cells were lysed in the plates with 1x Laemmli buffer. Lysates were analyzed by Western blotting using the indicated antibodies. A significant increase in phosphorylation of the substrate Hsp27 was only detected for WT MK5/PRAK in the presence of overexpressed p38 MAPK, ERK3 or ERK4. B) HEK293 cells were transfected with the expression constructs indicated. 24 h post transfection the cells were lysed and GFP-tagged MK5/PRAK or its exon 6 (Δex6) or exon 8 (Δex8) deletion mutants were immuno-precipitated by GFP nanobodies coupled to M270 epoxy beads. The beads were used in a kinase reaction with recombinant Hsp27 as a substrate. The reaction mixture was resolved by SDS-PAGE and Hsp27 phosphorylating activity was detected by phospho-imaging (autoradiogram) and Western blot against pS82-Hsp27. Increased Hsp27-kinase activity is only detected for WT MK5/PRAK (asterisk). C) In vitro MK5/PRAK kinase assay with Hsp27 as substrates. GST–p38 was used to activate MK5/PRAK in vitro. While Hsp27-kinase activity is only detected for WT MK5/PRAK, auto-phosphorylating activity in the absence of p38 MAPK (left panel) is detected for both WT MK5/PRAK and Δex8 (asterisk).

    Techniques Used: Activity Assay, Transfection, Construct, Expressing, Plasmid Preparation, Western Blot, Recombinant, SDS Page, Imaging, In Vitro, Kinase Assay

    11) Product Images from "Pervasive Transcription of a Herpesvirus Genome Generates Functionally Important RNAs"

    Article Title: Pervasive Transcription of a Herpesvirus Genome Generates Functionally Important RNAs

    Journal: mBio

    doi: 10.1128/mBio.01033-13

    EGR 26c ASO decreases specific genes of all kinetic classes. 3T12 cells transfected with GFP or EGR 26c ASO or untransfected (No ASO) were infected with MHV68 (MOI = 10) and analyzed for protein (A, C, and E) or transcript levels (B, D, and F). (A) Representative Western blots for M9 and actin at 18 hpi (2 experiments). (B) Representative Northern blot using a probe to M3 or actin at 14 hpi and corresponding quantification of M3 transcript levels normalized to those of actin (means and SEMs from 3 experiments). A 0.5-µg portion of RNA was used per lane for M3 Northern blots. (C) Representative Western blot for M3 protein at 18 hpi and corresponding quantification of M3 protein levels normalized to those of actin (means and SEMs from 4 experiments). (D) Representative Northern blot for ORF 6 and actin at 14 hpi and corresponding quantification of ORF 6 transcript levels normalized to actin for cells transfected with GFP or EGR 26 ASOs (means and SEMs from 3 to 5 experiments). (E) Representative Western blot for ORF 6 and actin at 18 hpi and corresponding quantification of ORF 6 protein normalized to actin (means and SEMs from 5 experiments). Representative Western blots for ORF 6 are the same as in Fig. 1 . (F) ORF 50 transcript levels at 14 hpi. RNA (1 µg) was reverse transcribed, and cDNA was analyzed by qPCR using primers designed to detect spliced ORF 50 transcripts or GAPDH. Data are relative ORF 50 abundances normalized to GAPDH transcript abundance and compared to untransfected cells by the ∆∆ C T method (means and SEMs from 4 experiments). Statistical analyses were performed by paired t test (A, B, C, and E) or one-way ANOVA with Dunnett’s posttest (D and F). *, P
    Figure Legend Snippet: EGR 26c ASO decreases specific genes of all kinetic classes. 3T12 cells transfected with GFP or EGR 26c ASO or untransfected (No ASO) were infected with MHV68 (MOI = 10) and analyzed for protein (A, C, and E) or transcript levels (B, D, and F). (A) Representative Western blots for M9 and actin at 18 hpi (2 experiments). (B) Representative Northern blot using a probe to M3 or actin at 14 hpi and corresponding quantification of M3 transcript levels normalized to those of actin (means and SEMs from 3 experiments). A 0.5-µg portion of RNA was used per lane for M3 Northern blots. (C) Representative Western blot for M3 protein at 18 hpi and corresponding quantification of M3 protein levels normalized to those of actin (means and SEMs from 4 experiments). (D) Representative Northern blot for ORF 6 and actin at 14 hpi and corresponding quantification of ORF 6 transcript levels normalized to actin for cells transfected with GFP or EGR 26 ASOs (means and SEMs from 3 to 5 experiments). (E) Representative Western blot for ORF 6 and actin at 18 hpi and corresponding quantification of ORF 6 protein normalized to actin (means and SEMs from 5 experiments). Representative Western blots for ORF 6 are the same as in Fig. 1 . (F) ORF 50 transcript levels at 14 hpi. RNA (1 µg) was reverse transcribed, and cDNA was analyzed by qPCR using primers designed to detect spliced ORF 50 transcripts or GAPDH. Data are relative ORF 50 abundances normalized to GAPDH transcript abundance and compared to untransfected cells by the ∆∆ C T method (means and SEMs from 4 experiments). Statistical analyses were performed by paired t test (A, B, C, and E) or one-way ANOVA with Dunnett’s posttest (D and F). *, P

    Techniques Used: Allele-specific Oligonucleotide, Transfection, Infection, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction

    Effect on immediate-early, early, and late genes by EGR 27. 3T12 cells transfected with GFP or ASOs targeting EGR 27 or left untransfected (No ASO) were infected with MHV68 (MOI = 10) and analyzed for protein (A and D) or transcript levels (B, C, and E). See Fig. 5 and also Table S1 in the supplemental material for ASO locations. (A) Representative Western blots for M9 and ORF 26 proteins at 18 hpi (2 or 3 experiments). (B) ORF 29 transcript levels at 14 hpi. RNA (1 µg) was reverse transcribed (RT), and cDNA was analyzed by qPCR using primers designed to detect spliced ORF 29 transcripts or GAPDH. Data are relative ORF 29 abundance normalized to GAPDH transcript abundance and compared to untransfected cells by the ∆∆ C T method (means and SEMs from 3 to 8 experiments). (C) Representative Northern blot for ORF 6 and actin at 14 hpi and corresponding quantification of ORF 6 transcript levels normalized to actin and compared to the value for untransfected cells (means and SEMs from 5 to 7 experiments). (D) Representative Western blot for ORF 6 and actin at 18 hpi (3 experiments). The representative experiment is the one whose results are shown in panel A. (E) ORF 50 transcript levels at 14 hpi measured by qRT-PCR, as for panel B. Statistical analyses were performed by one-way ANOVA with Dunnett’s posttest. *, P
    Figure Legend Snippet: Effect on immediate-early, early, and late genes by EGR 27. 3T12 cells transfected with GFP or ASOs targeting EGR 27 or left untransfected (No ASO) were infected with MHV68 (MOI = 10) and analyzed for protein (A and D) or transcript levels (B, C, and E). See Fig. 5 and also Table S1 in the supplemental material for ASO locations. (A) Representative Western blots for M9 and ORF 26 proteins at 18 hpi (2 or 3 experiments). (B) ORF 29 transcript levels at 14 hpi. RNA (1 µg) was reverse transcribed (RT), and cDNA was analyzed by qPCR using primers designed to detect spliced ORF 29 transcripts or GAPDH. Data are relative ORF 29 abundance normalized to GAPDH transcript abundance and compared to untransfected cells by the ∆∆ C T method (means and SEMs from 3 to 8 experiments). (C) Representative Northern blot for ORF 6 and actin at 14 hpi and corresponding quantification of ORF 6 transcript levels normalized to actin and compared to the value for untransfected cells (means and SEMs from 5 to 7 experiments). (D) Representative Western blot for ORF 6 and actin at 18 hpi (3 experiments). The representative experiment is the one whose results are shown in panel A. (E) ORF 50 transcript levels at 14 hpi measured by qRT-PCR, as for panel B. Statistical analyses were performed by one-way ANOVA with Dunnett’s posttest. *, P

    Techniques Used: Transfection, Allele-specific Oligonucleotide, Infection, Western Blot, Real-time Polymerase Chain Reaction, Northern Blot, Quantitative RT-PCR

    An antisense oligonucleotide to ORF 6 decreases ORF 6 transcript and protein expression and late-gene expression. 3T12 cells transfected with ASOs targeting ORF 6, M3, or GFP (negative control) or left untransfected (No ASO) were infected with MHV68. (A) Representative Northern blot for ORF 6 or actin transcripts at 14 hpi and corresponding quantification of ORF 6 monocistronic transcript levels normalized to those of actin (MOI = 10; values are means and standard errors of the means [SEMs] from 3 experiments; **, P
    Figure Legend Snippet: An antisense oligonucleotide to ORF 6 decreases ORF 6 transcript and protein expression and late-gene expression. 3T12 cells transfected with ASOs targeting ORF 6, M3, or GFP (negative control) or left untransfected (No ASO) were infected with MHV68. (A) Representative Northern blot for ORF 6 or actin transcripts at 14 hpi and corresponding quantification of ORF 6 monocistronic transcript levels normalized to those of actin (MOI = 10; values are means and standard errors of the means [SEMs] from 3 experiments; **, P

    Techniques Used: Expressing, Transfection, Negative Control, Allele-specific Oligonucleotide, Infection, Northern Blot

    12) Product Images from "Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression"

    Article Title: Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression

    Journal: Scientific Reports

    doi: 10.1038/srep19781

    PGC1 α1 levels increase in the soleus in trained mice and in transfected C2C12 cells upon transfection with pCMV-Entry-HSPD1 vector. ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P
    Figure Legend Snippet: PGC1 α1 levels increase in the soleus in trained mice and in transfected C2C12 cells upon transfection with pCMV-Entry-HSPD1 vector. ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P

    Techniques Used: Mouse Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

    13) Product Images from "Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression"

    Article Title: Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression

    Journal: Scientific Reports

    doi: 10.1038/srep19781

    PGC1 α1 levels increase in the soleus in trained mice and in transfected C2C12 cells upon transfection with pCMV-Entry-HSPD1 vector. ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P
    Figure Legend Snippet: PGC1 α1 levels increase in the soleus in trained mice and in transfected C2C12 cells upon transfection with pCMV-Entry-HSPD1 vector. ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P

    Techniques Used: Mouse Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

    14) Product Images from "Extracorporeal immune therapy with immobilized agonistic anti-Fas antibodies leads to transient reduction of circulating neutrophil numbers and limits tissue damage after hemorrhagic shock/resuscitation in a porcine model"

    Article Title: Extracorporeal immune therapy with immobilized agonistic anti-Fas antibodies leads to transient reduction of circulating neutrophil numbers and limits tissue damage after hemorrhagic shock/resuscitation in a porcine model

    Journal: Journal of Inflammation (London, England)

    doi: 10.1186/1476-9255-7-18

    Heme oxygenase-1 (HO-1) gene expression (A), and HO-1 protein expression (B) in control (white bars), SMC (grey bars), and LIM (black bars) animals .
    Figure Legend Snippet: Heme oxygenase-1 (HO-1) gene expression (A), and HO-1 protein expression (B) in control (white bars), SMC (grey bars), and LIM (black bars) animals .

    Techniques Used: Expressing

    15) Product Images from "Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion"

    Article Title: Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

    Journal: mBio

    doi: 10.1128/mBio.01802-14

    (A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.
    Figure Legend Snippet: (A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.

    Techniques Used: Infection, Staining, Purification, Mass Spectrometry, Immunoprecipitation, SDS Page, Sample Prep

    16) Product Images from "Functional role of ADAMTS5 in adiposity and metabolic health"

    Article Title: Functional role of ADAMTS5 in adiposity and metabolic health

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190595

    Effect of ADAMTS5 genotype on AT after cold exposure for 2 weeks. (A) Body weight of ADAMTS5-J WT (+/+; n = 7) or homozygous deficient (-/-; n = 9) mice at start and end of the experiment. (B) Mass of sWAT, GON AT and BAT for WT (white bars; n = 8) and Adamts5 -/- -J mice (black bars; n = 9). (C) Gene expression levels of markers of browning of sWAT, normalized to the housekeeping gene Tbp and shown relative to WT (n = 7–8). (D) sWAT western blots for UCP-1 (n = 7–8) and PGC1α (n = 7–9), and quantitation by densitometry, normalized to GAPDH and shown relative to WT. (E) sWAT sections of WT (+/+) and Adamts5 -/- -J mice were stained with haematoxylin eosin (upper panel) or with antibodies directed against UCP-1 (lower panel; brown color) to illustrate browning of sWAT. Magnification: 400x. Scale bar = 10 μm. Data are means ± SEM of n determinations. * p
    Figure Legend Snippet: Effect of ADAMTS5 genotype on AT after cold exposure for 2 weeks. (A) Body weight of ADAMTS5-J WT (+/+; n = 7) or homozygous deficient (-/-; n = 9) mice at start and end of the experiment. (B) Mass of sWAT, GON AT and BAT for WT (white bars; n = 8) and Adamts5 -/- -J mice (black bars; n = 9). (C) Gene expression levels of markers of browning of sWAT, normalized to the housekeeping gene Tbp and shown relative to WT (n = 7–8). (D) sWAT western blots for UCP-1 (n = 7–8) and PGC1α (n = 7–9), and quantitation by densitometry, normalized to GAPDH and shown relative to WT. (E) sWAT sections of WT (+/+) and Adamts5 -/- -J mice were stained with haematoxylin eosin (upper panel) or with antibodies directed against UCP-1 (lower panel; brown color) to illustrate browning of sWAT. Magnification: 400x. Scale bar = 10 μm. Data are means ± SEM of n determinations. * p

    Techniques Used: Mouse Assay, Expressing, Western Blot, Quantitation Assay, Staining

    Effect of ADAMTS5 deficiency on diet-induced obesity. (A-C) Expression of Adamts5 (A disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5), Ucp-1 (Uncoupling protein-1) and Cidea in subcutaneous (s) white adipose tissue (WAT) of ADAMTS5-J mice (n = 5–6). Data are corrected for the housekeeping gene β-actin and normalized to wild-type (WT) mice (+/+). Data are means ± SEM of n determinations. (D) Western blot analysis of UCP-1 protein levels in sWAT of Adamts5 +/+ -P (n = 2), Adamts5 -/- -P (-/-; n = 1), Adamts5 +/+ -J (n = 5) and Adamts5 -/- -J (-/-; n = 5) mice. The expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.
    Figure Legend Snippet: Effect of ADAMTS5 deficiency on diet-induced obesity. (A-C) Expression of Adamts5 (A disintegrin and metalloproteinase with thrombospondin type 1 motifs member 5), Ucp-1 (Uncoupling protein-1) and Cidea in subcutaneous (s) white adipose tissue (WAT) of ADAMTS5-J mice (n = 5–6). Data are corrected for the housekeeping gene β-actin and normalized to wild-type (WT) mice (+/+). Data are means ± SEM of n determinations. (D) Western blot analysis of UCP-1 protein levels in sWAT of Adamts5 +/+ -P (n = 2), Adamts5 -/- -P (-/-; n = 1), Adamts5 +/+ -J (n = 5) and Adamts5 -/- -J (-/-; n = 5) mice. The expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

    Techniques Used: Expressing, Mouse Assay, Western Blot

    Effect of ADAMTS5 genotype on AT during cold exposure for 72 h. (A, B) Gene expression levels of Ucp-1 (A) and Cidea (B) in sWAT of WT (+/+) and homozygous deficient (-/-) mice from the ADAMTS5-P (+/+: n = 10; -/-: n = 8) and ADAMTS5-J strains (+/+: n = 11; -/-: n = 7). Gene expression levels are normalized to the housekeeping gene Tbp and shown relative to WT mice at +4°C. (C,D) UCP-1 protein levels in sWAT. The Western blots (C) are quantitated by densitometry, normalized to β-actin and expressed relative to WT (D). (E-G) Relative ADAMTS5 gene expression in sWAT (E), GON AT (F) and BAT (G) of WT mice kept at 24°C or at 4°C. Gene expression levels are normalized to the housekeeping gene Tbp and shown relative to WT mice at 24°C. Data are means ± SEM of 4–8 and 5–11 determinations for panel D and panel E-G, respectively. ** p
    Figure Legend Snippet: Effect of ADAMTS5 genotype on AT during cold exposure for 72 h. (A, B) Gene expression levels of Ucp-1 (A) and Cidea (B) in sWAT of WT (+/+) and homozygous deficient (-/-) mice from the ADAMTS5-P (+/+: n = 10; -/-: n = 8) and ADAMTS5-J strains (+/+: n = 11; -/-: n = 7). Gene expression levels are normalized to the housekeeping gene Tbp and shown relative to WT mice at +4°C. (C,D) UCP-1 protein levels in sWAT. The Western blots (C) are quantitated by densitometry, normalized to β-actin and expressed relative to WT (D). (E-G) Relative ADAMTS5 gene expression in sWAT (E), GON AT (F) and BAT (G) of WT mice kept at 24°C or at 4°C. Gene expression levels are normalized to the housekeeping gene Tbp and shown relative to WT mice at 24°C. Data are means ± SEM of 4–8 and 5–11 determinations for panel D and panel E-G, respectively. ** p

    Techniques Used: Expressing, Mouse Assay, Western Blot

    17) Product Images from "Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor"

    Article Title: Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2012/278273

    Isorhamnetin potentiates the NGF-induced neurofilament expression. Cultured PC12 cells were treated with NGF (0.5 ng/mL), isorhamnetin (10 μ M), and NGF (0.5 ng/mL) + isorhamnetin (10 μ M) for 72 hours. NGF at 50 ng/mL was applied as a control. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. Quantification plot was shown in lower panel. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. ** where P
    Figure Legend Snippet: Isorhamnetin potentiates the NGF-induced neurofilament expression. Cultured PC12 cells were treated with NGF (0.5 ng/mL), isorhamnetin (10 μ M), and NGF (0.5 ng/mL) + isorhamnetin (10 μ M) for 72 hours. NGF at 50 ng/mL was applied as a control. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. Quantification plot was shown in lower panel. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. ** where P

    Techniques Used: Expressing, Cell Culture

    Isorhamnetin induces the neurofilament expression in cultured PC12 cells but not the neurite outgrowth. (a) The chemical structure of isorhamnetin is illustrated. (b) Cultured PC12 cells were treated with isorhamnetin (1 to10 μ M) for 72 hours. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. The lower panel shows the quantitation from the blots by a densitometer. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4, each with triplicate samples. (c) Cultures were treated with isorhamnetin (3 or 10 μ M) and NGF (50 ng/mL), as indicated, for 72 hours. Cells were fixed with ice-cold 4% paraformaldehyde. Bar = 10 μ m. Representative images were shown. (d) Cultured PC12 cell was treated as in (c). The % of differentiated cell (upper panel) and length of neurite (lower panel) were counted as described in the Materials and Methods section. Values are expressed as % of total cells in 100 counted cells, mean ± SEM, n = 4. ** P
    Figure Legend Snippet: Isorhamnetin induces the neurofilament expression in cultured PC12 cells but not the neurite outgrowth. (a) The chemical structure of isorhamnetin is illustrated. (b) Cultured PC12 cells were treated with isorhamnetin (1 to10 μ M) for 72 hours. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. The lower panel shows the quantitation from the blots by a densitometer. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4, each with triplicate samples. (c) Cultures were treated with isorhamnetin (3 or 10 μ M) and NGF (50 ng/mL), as indicated, for 72 hours. Cells were fixed with ice-cold 4% paraformaldehyde. Bar = 10 μ m. Representative images were shown. (d) Cultured PC12 cell was treated as in (c). The % of differentiated cell (upper panel) and length of neurite (lower panel) were counted as described in the Materials and Methods section. Values are expressed as % of total cells in 100 counted cells, mean ± SEM, n = 4. ** P

    Techniques Used: Expressing, Cell Culture, Quantitation Assay

    The potentiating effect of isorhamnetin on NGF-induced response could not be blocked by U0126. Cultured PC12 cells, serum starvation for 5 hours, were treated with NGF (0.5 ng/mL), isorhamnetin (Iso; 10 μ M), and NGF (0.5 ng/mL) + isorhamnetin (Iso; 10 μ M) for 72 hours with or without the pretreatment of U0126 (20 μ M) for 3 hours. NGF at 50 ng/mL served as a positive control. (a) The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. GADPH served as a loading control. (b) Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. **where P
    Figure Legend Snippet: The potentiating effect of isorhamnetin on NGF-induced response could not be blocked by U0126. Cultured PC12 cells, serum starvation for 5 hours, were treated with NGF (0.5 ng/mL), isorhamnetin (Iso; 10 μ M), and NGF (0.5 ng/mL) + isorhamnetin (Iso; 10 μ M) for 72 hours with or without the pretreatment of U0126 (20 μ M) for 3 hours. NGF at 50 ng/mL served as a positive control. (a) The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. GADPH served as a loading control. (b) Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. **where P

    Techniques Used: Cell Culture, Positive Control

    NGF induces the expression of neurofilaments in cultured PC12 cells. Cultured PC12 cells were treated with NGF (0.3 to 50 ng/mL) for 72 hours. The cell lysates were collected to determine the expressions of NF68 ( M r ~ 68 kDa), NF160 ( M r ~ 160 kDa), and NF200 ( M r ~ 200 kDa). GADPH ( M r ~ 38 kDa) served as a loading control (upper panel). Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in Mean ± SEM, n = 4, each with triplicate samples. Representative images were shown. ** P
    Figure Legend Snippet: NGF induces the expression of neurofilaments in cultured PC12 cells. Cultured PC12 cells were treated with NGF (0.3 to 50 ng/mL) for 72 hours. The cell lysates were collected to determine the expressions of NF68 ( M r ~ 68 kDa), NF160 ( M r ~ 160 kDa), and NF200 ( M r ~ 200 kDa). GADPH ( M r ~ 38 kDa) served as a loading control (upper panel). Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in Mean ± SEM, n = 4, each with triplicate samples. Representative images were shown. ** P

    Techniques Used: Expressing, Cell Culture

    18) Product Images from "Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1"

    Article Title: Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

    Journal: Journal of Virology

    doi: 10.1128/JVI.00517-14

    Nbs1 knockdown disrupts MRN complex formation. (A) Whole-cell lysates were harvested from HFK, CIN612, and CIN612 9E cells stably expressing shScramble or shNBS1 at T 0 and after 72 h of differentiation in high-calcium medium (Ca). Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. GAPDH was used as a loading control. (B) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from HFK and CIN612 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. (C) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from stable CIN612 9E shScramble and CIN612 9E shNBS1 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. For panels B and C, lamin A/C and tubulin were used to confirm nuclear and cytoplasmic fractionation, respectively. (D) DNA was harvested from CIN612 9E cells at T 0 and after 72 h of differentiation in high-calcium medium with dimethyl sulfoxide (DMSO) as a vehicle control or 50 μM Mre11 inhibitor Mirin. Southern blot analysis was performed to analyze viral genome amplification of DNA digested with BamHI (nonviral genome cutter; upper panel) or HindIII (cuts viral genome once; lower panel). All results are representative of observations of two or more independent experiments.
    Figure Legend Snippet: Nbs1 knockdown disrupts MRN complex formation. (A) Whole-cell lysates were harvested from HFK, CIN612, and CIN612 9E cells stably expressing shScramble or shNBS1 at T 0 and after 72 h of differentiation in high-calcium medium (Ca). Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. GAPDH was used as a loading control. (B) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from HFK and CIN612 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. (C) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from stable CIN612 9E shScramble and CIN612 9E shNBS1 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. For panels B and C, lamin A/C and tubulin were used to confirm nuclear and cytoplasmic fractionation, respectively. (D) DNA was harvested from CIN612 9E cells at T 0 and after 72 h of differentiation in high-calcium medium with dimethyl sulfoxide (DMSO) as a vehicle control or 50 μM Mre11 inhibitor Mirin. Southern blot analysis was performed to analyze viral genome amplification of DNA digested with BamHI (nonviral genome cutter; upper panel) or HindIII (cuts viral genome once; lower panel). All results are representative of observations of two or more independent experiments.

    Techniques Used: Stable Transfection, Expressing, Fractionation, Southern Blot, Amplification

    Phosphorylation of ATM and Chk2 is maintained with Nbs1 knockdown upon differentiation. (A) Whole-cell lysates were harvested from HFKs and CIN612 9E cells, as well as CIN612 9E cells stably expressing shScramble or shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated ATM (Ser1981) (pATM), total ATM, and Nbs1. Tubulin was used as a loading control. Protein levels were quantified using ImageJ, with phosphorylated protein levels normalized first to total levels and then to tubulin. Levels for this representative experiment are graphed as fold change compared to the T 0 HFK sample, which is set to 1. (B) Whole-cell lysates were harvested from HFK, CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated Chk2 (Thr68) (pChk2), total Chk2, and Nbs1. GAPDH was used as a loading control. Protein levels were quantified using ImageJ as indicated above. Shown is a representative experiment where levels are graphed as fold change compared to the T 0 HFK sample, which is set at 1. (C) DNA was harvested from CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and after 72 h of differentiation in high-calcium medium and linearized by digestion with HindIII. HPV episomes were visualized via Southern blot analysis. Results shown are representative observations of four or more independent experiments.
    Figure Legend Snippet: Phosphorylation of ATM and Chk2 is maintained with Nbs1 knockdown upon differentiation. (A) Whole-cell lysates were harvested from HFKs and CIN612 9E cells, as well as CIN612 9E cells stably expressing shScramble or shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated ATM (Ser1981) (pATM), total ATM, and Nbs1. Tubulin was used as a loading control. Protein levels were quantified using ImageJ, with phosphorylated protein levels normalized first to total levels and then to tubulin. Levels for this representative experiment are graphed as fold change compared to the T 0 HFK sample, which is set to 1. (B) Whole-cell lysates were harvested from HFK, CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated Chk2 (Thr68) (pChk2), total Chk2, and Nbs1. GAPDH was used as a loading control. Protein levels were quantified using ImageJ as indicated above. Shown is a representative experiment where levels are graphed as fold change compared to the T 0 HFK sample, which is set at 1. (C) DNA was harvested from CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and after 72 h of differentiation in high-calcium medium and linearized by digestion with HindIII. HPV episomes were visualized via Southern blot analysis. Results shown are representative observations of four or more independent experiments.

    Techniques Used: Stable Transfection, Expressing, Southern Blot

    19) Product Images from "Def-6, a Novel Regulator of Small GTPases in Podocytes, Acts Downstream of Atypical Protein Kinase C (aPKC) λ/ι"

    Article Title: Def-6, a Novel Regulator of Small GTPases in Podocytes, Acts Downstream of Atypical Protein Kinase C (aPKC) λ/ι

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.08.026

    Def-6 expression is enhanced in PKCλ/ι −/− glomeruli and localizes to cellular edges in podocytes. A–C : Staining of kidney cryosections from PKCλ/ι +/+ and PKCλ/ι −/− mice. A: Upper panels : Overview staining with an antibody against Def-6 (×10 magnification) reveals strong glomerular expression. Lower panels : Control staining with secondary antibody or Def-6 blocking peptide validated the specificity of the Def-6 antibody. Scale bars = 200 μm. B : Co-staining of Def-6 (red) and the podocyte marker podocalyxin (green) reveals enhanced podocytic expression of Def-6 in PKCλ/ι − / − glomeruli ( boxed areas in Merge views, and arrowhead s in Detail views). Nuclei are visualized with DAPI (blue). Scale bars = 25 μm (applies to all panels). C : For quantification of Def-6 expression, sections were double-stained against Def-6 (red) and nidogen (green). Nuclei were visualized using DAPI (blue). Def-6 expression is enhanced in the podocytic areas of PKCλ/ι − / − glomeruli ( boxed areas in Merge views, and arrowhead s in Detail views). Scale bars = 25 μm (applies to all panels). D : Images of PKCλ/ι +/+ and PKCλ/ι − / − glomeruli (40 glomeruli from four animals of each genotype) were obtained and analyzed using a semiquantitative score ranging from 0 (no expression) to 4 (strong expression). Compared with WT glomeruli, PKCλ/ι − / − glomeruli show a significantly higher score and an obvious change in score distribution. n = 4 mice of each genotype, ≥10 glomeruli per mouse. ∗∗∗ P
    Figure Legend Snippet: Def-6 expression is enhanced in PKCλ/ι −/− glomeruli and localizes to cellular edges in podocytes. A–C : Staining of kidney cryosections from PKCλ/ι +/+ and PKCλ/ι −/− mice. A: Upper panels : Overview staining with an antibody against Def-6 (×10 magnification) reveals strong glomerular expression. Lower panels : Control staining with secondary antibody or Def-6 blocking peptide validated the specificity of the Def-6 antibody. Scale bars = 200 μm. B : Co-staining of Def-6 (red) and the podocyte marker podocalyxin (green) reveals enhanced podocytic expression of Def-6 in PKCλ/ι − / − glomeruli ( boxed areas in Merge views, and arrowhead s in Detail views). Nuclei are visualized with DAPI (blue). Scale bars = 25 μm (applies to all panels). C : For quantification of Def-6 expression, sections were double-stained against Def-6 (red) and nidogen (green). Nuclei were visualized using DAPI (blue). Def-6 expression is enhanced in the podocytic areas of PKCλ/ι − / − glomeruli ( boxed areas in Merge views, and arrowhead s in Detail views). Scale bars = 25 μm (applies to all panels). D : Images of PKCλ/ι +/+ and PKCλ/ι − / − glomeruli (40 glomeruli from four animals of each genotype) were obtained and analyzed using a semiquantitative score ranging from 0 (no expression) to 4 (strong expression). Compared with WT glomeruli, PKCλ/ι − / − glomeruli show a significantly higher score and an obvious change in score distribution. n = 4 mice of each genotype, ≥10 glomeruli per mouse. ∗∗∗ P

    Techniques Used: Expressing, Staining, Mouse Assay, Blocking Assay, Marker

    20) Product Images from "ICAM-1-Targeted, Lcn2 siRNA-Encapsulating Liposomes are Potent Anti-angiogenic Agents for Triple Negative Breast Cancer"

    Article Title: ICAM-1-Targeted, Lcn2 siRNA-Encapsulating Liposomes are Potent Anti-angiogenic Agents for Triple Negative Breast Cancer

    Journal: Theranostics

    doi: 10.7150/thno.12167

    Conditioned media from MDA-MB-231 cells treated with ICAM-Lcn2-LP inhibits in vivo angiogenesis in the CAM assay. Representative micrographs are shown for CAMs treated with serum-free media (a), or CM from MDA-MB-231 cells treated with PBS (sham) (b), CM from MDA-MB-231 cells treated with IgG-Lcn2-LP (c), and CM from MDA-MB-231 cells treated with ICAM-Lcn2-LP (d). Scale bar is 1 mm, and arrows point to areas of newly formed blood vessels. CAM vascularity for each experimental group was scored on a scale of 0-4 (e). (NS: no significant difference,* p
    Figure Legend Snippet: Conditioned media from MDA-MB-231 cells treated with ICAM-Lcn2-LP inhibits in vivo angiogenesis in the CAM assay. Representative micrographs are shown for CAMs treated with serum-free media (a), or CM from MDA-MB-231 cells treated with PBS (sham) (b), CM from MDA-MB-231 cells treated with IgG-Lcn2-LP (c), and CM from MDA-MB-231 cells treated with ICAM-Lcn2-LP (d). Scale bar is 1 mm, and arrows point to areas of newly formed blood vessels. CAM vascularity for each experimental group was scored on a scale of 0-4 (e). (NS: no significant difference,* p

    Techniques Used: Multiple Displacement Amplification, In Vivo, Chick Chorioallantoic Membrane Assay

    siRNA knockdown of Lcn2 gene expression at the transcript level were determined by qRT-PCR (a). Lcn2 protein levels in MDA-MB-231 cells treated with immunoliposomes were determined by immunoblot assay (b), and quantified by densitometric analysis (c) (*** p
    Figure Legend Snippet: siRNA knockdown of Lcn2 gene expression at the transcript level were determined by qRT-PCR (a). Lcn2 protein levels in MDA-MB-231 cells treated with immunoliposomes were determined by immunoblot assay (b), and quantified by densitometric analysis (c) (*** p

    Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

    Schematic illustration of the designed ICAM-1 antibody-conjugated, Lcn2 siRNA-encapsulating liposome (ICAM-Lcn2-LP).
    Figure Legend Snippet: Schematic illustration of the designed ICAM-1 antibody-conjugated, Lcn2 siRNA-encapsulating liposome (ICAM-Lcn2-LP).

    Techniques Used:

    21) Product Images from "Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity"

    Article Title: Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2008-0211

    PTPRO expression augments tamoxifen sensitivity of breast cancer cell line. A, The WT or CS mutant of FLAG-tagged PTPRO was overexpressed in MCF-7 cells. The G418 selected pool was analyzed by Western blot analysis for PTPRO overexpression using anti-FLAG
    Figure Legend Snippet: PTPRO expression augments tamoxifen sensitivity of breast cancer cell line. A, The WT or CS mutant of FLAG-tagged PTPRO was overexpressed in MCF-7 cells. The G418 selected pool was analyzed by Western blot analysis for PTPRO overexpression using anti-FLAG

    Techniques Used: Expressing, Mutagenesis, Western Blot, Over Expression

    22) Product Images from "Arginine methylation of SMAD7 by PRMT1 in TGF-β–induced epithelial–mesenchymal transition and epithelial stem-cell generation"

    Article Title: Arginine methylation of SMAD7 by PRMT1 in TGF-β–induced epithelial–mesenchymal transition and epithelial stem-cell generation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002027

    PRMT1 regulates TGF-β–induced EMT. A , repression of PRMT1 expression using transfected siRNA attenuated the TGF-β–induced EMT morphology of HaCaT cells. Cells transfected with PRMT1 siRNA or control siRNA were treated with TGF-β for 48 or 72 h to induce EMT or left untreated. B and C , depletion of PRMT1 expression using transfected siRNA prevented the TGF-β–induced change in actin organization and down-regulation of E-cadherin expression, shown by immunofluorescence ( B ), as well as the EMT-associated decrease in Claudin1 expression and increase in Vimentin expression, shown by immunoblotting ( C ). HaCaT cells were transfected with PRMT1 siRNA or control siRNA and treated with TGF-β as in A. D , down-regulating PRMT1 expression using lentiviral shRNA reduced the TGF-β–induced EMT morphology in HMLE cells. HMLE cells were infected with lentiviral vectors expressing one of two different PRMT1 shRNAs, shRNA 1 and shRNA 3, or a control shRNA and treated with TGF-β for 6 days to induce EMT. E and F , down-regulating PRMT1 expression using lentiviral shRNA reduced TGF-β–induced changes in EMT marker expression. The HMLE cells expressing PRMT1 shRNA or control shRNA were treated with TGF-β as in D . Down-regulation of PRMT1 expression reduced the actin reorganization into stress fibers and the junctional localization of epithelial E-cadherin, shown by immunofluorescence ( IF ) ( E ), and attenuated the decreased expression of epithelial E-cadherin and the increased expression of mesenchymal N-cadherin, Fibronectin, Vimentin, and ZEB1, shown by immunoblotting ( IB ) ( F ). G , down-regulating PRMT1 expression using lentiviral shRNA reduced the TGF-β–induced expression of EMT markers, assessed by qRT-PCR analyses of mRNA. HMLE cells expressing PRMT1 shRNA or control shRNA were treated with TGF-β as in D . Down-regulation of PRMT1 reduced the suppression of epithelial marker gene CDH1 , which encodes E-cadherin, and induction of mRNAs encoding mesenchymal marker genes NCAD , FN1 , VIM , ZEB1 , ZEB2 , SNAI1 , and SNAI2 . #, p
    Figure Legend Snippet: PRMT1 regulates TGF-β–induced EMT. A , repression of PRMT1 expression using transfected siRNA attenuated the TGF-β–induced EMT morphology of HaCaT cells. Cells transfected with PRMT1 siRNA or control siRNA were treated with TGF-β for 48 or 72 h to induce EMT or left untreated. B and C , depletion of PRMT1 expression using transfected siRNA prevented the TGF-β–induced change in actin organization and down-regulation of E-cadherin expression, shown by immunofluorescence ( B ), as well as the EMT-associated decrease in Claudin1 expression and increase in Vimentin expression, shown by immunoblotting ( C ). HaCaT cells were transfected with PRMT1 siRNA or control siRNA and treated with TGF-β as in A. D , down-regulating PRMT1 expression using lentiviral shRNA reduced the TGF-β–induced EMT morphology in HMLE cells. HMLE cells were infected with lentiviral vectors expressing one of two different PRMT1 shRNAs, shRNA 1 and shRNA 3, or a control shRNA and treated with TGF-β for 6 days to induce EMT. E and F , down-regulating PRMT1 expression using lentiviral shRNA reduced TGF-β–induced changes in EMT marker expression. The HMLE cells expressing PRMT1 shRNA or control shRNA were treated with TGF-β as in D . Down-regulation of PRMT1 expression reduced the actin reorganization into stress fibers and the junctional localization of epithelial E-cadherin, shown by immunofluorescence ( IF ) ( E ), and attenuated the decreased expression of epithelial E-cadherin and the increased expression of mesenchymal N-cadherin, Fibronectin, Vimentin, and ZEB1, shown by immunoblotting ( IB ) ( F ). G , down-regulating PRMT1 expression using lentiviral shRNA reduced the TGF-β–induced expression of EMT markers, assessed by qRT-PCR analyses of mRNA. HMLE cells expressing PRMT1 shRNA or control shRNA were treated with TGF-β as in D . Down-regulation of PRMT1 reduced the suppression of epithelial marker gene CDH1 , which encodes E-cadherin, and induction of mRNAs encoding mesenchymal marker genes NCAD , FN1 , VIM , ZEB1 , ZEB2 , SNAI1 , and SNAI2 . #, p

    Techniques Used: Expressing, Transfection, Immunofluorescence, shRNA, Infection, Marker, Quantitative RT-PCR

    23) Product Images from "α-Synuclein binds to the ER–mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production"

    Article Title: α-Synuclein binds to the ER–mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-017-1704-z

    Expression of wild-type and familial Parkinson’s disease mutant α-synuclein reduce ER–mitochondria associations in SH-SY5Y cells. a Expression of α-synuclein does not alter expression of VAPB, PTPIP51 mitofusin-2 (MFN2) or the Sigma-1 receptor in stably transfected SH-SY5Y cells. Immunoblots of SH-SY5Y cells stably transfected with EGFP as a control, EGFP-α-synuclein, EGFP-α-synucleinA53T or EGFP-α-synucleinA30P and probed on immunoblots as indicated; GAPDH is shown as a loading control. Molecular masses in kD are shown on the right. b Representative electron micrographs of ER–mitochondria associations in SH-SY5Y cells expressing control EGFP vector (CTRL), EGFP-α-synuclein, EGFP-α-synucleinA53T or EGFP-α-synucleinA30P; arrowheads with loops show regions of association. Scale bar is 200 nm. Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. N = 30–35 cells and 107–155 mitochondria; error bars are SEM; *** p
    Figure Legend Snippet: Expression of wild-type and familial Parkinson’s disease mutant α-synuclein reduce ER–mitochondria associations in SH-SY5Y cells. a Expression of α-synuclein does not alter expression of VAPB, PTPIP51 mitofusin-2 (MFN2) or the Sigma-1 receptor in stably transfected SH-SY5Y cells. Immunoblots of SH-SY5Y cells stably transfected with EGFP as a control, EGFP-α-synuclein, EGFP-α-synucleinA53T or EGFP-α-synucleinA30P and probed on immunoblots as indicated; GAPDH is shown as a loading control. Molecular masses in kD are shown on the right. b Representative electron micrographs of ER–mitochondria associations in SH-SY5Y cells expressing control EGFP vector (CTRL), EGFP-α-synuclein, EGFP-α-synucleinA53T or EGFP-α-synucleinA30P; arrowheads with loops show regions of association. Scale bar is 200 nm. Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. N = 30–35 cells and 107–155 mitochondria; error bars are SEM; *** p

    Techniques Used: Expressing, Mutagenesis, Stable Transfection, Transfection, Western Blot, Plasmid Preparation

    24) Product Images from "Epigenetic reprogramming converts human Wharton’s jelly mesenchymal stem cells into functional cardiomyocytes by differential regulation of Wnt mediators"

    Article Title: Epigenetic reprogramming converts human Wharton’s jelly mesenchymal stem cells into functional cardiomyocytes by differential regulation of Wnt mediators

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0638-7

    a Study of cardiac morphology after induction with different epigenetic modifiers. Differentiation was performed with DC301, DC302, and DC303, and various combinations of these inhibitors, and the appearance of cardiac morphology was assessed by phase-contrast microscopy ( scale bars = 100 μm, n = 3). Treatment with DC301 + DC302 indicates clear cardiac morphology ( scale bar = 50 μm). b Analysis of cardiac markers by quantitative RT-PCR after cardiac induction with different epigenetic modifiers. Cardiac-specific genes GATA4 , Nkx2.5 , MLC , TnT , and cardiac actin were studied for their expression after cardiac induction with DC301, DC302, and DC303, and various combinations of these inhibitors as indicated. Results are mean ± SD of three independent experiments performed in triplicate (* p
    Figure Legend Snippet: a Study of cardiac morphology after induction with different epigenetic modifiers. Differentiation was performed with DC301, DC302, and DC303, and various combinations of these inhibitors, and the appearance of cardiac morphology was assessed by phase-contrast microscopy ( scale bars = 100 μm, n = 3). Treatment with DC301 + DC302 indicates clear cardiac morphology ( scale bar = 50 μm). b Analysis of cardiac markers by quantitative RT-PCR after cardiac induction with different epigenetic modifiers. Cardiac-specific genes GATA4 , Nkx2.5 , MLC , TnT , and cardiac actin were studied for their expression after cardiac induction with DC301, DC302, and DC303, and various combinations of these inhibitors as indicated. Results are mean ± SD of three independent experiments performed in triplicate (* p

    Techniques Used: Microscopy, Quantitative RT-PCR, Expressing

    Nkx2.5 promoter CpG island demethylation. a Position of Nkx2.5 gene on chromosome 5, sequence of the promoter region indicated. b After bisulfite conversion, the 160-bp promoter region of Nkx2.5 was amplified and cloned for bisulfite sequencing for control ( U ) and differentiated cardiomyocytes ( D ); shaded regions indicate the CpG islands that are modified after cardiac differentiation. c CpG islands were evaluated as methylated ( orange dot ) and unmethylated ( red dot ) sites by bisulfite sequencing. d After bisulfite conversion, methylation-specific PCR was performed for Nkx2.5 using unmethylated and methylated primers in control (U) and differentiated cardiomyocytes (D) (** p
    Figure Legend Snippet: Nkx2.5 promoter CpG island demethylation. a Position of Nkx2.5 gene on chromosome 5, sequence of the promoter region indicated. b After bisulfite conversion, the 160-bp promoter region of Nkx2.5 was amplified and cloned for bisulfite sequencing for control ( U ) and differentiated cardiomyocytes ( D ); shaded regions indicate the CpG islands that are modified after cardiac differentiation. c CpG islands were evaluated as methylated ( orange dot ) and unmethylated ( red dot ) sites by bisulfite sequencing. d After bisulfite conversion, methylation-specific PCR was performed for Nkx2.5 using unmethylated and methylated primers in control (U) and differentiated cardiomyocytes (D) (** p

    Techniques Used: Sequencing, Amplification, Clone Assay, Methylation Sequencing, Modification, Methylation, Polymerase Chain Reaction

    25) Product Images from "Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells"

    Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19051498

    Effect of Spns2-GFP overexpression on localization, S1P release, and CTGF secretion in HK2 cells. Cells were transiently transfected with an empty GFP vector (Ctrl) or a Spns2-GFP vector. ( A ) Confocal picture 48 h post transfection, showing diamidine-phenylindole (DAPI) stained nuclei (blue), or Spns2-GFP (green), or a merged picture. Bars represent 10 μm. ( B ) Supernatants of ctrl cells or Spns2-GFP overexpressing cells, stimulated for 24 h with either vehicle (−) or TGFβ 2 (5 ng/mL, +), were taken for a reporter assay to determine extracellular signal-regulated kinase (ERK) phosphorylation as a readout of S1P receptor activation, as described in the Methods Section. ( C , D ) Supernatants of vehicle (−) or TGFβ 2 (+) stimulated cells were taken for protein precipitation, and proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and then subjected to Western blotting using antibodies against CTGF. Bands were densitometrically evaluated. Data are expressed as % of control and are means ± S.D. ( n = 3–4); ** p
    Figure Legend Snippet: Effect of Spns2-GFP overexpression on localization, S1P release, and CTGF secretion in HK2 cells. Cells were transiently transfected with an empty GFP vector (Ctrl) or a Spns2-GFP vector. ( A ) Confocal picture 48 h post transfection, showing diamidine-phenylindole (DAPI) stained nuclei (blue), or Spns2-GFP (green), or a merged picture. Bars represent 10 μm. ( B ) Supernatants of ctrl cells or Spns2-GFP overexpressing cells, stimulated for 24 h with either vehicle (−) or TGFβ 2 (5 ng/mL, +), were taken for a reporter assay to determine extracellular signal-regulated kinase (ERK) phosphorylation as a readout of S1P receptor activation, as described in the Methods Section. ( C , D ) Supernatants of vehicle (−) or TGFβ 2 (+) stimulated cells were taken for protein precipitation, and proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and then subjected to Western blotting using antibodies against CTGF. Bands were densitometrically evaluated. Data are expressed as % of control and are means ± S.D. ( n = 3–4); ** p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Staining, Reporter Assay, Activation Assay, SDS Page, Western Blot

    26) Product Images from "Calcineurin in Reactive Astrocytes Plays a Key Role in the Interplay between Proinflammatory and Anti-Inflammatory Signals"

    Article Title: Calcineurin in Reactive Astrocytes Plays a Key Role in the Interplay between Proinflammatory and Anti-Inflammatory Signals

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1002-07.2007

    Astrocytic calcineurin and neuroinflammatory damage. A , Astrocytes transduced with ΔCnA show increased activity of calcineurin (measured as release of PO 4 ) compared with mock-transfected (CMV) or wt astrocytes (histograms). Only ΔCnA-transfected astrocytes produced the truncated mutant form of calcineurin (ΔCnA; blots). Levels of endogenous calcineurin remained unaffected. B , Schedule followed for coculture of astrocytes and neurons. Astrocytes were plated and transfected with corresponding DNAs, and neurons were added 24 h later. Thereafter, cocultures were challenged with inflammatory stimuli (LPS or TNF-α) for various times before analysis of neuronal death after a total of 4 d in coculture. Inhibitors such as CsA or MG-132 were added at indicated times. C , Photomicrographs, Representative double immunocytochemical staining used to identify apoptotic (activated caspase 3 + cells; red) neurons (β3-tubulin + cells; green) after inflammatory challenge. Histograms, Neurons cocultured with mock-transfected astrocytes (CMV; striped left histograms) die soon after LPS (top histograms) or TNF-α addition (bottom histograms), whereas when cocultured with astrocytes expressing ΔCnA (black right histograms), they show a significantly greater resistance to these inflammatory stimuli (** p
    Figure Legend Snippet: Astrocytic calcineurin and neuroinflammatory damage. A , Astrocytes transduced with ΔCnA show increased activity of calcineurin (measured as release of PO 4 ) compared with mock-transfected (CMV) or wt astrocytes (histograms). Only ΔCnA-transfected astrocytes produced the truncated mutant form of calcineurin (ΔCnA; blots). Levels of endogenous calcineurin remained unaffected. B , Schedule followed for coculture of astrocytes and neurons. Astrocytes were plated and transfected with corresponding DNAs, and neurons were added 24 h later. Thereafter, cocultures were challenged with inflammatory stimuli (LPS or TNF-α) for various times before analysis of neuronal death after a total of 4 d in coculture. Inhibitors such as CsA or MG-132 were added at indicated times. C , Photomicrographs, Representative double immunocytochemical staining used to identify apoptotic (activated caspase 3 + cells; red) neurons (β3-tubulin + cells; green) after inflammatory challenge. Histograms, Neurons cocultured with mock-transfected astrocytes (CMV; striped left histograms) die soon after LPS (top histograms) or TNF-α addition (bottom histograms), whereas when cocultured with astrocytes expressing ΔCnA (black right histograms), they show a significantly greater resistance to these inflammatory stimuli (** p

    Techniques Used: Transduction, Activity Assay, Transfection, Produced, Mutagenesis, Staining, Expressing

    Astrocytic calcineurin protects against LPS-induced inflammatory damage. A , Three days after intraparenchymael injection of LPS, reactive astrocytes (GFAP + ) in AIC mice not treated with Dox show negligible iNOS2 immunoreactivity. Scale bar, 100 μm. B , This was paralleled by a drastic reduction in levels of Cox2 and iNOS2 in the injected area ( p
    Figure Legend Snippet: Astrocytic calcineurin protects against LPS-induced inflammatory damage. A , Three days after intraparenchymael injection of LPS, reactive astrocytes (GFAP + ) in AIC mice not treated with Dox show negligible iNOS2 immunoreactivity. Scale bar, 100 μm. B , This was paralleled by a drastic reduction in levels of Cox2 and iNOS2 in the injected area ( p

    Techniques Used: Injection, Mouse Assay

    Calcineurin inhibits the NFκB/NFAT proinflammatory pathway in astrocytes. A , LPS failed to stimulate Cox2 and iNOS2 in astrocytes expressing ΔCnA. A representative blot is shown ( n = 6). B , LPS- or TNF-α-induced activation of NFκB and NFAT was abrogated in ΔCnA-transduced astrocytes. The activity of these transcription factors was inhibited after exposure to LPS/TNF-α in ΔCnA-expressing astrocytes. Note that expression of ΔCnA in unstimulated astrocytes reduced the activity of NFκB and NFAT. CMV, Astrocytes transfected with the empty vector; ΔCnA, astrocytes transfected with constitutively active calcineurin; CMV+NFκB or NFAT, mock-transfected astrocytes expressing the gene-reporter system for either transcription factor; ΔCnA+NFκB or NFAT, ΔCnA-transfected astrocytes expressing the gene-reporter system. *** p
    Figure Legend Snippet: Calcineurin inhibits the NFκB/NFAT proinflammatory pathway in astrocytes. A , LPS failed to stimulate Cox2 and iNOS2 in astrocytes expressing ΔCnA. A representative blot is shown ( n = 6). B , LPS- or TNF-α-induced activation of NFκB and NFAT was abrogated in ΔCnA-transduced astrocytes. The activity of these transcription factors was inhibited after exposure to LPS/TNF-α in ΔCnA-expressing astrocytes. Note that expression of ΔCnA in unstimulated astrocytes reduced the activity of NFκB and NFAT. CMV, Astrocytes transfected with the empty vector; ΔCnA, astrocytes transfected with constitutively active calcineurin; CMV+NFκB or NFAT, mock-transfected astrocytes expressing the gene-reporter system for either transcription factor; ΔCnA+NFκB or NFAT, ΔCnA-transfected astrocytes expressing the gene-reporter system. *** p

    Techniques Used: Expressing, Activation Assay, Activity Assay, Transfection, Plasmid Preparation

    Regulated expression of ΔCnA in astrocytes. A , Coexpression of GFAP–tTA (GFAP) and TetO–ΔCnA (ΔCnA) in cultured wt astrocytes allows Dox-regulated activation of calcineurin. Both control (wt) and GFAP–tTA-transduced astrocytes show lower and Dox-independent calcineurin activity. *** p
    Figure Legend Snippet: Regulated expression of ΔCnA in astrocytes. A , Coexpression of GFAP–tTA (GFAP) and TetO–ΔCnA (ΔCnA) in cultured wt astrocytes allows Dox-regulated activation of calcineurin. Both control (wt) and GFAP–tTA-transduced astrocytes show lower and Dox-independent calcineurin activity. *** p

    Techniques Used: Expressing, Cell Culture, Activation Assay, Activity Assay

    Stages of the neuroinflammatory process in which astrocyte calcineurin may participate. Initiation, Inflammatory signals set in motion by the neuropathological process activate calcineurin, which in turn activate the canonical NFκB/NFAT pathway. Activation of local and peripheral proinflammatory mechanisms together with the recruitment of autocrine and paracrine neuroprotective mediators follows. The time course of this simultaneous anti-inflammatory and proinflammatory cascade may be critical to the eventual outcome of the inflammatory response. Both agonistic and antagonistic inflammatory signals are produced by reactive astrocytes and microglia, damaged neurons and activated endothelia, and eventually from peripheral cells recruited to the lesion site. Resolution, If already activated calcineurin is stimulated by signals such as IGF-I, a neuroprotective network is activated; Progression, if calcineurin continues to be activated by inflammatory signals, the inflammation proceeds and neurons die. Both phases may be reversibly interrelated depending on the time course of the pathological process. Mechanisms whereby calcineurin is recruited toward either inflammation or neuroprotection, which involve differential interactions with transcription factors such as PPARγ and GATA3 or proteasome degradation and which depend on the upstream signal stimulating calcineurin, warrant additional analysis.
    Figure Legend Snippet: Stages of the neuroinflammatory process in which astrocyte calcineurin may participate. Initiation, Inflammatory signals set in motion by the neuropathological process activate calcineurin, which in turn activate the canonical NFκB/NFAT pathway. Activation of local and peripheral proinflammatory mechanisms together with the recruitment of autocrine and paracrine neuroprotective mediators follows. The time course of this simultaneous anti-inflammatory and proinflammatory cascade may be critical to the eventual outcome of the inflammatory response. Both agonistic and antagonistic inflammatory signals are produced by reactive astrocytes and microglia, damaged neurons and activated endothelia, and eventually from peripheral cells recruited to the lesion site. Resolution, If already activated calcineurin is stimulated by signals such as IGF-I, a neuroprotective network is activated; Progression, if calcineurin continues to be activated by inflammatory signals, the inflammation proceeds and neurons die. Both phases may be reversibly interrelated depending on the time course of the pathological process. Mechanisms whereby calcineurin is recruited toward either inflammation or neuroprotection, which involve differential interactions with transcription factors such as PPARγ and GATA3 or proteasome degradation and which depend on the upstream signal stimulating calcineurin, warrant additional analysis.

    Techniques Used: Activation Assay, Produced

    Astrocytic calcineurin participates in proinflammatory and in anti-inflammatory signaling. A , In wt astrocytes, calcineurin activity was incremented by proinflammatory stimuli such as LPS/TNF-α and by neuroprotective signals such as IGF-I. ** p
    Figure Legend Snippet: Astrocytic calcineurin participates in proinflammatory and in anti-inflammatory signaling. A , In wt astrocytes, calcineurin activity was incremented by proinflammatory stimuli such as LPS/TNF-α and by neuroprotective signals such as IGF-I. ** p

    Techniques Used: Activity Assay

    27) Product Images from "Neuronal Wiskott–Aldrich syndrome protein regulates TGF-β1–mediated lung vascular permeability"

    Article Title: Neuronal Wiskott–Aldrich syndrome protein regulates TGF-β1–mediated lung vascular permeability

    Journal: The FASEB Journal

    doi: 10.1096/fj.201600102R

    Activation of Rho and FAK is necessary for Y256 phosphorylation of N-WASP induced by TGF-β1 in RMVECs. A ) Proposed mechanism of N-WASP activation by TGF-β1. Previous data indicate that Rho GTPase activation unlocks N-WASP from an autoinhibitory,
    Figure Legend Snippet: Activation of Rho and FAK is necessary for Y256 phosphorylation of N-WASP induced by TGF-β1 in RMVECs. A ) Proposed mechanism of N-WASP activation by TGF-β1. Previous data indicate that Rho GTPase activation unlocks N-WASP from an autoinhibitory,

    Techniques Used: Activation Assay

    TGF-β1 induces phosphorylation of Y256 of N-WASP. N-WASP Y256 plays a critical role in TGF-β1–induced paracellular permeability and actin stress fiber formation in RMVECs. A ) TGF-β1 induces phosphorylation of Y256 of N-WASP
    Figure Legend Snippet: TGF-β1 induces phosphorylation of Y256 of N-WASP. N-WASP Y256 plays a critical role in TGF-β1–induced paracellular permeability and actin stress fiber formation in RMVECs. A ) TGF-β1 induces phosphorylation of Y256 of N-WASP

    Techniques Used: Permeability

    28) Product Images from "Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor"

    Article Title: Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001951

    Effect of BZL on transporter expression in cellular lysates. P-gp (panel A) and MRP2 (panel B) were detected by western blotting in HepG2 total lysates after 48 h of treatment with BZL (2, 20, 200 and 1000 µM) or vehicle (C). Equal amounts of total protein (15 µg) were loaded in the gels. MRP2 and P-gp O.D. were related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p
    Figure Legend Snippet: Effect of BZL on transporter expression in cellular lysates. P-gp (panel A) and MRP2 (panel B) were detected by western blotting in HepG2 total lysates after 48 h of treatment with BZL (2, 20, 200 and 1000 µM) or vehicle (C). Equal amounts of total protein (15 µg) were loaded in the gels. MRP2 and P-gp O.D. were related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p

    Techniques Used: Expressing, Western Blot

    Effect of BZL on transporter expression in crude plasma membranes. P-gp (panel A) and MRP2 (panel B) were detected by western blotting in HepG2 plasma membranes after 48 h of treatment with BZL (200 µM) or vehicle (C). Equal amounts of total protein (5 µg) were loaded in the gels. MRP2 and P-gp O.D. were related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p
    Figure Legend Snippet: Effect of BZL on transporter expression in crude plasma membranes. P-gp (panel A) and MRP2 (panel B) were detected by western blotting in HepG2 plasma membranes after 48 h of treatment with BZL (200 µM) or vehicle (C). Equal amounts of total protein (5 µg) were loaded in the gels. MRP2 and P-gp O.D. were related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p

    Techniques Used: Expressing, Western Blot

    Effect of BZL on CYP3A4 and GST expression. Cells were exposed either to vehicle (C) or BZL (200 µM) for 48 h. CYP3A4 (panel A), GSTα (panel B), GSTμ (panel C), and GSTπ (panel D) levels were estimated by western blotting. Equal amounts of total protein (15 µg) were loaded in the gels. CYP3A4 or GST O.D. was related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p
    Figure Legend Snippet: Effect of BZL on CYP3A4 and GST expression. Cells were exposed either to vehicle (C) or BZL (200 µM) for 48 h. CYP3A4 (panel A), GSTα (panel B), GSTμ (panel C), and GSTπ (panel D) levels were estimated by western blotting. Equal amounts of total protein (15 µg) were loaded in the gels. CYP3A4 or GST O.D. was related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p

    Techniques Used: Expressing, Western Blot

    29) Product Images from "Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations *"

    Article Title: Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M110.003129

    Antibody staining confirms novel NET identifications. A , Western blots testing antibodies generated to novel muscle NETs TMEM38A, LOC203547, and POPDC2, control NET SUN2, and a NET previously identified in liver that appears to be much more abundant in muscle, TMEM201/NET5. The correct size for each NET is indicated by an asterisk. B , comparison of the relative amounts of proteins in ER (microsome) and NE fractions. Lamin A was used as a control for a NE-specific protein, whereas Calreticulin was used as a control for an ER protein. Western blots of the two fractions similarly loaded for total protein were quantified on a LI-COR Odyssey imaging system, and the percentage of the total signal between both microsome and NE lanes was plotted with NE signal in blue and microsome signal in yellow (three repeats were averaged). The new muscle NETs were more enriched in the NE fraction than was the control NET SUN2. C , cryosections of rat muscle stained with NET antibodies. NE staining was clearly observed for all NETs tested as determined by a rim around the DAPI-stained DNA. Leg muscle is shown for TMEM38A, and heart is shown for the other two NETs. Bars , 10 μm. D , antibody staining on pre-extracted cells. All cells shown are C2C12 mouse myoblasts except in the case of TMEM201 where HeLa cells are shown. Cells were pre-extracted with detergent (1% Triton X-100) to remove membranes and most soluble cytoplasmic material and then fixed and incubated with NET antibodies. A nuclear rim staining was observed for the control NET SUN2 and all novel NETs tested. The resistance to detergent pre-extraction is consistent with the INM localization indicated by OMX results in Fig. 4 .
    Figure Legend Snippet: Antibody staining confirms novel NET identifications. A , Western blots testing antibodies generated to novel muscle NETs TMEM38A, LOC203547, and POPDC2, control NET SUN2, and a NET previously identified in liver that appears to be much more abundant in muscle, TMEM201/NET5. The correct size for each NET is indicated by an asterisk. B , comparison of the relative amounts of proteins in ER (microsome) and NE fractions. Lamin A was used as a control for a NE-specific protein, whereas Calreticulin was used as a control for an ER protein. Western blots of the two fractions similarly loaded for total protein were quantified on a LI-COR Odyssey imaging system, and the percentage of the total signal between both microsome and NE lanes was plotted with NE signal in blue and microsome signal in yellow (three repeats were averaged). The new muscle NETs were more enriched in the NE fraction than was the control NET SUN2. C , cryosections of rat muscle stained with NET antibodies. NE staining was clearly observed for all NETs tested as determined by a rim around the DAPI-stained DNA. Leg muscle is shown for TMEM38A, and heart is shown for the other two NETs. Bars , 10 μm. D , antibody staining on pre-extracted cells. All cells shown are C2C12 mouse myoblasts except in the case of TMEM201 where HeLa cells are shown. Cells were pre-extracted with detergent (1% Triton X-100) to remove membranes and most soluble cytoplasmic material and then fixed and incubated with NET antibodies. A nuclear rim staining was observed for the control NET SUN2 and all novel NETs tested. The resistance to detergent pre-extraction is consistent with the INM localization indicated by OMX results in Fig. 4 .

    Techniques Used: Staining, Western Blot, Generated, Imaging, Incubation

    Confirmation of NE residence for NETs by targeting tagged fusion proteins. A , NETs were tested as tagged fusions for NE targeting, which is determined by their enrichment in a rim at the limits of DNA staining. NET fusions are visualized in white in the left panels and in red in the right panels , whereas DNA is visualized in white in the right panels . NETs were sometimes expressed or targeted better in certain cell types: all panels shown are U2OS osteosarcoma cells except for TMEM38A in mouse C2C12 cells and TMEM70 and CKAP4 in HeLa cells. All micrographs are on the same scale except for CKAP4 with all scale bars at 10 μm. B and C , inner versus outer nuclear membrane targeting. Structured illumination microscopy can distinguish proteins in the INM from those in the ONM when co-stained with nuclear basket protein Nup153 and cytoplasmic filament protein Nup358. B , an ONM NET or ER/ONM protein ( red ) should be in the same plane with Nup358 ( green ) but should be external to Nup153 ( green ), and this is observed for the control ER protein Sec61β ( upper schematic and images ). Correspondingly, an INM NET should be in the same plane as Nup153 and internal to Nup358 as is observed for the control NET LAP2β ( lower schematic and images ). C , all new NETs tested appeared in the same plane of the INM with Nup153 and formed an internal ring to Nup358, indicating INM residence. Bars , 5 μm.
    Figure Legend Snippet: Confirmation of NE residence for NETs by targeting tagged fusion proteins. A , NETs were tested as tagged fusions for NE targeting, which is determined by their enrichment in a rim at the limits of DNA staining. NET fusions are visualized in white in the left panels and in red in the right panels , whereas DNA is visualized in white in the right panels . NETs were sometimes expressed or targeted better in certain cell types: all panels shown are U2OS osteosarcoma cells except for TMEM38A in mouse C2C12 cells and TMEM70 and CKAP4 in HeLa cells. All micrographs are on the same scale except for CKAP4 with all scale bars at 10 μm. B and C , inner versus outer nuclear membrane targeting. Structured illumination microscopy can distinguish proteins in the INM from those in the ONM when co-stained with nuclear basket protein Nup153 and cytoplasmic filament protein Nup358. B , an ONM NET or ER/ONM protein ( red ) should be in the same plane with Nup358 ( green ) but should be external to Nup153 ( green ), and this is observed for the control ER protein Sec61β ( upper schematic and images ). Correspondingly, an INM NET should be in the same plane as Nup153 and internal to Nup358 as is observed for the control NET LAP2β ( lower schematic and images ). C , all new NETs tested appeared in the same plane of the INM with Nup153 and formed an internal ring to Nup358, indicating INM residence. Bars , 5 μm.

    Techniques Used: Staining, Microscopy

    30) Product Images from "Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology"

    Article Title: Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201008050

    The JNK pathway and microtubule stability are downstream of Sarm1 and Sdc2. (A) Sarm1 overexpression increases the acetylation levels of tubulin. COS-1 cells were transfected with vector control or Sarm1 and were immunoblotted with acetyl-tubulin and α-tubulin antibodies. The levels of tubulin acetylation were normalized to total α-tubulin. (B) Sarm1 knockdown reduces tubulin acetylation in mouse neuroblastoma Neuro-2A cells. 2 d after transfection, the acetylation levels of tubulin were determined by immunoblotting. (C) A JNK kinase dead mutant (JNK KR ) and an MKK4 dominant-negative mutant (MKK4 DN ) suppress the effect of Sarm1 overexpression in dendritic arborization. (D) Sarm1-expressing neurons treated with JNK inhibitor SP600125 (SP) have shorter dendrites compared with Sarm1-expressing cells. In C and D, transfection was performed at 9 DIV and cells were harvested for immunostaining at 12 DIV. (E) Co-expression of JNK KR abolishes Sdc2–induced dendrite outgrowth. At 2 DIV, cultured hippocampal neurons were transfected with the indicated constructs, then fixed for immunostaining at 5 DIV. Three independent experiments were performed. Equal numbers of transfected neurons were analyzed for each experiment. The data represent mean values ± SEM (error bars). *, P
    Figure Legend Snippet: The JNK pathway and microtubule stability are downstream of Sarm1 and Sdc2. (A) Sarm1 overexpression increases the acetylation levels of tubulin. COS-1 cells were transfected with vector control or Sarm1 and were immunoblotted with acetyl-tubulin and α-tubulin antibodies. The levels of tubulin acetylation were normalized to total α-tubulin. (B) Sarm1 knockdown reduces tubulin acetylation in mouse neuroblastoma Neuro-2A cells. 2 d after transfection, the acetylation levels of tubulin were determined by immunoblotting. (C) A JNK kinase dead mutant (JNK KR ) and an MKK4 dominant-negative mutant (MKK4 DN ) suppress the effect of Sarm1 overexpression in dendritic arborization. (D) Sarm1-expressing neurons treated with JNK inhibitor SP600125 (SP) have shorter dendrites compared with Sarm1-expressing cells. In C and D, transfection was performed at 9 DIV and cells were harvested for immunostaining at 12 DIV. (E) Co-expression of JNK KR abolishes Sdc2–induced dendrite outgrowth. At 2 DIV, cultured hippocampal neurons were transfected with the indicated constructs, then fixed for immunostaining at 5 DIV. Three independent experiments were performed. Equal numbers of transfected neurons were analyzed for each experiment. The data represent mean values ± SEM (error bars). *, P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Mutagenesis, Dominant Negative Mutation, Expressing, Immunostaining, Cell Culture, Construct

    Sarm1 is widely expressed in rodent brain and neurons. (A) Immunoblot of Sarm1 in different mouse organs. GAPDH is used as an internal control. (B) Regional distribution of Sarm1 in mouse brain. Cx, cerebral cortex; Hi, hippocampus; St, striatum; Th, thalamus; Cb, cerebellum; BS, brain stem. α-Tubulin was used as an internal control. (C) Staining patterns of Sarm1 in mouse brain. The top right shows the merged image of the MAP2/Sarm1 double stain in the CA1 region of the hippocampus. The top left and bottom panels depict the Sarm1 patterns in brain regions including layer five of the somatosensory cortex (Cx), the posterior thalamic nuclear group (Th), and the caudate putamen of the striatum (St). 2-mo-old mice were used in A–C. (D) Developmental expression profile of Sarm1. The plotted relative Sarm1 protein expression levels were obtained by normalization to the corresponding α-tubulin protein amounts. The results are the means of three independent experiments. Error bars indicate SEM. (E) Distribution of Sarm1 protein in biochemical subcellular fractions of adult mouse brain. H, total homogenate; P1, nuclei and cell debris; S1, supernatant of P1; P2, crude synaptosomal fraction; S2, supernatant of P2; LP1, lysed synaptosomal membrane; LS1, supernatant of LP1; P3, light membrane fraction; S3, soluble cytosolic fraction. PSD-95 enriched in the P2 and LP1 fractions was used as a quality control of fraction preparation. Molecular mass standards (kD) are indicated next to the gel blots. (F) Distribution of PSD-95 (red) and Sarm1 (green) in cultured hippocampal neurons at 21 DIV. Representative high-magnification images are shown on the top right. Arrowheads indicate the Sarm1 puncta overlapping with PSD-95; arrows indicate the Sarm1 puncta adjacent to PSD-95 puncta. The percentage of overlapped Sarm1 and PSD-95 is shown on the bottom right. The original images and the overlays shifted for 1 and 1.66 µm were analyzed. Error bars indicate mean values ± SEM. **, P
    Figure Legend Snippet: Sarm1 is widely expressed in rodent brain and neurons. (A) Immunoblot of Sarm1 in different mouse organs. GAPDH is used as an internal control. (B) Regional distribution of Sarm1 in mouse brain. Cx, cerebral cortex; Hi, hippocampus; St, striatum; Th, thalamus; Cb, cerebellum; BS, brain stem. α-Tubulin was used as an internal control. (C) Staining patterns of Sarm1 in mouse brain. The top right shows the merged image of the MAP2/Sarm1 double stain in the CA1 region of the hippocampus. The top left and bottom panels depict the Sarm1 patterns in brain regions including layer five of the somatosensory cortex (Cx), the posterior thalamic nuclear group (Th), and the caudate putamen of the striatum (St). 2-mo-old mice were used in A–C. (D) Developmental expression profile of Sarm1. The plotted relative Sarm1 protein expression levels were obtained by normalization to the corresponding α-tubulin protein amounts. The results are the means of three independent experiments. Error bars indicate SEM. (E) Distribution of Sarm1 protein in biochemical subcellular fractions of adult mouse brain. H, total homogenate; P1, nuclei and cell debris; S1, supernatant of P1; P2, crude synaptosomal fraction; S2, supernatant of P2; LP1, lysed synaptosomal membrane; LS1, supernatant of LP1; P3, light membrane fraction; S3, soluble cytosolic fraction. PSD-95 enriched in the P2 and LP1 fractions was used as a quality control of fraction preparation. Molecular mass standards (kD) are indicated next to the gel blots. (F) Distribution of PSD-95 (red) and Sarm1 (green) in cultured hippocampal neurons at 21 DIV. Representative high-magnification images are shown on the top right. Arrowheads indicate the Sarm1 puncta overlapping with PSD-95; arrows indicate the Sarm1 puncta adjacent to PSD-95 puncta. The percentage of overlapped Sarm1 and PSD-95 is shown on the bottom right. The original images and the overlays shifted for 1 and 1.66 µm were analyzed. Error bars indicate mean values ± SEM. **, P

    Techniques Used: Staining, Mouse Assay, Expressing, Cell Culture

    Sarm1 is critical for dendritic arborization in cultured hippocampal neurons. (A) Sarm1 knockdown in COS-1 cells via cotransfection of Sarm1i 1 , Sarm1i 2 , or pSuper control and Myc-tagged wild-type Sarm1 or specific silent mutants resistant to Sarm1i 1 and Sarm1i 2 (mt1 and mt2). Immunoblotting was performed with Myc tag and α-tubulin antibodies. Molecular mass standards (kD) are indicated next to the gel blots. (B and C) Knockdown of Sarm1 in cultured hippocampal neurons. Neurons were transfected with the indicated plasmids at 0 (B) or 5 DIV (C) and immunostained with Sarm1 and GFP antibodies at 2 (B) or 9 DIV (C). Arrows point to transfected neurons. (D) Sarm1 knockdown affects dendritic arbors. At 13 DIV, cultured hippocampal neurons were transfected using the plasmids indicated. Neuronal morphology was monitored by GFP signals at 17 DIV. (E) Sholl analysis of the effect of Sarm1 knockdown. ***, P
    Figure Legend Snippet: Sarm1 is critical for dendritic arborization in cultured hippocampal neurons. (A) Sarm1 knockdown in COS-1 cells via cotransfection of Sarm1i 1 , Sarm1i 2 , or pSuper control and Myc-tagged wild-type Sarm1 or specific silent mutants resistant to Sarm1i 1 and Sarm1i 2 (mt1 and mt2). Immunoblotting was performed with Myc tag and α-tubulin antibodies. Molecular mass standards (kD) are indicated next to the gel blots. (B and C) Knockdown of Sarm1 in cultured hippocampal neurons. Neurons were transfected with the indicated plasmids at 0 (B) or 5 DIV (C) and immunostained with Sarm1 and GFP antibodies at 2 (B) or 9 DIV (C). Arrows point to transfected neurons. (D) Sarm1 knockdown affects dendritic arbors. At 13 DIV, cultured hippocampal neurons were transfected using the plasmids indicated. Neuronal morphology was monitored by GFP signals at 17 DIV. (E) Sholl analysis of the effect of Sarm1 knockdown. ***, P

    Techniques Used: Cell Culture, Cotransfection, Transfection

    31) Product Images from "Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor"

    Article Title: Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001951

    Effect of BZL on CYP3A4 and GST expression. Cells were exposed either to vehicle (C) or BZL (200 µM) for 48 h. CYP3A4 (panel A), GSTα (panel B), GSTμ (panel C), and GSTπ (panel D) levels were estimated by western blotting. Equal amounts of total protein (15 µg) were loaded in the gels. CYP3A4 or GST O.D. was related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p
    Figure Legend Snippet: Effect of BZL on CYP3A4 and GST expression. Cells were exposed either to vehicle (C) or BZL (200 µM) for 48 h. CYP3A4 (panel A), GSTα (panel B), GSTμ (panel C), and GSTπ (panel D) levels were estimated by western blotting. Equal amounts of total protein (15 µg) were loaded in the gels. CYP3A4 or GST O.D. was related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p

    Techniques Used: Expressing, Western Blot

    Effect of PXR knock down on BZL mediated P-gp, MRP2, CYP3A4 and GSTπ induction. P-gp (panel A), MRP2 (panel B), CYP3A4 (panel C) and GSTπ (panel D) levels were estimated by western blotting in lysates from HepG2 cells transfected either with 100 nM Control siRNA-A (PXR + ) or 100 nM PXR siRNA (h) (PXR − ) and exposed to BZL (200 µM, 48 h) or vehicle (C). Equal amounts of total protein (7 µg) were loaded in the gels. O.D. from each protein was related to GAPDH O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. Typical western blot detections from each group are shown at the bottom of bar graphics. The results (% of each control) are expressed as mean ± S.D. (n = 3). *Significantly different from C, p
    Figure Legend Snippet: Effect of PXR knock down on BZL mediated P-gp, MRP2, CYP3A4 and GSTπ induction. P-gp (panel A), MRP2 (panel B), CYP3A4 (panel C) and GSTπ (panel D) levels were estimated by western blotting in lysates from HepG2 cells transfected either with 100 nM Control siRNA-A (PXR + ) or 100 nM PXR siRNA (h) (PXR − ) and exposed to BZL (200 µM, 48 h) or vehicle (C). Equal amounts of total protein (7 µg) were loaded in the gels. O.D. from each protein was related to GAPDH O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. Typical western blot detections from each group are shown at the bottom of bar graphics. The results (% of each control) are expressed as mean ± S.D. (n = 3). *Significantly different from C, p

    Techniques Used: Western Blot, Transfection

    32) Product Images from "Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia"

    Article Title: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia

    Journal: Science signaling

    doi: 10.1126/scisignal.aao5617

    Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.
    Figure Legend Snippet: Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.

    Techniques Used: Inhibition, Mutagenesis

    TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.
    Figure Legend Snippet: TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.

    Techniques Used: Mutagenesis

    A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.
    Figure Legend Snippet: A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.

    Techniques Used: Mutagenesis

    Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.
    Figure Legend Snippet: Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.

    Techniques Used: Inhibition

    Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.
    Figure Legend Snippet: Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.

    Techniques Used: Functional Assay, Transformation Assay, Inhibition

    33) Product Images from "Astroglial changes in the zona incerta in response to motor cortex stimulation in a rat model of chronic neuropathy"

    Article Title: Astroglial changes in the zona incerta in response to motor cortex stimulation in a rat model of chronic neuropathy

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-57797-y

    Astrocyte expression in the ZI. ( A ) Location of the ZI in the subthalamus. Box indicates the location of the ZI. ( B ) Astrocytes (GFAP, green) in the ZI of a control rat. ( C ) Astrocytes in the ZI of an NP rat 24 days after nerve injury, showing decreased GFAP expression compared to the control. ( D ) After 10 days of MCS sessions, GFAP expression levels in the ZI were compared. Repetitive M1 stimulation contributed to the restoration of GFAP expression in the ZI. (Scale bars = 200 µm.) ( E ) GFAP expression was compared among groups. Activity was normalized to the protein concentration. ( F,G ) Expression levels of GFAP and NeuN are shown as % changes compared to the control rats. Error bars indicate the standard errors calculated from three independent experiments. After nerve injury, GFAP protein levels were significantly reduced in the ZI. Following repetitive MCS, GFAP protein expression increased (p
    Figure Legend Snippet: Astrocyte expression in the ZI. ( A ) Location of the ZI in the subthalamus. Box indicates the location of the ZI. ( B ) Astrocytes (GFAP, green) in the ZI of a control rat. ( C ) Astrocytes in the ZI of an NP rat 24 days after nerve injury, showing decreased GFAP expression compared to the control. ( D ) After 10 days of MCS sessions, GFAP expression levels in the ZI were compared. Repetitive M1 stimulation contributed to the restoration of GFAP expression in the ZI. (Scale bars = 200 µm.) ( E ) GFAP expression was compared among groups. Activity was normalized to the protein concentration. ( F,G ) Expression levels of GFAP and NeuN are shown as % changes compared to the control rats. Error bars indicate the standard errors calculated from three independent experiments. After nerve injury, GFAP protein levels were significantly reduced in the ZI. Following repetitive MCS, GFAP protein expression increased (p

    Techniques Used: Expressing, Activity Assay, Protein Concentration

    34) Product Images from "Astroglial changes in the zona incerta in response to motor cortex stimulation in a rat model of chronic neuropathy"

    Article Title: Astroglial changes in the zona incerta in response to motor cortex stimulation in a rat model of chronic neuropathy

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-57797-y

    CD68 expression in the ZI of the rats. In order to identify protein expression by microglia, brain slices were labeled with CD68 (red), GFAP (green) antibodies, and DAPI (blue). CD68-positive signals were compared in each group. NP + MCS rats had no effect on the morphological change in CD68-positive signal. Frequent co-occurrence of astrocytes and microglial cells was observed in NP + Sham stim. rats. (Scale bars = 20 µm).
    Figure Legend Snippet: CD68 expression in the ZI of the rats. In order to identify protein expression by microglia, brain slices were labeled with CD68 (red), GFAP (green) antibodies, and DAPI (blue). CD68-positive signals were compared in each group. NP + MCS rats had no effect on the morphological change in CD68-positive signal. Frequent co-occurrence of astrocytes and microglial cells was observed in NP + Sham stim. rats. (Scale bars = 20 µm).

    Techniques Used: Expressing, Labeling

    35) Product Images from "Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells"

    Article Title: Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.110.064840

    BK induces complex formation between EGFR and α5β1 integrin. Lysates from mIMCD-3 cells treated with vehicle, 100 nM BK, or 1 ng/ml EGF were immunoprecipitated with anti-α5β1 integrin antibody as described under Materials and Methods . Immunoblotting was performed with antibodies against EGFR (A) and BK B 2 receptor (B). The blots shown are representative of four experiments. A, coimmunoprecipitation experiments show that α5β1 integrin and EGFR coimmunoprecipitate and that their association can be increased by stimulation of mIMCD-3 cells with 100 nM BK but not with EGF. Inset, representative Western blot with antibody against EGFR showing immunoreactive band at 175 kDa. Blot was stripped and re-probed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. B, BK B 2 receptor coimmunoprecipitates with α5β1 integrin. Inset, representative Western blot with antibody against BK B 2 receptor showing immunoreactive duplet at 42/40 kDa. Blot was stripped and reprobed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. IP, immunoprecipitation; IB, immunoblot.
    Figure Legend Snippet: BK induces complex formation between EGFR and α5β1 integrin. Lysates from mIMCD-3 cells treated with vehicle, 100 nM BK, or 1 ng/ml EGF were immunoprecipitated with anti-α5β1 integrin antibody as described under Materials and Methods . Immunoblotting was performed with antibodies against EGFR (A) and BK B 2 receptor (B). The blots shown are representative of four experiments. A, coimmunoprecipitation experiments show that α5β1 integrin and EGFR coimmunoprecipitate and that their association can be increased by stimulation of mIMCD-3 cells with 100 nM BK but not with EGF. Inset, representative Western blot with antibody against EGFR showing immunoreactive band at 175 kDa. Blot was stripped and re-probed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. B, BK B 2 receptor coimmunoprecipitates with α5β1 integrin. Inset, representative Western blot with antibody against BK B 2 receptor showing immunoreactive duplet at 42/40 kDa. Blot was stripped and reprobed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. IP, immunoprecipitation; IB, immunoblot.

    Techniques Used: Immunoprecipitation, Western Blot

    Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p
    Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p

    Techniques Used: Transfection, Activation Assay

    Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p
    Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p

    Techniques Used: Transfection

    36) Product Images from "Molecular basis of arrhythmic substrate in ageing murine peroxisome proliferator-activated receptor γ co-activator deficient hearts modelling mitochondrial dysfunction"

    Article Title: Molecular basis of arrhythmic substrate in ageing murine peroxisome proliferator-activated receptor γ co-activator deficient hearts modelling mitochondrial dysfunction

    Journal: Bioscience Reports

    doi: 10.1042/BSR20190403

    IF analysis of atrial Cx43 expression ( A ) Representative micrographs of Cx43 signal in stained atrial sections visualised at 40×. ( B ) Expression levels of atrial Cx43 as obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.
    Figure Legend Snippet: IF analysis of atrial Cx43 expression ( A ) Representative micrographs of Cx43 signal in stained atrial sections visualised at 40×. ( B ) Expression levels of atrial Cx43 as obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.

    Techniques Used: Expressing, Staining, Mouse Assay

    WB analysis of atrial Na V 1.5, Cx40 and Cx43 expression ( A ) Representative Western blots of Na V 1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. ( B ) Expression levels of atrial Na V 1.5 obtained by densitometric analysis. ( C ) Expression levels of atrial Cx40 obtained by densitometric analysis. ( D ) Expression levels of atrial Cx43 expression obtained by densitometric analysis. ( E ) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. In (B–D), red boxes indicate young mice and blue boxes indicate aged mice. Primary monoclonal rabbit anti-Na V 1.5 antibodies used at dilution 1:500; polyclonal goat anti-Cx40 used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used in blots staining for Cx40 at dilution 1:15000. Donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for Na V 1.5, Cx43 and GAPDH. Significant P -values obtained by post-hoc testing with Tukey’s HSD test are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-A, wild type young atrial.
    Figure Legend Snippet: WB analysis of atrial Na V 1.5, Cx40 and Cx43 expression ( A ) Representative Western blots of Na V 1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. ( B ) Expression levels of atrial Na V 1.5 obtained by densitometric analysis. ( C ) Expression levels of atrial Cx40 obtained by densitometric analysis. ( D ) Expression levels of atrial Cx43 expression obtained by densitometric analysis. ( E ) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. In (B–D), red boxes indicate young mice and blue boxes indicate aged mice. Primary monoclonal rabbit anti-Na V 1.5 antibodies used at dilution 1:500; polyclonal goat anti-Cx40 used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used in blots staining for Cx40 at dilution 1:15000. Donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for Na V 1.5, Cx43 and GAPDH. Significant P -values obtained by post-hoc testing with Tukey’s HSD test are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-A, wild type young atrial.

    Techniques Used: Western Blot, Expressing, Mouse Assay, Staining

    WB analysis of ventricular Na V 1.5 and Cx43 expression ( A ) Representative Western blots of Na V 1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. ( B ) Expression levels of ventricular Na V 1.5 obtained by densitometric analysis. ( C ) Expression levels of ventricular Cx43 obtained by densitometric analysis. In (B,C), red boxes indicate young mice and blue boxes indicate aged mice. ( D ) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. Primary monoclonal rabbit anti-Na V 1.5 antibody used at dilution 1:500; polyclonal goat anti-Cx40 antibody used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used at dilution 1:15000 in blots staining for Cx40, and donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for Na V 1.5, Cx43 and GAPDH. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-V, wild type young ventricular.
    Figure Legend Snippet: WB analysis of ventricular Na V 1.5 and Cx43 expression ( A ) Representative Western blots of Na V 1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. ( B ) Expression levels of ventricular Na V 1.5 obtained by densitometric analysis. ( C ) Expression levels of ventricular Cx43 obtained by densitometric analysis. In (B,C), red boxes indicate young mice and blue boxes indicate aged mice. ( D ) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. Primary monoclonal rabbit anti-Na V 1.5 antibody used at dilution 1:500; polyclonal goat anti-Cx40 antibody used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used at dilution 1:15000 in blots staining for Cx40, and donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for Na V 1.5, Cx43 and GAPDH. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-V, wild type young ventricular.

    Techniques Used: Western Blot, Expressing, Mouse Assay, Staining

    IF analysis of ventricular Cx43 expression ( A ) Representative micrographs of Cx43 signal in stained ventricular sections visualised at 40×. ( B ) Expression levels of ventricular Cx43 obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.
    Figure Legend Snippet: IF analysis of ventricular Cx43 expression ( A ) Representative micrographs of Cx43 signal in stained ventricular sections visualised at 40×. ( B ) Expression levels of ventricular Cx43 obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.

    Techniques Used: Expressing, Staining, Mouse Assay

    37) Product Images from "Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity"

    Article Title: Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2008-0211

    PTPRO expression augments tamoxifen sensitivity of breast cancer cell line. A, The WT or CS mutant of FLAG-tagged PTPRO was overexpressed in MCF-7 cells. The G418 selected pool was analyzed by Western blot analysis for PTPRO overexpression using anti-FLAG
    Figure Legend Snippet: PTPRO expression augments tamoxifen sensitivity of breast cancer cell line. A, The WT or CS mutant of FLAG-tagged PTPRO was overexpressed in MCF-7 cells. The G418 selected pool was analyzed by Western blot analysis for PTPRO overexpression using anti-FLAG

    Techniques Used: Expressing, Mutagenesis, Western Blot, Over Expression

    38) Product Images from "Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression"

    Article Title: Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01126-12

    2-DG but not 2-FDG induces ER stress and a UPR in lytically infected cells. (A) 293rKSHV cells were lytically induced with butyrate (3 mM) in the presence of 2-DG (1 mM) or 2-FDG (1 mM). At the indicated times postinduction, cells were harvested and immunoblotting
    Figure Legend Snippet: 2-DG but not 2-FDG induces ER stress and a UPR in lytically infected cells. (A) 293rKSHV cells were lytically induced with butyrate (3 mM) in the presence of 2-DG (1 mM) or 2-FDG (1 mM). At the indicated times postinduction, cells were harvested and immunoblotting

    Techniques Used: Infection

    39) Product Images from "Regulation of Gene Transcription by Voltage-gated L-type Calcium Channel, Cav1.3 *"

    Article Title: Regulation of Gene Transcription by Voltage-gated L-type Calcium Channel, Cav1.3 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.586883

    Subcellular localization of SK2 channels was altered in atrial myocytes isolated from homozygous Ca v 1.3 null mutant mice. The subcellular distribution of SK2 channels in atrial myocytes isolated from Ca v 1.3 −/− mice (KO) ( a ) as compared with those of the WT animals ( b ). Anti-SK2 antibodies were used for single staining. The double staining showed SK2 channels and α-actinin2 ( Actn2 ) localization patterns. The right panels in a and b show single staining at higher magnification. Scale bars are 10 μm. Atrial myocytes were obtained from three pairs of Ca v 1.3 −/− mice compared with WT littermates. All experiments were repeated independently three times and consistent data were obtained as shown.
    Figure Legend Snippet: Subcellular localization of SK2 channels was altered in atrial myocytes isolated from homozygous Ca v 1.3 null mutant mice. The subcellular distribution of SK2 channels in atrial myocytes isolated from Ca v 1.3 −/− mice (KO) ( a ) as compared with those of the WT animals ( b ). Anti-SK2 antibodies were used for single staining. The double staining showed SK2 channels and α-actinin2 ( Actn2 ) localization patterns. The right panels in a and b show single staining at higher magnification. Scale bars are 10 μm. Atrial myocytes were obtained from three pairs of Ca v 1.3 −/− mice compared with WT littermates. All experiments were repeated independently three times and consistent data were obtained as shown.

    Techniques Used: Isolation, Mutagenesis, Mouse Assay, Staining, Double Staining

    Subcellular distribution of SK2 channel proteins in atrial tissues from Ca v 1.3 −/− (KO) and WT mice. a, Western blot analyses showing similar expression levels of SK2 channel protein in whole atrial tissue lysates isolated from Ca v 1.3 −/− and WT animals. GAPDH was used as a loading control. b , summary data for SK2 protein expression levels normalized to GAPDH. c, abnormal distribution of SK2 channels was observed in Ca v 1.3 −/− mice using a discontinuous sucrose density gradient ultracentrifugation. The purity of membrane fractionation in mouse atrial tissues was tested and normalized by Na,K-ATPase as the plasma membrane marker. Each sample of atrial myocytes was isolated from 5 animals and the experiments were repeated independently three times.
    Figure Legend Snippet: Subcellular distribution of SK2 channel proteins in atrial tissues from Ca v 1.3 −/− (KO) and WT mice. a, Western blot analyses showing similar expression levels of SK2 channel protein in whole atrial tissue lysates isolated from Ca v 1.3 −/− and WT animals. GAPDH was used as a loading control. b , summary data for SK2 protein expression levels normalized to GAPDH. c, abnormal distribution of SK2 channels was observed in Ca v 1.3 −/− mice using a discontinuous sucrose density gradient ultracentrifugation. The purity of membrane fractionation in mouse atrial tissues was tested and normalized by Na,K-ATPase as the plasma membrane marker. Each sample of atrial myocytes was isolated from 5 animals and the experiments were repeated independently three times.

    Techniques Used: Mouse Assay, Western Blot, Expressing, Isolation, Fractionation, Marker

    The normal expression of MLC2 is required for proper membrane localization of SK2 channels. a, expression of mRNAs of Myl2 was significantly decreased in Ca v 1.3 −/− (KO) compared with WT mice ( A , atria). b, Western blot analysis showing a significant decrease in MLC2 protein in Ca v 1.3 −/− mice compared with WT animals ( n = 3 animals for each group were used for Western blot analyses). c, MLC2 interacted directly with the SK2 channel as assessed using Y2H assays. d , left panels show the SK2 staining pattern in atrial myocytes treated with control siRNA. Right panels : treatment of atrial myocytes with siRNA specific to MLC2 resulted in a significant decrease in MLC2 expression. Moreover, there was a significant decrease in SK2 membrane expression. Lower panels compare the SK2 staining of the regions outlined in white boxes at higher magnification. A total of three animals were used for each group. e, apamin-sensitive currents recorded from HEK293 cells transfected with human SK2 channel alone compared with SK2 channel co-expressed with hMLC2. Data from non-transfected cells are shown in the lower left panel. Right lower panel compared the current density in pA/pF of apamin-sensitive currents. A total of 6 cells were performed for each group with similar results. f, a schematic model illustrating the C terminus of Ca v 1.3 as a transcriptional regulator for MLC2 expression. MLC2 physically interacts with SK2 channels and facilitates the targeting of SK2 channels to the plasma membrane. SK2 channels are shown to interact with filamin A ( FLNA ) and α-actinin2 ( Actn2 ) cytoskeletal proteins via the N and C termini, respectively. CaMBD refers to calmodulin binding domain within the C terminus of SK2 channels.
    Figure Legend Snippet: The normal expression of MLC2 is required for proper membrane localization of SK2 channels. a, expression of mRNAs of Myl2 was significantly decreased in Ca v 1.3 −/− (KO) compared with WT mice ( A , atria). b, Western blot analysis showing a significant decrease in MLC2 protein in Ca v 1.3 −/− mice compared with WT animals ( n = 3 animals for each group were used for Western blot analyses). c, MLC2 interacted directly with the SK2 channel as assessed using Y2H assays. d , left panels show the SK2 staining pattern in atrial myocytes treated with control siRNA. Right panels : treatment of atrial myocytes with siRNA specific to MLC2 resulted in a significant decrease in MLC2 expression. Moreover, there was a significant decrease in SK2 membrane expression. Lower panels compare the SK2 staining of the regions outlined in white boxes at higher magnification. A total of three animals were used for each group. e, apamin-sensitive currents recorded from HEK293 cells transfected with human SK2 channel alone compared with SK2 channel co-expressed with hMLC2. Data from non-transfected cells are shown in the lower left panel. Right lower panel compared the current density in pA/pF of apamin-sensitive currents. A total of 6 cells were performed for each group with similar results. f, a schematic model illustrating the C terminus of Ca v 1.3 as a transcriptional regulator for MLC2 expression. MLC2 physically interacts with SK2 channels and facilitates the targeting of SK2 channels to the plasma membrane. SK2 channels are shown to interact with filamin A ( FLNA ) and α-actinin2 ( Actn2 ) cytoskeletal proteins via the N and C termini, respectively. CaMBD refers to calmodulin binding domain within the C terminus of SK2 channels.

    Techniques Used: Expressing, Mouse Assay, Western Blot, Staining, Transfection, Binding Assay

    40) Product Images from "Thrombin-dependent MMP-2 Activity Is Regulated by Heparan Sulfate *"

    Article Title: Thrombin-dependent MMP-2 Activity Is Regulated by Heparan Sulfate *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.171595

    Heparan sulfate proteoglycans are required for thrombin-mediated activation of pro-MMP-2. A , Western blot analysis using an anti-MMP-2 antibody of 20 μg/ml pro-MMP-was 2 incubated with 50 n m thrombin in HBMEC cells for the indicated times. Cells
    Figure Legend Snippet: Heparan sulfate proteoglycans are required for thrombin-mediated activation of pro-MMP-2. A , Western blot analysis using an anti-MMP-2 antibody of 20 μg/ml pro-MMP-was 2 incubated with 50 n m thrombin in HBMEC cells for the indicated times. Cells

    Techniques Used: Activation Assay, Western Blot, Incubation

    Binding of heparan sulfate to thrombin elicits a decrease in thrombin-mediated MMP-2 degradation and a concomitant increase in activation. A , shown is the effect of heparan sulfate on the activation and degradation of MMP-2 and fibrinogen cleavage by
    Figure Legend Snippet: Binding of heparan sulfate to thrombin elicits a decrease in thrombin-mediated MMP-2 degradation and a concomitant increase in activation. A , shown is the effect of heparan sulfate on the activation and degradation of MMP-2 and fibrinogen cleavage by

    Techniques Used: Binding Assay, Activation Assay

    Simultaneous binding of MMP-2 to exosite 1 and 2 is essential for its degradation by thrombin. A , Western blot ( IB ) analysis using an anti-myc antibody of 20 μg/ml pro-MMP-2 incubated with 50 n m thrombin in the presence of heparan sulfate (40
    Figure Legend Snippet: Simultaneous binding of MMP-2 to exosite 1 and 2 is essential for its degradation by thrombin. A , Western blot ( IB ) analysis using an anti-myc antibody of 20 μg/ml pro-MMP-2 incubated with 50 n m thrombin in the presence of heparan sulfate (40

    Techniques Used: Binding Assay, Western Blot, Incubation

    Expression of syndecan-1 increases thrombin-mediated activation of pro-MMP-2 in K562 cells. A , flow cytometric analysis shows cell surface expression of heparan sulfate in K562 cells stably transfected with syndecan-1 is shown. A sample lacking primary
    Figure Legend Snippet: Expression of syndecan-1 increases thrombin-mediated activation of pro-MMP-2 in K562 cells. A , flow cytometric analysis shows cell surface expression of heparan sulfate in K562 cells stably transfected with syndecan-1 is shown. A sample lacking primary

    Techniques Used: Expressing, Activation Assay, Flow Cytometry, Stable Transfection, Transfection

    Heparan sulfate increases thrombin-mediated activation of pro-MMP-2 under cell-free conditions. A , Western blotting using an anti-MMP-2 antibody of 20 μg/ml pro-MMP-2 incubated with 50 n m thrombin in the presence of heparan sulfate under cell-free
    Figure Legend Snippet: Heparan sulfate increases thrombin-mediated activation of pro-MMP-2 under cell-free conditions. A , Western blotting using an anti-MMP-2 antibody of 20 μg/ml pro-MMP-2 incubated with 50 n m thrombin in the presence of heparan sulfate under cell-free

    Techniques Used: Activation Assay, Western Blot, Incubation

    41) Product Images from "scyllo-Inositol Promotes Robust Mutant Huntingtin Protein Degradation *"

    Article Title: scyllo-Inositol Promotes Robust Mutant Huntingtin Protein Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.501635

    SI dose-dependently decreases accumulation of Htt inclusions in PC12 cells. Expression and accumulation of Htt containing expanded polyglutamine (polyQ) were induced by 5 μ m ponasterone for 24 h. Induced cultures were treated with either vehicle
    Figure Legend Snippet: SI dose-dependently decreases accumulation of Htt inclusions in PC12 cells. Expression and accumulation of Htt containing expanded polyglutamine (polyQ) were induced by 5 μ m ponasterone for 24 h. Induced cultures were treated with either vehicle

    Techniques Used: Expressing

    Autophagy is not the main mechanism involved in the clearance of polyQ-containing Htt mediated by SI. A, representative immunoblots of LC3 conversion from LC3-I to LC3-II and polyQ-Htt in untreated/uninduced cultures ( Untr ) and 5 μ m ponasterone-induced
    Figure Legend Snippet: Autophagy is not the main mechanism involved in the clearance of polyQ-containing Htt mediated by SI. A, representative immunoblots of LC3 conversion from LC3-I to LC3-II and polyQ-Htt in untreated/uninduced cultures ( Untr ) and 5 μ m ponasterone-induced

    Techniques Used: Western Blot

    SI dose-dependently promotes clearance of polyQ-Htt. PC12 cultures were induced with 5 μ m ponasterone and were treated with either vehicle, 1–100 μ m SI, or 50 μ m cystamine. A, representative immunoblots; B, quantification
    Figure Legend Snippet: SI dose-dependently promotes clearance of polyQ-Htt. PC12 cultures were induced with 5 μ m ponasterone and were treated with either vehicle, 1–100 μ m SI, or 50 μ m cystamine. A, representative immunoblots; B, quantification

    Techniques Used: Western Blot

    Intrinsic degradation pathways activated in the absence of SI. A, representative immunoblots and B, quantification of polyQ-Htt expression 0 h (T0) to 48 h (T48) after removal of ponasterone induction. The cells were first induced with ponasterone for
    Figure Legend Snippet: Intrinsic degradation pathways activated in the absence of SI. A, representative immunoblots and B, quantification of polyQ-Htt expression 0 h (T0) to 48 h (T48) after removal of ponasterone induction. The cells were first induced with ponasterone for

    Techniques Used: Western Blot, Expressing

    Involvement of lysosomal proteolysis in SI-mediated clearance of polyQ-containing Htt. A, representative immunoblots; B, quantification of cathepsin B ( CatB ) conversion from pro-cathepsin B to active cathepsin B in untreated/uninduced ( Untr ) cultures
    Figure Legend Snippet: Involvement of lysosomal proteolysis in SI-mediated clearance of polyQ-containing Htt. A, representative immunoblots; B, quantification of cathepsin B ( CatB ) conversion from pro-cathepsin B to active cathepsin B in untreated/uninduced ( Untr ) cultures

    Techniques Used: Western Blot

    42) Product Images from "Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells"

    Article Title: Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.110.064840

    Western blot analysis of mIMCD-3 lysates. Western blot analyses of mIMCD-3 lysates (40 μg of total protein) were performed with commercially available anti-integrin antibodies to demonstrate the expression of these integrins on a protein level. The bands of α2 (155 kDa), α3 (150 and 130 kDa), α5 (150 kDa), α6 (140 kDa), αV (150 kDa), β1 (130 kDa), and β6 (97 kDa) are indicated.
    Figure Legend Snippet: Western blot analysis of mIMCD-3 lysates. Western blot analyses of mIMCD-3 lysates (40 μg of total protein) were performed with commercially available anti-integrin antibodies to demonstrate the expression of these integrins on a protein level. The bands of α2 (155 kDa), α3 (150 and 130 kDa), α5 (150 kDa), α6 (140 kDa), αV (150 kDa), β1 (130 kDa), and β6 (97 kDa) are indicated.

    Techniques Used: Western Blot, Expressing

    43) Product Images from "SLC25A22 Promotes Proliferation and Metastasis of Osteosarcoma Cells via the PTEN Signaling Pathway"

    Article Title: SLC25A22 Promotes Proliferation and Metastasis of Osteosarcoma Cells via the PTEN Signaling Pathway

    Journal: Technology in Cancer Research & Treatment

    doi: 10.1177/1533033818811143

    SLC25A22 accelerates cell cycle progression and inhibits cell apoptosis of osteosarcoma cells. A, SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells were subjected to cell cycle detection by flow cytometry. The results were analyzed with Modifit software and the proportions for each period were calculated. B, SLC25A22, cyclin B1, cdc25c, and cyclin D1 protein levels were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells. C, Forty-eight hours after U2OS and Saos-2 cells were transfected with shRNA (SLC25A22 KD or control), cells were stained with FITC-Annexin V and PE-PI, and apoptotic cells were detected by flow cytometry and subsequently analyzed with Flowjo software. D, Cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Bad were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells.
    Figure Legend Snippet: SLC25A22 accelerates cell cycle progression and inhibits cell apoptosis of osteosarcoma cells. A, SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells were subjected to cell cycle detection by flow cytometry. The results were analyzed with Modifit software and the proportions for each period were calculated. B, SLC25A22, cyclin B1, cdc25c, and cyclin D1 protein levels were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells. C, Forty-eight hours after U2OS and Saos-2 cells were transfected with shRNA (SLC25A22 KD or control), cells were stained with FITC-Annexin V and PE-PI, and apoptotic cells were detected by flow cytometry and subsequently analyzed with Flowjo software. D, Cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Bad were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells.

    Techniques Used: Flow Cytometry, Cytometry, Software, Western Blot, Transfection, shRNA, Staining

    44) Product Images from "Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival"

    Article Title: Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00937-14

    Syk expression protects MDA-MB-231 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MDA-MB-231-TR (TR) cells lacking Syk or MDA-MB-231-TRS (TRS) cells either induced (+) or not induced (−) with doxycycline (Tet) to express Syk-EGFP were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (B) Comparison of relative levels of Bcl-x L mRNA to Bcl-x S mRNA. Ratios were normalized to a value of 1.0 for Syk-deficient cells at time zero. Bars represent means ± SEMs from three replicate experiments. *, P
    Figure Legend Snippet: Syk expression protects MDA-MB-231 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MDA-MB-231-TR (TR) cells lacking Syk or MDA-MB-231-TRS (TRS) cells either induced (+) or not induced (−) with doxycycline (Tet) to express Syk-EGFP were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (B) Comparison of relative levels of Bcl-x L mRNA to Bcl-x S mRNA. Ratios were normalized to a value of 1.0 for Syk-deficient cells at time zero. Bars represent means ± SEMs from three replicate experiments. *, P

    Techniques Used: Expressing, Multiple Displacement Amplification, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Nucleolin is required for Syk-dependent stabilization of Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells were untreated (control [Ctrl]) or infected with one of a set of lentiviruses encoding shRNAs for nucleolin (shNCL). Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. Results from three different populations of infected cells are shown. (B) Tet-responsive MDA-MB-231 cells and two of the three sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL2 and shNCL3) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (C) DG75 B cells were untreated (control) or infected with a lentivirus encoding shRNA directed against nucleolin. Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. (D) DG75 cells either infected (+) or not infected (−) with the lentivirus carrying the nucleolin shRNA were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (E) Tet-responsive MCF7 cells were untreated (control) or infected with a set of lentiviruses encoding shRNAs for nucleolin. Nucleolin levels were measured by Western blotting. Results from two different populations of infected cells are shown. (F) Tet-responsive MCF7 cells and the two sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL1 and shNCL2) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA.
    Figure Legend Snippet: Nucleolin is required for Syk-dependent stabilization of Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells were untreated (control [Ctrl]) or infected with one of a set of lentiviruses encoding shRNAs for nucleolin (shNCL). Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. Results from three different populations of infected cells are shown. (B) Tet-responsive MDA-MB-231 cells and two of the three sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL2 and shNCL3) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (C) DG75 B cells were untreated (control) or infected with a lentivirus encoding shRNA directed against nucleolin. Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. (D) DG75 cells either infected (+) or not infected (−) with the lentivirus carrying the nucleolin shRNA were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (E) Tet-responsive MCF7 cells were untreated (control) or infected with a set of lentiviruses encoding shRNAs for nucleolin. Nucleolin levels were measured by Western blotting. Results from two different populations of infected cells are shown. (F) Tet-responsive MCF7 cells and the two sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL1 and shNCL2) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA.

    Techniques Used: Multiple Displacement Amplification, Infection, Western Blot, shRNA, Reverse Transcription Polymerase Chain Reaction

    Syk interacts with nucleolin. (A) MDA-MB-231 cells expressing rtTA but not Syk or MDA-MB-231 cells with Tet-regulated expression of Syk-EGFP or Syk-EGFP(K396R) (Lenti-X Tet-On) pretreated with doxycycline (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting with anti-Syk antibodies to detect Syk-EGFP or Syk-EGFP(K396R) (bottom). (B) Tet-responsive MDA-MB-231 cells not induced (−) or induced with doxycycline to express Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Syk-EGFP was immunoprecipitated (IP) from cell lysates with GFP-nanotrap beads. Anti-GFP immune complexes were separated by SDS-PAGE and analyzed by Western blotting (WB) with antibodies against NCL, γ-tubulin, or GFP (to detect Syk-EGFP). Whole-cell lysates (WCL) were analyzed by Western blotting with antibodies against phosphotyrosine (pTyr) (bottom). The migration position of the 50-kDa molecular mass marker is indicated. (C) Syk-EGFP was immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP. Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL or γ-tubulin. (D) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) and, to detect Syk-GFP, antibodies against GFP (bottom). (E) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express EGFP (lane GFP), Syk-EGFP (lane Syk), Syk-EGFP(Y342F/Y346F) (lane 2F), Syk-EGFP(Y317F/Y342F/Y346F) (lane 3F), Syk-EGFP(Y317F) (lane 317), Syk-EGFP(Y342F) (lane 342), or Syk-EGFP(Y346F) (lane 346) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) or GFP (bottom).
    Figure Legend Snippet: Syk interacts with nucleolin. (A) MDA-MB-231 cells expressing rtTA but not Syk or MDA-MB-231 cells with Tet-regulated expression of Syk-EGFP or Syk-EGFP(K396R) (Lenti-X Tet-On) pretreated with doxycycline (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting with anti-Syk antibodies to detect Syk-EGFP or Syk-EGFP(K396R) (bottom). (B) Tet-responsive MDA-MB-231 cells not induced (−) or induced with doxycycline to express Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Syk-EGFP was immunoprecipitated (IP) from cell lysates with GFP-nanotrap beads. Anti-GFP immune complexes were separated by SDS-PAGE and analyzed by Western blotting (WB) with antibodies against NCL, γ-tubulin, or GFP (to detect Syk-EGFP). Whole-cell lysates (WCL) were analyzed by Western blotting with antibodies against phosphotyrosine (pTyr) (bottom). The migration position of the 50-kDa molecular mass marker is indicated. (C) Syk-EGFP was immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP. Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL or γ-tubulin. (D) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) and, to detect Syk-GFP, antibodies against GFP (bottom). (E) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express EGFP (lane GFP), Syk-EGFP (lane Syk), Syk-EGFP(Y342F/Y346F) (lane 2F), Syk-EGFP(Y317F/Y342F/Y346F) (lane 3F), Syk-EGFP(Y317F) (lane 317), Syk-EGFP(Y342F) (lane 342), or Syk-EGFP(Y346F) (lane 346) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) or GFP (bottom).

    Techniques Used: Multiple Displacement Amplification, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, SDS Page, Migration, Marker

    Syk expression protects MCF7 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against PARP (top). The cleaved form of PARP is indicated by the arrow. The expression of Syk-EGFP was visualized by Western blotting of cell lysates (bottom). (B) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were exposed to 5 mM H 2 O 2 for 30 min (pulse) and then moved to fresh medium for the indicated total incubation times or treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (C) Comparison of relative levels of Bcl-x L mRNA. Changes in the ratio of Bcl-x L mRNA to Bcl-x S mRNA were normalized to their relative levels of expression in Syk-deficient cells at time zero, which was set equal to a value of 1.0. Bars represent means ± SEMs from three replicate experiments. Significant differences between pairs were determined using an unpaired, two-tailed Student's t test. *, P
    Figure Legend Snippet: Syk expression protects MCF7 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against PARP (top). The cleaved form of PARP is indicated by the arrow. The expression of Syk-EGFP was visualized by Western blotting of cell lysates (bottom). (B) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were exposed to 5 mM H 2 O 2 for 30 min (pulse) and then moved to fresh medium for the indicated total incubation times or treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (C) Comparison of relative levels of Bcl-x L mRNA. Changes in the ratio of Bcl-x L mRNA to Bcl-x S mRNA were normalized to their relative levels of expression in Syk-deficient cells at time zero, which was set equal to a value of 1.0. Bars represent means ± SEMs from three replicate experiments. Significant differences between pairs were determined using an unpaired, two-tailed Student's t test. *, P

    Techniques Used: Expressing, Stable Transfection, SDS Page, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    Nucleolin is required for Syk-dependent protection of cells from stress-induced apoptosis. (A) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (B) DG75 cells or DG75 cells expressing the shRNA targeting either Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (C) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 1 μg/ml doxorubicin (Dox) for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (D) DG75 cells or DG75 cells expressing either the shRNA targeting Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 1 μg/ml doxorubicin for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (E) The degree of PARP cleavage was quantified from Western blots of lysates of MCF7 cells lacking Syk (no Syk), expressing Syk-EGFP (SykGFP), or expressing Syk-EGFP and shRNA for nucleolin (shNCL) and treated for 24 h with 5 mM H 2 O 2 (left) or 1 μg/ml doxorubicin (right). The data represent means ± SEMs from three replicate experiments. **, P
    Figure Legend Snippet: Nucleolin is required for Syk-dependent protection of cells from stress-induced apoptosis. (A) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (B) DG75 cells or DG75 cells expressing the shRNA targeting either Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (C) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 1 μg/ml doxorubicin (Dox) for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (D) DG75 cells or DG75 cells expressing either the shRNA targeting Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 1 μg/ml doxorubicin for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (E) The degree of PARP cleavage was quantified from Western blots of lysates of MCF7 cells lacking Syk (no Syk), expressing Syk-EGFP (SykGFP), or expressing Syk-EGFP and shRNA for nucleolin (shNCL) and treated for 24 h with 5 mM H 2 O 2 (left) or 1 μg/ml doxorubicin (right). The data represent means ± SEMs from three replicate experiments. **, P

    Techniques Used: shRNA, SDS Page, Expressing, Western Blot

    Syk phosphorylates nucleolin and promotes its binding to Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells pretreated without or with doxycycline to induce Syk-EGFP were treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (WCL; bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (B) DG75 B cells were pretreated with 50 μM piceatannol (PIC; +) or dimethyl sulfoxide carrier alone (−) and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (C) DG75 cells were pretreated with the indicated concentrations of R406 and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (D) MDA-MB-231-TR (TR) or MDA-MB-231-TRS cells induced to express Syk-EGFP (TRS) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (E) DG75 B cells (−) or DG75 cells stably expressing shRNA targeted against Syk (+) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (F) Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) was immunoprecipitated from the corresponding doxycycline-induced lines of MDA-MB-231 cells using GFP-nanotrap beads. The resulting immune complexes were incubated with buffer containing (+) or lacking (−) ATP. The immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (G) Nucleolin was immunoprecipitated from Tet-responsive MDA-MB-231 cells either uninduced (−) or induced (+) with doxycycline to express Syk-EGFP and either treated with 5 mM H 2 O 2 for 3 h or not treated. Immune complexes were examined for the presence of Bcl-x L mRNA by RT-PCR (top) and nucleolin by Western blotting (middle). The expression of Syk-EGFP was determined by Western blotting of whole-cell lysates with antibodies against Syk (bottom). (H) The relative amount of Bcl-x L mRNA associated with nucleolin, analyzed as described in the legend to panel G, was quantified. The data represent means ± SEMs from three replicate experiments. The level of mRNA bound to nucleolin in Syk-EGFP-expressing cells not treated with H 2 O 2 was set equal to a value of 1.0.
    Figure Legend Snippet: Syk phosphorylates nucleolin and promotes its binding to Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells pretreated without or with doxycycline to induce Syk-EGFP were treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (WCL; bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (B) DG75 B cells were pretreated with 50 μM piceatannol (PIC; +) or dimethyl sulfoxide carrier alone (−) and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (C) DG75 cells were pretreated with the indicated concentrations of R406 and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (D) MDA-MB-231-TR (TR) or MDA-MB-231-TRS cells induced to express Syk-EGFP (TRS) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (E) DG75 B cells (−) or DG75 cells stably expressing shRNA targeted against Syk (+) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (F) Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) was immunoprecipitated from the corresponding doxycycline-induced lines of MDA-MB-231 cells using GFP-nanotrap beads. The resulting immune complexes were incubated with buffer containing (+) or lacking (−) ATP. The immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (G) Nucleolin was immunoprecipitated from Tet-responsive MDA-MB-231 cells either uninduced (−) or induced (+) with doxycycline to express Syk-EGFP and either treated with 5 mM H 2 O 2 for 3 h or not treated. Immune complexes were examined for the presence of Bcl-x L mRNA by RT-PCR (top) and nucleolin by Western blotting (middle). The expression of Syk-EGFP was determined by Western blotting of whole-cell lysates with antibodies against Syk (bottom). (H) The relative amount of Bcl-x L mRNA associated with nucleolin, analyzed as described in the legend to panel G, was quantified. The data represent means ± SEMs from three replicate experiments. The level of mRNA bound to nucleolin in Syk-EGFP-expressing cells not treated with H 2 O 2 was set equal to a value of 1.0.

    Techniques Used: Binding Assay, Multiple Displacement Amplification, Immunoprecipitation, SDS Page, Western Blot, Stable Transfection, Expressing, shRNA, Incubation, Reverse Transcription Polymerase Chain Reaction

    45) Product Images from "Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamateric synapses"

    Article Title: Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamateric synapses

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.12436

    Dependence of the ACM from nicotine-treated astrocyte (A/Nic) effect on astrocyte α7-nAChRs, longevity of the induced increase in GluA1 puncta, and the continuing responsiveness of cultures. (a) A/Nic obtained from astrocytes treated with methyllycaconitine
    Figure Legend Snippet: Dependence of the ACM from nicotine-treated astrocyte (A/Nic) effect on astrocyte α7-nAChRs, longevity of the induced increase in GluA1 puncta, and the continuing responsiveness of cultures. (a) A/Nic obtained from astrocytes treated with methyllycaconitine

    Techniques Used:

    ACM from nicotine-treated astrocyte (A/Nic) increases the number of GluA1 and GluA2 puncta on the neuron surface without changing the number of VGluT, post-synaptic density protein 95 (PSD-95), or NR1 puncta. One-week-old hippocampal cultures were incubated
    Figure Legend Snippet: ACM from nicotine-treated astrocyte (A/Nic) increases the number of GluA1 and GluA2 puncta on the neuron surface without changing the number of VGluT, post-synaptic density protein 95 (PSD-95), or NR1 puncta. One-week-old hippocampal cultures were incubated

    Techniques Used: Incubation

    46) Product Images from "Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells"

    Article Title: Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v19.i25.3969

    The effect of BN52021 on platelet-activating factor receptor signaling molecules at the mRNA level under lipopolysaccharide-induced inflammation. The mRNA level of adenylate cyclase (AC) (A), G protein-coupled receptor kinases (GRK) (B), phospholipase A 2 (PLA 2 ) (C), phospholipase Cβ (PLCβ) (D), p38-mitogen-activated protein kinase (p38 MAPK) (E) and protein tyrosine kinase (PTK) (F) was up-regulated after lipopolysaccharide (LPS) stimulation. The up-regulation of AC, GRK, p38 MAPK, PLCβ and PLA 2 mRNA was significantly suppressed by BN52021 except for that of PTK. a P
    Figure Legend Snippet: The effect of BN52021 on platelet-activating factor receptor signaling molecules at the mRNA level under lipopolysaccharide-induced inflammation. The mRNA level of adenylate cyclase (AC) (A), G protein-coupled receptor kinases (GRK) (B), phospholipase A 2 (PLA 2 ) (C), phospholipase Cβ (PLCβ) (D), p38-mitogen-activated protein kinase (p38 MAPK) (E) and protein tyrosine kinase (PTK) (F) was up-regulated after lipopolysaccharide (LPS) stimulation. The up-regulation of AC, GRK, p38 MAPK, PLCβ and PLA 2 mRNA was significantly suppressed by BN52021 except for that of PTK. a P

    Techniques Used: Proximity Ligation Assay

    The effect of BN52021 on platelet-activating factor receptor signaling molecules at the protein level under lipopolysaccharide-induced inflammation. The protein level of p-adenylate cyclase (p-AC) (A), G protein-coupled receptor kinases (GRK) (B), p-phospholipase A 2 (p-PLA 2 ) (C), phospholipase Cβ (PLCβ) (D) and p-p38-mitogen-activated protein kinase (p-p38 MAPK) (E) was up-regulated after lipopolysaccharide (LPS) stimulation vs the blank control ( a P
    Figure Legend Snippet: The effect of BN52021 on platelet-activating factor receptor signaling molecules at the protein level under lipopolysaccharide-induced inflammation. The protein level of p-adenylate cyclase (p-AC) (A), G protein-coupled receptor kinases (GRK) (B), p-phospholipase A 2 (p-PLA 2 ) (C), phospholipase Cβ (PLCβ) (D) and p-p38-mitogen-activated protein kinase (p-p38 MAPK) (E) was up-regulated after lipopolysaccharide (LPS) stimulation vs the blank control ( a P

    Techniques Used: Proximity Ligation Assay

    The dose effect of BN52021 on lipopolysaccharide-induced inflammation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and Hoechst 33342/propidium iodide staining. MS1 cell activity at A 490 nm was significantly decreased 24 h after administration of 10 μg/mL lipopolysaccharide (LPS) vs the control group ( a P
    Figure Legend Snippet: The dose effect of BN52021 on lipopolysaccharide-induced inflammation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and Hoechst 33342/propidium iodide staining. MS1 cell activity at A 490 nm was significantly decreased 24 h after administration of 10 μg/mL lipopolysaccharide (LPS) vs the control group ( a P

    Techniques Used: Staining, Activity Assay

    47) Product Images from "Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells"

    Article Title: Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v19.i25.3969

    The optimal dose and duration of lipopolysaccharide stimulation were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The cell survival rate was determined after incubation with 0 (saline) and 0.1, 1 and 10 μg/mL lipopolysaccharide (LPS) for 3, 6, 12 and 24 h. a P
    Figure Legend Snippet: The optimal dose and duration of lipopolysaccharide stimulation were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The cell survival rate was determined after incubation with 0 (saline) and 0.1, 1 and 10 μg/mL lipopolysaccharide (LPS) for 3, 6, 12 and 24 h. a P

    Techniques Used: Incubation

    The effect of BN52021 on platelet-activating factor receptor signaling molecules at the mRNA level under lipopolysaccharide-induced inflammation. The mRNA level of adenylate cyclase (AC) (A), G protein-coupled receptor kinases (GRK) (B), phospholipase A 2 (PLA 2 ) (C), phospholipase Cβ (PLCβ) (D), p38-mitogen-activated protein kinase (p38 MAPK) (E) and protein tyrosine kinase (PTK) (F) was up-regulated after lipopolysaccharide (LPS) stimulation. The up-regulation of AC, GRK, p38 MAPK, PLCβ and PLA 2 mRNA was significantly suppressed by BN52021 except for that of PTK. a P
    Figure Legend Snippet: The effect of BN52021 on platelet-activating factor receptor signaling molecules at the mRNA level under lipopolysaccharide-induced inflammation. The mRNA level of adenylate cyclase (AC) (A), G protein-coupled receptor kinases (GRK) (B), phospholipase A 2 (PLA 2 ) (C), phospholipase Cβ (PLCβ) (D), p38-mitogen-activated protein kinase (p38 MAPK) (E) and protein tyrosine kinase (PTK) (F) was up-regulated after lipopolysaccharide (LPS) stimulation. The up-regulation of AC, GRK, p38 MAPK, PLCβ and PLA 2 mRNA was significantly suppressed by BN52021 except for that of PTK. a P

    Techniques Used: Proximity Ligation Assay

    The effect of BN52021 on platelet-activating factor receptor signaling molecules at the protein level under lipopolysaccharide-induced inflammation. The protein level of p-adenylate cyclase (p-AC) (A), G protein-coupled receptor kinases (GRK) (B), p-phospholipase A 2 (p-PLA 2 ) (C), phospholipase Cβ (PLCβ) (D) and p-p38-mitogen-activated protein kinase (p-p38 MAPK) (E) was up-regulated after lipopolysaccharide (LPS) stimulation vs the blank control ( a P
    Figure Legend Snippet: The effect of BN52021 on platelet-activating factor receptor signaling molecules at the protein level under lipopolysaccharide-induced inflammation. The protein level of p-adenylate cyclase (p-AC) (A), G protein-coupled receptor kinases (GRK) (B), p-phospholipase A 2 (p-PLA 2 ) (C), phospholipase Cβ (PLCβ) (D) and p-p38-mitogen-activated protein kinase (p-p38 MAPK) (E) was up-regulated after lipopolysaccharide (LPS) stimulation vs the blank control ( a P

    Techniques Used: Proximity Ligation Assay

    The dose effect of BN52021 on lipopolysaccharide-induced inflammation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and Hoechst 33342/propidium iodide staining. MS1 cell activity at A 490 nm was significantly decreased 24 h after administration of 10 μg/mL lipopolysaccharide (LPS) vs the control group ( a P
    Figure Legend Snippet: The dose effect of BN52021 on lipopolysaccharide-induced inflammation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and Hoechst 33342/propidium iodide staining. MS1 cell activity at A 490 nm was significantly decreased 24 h after administration of 10 μg/mL lipopolysaccharide (LPS) vs the control group ( a P

    Techniques Used: Staining, Activity Assay

    48) Product Images from "Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc"

    Article Title: Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-07-0690

    Knockdown of nebulin in primary cultures of chick skeletal myotubes results in a loss of CapZ at the Z-disc. (A) Myotubes were triple stained with antibodies to N-terminal nebulin, CapZ, and α-actinin 3 d after siRNA treatment. Treatment with nebulin-specific siRNA resulted in a dramatic decrease in nebulin staining and a reduction in the amount of CapZ at the Z-disc, with α-actinin only partially perturbed. (B) Knockdown of nebulin also resulted in phalloidin staining along the entire thin filament, not just at the Z-disc (arrowheads) and pointed ends (arrows) as observed in control cells. Myotubes were costained with an anti-C-terminal nebulin antibody. Bar, 10 μm. (C) RT-PCR consistently showed a ≥70% reduction in nebulin transcript levels in chick skeletal myotubes 1 d after treatment with nebulin-specific (KD) versus control (C) siRNA, whereas GAPDH transcript levels were similar in both samples. (D) Western blot analysis revealed a ≥90% decrease in nebulin protein levels (arrow) in chick skeletal myotubes two days after treatment with nebulin-specific (KD) versus control (C) siRNA by using an anti-N-terminal nebulin antibody.
    Figure Legend Snippet: Knockdown of nebulin in primary cultures of chick skeletal myotubes results in a loss of CapZ at the Z-disc. (A) Myotubes were triple stained with antibodies to N-terminal nebulin, CapZ, and α-actinin 3 d after siRNA treatment. Treatment with nebulin-specific siRNA resulted in a dramatic decrease in nebulin staining and a reduction in the amount of CapZ at the Z-disc, with α-actinin only partially perturbed. (B) Knockdown of nebulin also resulted in phalloidin staining along the entire thin filament, not just at the Z-disc (arrowheads) and pointed ends (arrows) as observed in control cells. Myotubes were costained with an anti-C-terminal nebulin antibody. Bar, 10 μm. (C) RT-PCR consistently showed a ≥70% reduction in nebulin transcript levels in chick skeletal myotubes 1 d after treatment with nebulin-specific (KD) versus control (C) siRNA, whereas GAPDH transcript levels were similar in both samples. (D) Western blot analysis revealed a ≥90% decrease in nebulin protein levels (arrow) in chick skeletal myotubes two days after treatment with nebulin-specific (KD) versus control (C) siRNA by using an anti-N-terminal nebulin antibody.

    Techniques Used: Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

    49) Product Images from "Foxp1 regulation of neonatal vocalizations via cortical development"

    Article Title: Foxp1 regulation of neonatal vocalizations via cortical development

    Journal: Genes & Development

    doi: 10.1101/gad.305037.117

    Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.
    Figure Legend Snippet: Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.

    Techniques Used: Western Blot, Immunoprecipitation

    50) Product Images from "Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket"

    Article Title: Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket

    Journal: eLife

    doi: 10.7554/eLife.18492

    Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004
    Figure Legend Snippet: Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004

    Techniques Used: Activation Assay, Staining, Western Blot, Mouse Assay, Expressing

    51) Product Images from "ERBB Receptor Activation Is Required for Profibrotic Responses to Transforming Growth Factor ?"

    Article Title: ERBB Receptor Activation Is Required for Profibrotic Responses to Transforming Growth Factor ?

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-0232

    Activation of the ERBB axis by TGF-β is SMAD dependent. A, AKR-2B cells stably expressing shRNA targeting Smad2 or Smad3 were treated with TGF-β (10 ng/mL) for 6 or 12 h and harvested for total RNA extraction. Untransduced AKR-2B cells (Untr) and cells transduced with nontargeting sequences (NT-Ctrl) were used as controls. Samples were subjected to RT-PCR analysis using primers specific for Areg, Ereg , or Hbegf. Rpl13a was used as an internal control. B, cells were treated as in A, and total proteins (500 μg) were subjected to immunoprecipitation (IP) using ERBB1-specific antibodies. Immunoprecipitates were analyzed by Western blotting using phosphotyrosine (pY)–specific antibodies. Membranes were stripped and reprobed with ERBB1 antibodies. Equivalent protein aliquots were subjected to Western blot analysis using antibodies specific for ERBB1, phosphorylated SMAD2 (p-SMAD2), and phosphorylated SMAD3 (p-SMAD3). SMAD2 and SMAD3 antibodies were used to determine silencing efficiency for both genes. GAPDH served as an internal control. C, murine embryonic fibroblast cells, lacking expression of Smad2 ( Smad2 −/− ) as well as the WT counterpart, were treated with TGF-β (10 ng/mL), harvested at various time points, and subjected to RT-PCR analysis using primers specific for Areg, Ereg , or Hbegf. Gapdh was used as an internal control. D, cells were treated with TGF-β as in C, and total proteins (50 μg) were analyzed by Western blotting using antibodies specific for phosphorylated SMAD2 (p-SMAD2), phosphorylated SMAD3 (p-SMAD3), or total SMAD2/3.
    Figure Legend Snippet: Activation of the ERBB axis by TGF-β is SMAD dependent. A, AKR-2B cells stably expressing shRNA targeting Smad2 or Smad3 were treated with TGF-β (10 ng/mL) for 6 or 12 h and harvested for total RNA extraction. Untransduced AKR-2B cells (Untr) and cells transduced with nontargeting sequences (NT-Ctrl) were used as controls. Samples were subjected to RT-PCR analysis using primers specific for Areg, Ereg , or Hbegf. Rpl13a was used as an internal control. B, cells were treated as in A, and total proteins (500 μg) were subjected to immunoprecipitation (IP) using ERBB1-specific antibodies. Immunoprecipitates were analyzed by Western blotting using phosphotyrosine (pY)–specific antibodies. Membranes were stripped and reprobed with ERBB1 antibodies. Equivalent protein aliquots were subjected to Western blot analysis using antibodies specific for ERBB1, phosphorylated SMAD2 (p-SMAD2), and phosphorylated SMAD3 (p-SMAD3). SMAD2 and SMAD3 antibodies were used to determine silencing efficiency for both genes. GAPDH served as an internal control. C, murine embryonic fibroblast cells, lacking expression of Smad2 ( Smad2 −/− ) as well as the WT counterpart, were treated with TGF-β (10 ng/mL), harvested at various time points, and subjected to RT-PCR analysis using primers specific for Areg, Ereg , or Hbegf. Gapdh was used as an internal control. D, cells were treated with TGF-β as in C, and total proteins (50 μg) were analyzed by Western blotting using antibodies specific for phosphorylated SMAD2 (p-SMAD2), phosphorylated SMAD3 (p-SMAD3), or total SMAD2/3.

    Techniques Used: Activation Assay, Stable Transfection, Expressing, shRNA, RNA Extraction, Transduction, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot

    52) Product Images from "Multiple Orientia tsutsugamushi Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α"

    Article Title: Multiple Orientia tsutsugamushi Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105652

    Downregulation of EF1α in various types of host cell infected with O. tsutsugamushi . (A) The results of immunoblot analysis of the total cellular protein isolated from ECV304 cells infected with O. tsutsugamushi at the indicated time points are shown. Protein levels were standardized to GAPDH, and the level of TSA56, a type-specific antigen of O. tsutsugamushi , was monitored to confirm bacterial replication during the infection periods. EF1α levels during the infection were analyzed using anti-EF1α antibody. (B) EF1α levels were analyzed in lysates prepared from different types of cell lines at 2 d after the infection with O. tsutsugamushi and compared with that of uninfected cells. GAPDH and TSA56 were monitored simultaneously as protein loading and bacterial infection controls, respectively. UI, uninfected; I, infected. (C) EF1α levels were analyzed in HeLa cells treated with MG132 (10 µM) for 4 h at 2 d after infection with O. tsutsugamushi . GAPDH was used as protein loading control. (D) EF1α mRNA levels were examined by real-time RT-PCR and normalized to β-actin mRNAs in HeLa cells infected with O. tsutsugamushi . The data are presented as mean+SD of three independent experiments.
    Figure Legend Snippet: Downregulation of EF1α in various types of host cell infected with O. tsutsugamushi . (A) The results of immunoblot analysis of the total cellular protein isolated from ECV304 cells infected with O. tsutsugamushi at the indicated time points are shown. Protein levels were standardized to GAPDH, and the level of TSA56, a type-specific antigen of O. tsutsugamushi , was monitored to confirm bacterial replication during the infection periods. EF1α levels during the infection were analyzed using anti-EF1α antibody. (B) EF1α levels were analyzed in lysates prepared from different types of cell lines at 2 d after the infection with O. tsutsugamushi and compared with that of uninfected cells. GAPDH and TSA56 were monitored simultaneously as protein loading and bacterial infection controls, respectively. UI, uninfected; I, infected. (C) EF1α levels were analyzed in HeLa cells treated with MG132 (10 µM) for 4 h at 2 d after infection with O. tsutsugamushi . GAPDH was used as protein loading control. (D) EF1α mRNA levels were examined by real-time RT-PCR and normalized to β-actin mRNAs in HeLa cells infected with O. tsutsugamushi . The data are presented as mean+SD of three independent experiments.

    Techniques Used: Infection, Isolation, Quantitative RT-PCR

    Identification of cellular proteins that interact with Ank proteins. (A) Glutathione-Sepharose beads containing GST or one of the nine Ank proteins fused with GST were mixed with ECV304 cell lysate. Cellular interacting proteins were resolved by SDS-PAGE and visualized by Coomassie brilliant blue staining. Arrows indicate Cullin1 and EF1α, which were identified by mass spectrometry. (B) Immunoblot analyses were performed using specific antibodies and the cellular protein precipitates obtained from GST pull-down assays. At the bottom of the image, GST and the recombinant Ank proteins used in the pull-down assays are visualized after Coomassie brilliant blue (CBB).
    Figure Legend Snippet: Identification of cellular proteins that interact with Ank proteins. (A) Glutathione-Sepharose beads containing GST or one of the nine Ank proteins fused with GST were mixed with ECV304 cell lysate. Cellular interacting proteins were resolved by SDS-PAGE and visualized by Coomassie brilliant blue staining. Arrows indicate Cullin1 and EF1α, which were identified by mass spectrometry. (B) Immunoblot analyses were performed using specific antibodies and the cellular protein precipitates obtained from GST pull-down assays. At the bottom of the image, GST and the recombinant Ank proteins used in the pull-down assays are visualized after Coomassie brilliant blue (CBB).

    Techniques Used: SDS Page, Staining, Mass Spectrometry, Recombinant

    Ank1U5-mediated ubiquitination and downregulation of EF1α. (A) HeLa cells transfected with Flag-Ank1U5 for 18 h were fixed, permeabilized, and stained with anti-Flag antibody together with anti-Cullin1 or anti-EF1α antibody. Merged images show the colocalization of Ank1U5 (red) with endogenous Cullin1 and EF1α (green) in the nucleus. (B) HeLa cells transfected with either vector DNA or plasmid encoding Flag-Ank1U5 were subjected to immunoblot to monitor EF1α levels 48 h after transfection. Levels of GAPDH indicate equal protein loading. (C) The effect of Ank1U5 on EF1α ubiquitination was examined by in vitro ubiquitination reaction. EF1α was immunoprecipitated from the reaction mixture in the absence or presence of the recombinant GST-Ank1U5 at the indicated amounts and subjected to immunoblotting with anti-ubiquitin antibody.
    Figure Legend Snippet: Ank1U5-mediated ubiquitination and downregulation of EF1α. (A) HeLa cells transfected with Flag-Ank1U5 for 18 h were fixed, permeabilized, and stained with anti-Flag antibody together with anti-Cullin1 or anti-EF1α antibody. Merged images show the colocalization of Ank1U5 (red) with endogenous Cullin1 and EF1α (green) in the nucleus. (B) HeLa cells transfected with either vector DNA or plasmid encoding Flag-Ank1U5 were subjected to immunoblot to monitor EF1α levels 48 h after transfection. Levels of GAPDH indicate equal protein loading. (C) The effect of Ank1U5 on EF1α ubiquitination was examined by in vitro ubiquitination reaction. EF1α was immunoprecipitated from the reaction mixture in the absence or presence of the recombinant GST-Ank1U5 at the indicated amounts and subjected to immunoblotting with anti-ubiquitin antibody.

    Techniques Used: Transfection, Staining, Plasmid Preparation, In Vitro, Immunoprecipitation, Recombinant

    53) Product Images from "Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology"

    Article Title: Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201008050

    Sarm1 is widely expressed in rodent brain and neurons. (A) Immunoblot of Sarm1 in different mouse organs. GAPDH is used as an internal control. (B) Regional distribution of Sarm1 in mouse brain. Cx, cerebral cortex; Hi, hippocampus; St, striatum; Th, thalamus; Cb, cerebellum; BS, brain stem. α-Tubulin was used as an internal control. (C) Staining patterns of Sarm1 in mouse brain. The top right shows the merged image of the MAP2/Sarm1 double stain in the CA1 region of the hippocampus. The top left and bottom panels depict the Sarm1 patterns in brain regions including layer five of the somatosensory cortex (Cx), the posterior thalamic nuclear group (Th), and the caudate putamen of the striatum (St). 2-mo-old mice were used in A–C. (D) Developmental expression profile of Sarm1. The plotted relative Sarm1 protein expression levels were obtained by normalization to the corresponding α-tubulin protein amounts. The results are the means of three independent experiments. Error bars indicate SEM. (E) Distribution of Sarm1 protein in biochemical subcellular fractions of adult mouse brain. H, total homogenate; P1, nuclei and cell debris; S1, supernatant of P1; P2, crude synaptosomal fraction; S2, supernatant of P2; LP1, lysed synaptosomal membrane; LS1, supernatant of LP1; P3, light membrane fraction; S3, soluble cytosolic fraction. PSD-95 enriched in the P2 and LP1 fractions was used as a quality control of fraction preparation. Molecular mass standards (kD) are indicated next to the gel blots. (F) Distribution of PSD-95 (red) and Sarm1 (green) in cultured hippocampal neurons at 21 DIV. Representative high-magnification images are shown on the top right. Arrowheads indicate the Sarm1 puncta overlapping with PSD-95; arrows indicate the Sarm1 puncta adjacent to PSD-95 puncta. The percentage of overlapped Sarm1 and PSD-95 is shown on the bottom right. The original images and the overlays shifted for 1 and 1.66 µm were analyzed. Error bars indicate mean values ± SEM. **, P
    Figure Legend Snippet: Sarm1 is widely expressed in rodent brain and neurons. (A) Immunoblot of Sarm1 in different mouse organs. GAPDH is used as an internal control. (B) Regional distribution of Sarm1 in mouse brain. Cx, cerebral cortex; Hi, hippocampus; St, striatum; Th, thalamus; Cb, cerebellum; BS, brain stem. α-Tubulin was used as an internal control. (C) Staining patterns of Sarm1 in mouse brain. The top right shows the merged image of the MAP2/Sarm1 double stain in the CA1 region of the hippocampus. The top left and bottom panels depict the Sarm1 patterns in brain regions including layer five of the somatosensory cortex (Cx), the posterior thalamic nuclear group (Th), and the caudate putamen of the striatum (St). 2-mo-old mice were used in A–C. (D) Developmental expression profile of Sarm1. The plotted relative Sarm1 protein expression levels were obtained by normalization to the corresponding α-tubulin protein amounts. The results are the means of three independent experiments. Error bars indicate SEM. (E) Distribution of Sarm1 protein in biochemical subcellular fractions of adult mouse brain. H, total homogenate; P1, nuclei and cell debris; S1, supernatant of P1; P2, crude synaptosomal fraction; S2, supernatant of P2; LP1, lysed synaptosomal membrane; LS1, supernatant of LP1; P3, light membrane fraction; S3, soluble cytosolic fraction. PSD-95 enriched in the P2 and LP1 fractions was used as a quality control of fraction preparation. Molecular mass standards (kD) are indicated next to the gel blots. (F) Distribution of PSD-95 (red) and Sarm1 (green) in cultured hippocampal neurons at 21 DIV. Representative high-magnification images are shown on the top right. Arrowheads indicate the Sarm1 puncta overlapping with PSD-95; arrows indicate the Sarm1 puncta adjacent to PSD-95 puncta. The percentage of overlapped Sarm1 and PSD-95 is shown on the bottom right. The original images and the overlays shifted for 1 and 1.66 µm were analyzed. Error bars indicate mean values ± SEM. **, P

    Techniques Used: Staining, Mouse Assay, Expressing, Cell Culture

    54) Product Images from "Sequential Phosphorylation of Smoothened Transduces Graded Hedgehog Signaling"

    Article Title: Sequential Phosphorylation of Smoothened Transduces Graded Hedgehog Signaling

    Journal: Science signaling

    doi: 10.1126/scisignal.2001747

    Smo forms complexes with PP1c and Wdb-PP2A. ( A to C ) Immunocomplexes formed in (A) cl-8– gfp , (B) cl-8– gsmo , or (C) cl-8– gsmo cells transfected with individual pp1c-V5 plasmids. GFP or GFP-Smo was immunoprecipitated (IP) with an antibody against GFP conjugated to agarose. GFP-Smo formed complexes with PP2A catalytic (Mts) and regulatory (Wdb) subunits as well as with PP1c subunits. GAPDH served as a negative control. ( D and E ). ( F to H ) A direct interaction between GST-Smo and PP2A or PP1c. GST-Smo bound on glutathione beads was incubated with (F) the purified PP2A A:C (Mts) dimer, (G) MBP-Wdb, or (H) recombinant PP1c. Compared with the shorter GST fusions, GST-Smo 557–1036 showed stronger binding to Mts (panel F, top). The binding between GST-Smo and the PP2A A scaffolding subunit was detectable only in a low-stringency wash condition (panel F, bottom). (G) MBP-Wdb efficiently pulled down GST-Smo 600–800 and GST-Smo 557–1036 (asterisk). (H) Only GST-Smo 557–1036, which contains the PP1-binding site, pulled down recombinant PP1c. GST served as a negative control.
    Figure Legend Snippet: Smo forms complexes with PP1c and Wdb-PP2A. ( A to C ) Immunocomplexes formed in (A) cl-8– gfp , (B) cl-8– gsmo , or (C) cl-8– gsmo cells transfected with individual pp1c-V5 plasmids. GFP or GFP-Smo was immunoprecipitated (IP) with an antibody against GFP conjugated to agarose. GFP-Smo formed complexes with PP2A catalytic (Mts) and regulatory (Wdb) subunits as well as with PP1c subunits. GAPDH served as a negative control. ( D and E ). ( F to H ) A direct interaction between GST-Smo and PP2A or PP1c. GST-Smo bound on glutathione beads was incubated with (F) the purified PP2A A:C (Mts) dimer, (G) MBP-Wdb, or (H) recombinant PP1c. Compared with the shorter GST fusions, GST-Smo 557–1036 showed stronger binding to Mts (panel F, top). The binding between GST-Smo and the PP2A A scaffolding subunit was detectable only in a low-stringency wash condition (panel F, bottom). (G) MBP-Wdb efficiently pulled down GST-Smo 600–800 and GST-Smo 557–1036 (asterisk). (H) Only GST-Smo 557–1036, which contains the PP1-binding site, pulled down recombinant PP1c. GST served as a negative control.

    Techniques Used: Transfection, Immunoprecipitation, Negative Control, Incubation, Purification, Recombinant, Binding Assay, Scaffolding

    PP1 and PP2A dephosphorylate distinct species of pSmo. ( A and B ) PP1 or PP2A alone incompletely dephosphorylated GFP-pSmo. Hh-induced cl-8– gsmo lysates were treated with increasing amounts of (A) PP1 or (B) PP2A. Western blotting analysis with an antibody against GFP (top panels) detected all forms of GFP-pSmo (box brackets), which were completely removed by CIP. The α-Smo-pS 667 antibody (middle panels) specifically detected PKA-phosphorylated GFP-Smo (GFP-PKA-pSmo). PP1 efficiently dephosphorylated GFP-PKA-pSmo, whereas PP2A treatment enriched for the α-Smo-pS 667 antibody–specific GFP-PKA-pSmo. GFP-PKA-pSmo species treated with PP2A were greatly reduced in abundance by additional PP1 activity (panel B, compare lane 6 with lanes 2 to 4). The Western blot analyzed with the α-Smo-pS 667 antibody (A, middle panel) was overexposed to reveal PKA-pSmo in lysates containing enhanced amounts of pSmo. β-Tubulin served as the loading control. (C and D) PP1 dephosphorylates PKA-phosphorylated Smo. GST-PKA-pSmo labeled with cold ATP was dephosphorylated by PP1, but not by PP2A, as indicated by the decrease in the extent of detection of GST-PKA-pSmo with α-Smo-pS 667 (C). This PP1 response was inhibited by PP1-I2, a specific PP1 inhibitor. Radiolabeled PKA–phosphorylated GST-Smo (GST-PKA- 32 pSmo) was dephosphorylated by PP1, but not by PP2A (D). λ-Phosphatase (λPpase) was used as a positive control in panels C to F. (E) GST-Smo (lane 1) was phosphorylated with PKA alone (lanes 2 and 3) or PKA and CKI (lanes 4 to 8) in the presence of cold ATP. Treating GST-CKI-pSmo with PP2A (lane 5), but not PP1 (lane 6), enriched for the α-Smo-pS 667 –reactive GST-PKA-pSmo, which was reversed by additional PP1 activity (lane 7). (F) PP2A specifically targets CKI consensus modifications. Individual PKA-CKI clusters in GST-Smo were phosphorylated by PKA in the presence of ATP followed by CKI-mediated phosphorylation in the presence of [γ- 32 P]ATP (lanes 1, 3, and 5), resulting in selective incorporation of [γ- 32 P]ATP only at CKI consensus serines within a single cluster, as schematically shown. PP2A removed CKI phosphorylation–specific 32 P-phosphates from individual PKA-CKI clusters (lanes 2, 4, and 6).
    Figure Legend Snippet: PP1 and PP2A dephosphorylate distinct species of pSmo. ( A and B ) PP1 or PP2A alone incompletely dephosphorylated GFP-pSmo. Hh-induced cl-8– gsmo lysates were treated with increasing amounts of (A) PP1 or (B) PP2A. Western blotting analysis with an antibody against GFP (top panels) detected all forms of GFP-pSmo (box brackets), which were completely removed by CIP. The α-Smo-pS 667 antibody (middle panels) specifically detected PKA-phosphorylated GFP-Smo (GFP-PKA-pSmo). PP1 efficiently dephosphorylated GFP-PKA-pSmo, whereas PP2A treatment enriched for the α-Smo-pS 667 antibody–specific GFP-PKA-pSmo. GFP-PKA-pSmo species treated with PP2A were greatly reduced in abundance by additional PP1 activity (panel B, compare lane 6 with lanes 2 to 4). The Western blot analyzed with the α-Smo-pS 667 antibody (A, middle panel) was overexposed to reveal PKA-pSmo in lysates containing enhanced amounts of pSmo. β-Tubulin served as the loading control. (C and D) PP1 dephosphorylates PKA-phosphorylated Smo. GST-PKA-pSmo labeled with cold ATP was dephosphorylated by PP1, but not by PP2A, as indicated by the decrease in the extent of detection of GST-PKA-pSmo with α-Smo-pS 667 (C). This PP1 response was inhibited by PP1-I2, a specific PP1 inhibitor. Radiolabeled PKA–phosphorylated GST-Smo (GST-PKA- 32 pSmo) was dephosphorylated by PP1, but not by PP2A (D). λ-Phosphatase (λPpase) was used as a positive control in panels C to F. (E) GST-Smo (lane 1) was phosphorylated with PKA alone (lanes 2 and 3) or PKA and CKI (lanes 4 to 8) in the presence of cold ATP. Treating GST-CKI-pSmo with PP2A (lane 5), but not PP1 (lane 6), enriched for the α-Smo-pS 667 –reactive GST-PKA-pSmo, which was reversed by additional PP1 activity (lane 7). (F) PP2A specifically targets CKI consensus modifications. Individual PKA-CKI clusters in GST-Smo were phosphorylated by PKA in the presence of ATP followed by CKI-mediated phosphorylation in the presence of [γ- 32 P]ATP (lanes 1, 3, and 5), resulting in selective incorporation of [γ- 32 P]ATP only at CKI consensus serines within a single cluster, as schematically shown. PP2A removed CKI phosphorylation–specific 32 P-phosphates from individual PKA-CKI clusters (lanes 2, 4, and 6).

    Techniques Used: Western Blot, Activity Assay, Labeling, Positive Control

    55) Product Images from "Vimentin-Mediated Steroidogenesis Induced by Phthalate Esters: Involvement of DNA Demethylation and Nuclear Factor κB"

    Article Title: Vimentin-Mediated Steroidogenesis Induced by Phthalate Esters: Involvement of DNA Demethylation and Nuclear Factor κB

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0146138

    Effects of MBP and hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were exposed to1000 nM MBP in the absence or presence of 100 U/L hCG or 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p
    Figure Legend Snippet: Effects of MBP and hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were exposed to1000 nM MBP in the absence or presence of 100 U/L hCG or 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p

    Techniques Used: Western Blot

    Effects of MBP on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were treated by 1000 nM MBP for 24 h. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p
    Figure Legend Snippet: Effects of MBP on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were treated by 1000 nM MBP for 24 h. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p

    Techniques Used: Western Blot

    Effects of hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were exposed to 100 U/L hCG or to 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. *p
    Figure Legend Snippet: Effects of hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were exposed to 100 U/L hCG or to 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. *p

    Techniques Used: Western Blot

    56) Product Images from "Regeneration-associated WNT Signaling Is Activated in Long-term Reconstituting AC133bright Acute Myeloid Leukemia Cells 1 Acute Myeloid Leukemia Cells 1 2"

    Article Title: Regeneration-associated WNT Signaling Is Activated in Long-term Reconstituting AC133bright Acute Myeloid Leukemia Cells 1 Acute Myeloid Leukemia Cells 1 2

    Journal: Neoplasia (New York, N.Y.)

    doi:

    β-Catenin activation in the subpopulation of AC133 bright ). Cell nuclei are shown
    Figure Legend Snippet: β-Catenin activation in the subpopulation of AC133 bright ). Cell nuclei are shown

    Techniques Used: Activation Assay

    57) Product Images from "14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection"

    Article Title: 14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection

    Journal: Veterinary Research

    doi: 10.1186/s13567-019-0636-0

    Identification of interactions between 14-3-3 subtypes and NSP2 by confocal microscopy (A and C) and IP (B). A Co-localization of NSP2 with 14-3-3 subtypes, confirmed by immunofluorescence microscopy. Co-localization of EGFP–NSP2 (green) with 14-3-3 β, ε, γ, and τ/ζ (red) was visualized in transfected 293T cells. B 293T cells were transfected with GFP–nsp2 and an empty vector. Proteins associated with NSP2 were pulled down using GFP-Trap and analyzed by Western blot using specific antibodies against 14-3-3β, ε, γ, and τ/ζ. C Co-localization of HP-PRRSV with 14-3-3 subtypes, confirmed by immunofluorescence microscopy. Co-localization of NSP2 of TA-12 (green) with 14-3-3 α/β, ε, γ, and τ/ζ (red) was visualized in Marc-145 cells. Co-localization was determined by the yellow signal in merged images. WCL: whole-cell lysates.
    Figure Legend Snippet: Identification of interactions between 14-3-3 subtypes and NSP2 by confocal microscopy (A and C) and IP (B). A Co-localization of NSP2 with 14-3-3 subtypes, confirmed by immunofluorescence microscopy. Co-localization of EGFP–NSP2 (green) with 14-3-3 β, ε, γ, and τ/ζ (red) was visualized in transfected 293T cells. B 293T cells were transfected with GFP–nsp2 and an empty vector. Proteins associated with NSP2 were pulled down using GFP-Trap and analyzed by Western blot using specific antibodies against 14-3-3β, ε, γ, and τ/ζ. C Co-localization of HP-PRRSV with 14-3-3 subtypes, confirmed by immunofluorescence microscopy. Co-localization of NSP2 of TA-12 (green) with 14-3-3 α/β, ε, γ, and τ/ζ (red) was visualized in Marc-145 cells. Co-localization was determined by the yellow signal in merged images. WCL: whole-cell lysates.

    Techniques Used: Confocal Microscopy, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Western Blot

    58) Product Images from "Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton"

    Article Title: Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126214

    Distinct Tpm isoforms differentially impact on the elastic modulus of the cell. Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from n = 3 independent experiments. * P
    Figure Legend Snippet: Distinct Tpm isoforms differentially impact on the elastic modulus of the cell. Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from n = 3 independent experiments. * P

    Techniques Used: Clone Assay, Generated, Stable Transfection, Isolation, SDS Page, Western Blot, Whisker Assay

    59) Product Images from "Hairy and Enhancer of Split-related with YRPW Motif (HEY)2 Regulates Bone Remodeling in Mice"

    Article Title: Hairy and Enhancer of Split-related with YRPW Motif (HEY)2 Regulates Bone Remodeling in Mice

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.489435

    HEY2 overexpression suppresses osteoblast function in vitro . A and B , osteoblast-enriched cells were harvested from the calvariae of male ( A ) or female ( B ) Col3.6-Hey2 transgenic mice ( Hey2 , black bars ) or littermate wild type controls of the same sex
    Figure Legend Snippet: HEY2 overexpression suppresses osteoblast function in vitro . A and B , osteoblast-enriched cells were harvested from the calvariae of male ( A ) or female ( B ) Col3.6-Hey2 transgenic mice ( Hey2 , black bars ) or littermate wild type controls of the same sex

    Techniques Used: Over Expression, In Vitro, Transgenic Assay, Mouse Assay

    Hey2 inactivation inhibits osteoblast function in vitro . Osteoblast-enriched cells were harvested from calvariae of male or female Oc-Cre +/− ; Hey2 Δ/Δ mice ( Hey2 null , black bars ), or littermate Hey2 loxP/loxP controls of the same
    Figure Legend Snippet: Hey2 inactivation inhibits osteoblast function in vitro . Osteoblast-enriched cells were harvested from calvariae of male or female Oc-Cre +/− ; Hey2 Δ/Δ mice ( Hey2 null , black bars ), or littermate Hey2 loxP/loxP controls of the same

    Techniques Used: In Vitro, Mouse Assay

    HEY2 overexpression in osteoblasts from male mice induces resorption by co-cultured splenocytes and IL6 expression in vitro . Osteoblast-enriched cells were harvested from calvariae of male or female Col3.6-Hey2 transgenics ( Hey2 , black bars ), or littermate
    Figure Legend Snippet: HEY2 overexpression in osteoblasts from male mice induces resorption by co-cultured splenocytes and IL6 expression in vitro . Osteoblast-enriched cells were harvested from calvariae of male or female Col3.6-Hey2 transgenics ( Hey2 , black bars ), or littermate

    Techniques Used: Over Expression, Mouse Assay, Cell Culture, Expressing, In Vitro

    60) Product Images from "Genetic Ablation of Tau Mitigates Cognitive Impairment Induced by Type 1 Diabetes"

    Article Title: Genetic Ablation of Tau Mitigates Cognitive Impairment Induced by Type 1 Diabetes

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.11.021

    Streptozotocin treatment alters the IR/PI3K/AKT pathway in Ntg and tauKO mice. A: Immunoblot analyses of pIR, IR, pPI3k(p85), PI3k, pAKT(Ser473), AKT, GSK3β (Ser9), and GSK3β of protein extracts from whole-brain homogenates of Ntg, Ntg-STZ, tauKO, and tauKO-STZ mice at 5 months of age are shown on alternating lanes. B: Quantification normalized to GAPDH and expressed as percentage of control. Pairwise comparisons: ∗ P
    Figure Legend Snippet: Streptozotocin treatment alters the IR/PI3K/AKT pathway in Ntg and tauKO mice. A: Immunoblot analyses of pIR, IR, pPI3k(p85), PI3k, pAKT(Ser473), AKT, GSK3β (Ser9), and GSK3β of protein extracts from whole-brain homogenates of Ntg, Ntg-STZ, tauKO, and tauKO-STZ mice at 5 months of age are shown on alternating lanes. B: Quantification normalized to GAPDH and expressed as percentage of control. Pairwise comparisons: ∗ P

    Techniques Used: Mouse Assay

    61) Product Images from "Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells"

    Article Title: Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.110.064840

    Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p
    Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p

    Techniques Used: Transfection, Activation Assay

    Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p
    Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p

    Techniques Used: Transfection

    62) Product Images from "Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells"

    Article Title: Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.110.064840

    BK induces complex formation between EGFR and α5β1 integrin. Lysates from mIMCD-3 cells treated with vehicle, 100 nM BK, or 1 ng/ml EGF were immunoprecipitated with anti-α5β1 integrin antibody as described under Materials and Methods . Immunoblotting was performed with antibodies against EGFR (A) and BK B 2 receptor (B). The blots shown are representative of four experiments. A, coimmunoprecipitation experiments show that α5β1 integrin and EGFR coimmunoprecipitate and that their association can be increased by stimulation of mIMCD-3 cells with 100 nM BK but not with EGF. Inset, representative Western blot with antibody against EGFR showing immunoreactive band at 175 kDa. Blot was stripped and re-probed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. B, BK B 2 receptor coimmunoprecipitates with α5β1 integrin. Inset, representative Western blot with antibody against BK B 2 receptor showing immunoreactive duplet at 42/40 kDa. Blot was stripped and reprobed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. IP, immunoprecipitation; IB, immunoblot.
    Figure Legend Snippet: BK induces complex formation between EGFR and α5β1 integrin. Lysates from mIMCD-3 cells treated with vehicle, 100 nM BK, or 1 ng/ml EGF were immunoprecipitated with anti-α5β1 integrin antibody as described under Materials and Methods . Immunoblotting was performed with antibodies against EGFR (A) and BK B 2 receptor (B). The blots shown are representative of four experiments. A, coimmunoprecipitation experiments show that α5β1 integrin and EGFR coimmunoprecipitate and that their association can be increased by stimulation of mIMCD-3 cells with 100 nM BK but not with EGF. Inset, representative Western blot with antibody against EGFR showing immunoreactive band at 175 kDa. Blot was stripped and re-probed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. B, BK B 2 receptor coimmunoprecipitates with α5β1 integrin. Inset, representative Western blot with antibody against BK B 2 receptor showing immunoreactive duplet at 42/40 kDa. Blot was stripped and reprobed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. IP, immunoprecipitation; IB, immunoblot.

    Techniques Used: Immunoprecipitation, Western Blot

    Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p
    Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p

    Techniques Used: Transfection, Activation Assay

    Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p
    Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p

    Techniques Used: Transfection

    63) Product Images from "Altered Connexin 43 and Connexin 45 Protein Expression in the Heart as a Function of Social and Environmental Stress in the Prairie Vole"

    Article Title: Altered Connexin 43 and Connexin 45 Protein Expression in the Heart as a Function of Social and Environmental Stress in the Prairie Vole

    Journal: Stress (Amsterdam, Netherlands)

    doi: 10.3109/10253890.2014.979785

    A representative Western blot of connexin43 (Cx43) and connexin45 (Cx45) expression in the left ventricle, relative to the loading control (glyceraldehyde 3-phosphate dehydrogenase, GAPDH), for a set of paired (P) and isolated (I) prairie voles in the
    Figure Legend Snippet: A representative Western blot of connexin43 (Cx43) and connexin45 (Cx45) expression in the left ventricle, relative to the loading control (glyceraldehyde 3-phosphate dehydrogenase, GAPDH), for a set of paired (P) and isolated (I) prairie voles in the

    Techniques Used: Western Blot, Expressing, Isolation

    The impact of social isolation and chronic mild stress (CMS) on connexin43 (Cx43) and connexin45 (Cx45) expression in the left ventricle. Expression levels of Cx43 (A) and Cx45 (B) were determined by Western blot and normalized to the GAPDH loading control.
    Figure Legend Snippet: The impact of social isolation and chronic mild stress (CMS) on connexin43 (Cx43) and connexin45 (Cx45) expression in the left ventricle. Expression levels of Cx43 (A) and Cx45 (B) were determined by Western blot and normalized to the GAPDH loading control.

    Techniques Used: Isolation, Expressing, Western Blot

    64) Product Images from "The Beneficial Effect of Praeruptorin C on Osteoporotic Bone in Ovariectomized Mice via Suppression of Osteoclast Formation and Bone Resorption"

    Article Title: The Beneficial Effect of Praeruptorin C on Osteoporotic Bone in Ovariectomized Mice via Suppression of Osteoclast Formation and Bone Resorption

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00627

    Pra-C suppresses RANKL-induced osteoclast-specific gene expression. BMMs were seeded in six-well plates, and then stimulated with 30 ng/mL M-CSF, 50 ng/mL RANKL, and 10 or 20 μM Pra-C. Once mature osteoclasts were observed, total RNA was extracted and the expression of osteoclast-specific mRNA [ (A) TRAP , (B) V-ATPase a3 , (C) V-ATPase d2 , (D) Cathepsin K , (E) c-fos , (F) calcitonin receptor , (G) DC-STAMP , and (H) NFATC1 ] was measured using quantitative reverse transcription polymerase chain reaction. All experiments were performed at least three times. All plotted data are the means ± SD. ∗ P
    Figure Legend Snippet: Pra-C suppresses RANKL-induced osteoclast-specific gene expression. BMMs were seeded in six-well plates, and then stimulated with 30 ng/mL M-CSF, 50 ng/mL RANKL, and 10 or 20 μM Pra-C. Once mature osteoclasts were observed, total RNA was extracted and the expression of osteoclast-specific mRNA [ (A) TRAP , (B) V-ATPase a3 , (C) V-ATPase d2 , (D) Cathepsin K , (E) c-fos , (F) calcitonin receptor , (G) DC-STAMP , and (H) NFATC1 ] was measured using quantitative reverse transcription polymerase chain reaction. All experiments were performed at least three times. All plotted data are the means ± SD. ∗ P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Pra-C prevents ovariectomized (OVX) mouse bone loss as assessed with histological and histomorphometric analyses. (A) Decalcified bone tissue was paraffin-embedded and sectioned for hematoxylin and eosin, as well as tartrate-resistant acid phosphatase (TRAP) staining. Representative microscopic images are shown at the indicated magnification. Increased bone loss and TRAP-positive osteoclasts were observed in vehicle-treated OVX mice, whereas Pra-C treatment reversed the pathology, bone loss, and the number of mature osteoclasts was obviously reduced. The black arrowheads represent TRAP-positive osteoclasts observed on the surface of trabecular bone below growth plate. (B) Bone volume to tissue volume (BV/TV) and (C) the number of TRAP-positive mature osteoclasts was counted in each sample using BioQuant software. All plotted data are the means ± SE ( n = 5). ∗ P
    Figure Legend Snippet: Pra-C prevents ovariectomized (OVX) mouse bone loss as assessed with histological and histomorphometric analyses. (A) Decalcified bone tissue was paraffin-embedded and sectioned for hematoxylin and eosin, as well as tartrate-resistant acid phosphatase (TRAP) staining. Representative microscopic images are shown at the indicated magnification. Increased bone loss and TRAP-positive osteoclasts were observed in vehicle-treated OVX mice, whereas Pra-C treatment reversed the pathology, bone loss, and the number of mature osteoclasts was obviously reduced. The black arrowheads represent TRAP-positive osteoclasts observed on the surface of trabecular bone below growth plate. (B) Bone volume to tissue volume (BV/TV) and (C) the number of TRAP-positive mature osteoclasts was counted in each sample using BioQuant software. All plotted data are the means ± SE ( n = 5). ∗ P

    Techniques Used: Staining, Mouse Assay, Software

    Praeruptorin C (Pra-C) inhibits osteoclast formation without cytotoxic and apoptotic effects. (A) Bone marrow-derived macrophages (BMMs) were treated with different concentrations of Pra-C (0, 5, 10, or 20 μM), followed by 30 ng/mL M-CSF and 50 ng/mL RANKL for 5–7 days. Then, cells were fixed with 4% paraformaldehyde and subjected to tartrate-resistant acid phosphatase (TRAP) staining. (B) Quantification of the area of TRAP+ cells. (C) Quantification of the number of TRAP+ cells. (D) BMMs were seeded in six-well plates and treated with different concentrations of Pra-C (0, 5, 10, or 20 μM) for another 48 h, and then subjected to staining with Annexin V-APC and propidium iodide. Cells were analyzed by flow cytometry. (E) Quantification of the cell apoptosis rate by flow cytometry. (F) BMMs were cultured in a 96-well plate and treated with 30 ng/mL M-CSF, and different concentrations of Pra-C, for 48 h. Cell viability was measured by CCK-8 assay. All plotted data are the means ± SD ( n = 3). ∗ P
    Figure Legend Snippet: Praeruptorin C (Pra-C) inhibits osteoclast formation without cytotoxic and apoptotic effects. (A) Bone marrow-derived macrophages (BMMs) were treated with different concentrations of Pra-C (0, 5, 10, or 20 μM), followed by 30 ng/mL M-CSF and 50 ng/mL RANKL for 5–7 days. Then, cells were fixed with 4% paraformaldehyde and subjected to tartrate-resistant acid phosphatase (TRAP) staining. (B) Quantification of the area of TRAP+ cells. (C) Quantification of the number of TRAP+ cells. (D) BMMs were seeded in six-well plates and treated with different concentrations of Pra-C (0, 5, 10, or 20 μM) for another 48 h, and then subjected to staining with Annexin V-APC and propidium iodide. Cells were analyzed by flow cytometry. (E) Quantification of the cell apoptosis rate by flow cytometry. (F) BMMs were cultured in a 96-well plate and treated with 30 ng/mL M-CSF, and different concentrations of Pra-C, for 48 h. Cell viability was measured by CCK-8 assay. All plotted data are the means ± SD ( n = 3). ∗ P

    Techniques Used: Derivative Assay, Staining, Flow Cytometry, Cytometry, Cell Culture, CCK-8 Assay

    65) Product Images from "Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II"

    Article Title: Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II

    Journal: Veterinary Research

    doi: 10.1186/s13567-018-0584-0

    Small interfering RNAs (siRNAs) against clathrin- and dynamin-mediated endocytosis reduce PSaV infection. A LLC-PK cells transfected with scrambled siRNA (Scram) or siRNAs against clathrin heavy chain (CHC), dynamin II, or caveolin-1 were harvested at 24 and 48 h post-transfection. The down-regulation of each protein by siRNA knock-down was evaluated by western blotting analysis using antibodies specific for each protein. The intensity of each target protein relative to GAPDH was determined by densitometric analysis and is indicated above each lane. The cells transfected with each siRNA and then incubated were infected with PSaV Cowden ( B ), CVB3 Nancy ( C ), or rotavirus Wa ( D ) strains. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, and normalized to the results obtained in the scrambled siRNA-transfected cells. Data for panels B – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. * P
    Figure Legend Snippet: Small interfering RNAs (siRNAs) against clathrin- and dynamin-mediated endocytosis reduce PSaV infection. A LLC-PK cells transfected with scrambled siRNA (Scram) or siRNAs against clathrin heavy chain (CHC), dynamin II, or caveolin-1 were harvested at 24 and 48 h post-transfection. The down-regulation of each protein by siRNA knock-down was evaluated by western blotting analysis using antibodies specific for each protein. The intensity of each target protein relative to GAPDH was determined by densitometric analysis and is indicated above each lane. The cells transfected with each siRNA and then incubated were infected with PSaV Cowden ( B ), CVB3 Nancy ( C ), or rotavirus Wa ( D ) strains. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, and normalized to the results obtained in the scrambled siRNA-transfected cells. Data for panels B – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. * P

    Techniques Used: Infection, Transfection, Western Blot, Incubation, Staining, Standard Deviation

    Dominant negative (DN) mutants against clathrin- and dynamin-mediated endocytosis reduce PSaV infection. The cells transfected with GFP-tagged wild type (WT) or dominant negative (DN) Eps15, caveolin-1, or dynamin II were incubated with PSaV Cowden ( A ), CVB3 Nancy ( B ), or rotavirus Wa ( C ) strains. Infected GFP-expressing cells were counted after being stained with antibodies specific for each virus. Data for panels A – C are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. * P
    Figure Legend Snippet: Dominant negative (DN) mutants against clathrin- and dynamin-mediated endocytosis reduce PSaV infection. The cells transfected with GFP-tagged wild type (WT) or dominant negative (DN) Eps15, caveolin-1, or dynamin II were incubated with PSaV Cowden ( A ), CVB3 Nancy ( B ), or rotavirus Wa ( C ) strains. Infected GFP-expressing cells were counted after being stained with antibodies specific for each virus. Data for panels A – C are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. * P

    Techniques Used: Dominant Negative Mutation, Infection, Transfection, Incubation, Expressing, Staining, Standard Deviation

    PSaV infection depends on Rab5 and Rab7. A – D LLC-PK, Caco-2, and MA104 cells were transfected with scrambled siRNA or siRNAs against Rab5 or Rab7, and then incubated with PSaV Cowden ( A ), CVB3 Nancy ( B ), or rotavirus Wa ( C ) strains, respectively. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, normalized to the results obtained in the scrambled siRNA-transfected cells. D The virus titer was determined by TCID 50 using the cell lysates harvested from the cells in the above experimental conditions. Data for panels A – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using the one-way ANOVA. * P
    Figure Legend Snippet: PSaV infection depends on Rab5 and Rab7. A – D LLC-PK, Caco-2, and MA104 cells were transfected with scrambled siRNA or siRNAs against Rab5 or Rab7, and then incubated with PSaV Cowden ( A ), CVB3 Nancy ( B ), or rotavirus Wa ( C ) strains, respectively. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, normalized to the results obtained in the scrambled siRNA-transfected cells. D The virus titer was determined by TCID 50 using the cell lysates harvested from the cells in the above experimental conditions. Data for panels A – D are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using the one-way ANOVA. * P

    Techniques Used: Infection, Transfection, Incubation, Staining, Standard Deviation

    Chemical inhibitors against clathrin-, dynamin-, and cholesterol-mediated endocytosis reduce PSaV infection. A – C Confluent monolayers of LLC-PK, Caco-2, and MA104 cells were treated with DMSO vehicle or different concentrations of chlorpromazine (CPZ), MβCD, nystatin, or dynasore prior to infection with PSaV Cowden ( A ), Coxsackievirus B3 (CVB3) Nancy ( B ), or human rotavirus Wa ( C ) strains. Soluble cholesterol was added to the medium to examine the effect of cholesterol replenishment following MβCD-mediated depletion and then cells were infected with PSaV Cowden, CVB3 Nancy, or human rotavirus Wa strains. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, and normalized to the result obtained with control DMSO-treated cells. Data for panels A – C are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. * P
    Figure Legend Snippet: Chemical inhibitors against clathrin-, dynamin-, and cholesterol-mediated endocytosis reduce PSaV infection. A – C Confluent monolayers of LLC-PK, Caco-2, and MA104 cells were treated with DMSO vehicle or different concentrations of chlorpromazine (CPZ), MβCD, nystatin, or dynasore prior to infection with PSaV Cowden ( A ), Coxsackievirus B3 (CVB3) Nancy ( B ), or human rotavirus Wa ( C ) strains. Soluble cholesterol was added to the medium to examine the effect of cholesterol replenishment following MβCD-mediated depletion and then cells were infected with PSaV Cowden, CVB3 Nancy, or human rotavirus Wa strains. Infected cells were counted after staining with antibodies specific for each virus, and nuclei were counted after staining with DAPI. For each virus, results are shown as the percentage of infected cells, and normalized to the result obtained with control DMSO-treated cells. Data for panels A – C are presented as mean ± standard deviation of the mean from three independent experiments. Differences were evaluated using one-way ANOVA. * P

    Techniques Used: Infection, Staining, Standard Deviation

    66) Product Images from "Altered expression of caveolin 2 and 3 in smooth muscle of rat urinary bladder by 17?-estradiol"

    Article Title: Altered expression of caveolin 2 and 3 in smooth muscle of rat urinary bladder by 17?-estradiol

    Journal: BMC Urology

    doi: 10.1186/1471-2490-13-44

    Immunofluorescence labeling for caveolin2 and caveolin3 in rat urinary bladder. Caveolin2 and caveolin3 expression (red) was noted throughout the cell membrane in the smooth muscle layers. Both caveolin 2 and caveolin 3 showed decreased immunoreactivity in the Ovx group but returned to the control value after estrogen treatment, as demonstrated by the Ovx?+?Est group. The horizontal scale bar at the bottom left of each figure indicates the magnification power. The right panels denote the means?±?SD of 5 experiments for each condition determined by relative densitometry. Con?=?control; Ovx?=?ovariectomy; Ovx?+?Est?=?ovariectomy plus 17β-estradiol treatment. *P?
    Figure Legend Snippet: Immunofluorescence labeling for caveolin2 and caveolin3 in rat urinary bladder. Caveolin2 and caveolin3 expression (red) was noted throughout the cell membrane in the smooth muscle layers. Both caveolin 2 and caveolin 3 showed decreased immunoreactivity in the Ovx group but returned to the control value after estrogen treatment, as demonstrated by the Ovx?+?Est group. The horizontal scale bar at the bottom left of each figure indicates the magnification power. The right panels denote the means?±?SD of 5 experiments for each condition determined by relative densitometry. Con?=?control; Ovx?=?ovariectomy; Ovx?+?Est?=?ovariectomy plus 17β-estradiol treatment. *P?

    Techniques Used: Immunofluorescence, Labeling, Expressing

    Immunoblotting of caveolin2 and caveolin3 in the rat urinary bladder. The anti-caveolin 2 and caveolin 3 antibodies recognized 18–20 kDa bands. Anti-GAPDH antibody recognized the 42 kDa band. Caveolin 2 and caveolin 3 protein expressions were significantly decreased in the Ovx group. However, this expression was restored to the level of the control after 17β-estradiol treatment in the Ovx?+?Est group. The lower panels denote the mean?±?standard deviation of 5 experiments for each condition determined by densitometry relative to GAPDH. Con?=?control; Ovx?=?ovariectomy; Ovx?+?Est?=?ovariectomy plus 17β-estradiol treatment; *P?
    Figure Legend Snippet: Immunoblotting of caveolin2 and caveolin3 in the rat urinary bladder. The anti-caveolin 2 and caveolin 3 antibodies recognized 18–20 kDa bands. Anti-GAPDH antibody recognized the 42 kDa band. Caveolin 2 and caveolin 3 protein expressions were significantly decreased in the Ovx group. However, this expression was restored to the level of the control after 17β-estradiol treatment in the Ovx?+?Est group. The lower panels denote the mean?±?standard deviation of 5 experiments for each condition determined by densitometry relative to GAPDH. Con?=?control; Ovx?=?ovariectomy; Ovx?+?Est?=?ovariectomy plus 17β-estradiol treatment; *P?

    Techniques Used: Expressing, Standard Deviation

    67) Product Images from "Effects of Phonation Time and Magnitude Dose on Vocal Fold Epithelial Genes, Barrier Integrity, and Function"

    Article Title: Effects of Phonation Time and Magnitude Dose on Vocal Fold Epithelial Genes, Barrier Integrity, and Function

    Journal: The Laryngoscope

    doi: 10.1002/lary.24827

    Gene transcript level changes in the inflammatory mediators Interleukin-1β (IL-1β) and Cyclooxygenase-2 (COX-2), the multifunctional peptide Transforming Growth Factorβ1 (TGFβ1), and the multifunctional extracellular matrix
    Figure Legend Snippet: Gene transcript level changes in the inflammatory mediators Interleukin-1β (IL-1β) and Cyclooxygenase-2 (COX-2), the multifunctional peptide Transforming Growth Factorβ1 (TGFβ1), and the multifunctional extracellular matrix

    Techniques Used:

    68) Product Images from "PHGDH as a key enzyme for serine biosynthesis in HIF2α-targeting therapy for renal cell carcinoma"

    Article Title: PHGDH as a key enzyme for serine biosynthesis in HIF2α-targeting therapy for renal cell carcinoma

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-17-1589

    PHGDH over expression in 786-o and A498 parental cells A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in 786-o and A498 cells. B, Representative images of parental and PHGDH overexpressed 786-o or A498 cells. C, Immunofluorescence analysis; PHGDH overexpressed 786-o or A498 cells showed morphological changes from a spindle to a round cell shape. The graph showed the ratio between spindle and round cells in PHGDH overexpressed 786-o or A498 cells. D, Representative image of colony formation in parental and PHGDH overexpressed 786-o or A498 cells. The graph showed the ratio of number of colonies between parental and PHGDH overexpressed cells (* P
    Figure Legend Snippet: PHGDH over expression in 786-o and A498 parental cells A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in 786-o and A498 cells. B, Representative images of parental and PHGDH overexpressed 786-o or A498 cells. C, Immunofluorescence analysis; PHGDH overexpressed 786-o or A498 cells showed morphological changes from a spindle to a round cell shape. The graph showed the ratio between spindle and round cells in PHGDH overexpressed 786-o or A498 cells. D, Representative image of colony formation in parental and PHGDH overexpressed 786-o or A498 cells. The graph showed the ratio of number of colonies between parental and PHGDH overexpressed cells (* P

    Techniques Used: Over Expression, Expressing, Immunofluorescence

    Establishment of HIF2α knock out SU-R-786-o cells A, Immunoblotting analysis showed that HIF2α was significantly depleted in sunitinib resistant 786-o cells. B, RSEM values of HIF targets genes from RNA-seq expression data between 786-o empty and HIF2α-KO-SU-R-786-o cells. C, Representative images of HIF2α-KO-SU-R-786-o cells. D, Cell proliferation assay between control and HIF2α-KO-SU-R-786-o (* P
    Figure Legend Snippet: Establishment of HIF2α knock out SU-R-786-o cells A, Immunoblotting analysis showed that HIF2α was significantly depleted in sunitinib resistant 786-o cells. B, RSEM values of HIF targets genes from RNA-seq expression data between 786-o empty and HIF2α-KO-SU-R-786-o cells. C, Representative images of HIF2α-KO-SU-R-786-o cells. D, Cell proliferation assay between control and HIF2α-KO-SU-R-786-o (* P

    Techniques Used: Knock-Out, RNA Sequencing Assay, Expressing, Proliferation Assay

    PHGDH inhibition by si-RNA and inhibitor A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in HIF2α-KO-SU-R-786-o cells. B, Cell proliferation assay by PHGDH si-RNA. C, Cell proliferation assay by PHGDH inhibitor (CBR-5884). (* P
    Figure Legend Snippet: PHGDH inhibition by si-RNA and inhibitor A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in HIF2α-KO-SU-R-786-o cells. B, Cell proliferation assay by PHGDH si-RNA. C, Cell proliferation assay by PHGDH inhibitor (CBR-5884). (* P

    Techniques Used: Inhibition, Expressing, Proliferation Assay

    69) Product Images from "Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits"

    Article Title: Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-12-74

    Neuronal markers in the cerebral cortex, striatum/mesencephalon, hippocampus, and cerebellum . (A, E) Densitometry analysis of immunoblots prepared from lysates of cerebral cortex of WT (white bars) and Ctsk -/- mice (grey bars) as indicated. Synaptotagmin levels were significantly elevated by approximately 45% in Ctsk -/- mice as compared to WT (A, Ctsk -/- n = 10; WT n = 13), while analysis of the same samples showed no significant difference in the amount of NF-M protein (E, Ctsk -/- n = 8; WT n = 6). (B, F) Densitometry analysis of immunoblots prepared from lysates of striatum/mesencephalon of WT (white bars) and Ctsk -/- mice (grey bars) as indicated. Synaptotagmin levels were elevated in Ctsk -/- mice by 95% as compared to WT mice (B, Ctsk -/- n = 10; WT n = 10), while no significant difference was observed in NF-M protein (F, Ctsk -/- n = 9; WT n = 11). (C, G) Densitometry analysis of immunoblots prepared from lysates of cerebellum of WT (white bars) and Ctsk -/- mice (grey bars) as indicated. Synaptotagmin levels were significantly down-regulated in Ctsk -/- mice by approximately 55% compared to WT animals (C, Ctsk -/- n = 10; WT n = 13), while no significant difference was observed in NF-M protein between the two genotypes (G, Ctsk -/- n = 9; WT n = 10). (D, H) Densitometry analysis of immunoblots prepared from lysates of hippocampus of WT (white bars) and Ctsk -/- mice (grey bars) as indicated. No significant difference was detected in the amount of synaptotagmin in Ctsk -/- mice compared to WT (D, Ctsk -/- n = 10; WT n = 13), while NF-M protein was elevated by approximately 50% in Ctsk -/- mice compared to WT (H, Ctsk -/- n = 11; WT n = 9). Protein levels were determined by densitometry and grey values per area were normalized to the loading control β-tubulin. Bar charts denote mean values expressed as percent of control ± standard error; levels of significance are denoted as * for p
    Figure Legend Snippet: Neuronal markers in the cerebral cortex, striatum/mesencephalon, hippocampus, and cerebellum . (A, E) Densitometry analysis of immunoblots prepared from lysates of cerebral cortex of WT (white bars) and Ctsk -/- mice (grey bars) as indicated. Synaptotagmin levels were significantly elevated by approximately 45% in Ctsk -/- mice as compared to WT (A, Ctsk -/- n = 10; WT n = 13), while analysis of the same samples showed no significant difference in the amount of NF-M protein (E, Ctsk -/- n = 8; WT n = 6). (B, F) Densitometry analysis of immunoblots prepared from lysates of striatum/mesencephalon of WT (white bars) and Ctsk -/- mice (grey bars) as indicated. Synaptotagmin levels were elevated in Ctsk -/- mice by 95% as compared to WT mice (B, Ctsk -/- n = 10; WT n = 10), while no significant difference was observed in NF-M protein (F, Ctsk -/- n = 9; WT n = 11). (C, G) Densitometry analysis of immunoblots prepared from lysates of cerebellum of WT (white bars) and Ctsk -/- mice (grey bars) as indicated. Synaptotagmin levels were significantly down-regulated in Ctsk -/- mice by approximately 55% compared to WT animals (C, Ctsk -/- n = 10; WT n = 13), while no significant difference was observed in NF-M protein between the two genotypes (G, Ctsk -/- n = 9; WT n = 10). (D, H) Densitometry analysis of immunoblots prepared from lysates of hippocampus of WT (white bars) and Ctsk -/- mice (grey bars) as indicated. No significant difference was detected in the amount of synaptotagmin in Ctsk -/- mice compared to WT (D, Ctsk -/- n = 10; WT n = 13), while NF-M protein was elevated by approximately 50% in Ctsk -/- mice compared to WT (H, Ctsk -/- n = 11; WT n = 9). Protein levels were determined by densitometry and grey values per area were normalized to the loading control β-tubulin. Bar charts denote mean values expressed as percent of control ± standard error; levels of significance are denoted as * for p

    Techniques Used: Western Blot, Mouse Assay

    70) Product Images from "Peptidylarginine deiminase type 2 is over expressed in the glaucomatous optic nerve"

    Article Title: Peptidylarginine deiminase type 2 is over expressed in the glaucomatous optic nerve

    Journal: Molecular Vision

    doi:

    Representative immunohistochemical analyses of NTG (70 F) and control (72F) donor optic nerve sections. A : Immunoreactivity for protein-bound citrulline detected by a rabbit polyclonal antibody to citrulline after monoxime modification as described in methods. B : Merged image of anti-citrulline with DAPI. C : Control donor section probed for immunoreacitivty with anti-citrulline. D : Merged image of anti-citrulline and DAPI for control donor section. E : Densitometric analysis of immunohistochemical detection of protein-bound citrulline in NTG eyes. The data was analyzed using Image J software.
    Figure Legend Snippet: Representative immunohistochemical analyses of NTG (70 F) and control (72F) donor optic nerve sections. A : Immunoreactivity for protein-bound citrulline detected by a rabbit polyclonal antibody to citrulline after monoxime modification as described in methods. B : Merged image of anti-citrulline with DAPI. C : Control donor section probed for immunoreacitivty with anti-citrulline. D : Merged image of anti-citrulline and DAPI for control donor section. E : Densitometric analysis of immunohistochemical detection of protein-bound citrulline in NTG eyes. The data was analyzed using Image J software.

    Techniques Used: Immunohistochemistry, Modification, Software

    71) Product Images from "Mitochondrial aldehyde dehydrogenase (ALDH2) protects against streptozotocin-induced diabetic cardiomyopathy: role of GSK3? and mitochondrial function"

    Article Title: Mitochondrial aldehyde dehydrogenase (ALDH2) protects against streptozotocin-induced diabetic cardiomyopathy: role of GSK3? and mitochondrial function

    Journal: BMC Medicine

    doi: 10.1186/1741-7015-10-40

    Western blot analysis of the mitochondrial proteins UCP-2 and PGC1α as well as the Ca 2+ regulatory proteins SERCA2a, Na + -Ca 2+ exchanger and phospholamban in myocardium from FVB and ALDH2 mice treated with or without streptozotocin . (A) Representative gel blots of UCP-2, PGC1α, SERCA2a, Na + -Ca 2+ exchanger, phospholamban and GAPDH (loading control) using specific antibodies; (B) UCP-2; (C) PGC1α; (D) SERCA2a; (E) Na + -Ca 2+ exchanger; (F) phospholamban. All proteins were normalized to the loading control GAPDH. Mean ± SEM, n = 5 to 6 mice per group. * P
    Figure Legend Snippet: Western blot analysis of the mitochondrial proteins UCP-2 and PGC1α as well as the Ca 2+ regulatory proteins SERCA2a, Na + -Ca 2+ exchanger and phospholamban in myocardium from FVB and ALDH2 mice treated with or without streptozotocin . (A) Representative gel blots of UCP-2, PGC1α, SERCA2a, Na + -Ca 2+ exchanger, phospholamban and GAPDH (loading control) using specific antibodies; (B) UCP-2; (C) PGC1α; (D) SERCA2a; (E) Na + -Ca 2+ exchanger; (F) phospholamban. All proteins were normalized to the loading control GAPDH. Mean ± SEM, n = 5 to 6 mice per group. * P

    Techniques Used: Western Blot, Mouse Assay

    72) Product Images from "KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿"

    Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.05036-11

    Knockdown of KPNA6 inhibits nuclear import of Keap1. (A) NIH 3T3 cells were cotransfected with either scrambled control siRNA or KPNA6 siRNA, along with an expression vector for Keap1. At 48 h after transfection, cells were treated with 5 nM LMB for the
    Figure Legend Snippet: Knockdown of KPNA6 inhibits nuclear import of Keap1. (A) NIH 3T3 cells were cotransfected with either scrambled control siRNA or KPNA6 siRNA, along with an expression vector for Keap1. At 48 h after transfection, cells were treated with 5 nM LMB for the

    Techniques Used: Expressing, Plasmid Preparation, Transfection

    Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T
    Figure Legend Snippet: Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T

    Techniques Used: Over Expression, Expressing

    KPNA6 interacts with the Kelch domain of Keap1. (A) KPNA6 binds Keap1 in vitro . Purified His-tagged Keap1 proteins were incubated with the indicated in vitro -translated (IVT) 35 S-labeled proteins of the importin family, followed by pulldown with Ni-NTA
    Figure Legend Snippet: KPNA6 interacts with the Kelch domain of Keap1. (A) KPNA6 binds Keap1 in vitro . Purified His-tagged Keap1 proteins were incubated with the indicated in vitro -translated (IVT) 35 S-labeled proteins of the importin family, followed by pulldown with Ni-NTA

    Techniques Used: In Vitro, Purification, Incubation, Labeling

    Overexpression of KPNA6 facilitates nuclear import of Keap1. (A) NIH 3T3 cells were transfected with an expression vector for Keap1 with or without a vector for Myc-tagged KPNA6. Cells were treated with 5 nM LMB for the indicated times. After fixation
    Figure Legend Snippet: Overexpression of KPNA6 facilitates nuclear import of Keap1. (A) NIH 3T3 cells were transfected with an expression vector for Keap1 with or without a vector for Myc-tagged KPNA6. Cells were treated with 5 nM LMB for the indicated times. After fixation

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation

    KPNA6 modulates the antioxidant response by promoting ubiquitination and degradation of Nrf2. (A) KPNA6 is required for efficient repression of nuclear Nrf2 protein levels during the postinduction phase of the antioxidant response. MDA-MB-231 cells were
    Figure Legend Snippet: KPNA6 modulates the antioxidant response by promoting ubiquitination and degradation of Nrf2. (A) KPNA6 is required for efficient repression of nuclear Nrf2 protein levels during the postinduction phase of the antioxidant response. MDA-MB-231 cells were

    Techniques Used: Multiple Displacement Amplification

    73) Product Images from "Atg9A Protein, an Autophagy-related Membrane Protein, Is Localized in the Neurons of Mouse Brains"

    Article Title: Atg9A Protein, an Autophagy-related Membrane Protein, Is Localized in the Neurons of Mouse Brains

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/jhc.2010.955690

    Double immunostaining for Atg9Ap and vesicular GABA transporter (VGAT) in the Purkinje cell layer ( A ), GLAST ( B ), and glia fibrillary acidic protein (GFAP) ( C ) in the Purkinje cell layer, neurofilament-L (NF-L) in the white matter ( D ), and synaptophysin in the dentate nucleus ( E ) of the cerebellum. Immunostaining for Atg9Ap (green) is intensely detected in the initial segment of the Purkinje cell (arrowheads) ( A–C ), whereas it is weak but distinct in a dendrite (arrow) ( C ). Immunoreactivity for VGAT (red) is densely detected in axons and axon terminals of basket cells around Atg9Ap-positive initial axon terminals of Purkinje cells to form a pinceau, in which the immunosignal for Atg9Ap is colocalized ( A ). Moreover, the immunoreactivity for Atg9Ap is not colocalized in GLAST- or GFAP-positive astroglial cells (red) ( B,C ). In the white matter, Atg9Ap-immunopositive staining is longitudinally running in axons that are immunopositive for NF-L (red) ( D ). In the dentate nucleus, Atg9Ap-immunopositive large neuronal cell bodies are surrounded by synaptophysin-positive axon terminals (red), in which punctate signals for Atg9Ap overlap or are closely associated with synaptophysin (red) ( D ). Bars: A–C,E = 5 μm; D = 20 μm; inset in E = 2 μm.
    Figure Legend Snippet: Double immunostaining for Atg9Ap and vesicular GABA transporter (VGAT) in the Purkinje cell layer ( A ), GLAST ( B ), and glia fibrillary acidic protein (GFAP) ( C ) in the Purkinje cell layer, neurofilament-L (NF-L) in the white matter ( D ), and synaptophysin in the dentate nucleus ( E ) of the cerebellum. Immunostaining for Atg9Ap (green) is intensely detected in the initial segment of the Purkinje cell (arrowheads) ( A–C ), whereas it is weak but distinct in a dendrite (arrow) ( C ). Immunoreactivity for VGAT (red) is densely detected in axons and axon terminals of basket cells around Atg9Ap-positive initial axon terminals of Purkinje cells to form a pinceau, in which the immunosignal for Atg9Ap is colocalized ( A ). Moreover, the immunoreactivity for Atg9Ap is not colocalized in GLAST- or GFAP-positive astroglial cells (red) ( B,C ). In the white matter, Atg9Ap-immunopositive staining is longitudinally running in axons that are immunopositive for NF-L (red) ( D ). In the dentate nucleus, Atg9Ap-immunopositive large neuronal cell bodies are surrounded by synaptophysin-positive axon terminals (red), in which punctate signals for Atg9Ap overlap or are closely associated with synaptophysin (red) ( D ). Bars: A–C,E = 5 μm; D = 20 μm; inset in E = 2 μm.

    Techniques Used: Double Immunostaining, Immunostaining, Staining

    74) Product Images from "Role of Cathepsin D in U18666A-induced Neuronal Cell Death"

    Article Title: Role of Cathepsin D in U18666A-induced Neuronal Cell Death

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.412460

    Exogenous cathepsin D increased autophagy markers in cultured neurons. A–E , immunoblots ( A ) and quantifications ( B–E ) depicting increased levels of LC3-II, p62, Atg5, and Beclin-1 following treatment with cathepsin D and their reductions with 3-MA treatment in hippocampal cultured neurons. F , MTT assay showing that 3-MA treatment protected cultured hippocampal neurons against cathepsin D-induced toxicity. All results, which are presented as means ± S.E. ( error bars ), were obtained from three separate experiments. Cat D , cathepsin D; Ctrl , control. *, p
    Figure Legend Snippet: Exogenous cathepsin D increased autophagy markers in cultured neurons. A–E , immunoblots ( A ) and quantifications ( B–E ) depicting increased levels of LC3-II, p62, Atg5, and Beclin-1 following treatment with cathepsin D and their reductions with 3-MA treatment in hippocampal cultured neurons. F , MTT assay showing that 3-MA treatment protected cultured hippocampal neurons against cathepsin D-induced toxicity. All results, which are presented as means ± S.E. ( error bars ), were obtained from three separate experiments. Cat D , cathepsin D; Ctrl , control. *, p

    Techniques Used: Cell Culture, Western Blot, MTT Assay

    75) Product Images from "HDAC Inhibition Modulates Cardiac PPARs and Fatty Acid Metabolism in Diabetic Cardiomyopathy"

    Article Title: HDAC Inhibition Modulates Cardiac PPARs and Fatty Acid Metabolism in Diabetic Cardiomyopathy

    Journal: PPAR Research

    doi: 10.1155/2016/5938740

    Cardiac fatty acid metabolic proteins in control, diabetes mellitus (DM), and MPT0E014-treated DM (DM + MPT0E014) rats. Representative immunoblots and average data of (a) ratio of phosphorylated 5′ adenosine monophosphate-activated protein kinase 2 α (pAMPK2 α ) to total AMPK2 α , (b) peroxisome proliferator-activated receptor- (PPAR-) γ coactivator- (PGC-) 1 α , (c) phosphorylated acetyl coenzyme A carboxylase (pACC), (d) cluster of differentiation 36 (CD36), (e) diacylglycerol acyltransferase 1 (DGAT1), and (f) DGAT2 from control ( n = 4), DM ( n = 4), and DM + MPT0E014 ( n = 4) rats. Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.
    Figure Legend Snippet: Cardiac fatty acid metabolic proteins in control, diabetes mellitus (DM), and MPT0E014-treated DM (DM + MPT0E014) rats. Representative immunoblots and average data of (a) ratio of phosphorylated 5′ adenosine monophosphate-activated protein kinase 2 α (pAMPK2 α ) to total AMPK2 α , (b) peroxisome proliferator-activated receptor- (PPAR-) γ coactivator- (PGC-) 1 α , (c) phosphorylated acetyl coenzyme A carboxylase (pACC), (d) cluster of differentiation 36 (CD36), (e) diacylglycerol acyltransferase 1 (DGAT1), and (f) DGAT2 from control ( n = 4), DM ( n = 4), and DM + MPT0E014 ( n = 4) rats. Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

    Techniques Used: Western Blot, Pyrolysis Gas Chromatography

    Cardiac peroxisome proliferator-activated receptor (PPAR) proteins in control, diabetes mellitus (DM), and MPT0E014-treated DM (DM + MPT0E014) rats. Cardiac PPAR- α and PPAR- δ protein expressions significantly decreased in DM ( n = 4) compared to control ( n = 4) and DM + MPT0E014 ( n = 4) rats. Cardiac PPAR- γ protein expressions were enhanced in DM ( n = 4) compared to control ( n = 4) and DM + MPT0E014 ( n = 4) rats. Representative immunoblots and average data of (a) PPAR- α , (b) PPAR- γ , and (c) PPAR- δ in different groups. Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.
    Figure Legend Snippet: Cardiac peroxisome proliferator-activated receptor (PPAR) proteins in control, diabetes mellitus (DM), and MPT0E014-treated DM (DM + MPT0E014) rats. Cardiac PPAR- α and PPAR- δ protein expressions significantly decreased in DM ( n = 4) compared to control ( n = 4) and DM + MPT0E014 ( n = 4) rats. Cardiac PPAR- γ protein expressions were enhanced in DM ( n = 4) compared to control ( n = 4) and DM + MPT0E014 ( n = 4) rats. Representative immunoblots and average data of (a) PPAR- α , (b) PPAR- γ , and (c) PPAR- δ in different groups. Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

    Techniques Used: Western Blot

    Cardiac inflammatory proteins and ratio of phosphorylated Akt to total Akt in control, diabetes mellitus (DM), and MPT0E014-treated DM (DM + MPT0E014) rats. Cardiac tumor necrosis factor- (TNF-) α and interleukin- (IL-) 6 protein levels were significantly higher in DM rats ( n = 4) compared to control ( n = 4) and DM + MPT0E014 ( n = 4) rats. Representative immunoblots and average data of (a) TNF- α , (b) IL-6, and (c) ratio of pAkt to total Akt in different groups. Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.
    Figure Legend Snippet: Cardiac inflammatory proteins and ratio of phosphorylated Akt to total Akt in control, diabetes mellitus (DM), and MPT0E014-treated DM (DM + MPT0E014) rats. Cardiac tumor necrosis factor- (TNF-) α and interleukin- (IL-) 6 protein levels were significantly higher in DM rats ( n = 4) compared to control ( n = 4) and DM + MPT0E014 ( n = 4) rats. Representative immunoblots and average data of (a) TNF- α , (b) IL-6, and (c) ratio of pAkt to total Akt in different groups. Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

    Techniques Used: Western Blot

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    Centrifugation:

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *
    Article Snippet: Fractions of R2T and R6T were precipitated by 10% TCA for 30 min on ice, pelleted by centrifugation, and washed twice with acetone. .. TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling).

    Filtration:

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *
    Article Snippet: TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling). .. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.

    Synthesized:

    Article Title: Activation of p38/JNK Pathway Is Responsible for Embelin Induced Apoptosis in Lung Cancer Cells: Transitional Role of Reactive Oxygen Species
    Article Snippet: U0126 and FeTMPyP were purchased from Calbiochem. .. SMAC-N7-Ant peptide (AVPIAQK-P-RQIKIWFQNRRMKWKK) was synthesized by GenPro Biotech, Noida, India.

    Electrophoresis:

    Article Title: Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice
    Article Snippet: Equal amounts of protein (30 μ g) were then loaded onto 10% sodium dodecyl sulphate polyacrylamide electrophoresis gel and transferred onto polyvinylidene difluoride membranes at 125 mA for 90 min. .. Finally, for β -actin, the membranes were incubated with anti-β -actin polyclonal antibody (1 : 10000 dilution), obtained from Sigma-Aldrich.

    Incubation:

    Article Title: Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice
    Article Snippet: .. Finally, for β -actin, the membranes were incubated with anti-β -actin polyclonal antibody (1 : 10000 dilution), obtained from Sigma-Aldrich. .. The blots were washed and incubated with peroxidase-conjugate anti-rabbit, anti-mouse, or anti-goat immunoglobulin G (1 : 12000 dilution; Cayman).

    Article Title: Deregulation of Mitochondrial ATPsyn-? in Acute Myeloid Leukemia Cells and with Increased Drug Resistance
    Article Snippet: .. After blocked with 5%(ATPsyn-β and β-actin) or 2% fat-free milk(MRP1), the membranes were incubated with antibody against human mitochondrial ATPsyn-β (1∶800 dilutions; Abcam, UK) or MRP1 (1∶400 dilutions; Abcam, UK) or β-actin (1∶5 000 dilutions; Sigma, USA) at 4°C overnight. .. The bound antibodies were detected using horseradish peroxidase (HRP) - conjugated IgG and visualized with enhanced chemiluminescence (ECL) detection reagents (Thermo scientific, USA).

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *
    Article Snippet: .. TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling). .. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.

    Expressing:

    Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells
    Article Snippet: .. Immunoblotting To analyze protein expression, BeWo cells and primary vCTB were rinsed with PBS and lysed with ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris-HCl pH 7.4, 1% NP-40, 0,25% Na-deoxycholate, 150 mmol/l NaCl and 1 mmol/l EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich). .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Waltham, MA).

    BIA-KA:

    Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells
    Article Snippet: Immunoblotting To analyze protein expression, BeWo cells and primary vCTB were rinsed with PBS and lysed with ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris-HCl pH 7.4, 1% NP-40, 0,25% Na-deoxycholate, 150 mmol/l NaCl and 1 mmol/l EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich). .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Waltham, MA).

    Modification:

    Article Title: Activation of p38/JNK Pathway Is Responsible for Embelin Induced Apoptosis in Lung Cancer Cells: Transitional Role of Reactive Oxygen Species
    Article Snippet: Minimal essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS), penicillin, streptomycin, sulphorhodamine B (SRB), Ac-DEVD-7-AFC, Ac-LEHD-7-AFC, PD169316, SP600125, N-acetyl-L-cysteine (NAC), radioimmune precipitation assay buffer (RIPA) and protease inhibitor cocktail were purchased from Sigma-Aldrich, Germany. .. U0126 and FeTMPyP were purchased from Calbiochem.

    Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells
    Article Snippet: .. Immunoblotting To analyze protein expression, BeWo cells and primary vCTB were rinsed with PBS and lysed with ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris-HCl pH 7.4, 1% NP-40, 0,25% Na-deoxycholate, 150 mmol/l NaCl and 1 mmol/l EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich). .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Waltham, MA).

    Western Blot:

    Article Title: Fibroblast growth factor 2 inhibits the expression of stromal cell-derived factor 1? in periodontal ligament cells derived from human permanent teeth in vitro
    Article Snippet: Anti-SDF-1α polyclonal antibody for the western blot analysis was obtained from Abcam (ab9797, Cambridge, UK). .. SU5402 (10 μM), SP600125 (10 μM), U0126 (10 μM), SB203580 (10 μM) and LY294002 (10 μM) were purchased from EMD Chemicals, Inc. (Calbiochem; Gibbstown, NJ, USA).

    Article Title: Increased Cystatin F Levels Correlate with Decreased Cytotoxicity of Cytotoxic T Cells
    Article Snippet: Antibodies Rabbit anti-cystatin F antibody from Davids Biotechnologie GmbH (Regensburg, Germany) was used in all experiments except in western blots, where rabbit anti-cystatin F antibody from Sigma-Aldrich (St. Louis, MO, USA) was used. .. Other antibodies against human cathepsins were developed: 1D10 mouse anti-cathepsin H antibody , sheep anti-cathepsin H and sheep anti-cathepsin L. Rabbit and mouse anti-β-actin antibodies were from Sigma-Aldrich, rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology and mouse anti-GAPDH from Proteintech (Rosemont, IL, USA).

    Article Title: Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice
    Article Snippet: Paragraph title: 2.8. Western Blot Analysis for Cyclooxygenase-2, NF-κ B p65, and Phosphorylated Signal Transducer and Activator of Transcription (pSTAT)3 ... Finally, for β -actin, the membranes were incubated with anti-β -actin polyclonal antibody (1 : 10000 dilution), obtained from Sigma-Aldrich.

    Article Title: Deregulation of Mitochondrial ATPsyn-? in Acute Myeloid Leukemia Cells and with Increased Drug Resistance
    Article Snippet: Paragraph title: Western blot analysis for mitochondrial ATPsyn-β protein ... After blocked with 5%(ATPsyn-β and β-actin) or 2% fat-free milk(MRP1), the membranes were incubated with antibody against human mitochondrial ATPsyn-β (1∶800 dilutions; Abcam, UK) or MRP1 (1∶400 dilutions; Abcam, UK) or β-actin (1∶5 000 dilutions; Sigma, USA) at 4°C overnight.

    High Performance Liquid Chromatography:

    Article Title: 3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: β-ketoamphetamine modulation of neurotoxicity by the dopamine transporter
    Article Snippet: .. Methylone hydrochloride, MDPV hydrochloride, (±) mephedrone hydrochloride, and (±) MDMA hydrochloride were obtained from the NIDA Research Resources Drug Supply Program. (+) methamphetamine hydrochloride, D-amphetamine sulfate, MPTP hydrochloride, DA, polyclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and all buffers and HPLC reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. Bicinchoninic acid protein assay kits were obtained from Pierce (Rockford, IL, USA).

    Transfection:

    Article Title: Mirk/Dyrk1B mediates G0/G1 to S phase cell cycle progression and cell survival involving MAPK/ERK signaling in human cancer cells
    Article Snippet: Small interfering RNA treatment Cells were reverse transfected with small interfering RNAs (siRNAs) using lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. .. For combined treatment, cells were pretreated with U0126, an inhibitor of MEK purchased from CalBiochem-NovaBiochem Corporation (La Jolla, CA, USA) at dose escalation for 1 h followed by combination with a constant 20nM dose of siRNAs.

    Protease Inhibitor:

    Article Title: Activation of p38/JNK Pathway Is Responsible for Embelin Induced Apoptosis in Lung Cancer Cells: Transitional Role of Reactive Oxygen Species
    Article Snippet: Minimal essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS), penicillin, streptomycin, sulphorhodamine B (SRB), Ac-DEVD-7-AFC, Ac-LEHD-7-AFC, PD169316, SP600125, N-acetyl-L-cysteine (NAC), radioimmune precipitation assay buffer (RIPA) and protease inhibitor cocktail were purchased from Sigma-Aldrich, Germany. .. U0126 and FeTMPyP were purchased from Calbiochem.

    other:

    Article Title: Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization
    Article Snippet: SP600125, U0126 and SB202190 were from Calbiochem (La Jolla, CA).

    Protein Concentration:

    Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells
    Article Snippet: Immunoblotting To analyze protein expression, BeWo cells and primary vCTB were rinsed with PBS and lysed with ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris-HCl pH 7.4, 1% NP-40, 0,25% Na-deoxycholate, 150 mmol/l NaCl and 1 mmol/l EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich). .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Waltham, MA).

    Recombinant:

    Article Title: CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells
    Article Snippet: .. Recombinant human CXCL12, U0126, LY294002, and AG1478 were obtained from Sigma-Aldrich. .. PE-conjugated secondary antibody was purchased from R & D systems (Minneapolis, MN).

    Immunofluorescence:

    Article Title: Increased Cystatin F Levels Correlate with Decreased Cytotoxicity of Cytotoxic T Cells
    Article Snippet: Other antibodies against human cathepsins were developed: 1D10 mouse anti-cathepsin H antibody , sheep anti-cathepsin H and sheep anti-cathepsin L. Rabbit and mouse anti-β-actin antibodies were from Sigma-Aldrich, rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology and mouse anti-GAPDH from Proteintech (Rosemont, IL, USA). .. For immunofluorescence studies all secondary antibodies were from Thermo Fisher Scientific (Thermo Scientific, Rockford, IL, USA): donkey anti-rabbit Alexa Fluor 488, goat anti-rabbit Alexa Fluor 647, donkey anti-mouse Alexa Fluor 555 and donkey anti-sheep Alexa Fluor 488.

    Molecular Weight:

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *
    Article Snippet: TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling). .. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.

    Bicinchoninic Acid Protein Assay:

    Article Title: 3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: β-ketoamphetamine modulation of neurotoxicity by the dopamine transporter
    Article Snippet: Methylone hydrochloride, MDPV hydrochloride, (±) mephedrone hydrochloride, and (±) MDMA hydrochloride were obtained from the NIDA Research Resources Drug Supply Program. (+) methamphetamine hydrochloride, D-amphetamine sulfate, MPTP hydrochloride, DA, polyclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and all buffers and HPLC reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. Bicinchoninic acid protein assay kits were obtained from Pierce (Rockford, IL, USA).

    Size-exclusion Chromatography:

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *
    Article Snippet: Paragraph title: Size Exclusion Chromatography ... TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling).

    Purification:

    Article Title: Activation of p38/JNK Pathway Is Responsible for Embelin Induced Apoptosis in Lung Cancer Cells: Transitional Role of Reactive Oxygen Species
    Article Snippet: Materials Embelin was purified from the fruits of Embelia ribes as described previously , . .. U0126 and FeTMPyP were purchased from Calbiochem.

    Protein Extraction:

    Article Title: Deregulation of Mitochondrial ATPsyn-? in Acute Myeloid Leukemia Cells and with Increased Drug Resistance
    Article Snippet: Western blot analysis for mitochondrial ATPsyn-β protein For protein extraction, cells (including BMMCs from 80 AML patients, 20 controls; CD34+ cells from 5 AML patients and cell lines) were homogenized on ice in lysis buffer (50 mM Tris-HCL, PH7.5, 150 mM NaCl, 1% NP-40, 0.25% Na-desoxycholate, 5 mM EDTA, 1 mM NaF, 25 mM Na3 VO4 , 0.1 mM PMSF and 2 mg/ml Aprotinin) and cellular debris was pelleted at 13 000 g for 10 min at 4°C. .. After blocked with 5%(ATPsyn-β and β-actin) or 2% fat-free milk(MRP1), the membranes were incubated with antibody against human mitochondrial ATPsyn-β (1∶800 dilutions; Abcam, UK) or MRP1 (1∶400 dilutions; Abcam, UK) or β-actin (1∶5 000 dilutions; Sigma, USA) at 4°C overnight.

    Multiplex Assay:

    Article Title: An Azabisphosphonate-Capped Poly(phosphorhydrazone) Dendrimer for the Treatment of Endotoxin-Induced Uveitis
    Article Snippet: .. Pro-inflammatory T helper cytokines TNFα, IL-1β, IL-2, IL-6, IL-17 and IFNγ as well as anti-inflammatory cytokines IL-4 and IL-10 quantities were determined by Multiplex analysis (Milliplex Map Kit; Millipore, Saint-Quentin-en-Yvelines, France). ..

    Sulforhodamine B Assay:

    Article Title: Activation of p38/JNK Pathway Is Responsible for Embelin Induced Apoptosis in Lung Cancer Cells: Transitional Role of Reactive Oxygen Species
    Article Snippet: Minimal essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS), penicillin, streptomycin, sulphorhodamine B (SRB), Ac-DEVD-7-AFC, Ac-LEHD-7-AFC, PD169316, SP600125, N-acetyl-L-cysteine (NAC), radioimmune precipitation assay buffer (RIPA) and protease inhibitor cocktail were purchased from Sigma-Aldrich, Germany. .. U0126 and FeTMPyP were purchased from Calbiochem.

    Annexin V Assay:

    Article Title: Activation of p38/JNK Pathway Is Responsible for Embelin Induced Apoptosis in Lung Cancer Cells: Transitional Role of Reactive Oxygen Species
    Article Snippet: U0126 and FeTMPyP were purchased from Calbiochem. .. Annexin-V assay kit was purchased from Clontech Inc, USA.

    Small Interfering RNA:

    Article Title: Mirk/Dyrk1B mediates G0/G1 to S phase cell cycle progression and cell survival involving MAPK/ERK signaling in human cancer cells
    Article Snippet: Paragraph title: Small interfering RNA treatment ... For combined treatment, cells were pretreated with U0126, an inhibitor of MEK purchased from CalBiochem-NovaBiochem Corporation (La Jolla, CA, USA) at dose escalation for 1 h followed by combination with a constant 20nM dose of siRNAs.

    Produced:

    Article Title: 3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: β-ketoamphetamine modulation of neurotoxicity by the dopamine transporter
    Article Snippet: Methylone hydrochloride, MDPV hydrochloride, (±) mephedrone hydrochloride, and (±) MDMA hydrochloride were obtained from the NIDA Research Resources Drug Supply Program. (+) methamphetamine hydrochloride, D-amphetamine sulfate, MPTP hydrochloride, DA, polyclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and all buffers and HPLC reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. Polyclonal antibodies against rat tyrosine hydroxylase (TH) were produced as described previously ( ).

    Lysis:

    Article Title: Deregulation of Mitochondrial ATPsyn-? in Acute Myeloid Leukemia Cells and with Increased Drug Resistance
    Article Snippet: Western blot analysis for mitochondrial ATPsyn-β protein For protein extraction, cells (including BMMCs from 80 AML patients, 20 controls; CD34+ cells from 5 AML patients and cell lines) were homogenized on ice in lysis buffer (50 mM Tris-HCL, PH7.5, 150 mM NaCl, 1% NP-40, 0.25% Na-desoxycholate, 5 mM EDTA, 1 mM NaF, 25 mM Na3 VO4 , 0.1 mM PMSF and 2 mg/ml Aprotinin) and cellular debris was pelleted at 13 000 g for 10 min at 4°C. .. After blocked with 5%(ATPsyn-β and β-actin) or 2% fat-free milk(MRP1), the membranes were incubated with antibody against human mitochondrial ATPsyn-β (1∶800 dilutions; Abcam, UK) or MRP1 (1∶400 dilutions; Abcam, UK) or β-actin (1∶5 000 dilutions; Sigma, USA) at 4°C overnight.

    Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *
    Article Snippet: TAP lysis buffer was used for elution of 50 0.5-ml fractions with 0.4 ml/min. .. TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling).

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  • 90
    Millipore mouse anti bax monoclonal
    Effects of Gli-1 downregulation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two Gli-1-specific siRNAs (siGli-1-1 and siGli-1-2) for 24 h. Then, the expression of <t>pro-caspase-8/9/3</t> (A) and <t>Bcl-2/Bax</t> (B) was determined by western blot (WB) analysis. Shh protein (5 μg/mL) was simultaneously added to the non-transfected LN229 cells as a positive control. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments ( n = 5). * P
    Mouse Anti Bax Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti bax monoclonal/product/Millipore
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    mouse anti bax monoclonal - by Bioz Stars, 2020-04
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    92
    Millipore mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody
    Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to <t>glyceraldehyde-3-phosphate</t> dehydrogenase <t>(GAPDH)</t> at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.
    Mouse Monoclonal Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody/product/Millipore
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    mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody - by Bioz Stars, 2020-04
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    90
    Millipore rabbit anti bax
    Effects of coffee oil-algae oil nanoemulsions on protein expressions of <t>cyclin</t> B, CDK2, cyclin A, and CDK1 ( A ), p53 and p21 ( B ), and <t>Bax,</t> Bcl-2, cytochrome C ( C ). Notes: Control cells are incubated with medium only. Results are presented as mean ± standard deviation of triplicate determinations. Data with different letters (A–C) are significantly different at p
    Rabbit Anti Bax, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti bax/product/Millipore
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    Effects of Gli-1 downregulation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two Gli-1-specific siRNAs (siGli-1-1 and siGli-1-2) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. Shh protein (5 μg/mL) was simultaneously added to the non-transfected LN229 cells as a positive control. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments ( n = 5). * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Effects of Gli-1 downregulation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two Gli-1-specific siRNAs (siGli-1-1 and siGli-1-2) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. Shh protein (5 μg/mL) was simultaneously added to the non-transfected LN229 cells as a positive control. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments ( n = 5). * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Expressing, Cell Culture, Transfection, Western Blot, Positive Control

    Effects of mGluR4 activation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two mGluR4-specific siRNAs (simGluR4-1 and simGluR4-2) for 24 h, followed by treatment with the vehicle (Ctrl) or 30 μM of VU0155041 for 24 h. Then, the differential expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) , 9 (D) , 3 (E) , Bcl-2 (F) , Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) , each statistical value represents the mean ± SD of at least three independent experiments. * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Effects of mGluR4 activation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two mGluR4-specific siRNAs (simGluR4-1 and simGluR4-2) for 24 h, followed by treatment with the vehicle (Ctrl) or 30 μM of VU0155041 for 24 h. Then, the differential expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) , 9 (D) , 3 (E) , Bcl-2 (F) , Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) , each statistical value represents the mean ± SD of at least three independent experiments. * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Activation Assay, Expressing, Cell Culture, Transfection, Western Blot

    Gli-1 downregulation is involved in mGluR4-mediated dynamic expression of apoptosis-related proteins in LN229 cells. (A,B) LN229 cells were treated with the vehicle (Ctrl), 5 μg/mL shh, or 30 μM VU0155041 plus 5 μg/mL shh (VU + shh) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments. * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Gli-1 downregulation is involved in mGluR4-mediated dynamic expression of apoptosis-related proteins in LN229 cells. (A,B) LN229 cells were treated with the vehicle (Ctrl), 5 μg/mL shh, or 30 μM VU0155041 plus 5 μg/mL shh (VU + shh) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments. * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Expressing, Western Blot

    Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.

    Journal: Genes & Development

    Article Title: Foxp1 regulation of neonatal vocalizations via cortical development

    doi: 10.1101/gad.305037.117

    Figure Lengend Snippet: Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.

    Article Snippet: The following antibodies were used: mouse monoclonal anti-SUMO-1 (D-11) antibody (Santa Cruz Biotechnology, sc-5308), rabbit polyclonal anti-FOXP1 antibody , mouse monoclonal anti-FOXP1 (JC12) antibody (Abcam, ab32010), goat polyclonal anti-FOXP2 (N-16) antibody (Santa Cruz Biotechnology, sc-21068), mouse monoclonal anti-Flag M2 antibody (Sigma-Aldrich, F1804), mouse monoclonal anti-V5 antibody (Invitrogen, R960-25), goat polyclonal anti-GFP antibody (Rockland Immunochemicals, 600-101-215), chick polyclonal anti-GFP antibody (Aves Laboratories, GFP-1010), rabbit monoclonal anti-SUMO-2/3 (18H8) antibody (Cell Signaling Technology, 4971), rabbit polyclonal anti-PIAS2 antibody (Abcam, ab155556), rabbit polyclonal anti-PIAS3 (H-169) antibody (Santa Cruz Biotechnology, sc-14017), rabbit polyclonal anti-MAP2 antibody (Chemicon, AB5622), mouse monoclonal anti-CtBP (E-12) antibody (Santa Cruz Biotechnology, sc-17759), rabbit polyclonal anti-CDP (CUX1: M-222) antibody (Santa Cruz Biotechnology, sc-13024), rat anti-CTIP2 (Abcam, ab18465), rabbit polyclonal anti-HDAC1 antibody (Abcam, ab19845), mouse monoclonal anti-HDAC1 (10E2) antibody (Cell Signaling Technology, 5256), mouse monoclonal anti-HDAC2 (3F3) antibody (Cell Signaling Technology, 5113), rabbit monoclonal anti-MTA1 (D40D1) XP antibody (Cell Signaling Technology, 5647), rabbit polyclonal anti-MTA2 (H-170) antibody (Santa Cruz Biotechnology, sc-28731), rabbit polyclonal anti-p66β (GATAD2B) antibody (Novus Biologicals, NBP1-87358), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Millipore, MAB374), mouse (G3A1) mAb IgG1 isotype control (Cell Signaling Technology, 5415), normal rabbit IgG (Cell Signaling Technology, 2729), and normal goat IgG (Santa Cruz Biotechnology, sc-2028).

    Techniques: Western Blot, Immunoprecipitation

    Effects of coffee oil-algae oil nanoemulsions on protein expressions of cyclin B, CDK2, cyclin A, and CDK1 ( A ), p53 and p21 ( B ), and Bax, Bcl-2, cytochrome C ( C ). Notes: Control cells are incubated with medium only. Results are presented as mean ± standard deviation of triplicate determinations. Data with different letters (A–C) are significantly different at p

    Journal: International Journal of Nanomedicine

    Article Title: Preparation of coffee oil-algae oil-based nanoemulsions and the study of their inhibition effect on UVA-induced skin damage in mice and melanoma cell growth

    doi: 10.2147/IJN.S144705

    Figure Lengend Snippet: Effects of coffee oil-algae oil nanoemulsions on protein expressions of cyclin B, CDK2, cyclin A, and CDK1 ( A ), p53 and p21 ( B ), and Bax, Bcl-2, cytochrome C ( C ). Notes: Control cells are incubated with medium only. Results are presented as mean ± standard deviation of triplicate determinations. Data with different letters (A–C) are significantly different at p

    Article Snippet: The primary antibodies include mouse monoclonal anti-α-tubulin antibody (Sigma–Aldrich Co.), mouse anti-cyclin A and rabbit anti-Bax (EMD Millipore), mouse anti-CDK2, mouse anti-cytochrome C, mouse anti-p21, mouse anti-cyclin B, and mouse anti-Bcl-2 (BD Biosciences), as well as anti-cdc2 (CDK1) and mouse anti-p53 (Novus Biologicals Co., Littleton, CO, USA).

    Techniques: Incubation, Standard Deviation