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    Structured Review

    Millipore mouse β actin
    Effects of bradykinin on viability, levels, and functions of bradykinin receptor (BDKR) B1/2 in human malignant glioblastoma cells. Human U87 MG glioblastoma cells were stained with a fluorescent 4’,6-diamidino-2-phenylindole (DAPI) dye and reacted with a monoclonal antibody against glial fibrillary acidic protein (GFAP), a biomarker of astrocytes ( A ). Fluorescent signals were observed and analyzed using confocal microscopy. U87 MG cells were treated with 100 nM bradykinin for 6, 12, and 24 h or with 10, 50, and 100 nM bradykinin for 24 h. Cell morphologies were observed and photographed using a light microscope ( B ). Cell survival was analyzed using a trypan blue exclusion method ( C , D ). Levels of BDKRB1 and BDKRB2 were immunodetected ( E , top two panels). <t>β-Actin</t> was analyzed as an internal control (bottom panel). These protein bands were quantified and statistically analyzed ( F ). After exposure to bradykinin and Fluo3, dynamic changes in levels of intracellular calcium (Ca 2+ ) were immediately observed and recorded by confocal microscopy ( G ). Marked enhancement of fluorescent signals showed the increased intensities of intracellular Ca 2+ following bradykinin treatment ( H ). Each value represents the mean ± standard deviation (SD) for n = 9. Representative immunoblots and confocal images are shown. An asterisk (*) indicates that a value significantly ( p
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    Images

    1) Product Images from "The Bradykinin-BDKRB1 Axis Regulates Aquaporin 4 Gene Expression and Consequential Migration and Invasion of Malignant Glioblastoma Cells via a Ca2+-MEK1-ERK1/2-NF-κB Mechanism"

    Article Title: The Bradykinin-BDKRB1 Axis Regulates Aquaporin 4 Gene Expression and Consequential Migration and Invasion of Malignant Glioblastoma Cells via a Ca2+-MEK1-ERK1/2-NF-κB Mechanism

    Journal: Cancers

    doi: 10.3390/cancers12030667

    Effects of bradykinin on viability, levels, and functions of bradykinin receptor (BDKR) B1/2 in human malignant glioblastoma cells. Human U87 MG glioblastoma cells were stained with a fluorescent 4’,6-diamidino-2-phenylindole (DAPI) dye and reacted with a monoclonal antibody against glial fibrillary acidic protein (GFAP), a biomarker of astrocytes ( A ). Fluorescent signals were observed and analyzed using confocal microscopy. U87 MG cells were treated with 100 nM bradykinin for 6, 12, and 24 h or with 10, 50, and 100 nM bradykinin for 24 h. Cell morphologies were observed and photographed using a light microscope ( B ). Cell survival was analyzed using a trypan blue exclusion method ( C , D ). Levels of BDKRB1 and BDKRB2 were immunodetected ( E , top two panels). β-Actin was analyzed as an internal control (bottom panel). These protein bands were quantified and statistically analyzed ( F ). After exposure to bradykinin and Fluo3, dynamic changes in levels of intracellular calcium (Ca 2+ ) were immediately observed and recorded by confocal microscopy ( G ). Marked enhancement of fluorescent signals showed the increased intensities of intracellular Ca 2+ following bradykinin treatment ( H ). Each value represents the mean ± standard deviation (SD) for n = 9. Representative immunoblots and confocal images are shown. An asterisk (*) indicates that a value significantly ( p
    Figure Legend Snippet: Effects of bradykinin on viability, levels, and functions of bradykinin receptor (BDKR) B1/2 in human malignant glioblastoma cells. Human U87 MG glioblastoma cells were stained with a fluorescent 4’,6-diamidino-2-phenylindole (DAPI) dye and reacted with a monoclonal antibody against glial fibrillary acidic protein (GFAP), a biomarker of astrocytes ( A ). Fluorescent signals were observed and analyzed using confocal microscopy. U87 MG cells were treated with 100 nM bradykinin for 6, 12, and 24 h or with 10, 50, and 100 nM bradykinin for 24 h. Cell morphologies were observed and photographed using a light microscope ( B ). Cell survival was analyzed using a trypan blue exclusion method ( C , D ). Levels of BDKRB1 and BDKRB2 were immunodetected ( E , top two panels). β-Actin was analyzed as an internal control (bottom panel). These protein bands were quantified and statistically analyzed ( F ). After exposure to bradykinin and Fluo3, dynamic changes in levels of intracellular calcium (Ca 2+ ) were immediately observed and recorded by confocal microscopy ( G ). Marked enhancement of fluorescent signals showed the increased intensities of intracellular Ca 2+ following bradykinin treatment ( H ). Each value represents the mean ± standard deviation (SD) for n = 9. Representative immunoblots and confocal images are shown. An asterisk (*) indicates that a value significantly ( p

    Techniques Used: Staining, Biomarker Assay, Confocal Microscopy, Light Microscopy, Standard Deviation, Western Blot

    Effects of bradykinin on levels, translocation, and transactivation activity of nuclear factor-kappaB (NF-κB) in human malignant glioblastoma cells. Human U87 MG glioblastoma cells were treated with 100 nM bradykinin for 0.5, 1, and 3 h. Levels of cytosolic (c) and nuclear (n) NF-κB were immunodetected ( A , C , top panels). Amounts of β-actin and proliferating cell nuclear antigen (PCNA) were analyzed as internal controls for the cytosolic and nuclear proteins, respectively (bottom panels). These protein bands were quantified and statistically analyzed ( B , D ). A schematic diagram indicates the NF-κB-specific DNA binding element (−463 to −471) in the 5’-promoter region of the aqp4 gene ( E ). The NF-κB luciferase reporter plasmids (pNF-κB) and pUC18 control plasmids (pUC18) were transfected into human U87 MG cells. Transactivation activity of NF-κB was assayed with a reporter gene assay ( F ). Each value represents the mean ± standard deviation (SD), n = 9. An asterisk (*) indicates that a value significantly ( p
    Figure Legend Snippet: Effects of bradykinin on levels, translocation, and transactivation activity of nuclear factor-kappaB (NF-κB) in human malignant glioblastoma cells. Human U87 MG glioblastoma cells were treated with 100 nM bradykinin for 0.5, 1, and 3 h. Levels of cytosolic (c) and nuclear (n) NF-κB were immunodetected ( A , C , top panels). Amounts of β-actin and proliferating cell nuclear antigen (PCNA) were analyzed as internal controls for the cytosolic and nuclear proteins, respectively (bottom panels). These protein bands were quantified and statistically analyzed ( B , D ). A schematic diagram indicates the NF-κB-specific DNA binding element (−463 to −471) in the 5’-promoter region of the aqp4 gene ( E ). The NF-κB luciferase reporter plasmids (pNF-κB) and pUC18 control plasmids (pUC18) were transfected into human U87 MG cells. Transactivation activity of NF-κB was assayed with a reporter gene assay ( F ). Each value represents the mean ± standard deviation (SD), n = 9. An asterisk (*) indicates that a value significantly ( p

    Techniques Used: Translocation Assay, Activity Assay, Binding Assay, Luciferase, Transfection, Reporter Gene Assay, Standard Deviation

    Effects of bradykinin on aquaporin-4 (AQP4) mRNA and protein expressions in human malignant glioblastoma cells. Human U87 MG glioblastoma cells were treated with 100 nM bradykinin for 3, 6, 12, and 24 h. Levels of AQP4 mRNA were analyzed using an RT-PCR ( A , top panel). Amounts of β-actin mRNA were examined as an internal control (bottom panel). These DNA bands were quantified and statistically analyzed ( B ). Expression of AQP4 mRNA was further quantified using a real-time polymerase chain reaction (PCR) analysis ( C ). Human U87 MG cells were exposed to bradykinin for 24 h. Levels and distribution of AQP4 were immunodetected ( D , left panel). The nucleus was stained with 4’,6-diamidino-2-phenylindole (DAPI) (middle panel). The merged signals are shown in the right panel ( D ) and were quantified and statistically analyzed ( E ). Expression of the bradykinin receptor (BDKR) B1 was knocked-down using RNA interference. Control cells received scrambled siRNA. Levels of BDKRB1 were immunodetected ( F , top panel). β-Actin was immunodetected as an internal control. These protein bands were quantified and statistically analyzed (bottom panel). Human U87 MG cells were pretreated with BDKRB1 siRNA and then exposed to bradykinin. Expression of AQP4 mRNA was quantified with a real-time PCR ( G ). Each value represents the mean ± standard deviation (SD), n = 9. Symbols * and # indicate that the values significantly ( p
    Figure Legend Snippet: Effects of bradykinin on aquaporin-4 (AQP4) mRNA and protein expressions in human malignant glioblastoma cells. Human U87 MG glioblastoma cells were treated with 100 nM bradykinin for 3, 6, 12, and 24 h. Levels of AQP4 mRNA were analyzed using an RT-PCR ( A , top panel). Amounts of β-actin mRNA were examined as an internal control (bottom panel). These DNA bands were quantified and statistically analyzed ( B ). Expression of AQP4 mRNA was further quantified using a real-time polymerase chain reaction (PCR) analysis ( C ). Human U87 MG cells were exposed to bradykinin for 24 h. Levels and distribution of AQP4 were immunodetected ( D , left panel). The nucleus was stained with 4’,6-diamidino-2-phenylindole (DAPI) (middle panel). The merged signals are shown in the right panel ( D ) and were quantified and statistically analyzed ( E ). Expression of the bradykinin receptor (BDKR) B1 was knocked-down using RNA interference. Control cells received scrambled siRNA. Levels of BDKRB1 were immunodetected ( F , top panel). β-Actin was immunodetected as an internal control. These protein bands were quantified and statistically analyzed (bottom panel). Human U87 MG cells were pretreated with BDKRB1 siRNA and then exposed to bradykinin. Expression of AQP4 mRNA was quantified with a real-time PCR ( G ). Each value represents the mean ± standard deviation (SD), n = 9. Symbols * and # indicate that the values significantly ( p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Staining, Standard Deviation

    2) Product Images from "SUMOylation of DRIL1 Directs Its Transcriptional Activity Towards Leukocyte Lineage-Specific Genes"

    Article Title: SUMOylation of DRIL1 Directs Its Transcriptional Activity Towards Leukocyte Lineage-Specific Genes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005542

    DRIL1 is sumoylated in vitro and in vivo . (A) Table showing DRIL1 potential SUMO consensus motifs and SUMOplot™ score (Abgent; www.abgent.com/doc/sumoplot ). Below is the schematic structure of DRIL1 showing lysines 126, 398, 399, 453 and the functional domains. (B) In vitro sumoylation assay performed with 35 S-labeled in vitro -translated wt, SUMO point mutants (K126R, K398R, K399R, K453R) or the quadruple mutant (Kx4R) of DRIL1, incubated in a sumoylation mix containing purified E1, E2 and ATP in the absence or presence of SUMO1. (C) 293T cells transfected with wt DRIL1, K398R or Kx4R mutants. Lysates were Western blotted using antibodies against DRIL1 and β-actin as loading control. (Sh Exp) for short exposure and (Lg Exp) for long exposure. (D) 293T cells transfected with wt DRIL1, K398R or Kx4R. Lysate were immunoprecipited (IP) with antibody against DRIL1, and precipited proteins were western blotted (WB) using antibodies against DRIL1 (left panel) or SUMO1 (right panel). (E) Alignment of amino acid sequences of DRIL1 from human, mice and zebrafish spanning the conserved SUMO consensus motif using Clustal software.
    Figure Legend Snippet: DRIL1 is sumoylated in vitro and in vivo . (A) Table showing DRIL1 potential SUMO consensus motifs and SUMOplot™ score (Abgent; www.abgent.com/doc/sumoplot ). Below is the schematic structure of DRIL1 showing lysines 126, 398, 399, 453 and the functional domains. (B) In vitro sumoylation assay performed with 35 S-labeled in vitro -translated wt, SUMO point mutants (K126R, K398R, K399R, K453R) or the quadruple mutant (Kx4R) of DRIL1, incubated in a sumoylation mix containing purified E1, E2 and ATP in the absence or presence of SUMO1. (C) 293T cells transfected with wt DRIL1, K398R or Kx4R mutants. Lysates were Western blotted using antibodies against DRIL1 and β-actin as loading control. (Sh Exp) for short exposure and (Lg Exp) for long exposure. (D) 293T cells transfected with wt DRIL1, K398R or Kx4R. Lysate were immunoprecipited (IP) with antibody against DRIL1, and precipited proteins were western blotted (WB) using antibodies against DRIL1 (left panel) or SUMO1 (right panel). (E) Alignment of amino acid sequences of DRIL1 from human, mice and zebrafish spanning the conserved SUMO consensus motif using Clustal software.

    Techniques Used: In Vitro, In Vivo, Functional Assay, Labeling, Mutagenesis, Incubation, Purification, Transfection, Western Blot, Mouse Assay, Software

    PIASy is an E3 SUMO ligase for DRIL1. (A) In vitro sumoylation assay performed with 35 S-labeled in vitro -translated wt DRIL1, in vitro -translated PIAS1, PIAS3, PIASy, PIASxα, PIASxβ or recombinant RanBP2 and incubated in a sumoylation mix containing purified E1, E2, ATP and SUMO1. 10% of the in vitro -translated PIAS1 (P1), PIAS3 (P3), PIASy (Py), PIASxα (Pxα), PIASxβ (Pxβ) were 35 S-labeled in vitro -translated for input control (inset). (-) for empty vector. (B) 293T cells co-transfected with wt DRIL1, T7-PIAS1, T7-PIASy, SUMO1 or SUMO2 constructs. Lysates were Western blotted using antibodies against DRIL1, T7 and β-actin as loading control. (-) for empty vector.
    Figure Legend Snippet: PIASy is an E3 SUMO ligase for DRIL1. (A) In vitro sumoylation assay performed with 35 S-labeled in vitro -translated wt DRIL1, in vitro -translated PIAS1, PIAS3, PIASy, PIASxα, PIASxβ or recombinant RanBP2 and incubated in a sumoylation mix containing purified E1, E2, ATP and SUMO1. 10% of the in vitro -translated PIAS1 (P1), PIAS3 (P3), PIASy (Py), PIASxα (Pxα), PIASxβ (Pxβ) were 35 S-labeled in vitro -translated for input control (inset). (-) for empty vector. (B) 293T cells co-transfected with wt DRIL1, T7-PIAS1, T7-PIASy, SUMO1 or SUMO2 constructs. Lysates were Western blotted using antibodies against DRIL1, T7 and β-actin as loading control. (-) for empty vector.

    Techniques Used: In Vitro, Labeling, Recombinant, Incubation, Purification, Plasmid Preparation, Transfection, Construct, Western Blot

    3) Product Images from "Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells"

    Article Title: Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137958

    Effects of PI3K, MEK and lysosomal inhibitors on rapamycin-elicited autophagic responses in N2a-FK cells. (A) N2a-FK cells were co-treated with LY294002 (10 μM) or PD98059 (50 μM) along with rapamycin (1 μM) for 48 h. (B) N2a-FK cells were co-treated with NH 4 Cl (10 mM) together with rapamycin (1 μM) for 48 h. Upper: PK-treated or-untreated samples were applied at concentrations of 100 and 50 μg protein per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-PrP,-LC3 and -β-actin antibodies. Bottom: PrP Sc expression levels following drug treatment were quantified in N2a-FK cells. The results are representative of at least three independent experiments, with each experiment performed in triplicate. *p
    Figure Legend Snippet: Effects of PI3K, MEK and lysosomal inhibitors on rapamycin-elicited autophagic responses in N2a-FK cells. (A) N2a-FK cells were co-treated with LY294002 (10 μM) or PD98059 (50 μM) along with rapamycin (1 μM) for 48 h. (B) N2a-FK cells were co-treated with NH 4 Cl (10 mM) together with rapamycin (1 μM) for 48 h. Upper: PK-treated or-untreated samples were applied at concentrations of 100 and 50 μg protein per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-PrP,-LC3 and -β-actin antibodies. Bottom: PrP Sc expression levels following drug treatment were quantified in N2a-FK cells. The results are representative of at least three independent experiments, with each experiment performed in triplicate. *p

    Techniques Used: SDS Page, Western Blot, Expressing

    PrP Sc in N2a-FK cells is potently increased by a lysosomal but not by a proteasomal inhibitor. (A) N2aFK cells were treated for 48 h with 0.01 to 1 μM MG132 and 0.1 to 10 nM epoxomicin (Epo) as proteasome inhibitors and 0.1 to 10 mM NH 4 Cl as a lysosomal inhibitor. PK-treated and-untreated N2a-FK cells were loaded at concentrations of 100 and 30 μg protein per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-PrP and -β-actin antibodies. (B) For densitometric analysis, PrP Sc band intensities are expressed as a percentage of those of the negative controls. The results in the graph are the mean ± SD of at least three independent experiments. *p
    Figure Legend Snippet: PrP Sc in N2a-FK cells is potently increased by a lysosomal but not by a proteasomal inhibitor. (A) N2aFK cells were treated for 48 h with 0.01 to 1 μM MG132 and 0.1 to 10 nM epoxomicin (Epo) as proteasome inhibitors and 0.1 to 10 mM NH 4 Cl as a lysosomal inhibitor. PK-treated and-untreated N2a-FK cells were loaded at concentrations of 100 and 30 μg protein per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-PrP and -β-actin antibodies. (B) For densitometric analysis, PrP Sc band intensities are expressed as a percentage of those of the negative controls. The results in the graph are the mean ± SD of at least three independent experiments. *p

    Techniques Used: SDS Page, Western Blot

    PrP Sc in N2a-FK cells undergoes degradation via upstream intracellular signalling cascades associated with autophagy. (A) N2a-FK cells were treated with 0.1 to 10 μM of the the PI3K inhibitor LY294002 and 1 to 50 μM of the MEK inhibitor of PD98059 for 48 h. PK-treated or-untreated samples were applied at concentrations of 100 and 50 μg protein per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were analyzed by western blotting using anti-PrP, anti-Akt, anti-phosphorylated Akt (to determine the Akt activation level), anti-p44/p42 MAPK, anti-phosphorylated p44/p42 MAPK (to determine the p44/p42 MAPK activation level) and anti-β-actin antibodies. (B) The effect of these drugs on PrP Sc was determined by quantifying the PrP Sc band intensities as a percentage of those of the negative controls. The results in the graph are the mean ± SD of at least three independent experiments. *p
    Figure Legend Snippet: PrP Sc in N2a-FK cells undergoes degradation via upstream intracellular signalling cascades associated with autophagy. (A) N2a-FK cells were treated with 0.1 to 10 μM of the the PI3K inhibitor LY294002 and 1 to 50 μM of the MEK inhibitor of PD98059 for 48 h. PK-treated or-untreated samples were applied at concentrations of 100 and 50 μg protein per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were analyzed by western blotting using anti-PrP, anti-Akt, anti-phosphorylated Akt (to determine the Akt activation level), anti-p44/p42 MAPK, anti-phosphorylated p44/p42 MAPK (to determine the p44/p42 MAPK activation level) and anti-β-actin antibodies. (B) The effect of these drugs on PrP Sc was determined by quantifying the PrP Sc band intensities as a percentage of those of the negative controls. The results in the graph are the mean ± SD of at least three independent experiments. *p

    Techniques Used: SDS Page, Western Blot, Activation Assay

    PrP Sc levels in the lysosomal fraction are reduced following autophagy in starved cells. (A) Lysosomes were isolated by the fractionation of N2a-FK cells incubated or not in HBSS for 8 h, as an inducer of autophagy. PK-treated or-untreated gradient fractions were applied at one-tenth of their original volume per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-PrP, anti-Lamp1 (as a lysosomal marker) and anti-β-actin (as the internal standard) antibodies. Fractions 1 to 3 contained high-quality, purified native lysosomes. Positive controls consisted of cell lysates containing 15 μg of protein before purification (P). (B) To quantify ratio of the PrP Sc degradation in the isolated lysosomes, PrP Sc band intensities were measured as a percentage of those of the controls. Empty columns indicate the control, non-treated fractions and shaded columns the HBSS-treated fractions. The results in the graph are the mean ± SD of at least three independent experiments. *p
    Figure Legend Snippet: PrP Sc levels in the lysosomal fraction are reduced following autophagy in starved cells. (A) Lysosomes were isolated by the fractionation of N2a-FK cells incubated or not in HBSS for 8 h, as an inducer of autophagy. PK-treated or-untreated gradient fractions were applied at one-tenth of their original volume per lane onto a 15% polyacrylamide gel and subjected to SDS-PAGE. The proteins were detected by western blotting using anti-PrP, anti-Lamp1 (as a lysosomal marker) and anti-β-actin (as the internal standard) antibodies. Fractions 1 to 3 contained high-quality, purified native lysosomes. Positive controls consisted of cell lysates containing 15 μg of protein before purification (P). (B) To quantify ratio of the PrP Sc degradation in the isolated lysosomes, PrP Sc band intensities were measured as a percentage of those of the controls. Empty columns indicate the control, non-treated fractions and shaded columns the HBSS-treated fractions. The results in the graph are the mean ± SD of at least three independent experiments. *p

    Techniques Used: Isolation, Fractionation, Incubation, SDS Page, Western Blot, Marker, Purification

    4) Product Images from "Nerve growth factor signaling following unilateral pelvic ganglionectomy in the rat ventral prostate is age dependent"

    Article Title: Nerve growth factor signaling following unilateral pelvic ganglionectomy in the rat ventral prostate is age dependent

    Journal: Asian Journal of Andrology

    doi: 10.1038/aja.2013.59

    Quantification of NGF/β-ACTIN proteins in denervated, intact and sham control VP. ( a ) In P30 rats, there was no difference in NGF protein between the three groups ( P =0.9113). ( b ) In P60 rats, NGF increased 15% in the intact VP in comparison
    Figure Legend Snippet: Quantification of NGF/β-ACTIN proteins in denervated, intact and sham control VP. ( a ) In P30 rats, there was no difference in NGF protein between the three groups ( P =0.9113). ( b ) In P60 rats, NGF increased 15% in the intact VP in comparison

    Techniques Used:

    5) Product Images from "Arborization of Dendrites by Developing Neocortical Neurons Is Dependent on Primary Cilia and Type 3 Adenylyl Cyclase"

    Article Title: Arborization of Dendrites by Developing Neocortical Neurons Is Dependent on Primary Cilia and Type 3 Adenylyl Cyclase

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2906-12.2013

    GPCR overexpression induces upregulation of IFT proteins and premature cilia lengthening. A–D , Comparisons were made between protein expression in nonelectroporated control cortex ( A ), fetal cortex that was electroporated at E15.5 with either a vector encoding EGFP and mCherry:AU1 ( in utero electroporation control) ( B ), or mCherry:AU1 and either SSTR3:EGFP or 5HT6:EGFP ( C ). Expression of all transgenes was under the control of the EF1α promoter. Hyphenated lines indicate cortical regions of P14 brains that were used to prepare the protein lysates analyzed by Western blot. D , Western blots (10 μg of total protein/group) were probed for proteins associated with either anterograde (Kif3a) or retrograde (cytoplasmic dynein, D1 IC74) IFT complex B protein (IFT88), or GPCR trafficking into cilia (TULP3). β-Actin was used as a loading control. E , Cultured, nonelectroporated control cortical neuron immunostained for pericentrin (basal body, red), IFT88 (green), and the neuronal marker, MAP2 (blue). The arrow in the middle panel points to an IFT88 + cilium extending from a pericentrin + basal body (arrow left panel). F , Example of an abnormally long, branched 5HT6:EGFP + cilium synthesized by a cultured neuron expressing 5HT6:EGFP (green) under the control of the CMV promoter and mRFP (pseudocolored blue). IFT88 (red) and EGFP were colocalized along the length and branches of the cilium (white arrows). Scale bar, 5 μm. G , E15.5 brains were electroporated with a vector encoding mCherry(AU1)-2a-5HT6:EGFP. At P1, electroporated brains were sectioned and stained with an antibody against ACIII. Examination of the upper layers of the cortical plate revealed mCherry + neurons (red) that possessed longer 5HT6:EGFP + cilia (arrowheads) than their neighboring nonelectroporated cells whose ACIII-stained cilia appear punctate (blue, arrows). Scale bar, 10 μm. H , Comparison of the lengths of the cilia of neurons overexpressing 5HT6:EGFP and control neurons: ***Student's t test. ( I ) Section of brain electroporated and processed as described for G , but not including the red channel used to visualize mCherry. Numerous, often long cilia (arrows) were present in the upper layers of the cortical plate. J , P1 neurons in the upper cortical plate that were coelectroporated at E15.5 with vectors encoding mCherry and 5HT6:EGFP (mCherry(AU1)-2a-5HT6:EGFP) and mCherry and dnKif3a (mCherry(AU1)-2a-dnKif3a). The elongated 5HT6:EGFP + cilia of neurons expressing 5HT6:EGFP alone ( I ) are not observed in cells coexpressing 5HT6:EGFP and dnKif3a.
    Figure Legend Snippet: GPCR overexpression induces upregulation of IFT proteins and premature cilia lengthening. A–D , Comparisons were made between protein expression in nonelectroporated control cortex ( A ), fetal cortex that was electroporated at E15.5 with either a vector encoding EGFP and mCherry:AU1 ( in utero electroporation control) ( B ), or mCherry:AU1 and either SSTR3:EGFP or 5HT6:EGFP ( C ). Expression of all transgenes was under the control of the EF1α promoter. Hyphenated lines indicate cortical regions of P14 brains that were used to prepare the protein lysates analyzed by Western blot. D , Western blots (10 μg of total protein/group) were probed for proteins associated with either anterograde (Kif3a) or retrograde (cytoplasmic dynein, D1 IC74) IFT complex B protein (IFT88), or GPCR trafficking into cilia (TULP3). β-Actin was used as a loading control. E , Cultured, nonelectroporated control cortical neuron immunostained for pericentrin (basal body, red), IFT88 (green), and the neuronal marker, MAP2 (blue). The arrow in the middle panel points to an IFT88 + cilium extending from a pericentrin + basal body (arrow left panel). F , Example of an abnormally long, branched 5HT6:EGFP + cilium synthesized by a cultured neuron expressing 5HT6:EGFP (green) under the control of the CMV promoter and mRFP (pseudocolored blue). IFT88 (red) and EGFP were colocalized along the length and branches of the cilium (white arrows). Scale bar, 5 μm. G , E15.5 brains were electroporated with a vector encoding mCherry(AU1)-2a-5HT6:EGFP. At P1, electroporated brains were sectioned and stained with an antibody against ACIII. Examination of the upper layers of the cortical plate revealed mCherry + neurons (red) that possessed longer 5HT6:EGFP + cilia (arrowheads) than their neighboring nonelectroporated cells whose ACIII-stained cilia appear punctate (blue, arrows). Scale bar, 10 μm. H , Comparison of the lengths of the cilia of neurons overexpressing 5HT6:EGFP and control neurons: ***Student's t test. ( I ) Section of brain electroporated and processed as described for G , but not including the red channel used to visualize mCherry. Numerous, often long cilia (arrows) were present in the upper layers of the cortical plate. J , P1 neurons in the upper cortical plate that were coelectroporated at E15.5 with vectors encoding mCherry and 5HT6:EGFP (mCherry(AU1)-2a-5HT6:EGFP) and mCherry and dnKif3a (mCherry(AU1)-2a-dnKif3a). The elongated 5HT6:EGFP + cilia of neurons expressing 5HT6:EGFP alone ( I ) are not observed in cells coexpressing 5HT6:EGFP and dnKif3a.

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, In Utero, Electroporation, Western Blot, Cell Culture, Marker, Synthesized, Staining

    6) Product Images from "Aging Impairs Hippocampal- Dependent Recognition Memory and LTP and Prevents the Associated RyR Up-regulation"

    Article Title: Aging Impairs Hippocampal- Dependent Recognition Memory and LTP and Prevents the Associated RyR Up-regulation

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2017.00111

    The hippocampus from aged rats has RyR2 channels with higher oxidation levels. (A) The figure illustrates a representative blot of duplicate hippocampal samples from young or aged rats, treated first with the streptavidin reagent for NEM-biotin labeling and then with antibodies against RyR2 and β-actin. (B) Quantification of band densities revealed by NEM-biotin labeling and antibodies against RyR2; values expressed as ratios were normalized to the values exhibited by young rats. (C) Quantification of band densities revealed with antibodies against RyR2 and β-actin; values represent Mean ± SEM of RyR2/β-actin ratios, normalized to the values exhibited by young rats. The whole hippocampus from either young ( n = 6) or aged ( n = 6) animals was analyzed for each condition. Statistical analysis of results presented in (B,C) was performed with unpaired One-tailed Student’s t -test. ∗ p
    Figure Legend Snippet: The hippocampus from aged rats has RyR2 channels with higher oxidation levels. (A) The figure illustrates a representative blot of duplicate hippocampal samples from young or aged rats, treated first with the streptavidin reagent for NEM-biotin labeling and then with antibodies against RyR2 and β-actin. (B) Quantification of band densities revealed by NEM-biotin labeling and antibodies against RyR2; values expressed as ratios were normalized to the values exhibited by young rats. (C) Quantification of band densities revealed with antibodies against RyR2 and β-actin; values represent Mean ± SEM of RyR2/β-actin ratios, normalized to the values exhibited by young rats. The whole hippocampus from either young ( n = 6) or aged ( n = 6) animals was analyzed for each condition. Statistical analysis of results presented in (B,C) was performed with unpaired One-tailed Student’s t -test. ∗ p

    Techniques Used: Labeling, One-tailed Test

    7) Product Images from "Dendritic Cells Require PINK1-Mediated Phosphorylation of BCKDE1α to Promote Fatty Acid Oxidation for Immune Function"

    Article Title: Dendritic Cells Require PINK1-Mediated Phosphorylation of BCKDE1α to Promote Fatty Acid Oxidation for Immune Function

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02386

    PINK1 is required for OXPHOS in pRNA-stimulated DC. (A) Relative mRNA levels were analyzed after 12 h of pRNA stimulation by (qPCR) and normalized to β-actin expression by using the 2 ΔΔ CT method. Data represents Mean ± SEM of three independent experiments performed in triplicate. * p
    Figure Legend Snippet: PINK1 is required for OXPHOS in pRNA-stimulated DC. (A) Relative mRNA levels were analyzed after 12 h of pRNA stimulation by (qPCR) and normalized to β-actin expression by using the 2 ΔΔ CT method. Data represents Mean ± SEM of three independent experiments performed in triplicate. * p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    pRNA-stimulated DC maturation requires PINK1-dependent FAO. (A) Relative mRNA levels were analyzed after 12 h of pRNA stimulation by (qPCR) and normalized to β-actin expression by using the 2 ΔΔ CT method. Data represents Mean ± SEM of three independent experiments performed in triplicate. *** p
    Figure Legend Snippet: pRNA-stimulated DC maturation requires PINK1-dependent FAO. (A) Relative mRNA levels were analyzed after 12 h of pRNA stimulation by (qPCR) and normalized to β-actin expression by using the 2 ΔΔ CT method. Data represents Mean ± SEM of three independent experiments performed in triplicate. *** p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Kinetin primes immunosuppressive DC for maturation. (A) Expression of PINK1 in DCs was determined by western blotting. β-actin was used as a loading control. (B) Total BCAA concentrations were measured in DCs, stimulated for 12 h with pRNA in the presence or absence of 20 μM kinetin, using a colorimetric assay kit. Data represents Mean ± SEM of three independent experiments performed in triplicate. ** p
    Figure Legend Snippet: Kinetin primes immunosuppressive DC for maturation. (A) Expression of PINK1 in DCs was determined by western blotting. β-actin was used as a loading control. (B) Total BCAA concentrations were measured in DCs, stimulated for 12 h with pRNA in the presence or absence of 20 μM kinetin, using a colorimetric assay kit. Data represents Mean ± SEM of three independent experiments performed in triplicate. ** p

    Techniques Used: Expressing, Western Blot, Colorimetric Assay

    8) Product Images from "Glutathione Supplementation Attenuates Lipopolysaccharide-Induced Mitochondrial Dysfunction and Apoptosis in a Mouse Model of Acute Lung Injury"

    Article Title: Glutathione Supplementation Attenuates Lipopolysaccharide-Induced Mitochondrial Dysfunction and Apoptosis in a Mouse Model of Acute Lung Injury

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2012.00161

    GSH-EE supplementation prevents LPS-mediated mitochondrial dysfunction . Protein extracts prepared from peripheral lungs of vehicle, LPS, GSH-EE + LPS, and GSH-EE treated mice were analyzed by Western blot analysis using a specific antiserum raised against SOD1 (A) or SOD2 (B) . Protein levels were normalized for loading using β-actin. A representative blot and normalized densitometric values are shown. There was a significant decrease in SOD1 and SOD2 protein levels in LPS-treated mice and this was prevented by GHS-EE pre-treatment. The lactate/pyruvate ratio was also determined in all four groups (C) . LPS-exposed mice had a significantly higher lactate/pyruvate ratio (C) . Pre-treatment with GSH-EE preserved the lactate/pyruvate ratio. There was a significant reduction in lung ATP levels after LPS exposure (D) . However, the LPS-mediated decrease in ATP levels was not observed in mice pre-treated with GSH-EE (D) . Values are mean ± SEM; n = 6/group. * P
    Figure Legend Snippet: GSH-EE supplementation prevents LPS-mediated mitochondrial dysfunction . Protein extracts prepared from peripheral lungs of vehicle, LPS, GSH-EE + LPS, and GSH-EE treated mice were analyzed by Western blot analysis using a specific antiserum raised against SOD1 (A) or SOD2 (B) . Protein levels were normalized for loading using β-actin. A representative blot and normalized densitometric values are shown. There was a significant decrease in SOD1 and SOD2 protein levels in LPS-treated mice and this was prevented by GHS-EE pre-treatment. The lactate/pyruvate ratio was also determined in all four groups (C) . LPS-exposed mice had a significantly higher lactate/pyruvate ratio (C) . Pre-treatment with GSH-EE preserved the lactate/pyruvate ratio. There was a significant reduction in lung ATP levels after LPS exposure (D) . However, the LPS-mediated decrease in ATP levels was not observed in mice pre-treated with GSH-EE (D) . Values are mean ± SEM; n = 6/group. * P

    Techniques Used: Mouse Assay, Western Blot

    9) Product Images from "TRAF Family Proteins Link PKR with NF-?B Activation"

    Article Title: TRAF Family Proteins Link PKR with NF-?B Activation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.10.4502-4512.2004

    Absence of PKR-dependent NF-κB activation in 3T3 cells deficient in TRAF2 and TRAF5. (A) 3T3 cells grown in 10-cm-diameter plates, obtained from WT mice (WT 3T3) or mice deficient in both TRAF2 and TRAF5 (DKO 3T3), were mock infected (M) or infected with 5 PFU of VV per cell (VV) or 5 PFU of VV PKR per cell (PKR). Nuclear extracts were prepared at 20 hpi and analyzed by gel shift assay with a probe specific for NF-κB. (B) Extracts (50 μg) from the same cells were analyzed for PKR expression by Western blotting (WB). (C) Cells infected as described for panel A were analyzed for induction of apoptosis as indicated in Materials and Methods. The lane numbers in panels B and C are the same as those in panel A. O.D., optical density. DKO (D) or WT (E) 3T3 cells were treated with 10 μg of pIC per ml for the times indicated, and levels of IκBα and β-actin were analyzed by Western blotting. The arrows indicate specific proteins.
    Figure Legend Snippet: Absence of PKR-dependent NF-κB activation in 3T3 cells deficient in TRAF2 and TRAF5. (A) 3T3 cells grown in 10-cm-diameter plates, obtained from WT mice (WT 3T3) or mice deficient in both TRAF2 and TRAF5 (DKO 3T3), were mock infected (M) or infected with 5 PFU of VV per cell (VV) or 5 PFU of VV PKR per cell (PKR). Nuclear extracts were prepared at 20 hpi and analyzed by gel shift assay with a probe specific for NF-κB. (B) Extracts (50 μg) from the same cells were analyzed for PKR expression by Western blotting (WB). (C) Cells infected as described for panel A were analyzed for induction of apoptosis as indicated in Materials and Methods. The lane numbers in panels B and C are the same as those in panel A. O.D., optical density. DKO (D) or WT (E) 3T3 cells were treated with 10 μg of pIC per ml for the times indicated, and levels of IκBα and β-actin were analyzed by Western blotting. The arrows indicate specific proteins.

    Techniques Used: Activation Assay, Mouse Assay, Infection, Electrophoretic Mobility Shift Assay, Expressing, Western Blot

    10) Product Images from "Glucagon regulates intracellular distribution of adipose differentiation-related protein during triacylglycerol accumulation in the liver [S]"

    Article Title: Glucagon regulates intracellular distribution of adipose differentiation-related protein during triacylglycerol accumulation in the liver [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M004648

    Accumulation of ADRP in the liver of fasting mice . A: Effects of fasting on ADRP protein levels in the tissues were examined. Male C57BL/6 mice (10 weeks old) were either fed chow diet or fasted for 16 h, euthanized, and then brain, heart, liver, skeletal muscle, and WAT were recovered. Immunoblot analysis of ADRP protein levels in each type of mouse tissue was performed, and the intensity of the detected bands for ADRP and β-actin were quantified by densitometric scanning. The relative index of 1.0 is the ADRP/β-actin in the liver from chow-fed mice. Bar graph shows mean and standard deviation of three mice. B: Fasting altered levels of TIP47, another LD protein, and SCD-1, an enzyme involved in lipid metabolism. Among the PAT family proteins, perilipin was not detectable. The intensity of the detected bands of ADRP, TIP47, SCD-1, and β-actin were quantified by densitometric scanning. ADRP, adipose differentiation-related protein; LD, lipid droplet; SCD-1, stearoyl-CoA desaturase-1; TIP47; tail interacting protein of 47 kDa; WAT, white adipose tissue.
    Figure Legend Snippet: Accumulation of ADRP in the liver of fasting mice . A: Effects of fasting on ADRP protein levels in the tissues were examined. Male C57BL/6 mice (10 weeks old) were either fed chow diet or fasted for 16 h, euthanized, and then brain, heart, liver, skeletal muscle, and WAT were recovered. Immunoblot analysis of ADRP protein levels in each type of mouse tissue was performed, and the intensity of the detected bands for ADRP and β-actin were quantified by densitometric scanning. The relative index of 1.0 is the ADRP/β-actin in the liver from chow-fed mice. Bar graph shows mean and standard deviation of three mice. B: Fasting altered levels of TIP47, another LD protein, and SCD-1, an enzyme involved in lipid metabolism. Among the PAT family proteins, perilipin was not detectable. The intensity of the detected bands of ADRP, TIP47, SCD-1, and β-actin were quantified by densitometric scanning. ADRP, adipose differentiation-related protein; LD, lipid droplet; SCD-1, stearoyl-CoA desaturase-1; TIP47; tail interacting protein of 47 kDa; WAT, white adipose tissue.

    Techniques Used: Mouse Assay, Standard Deviation

    11) Product Images from "Muscle-specific inactivation of the IGF-I receptor induces compensatory hyperplasia in skeletal muscle"

    Article Title: Muscle-specific inactivation of the IGF-I receptor induces compensatory hyperplasia in skeletal muscle

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI13503

    Delayed activation of MyoD and myogenin in MKR mice. Hindlimb muscles were removed from WT and MKR mice at the ages of 0–3 weeks, 5 weeks, and 8 weeks, and subjected to Northern or Western blot analysis, as indicated below. ( a ) Northern blot analysis of MyoD levels in MKR and WT mice (upper panel). 18S RNA levels were similar in all groups (lower panel). ( b ) Western blot analysis of MyoD (upper panel) and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. ( c ) Northern blot analysis of myogenin levels in MKR and WT mice (upper panel). 18S RNA levels were similar in all groups (lower panel). ( d ) Western blot analysis of myogenin (upper panel) and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. Data are expressed as the mean ± SEM of three independent experiments, using five animals in each group.
    Figure Legend Snippet: Delayed activation of MyoD and myogenin in MKR mice. Hindlimb muscles were removed from WT and MKR mice at the ages of 0–3 weeks, 5 weeks, and 8 weeks, and subjected to Northern or Western blot analysis, as indicated below. ( a ) Northern blot analysis of MyoD levels in MKR and WT mice (upper panel). 18S RNA levels were similar in all groups (lower panel). ( b ) Western blot analysis of MyoD (upper panel) and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. ( c ) Northern blot analysis of myogenin levels in MKR and WT mice (upper panel). 18S RNA levels were similar in all groups (lower panel). ( d ) Western blot analysis of myogenin (upper panel) and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. Data are expressed as the mean ± SEM of three independent experiments, using five animals in each group.

    Techniques Used: Activation Assay, Mouse Assay, Northern Blot, Western Blot

    Comparison of ERK1/2, p44/42, p38, JNK, and Akt levels in WT versus MKR mice. Hindlimb muscles were removed from WT and MKR mice at the ages of 0–3 weeks, 5 weeks, and 8 weeks, and subjected to Western blot analysis. ( a ) Representative blots showing phospho-p44/42 MAP kinase (upper panel), ERK1/2 (middle panel), and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. ( b ) Representative blots showing phospho-p38 (upper panel), p38 (middle panel), and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. ( c ) Representative blots showing phospho-JNK (upper panel) and JNK (lower panel) immunoreactivity levels in MKR and WT mice. ( d ) Representative blots showing phospho-Akt (upper panel) and Akt (lower panel) immunoreactivity levels in MKR and WT mice. Each blot represents three independent experiments, using five animals in each group.
    Figure Legend Snippet: Comparison of ERK1/2, p44/42, p38, JNK, and Akt levels in WT versus MKR mice. Hindlimb muscles were removed from WT and MKR mice at the ages of 0–3 weeks, 5 weeks, and 8 weeks, and subjected to Western blot analysis. ( a ) Representative blots showing phospho-p44/42 MAP kinase (upper panel), ERK1/2 (middle panel), and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. ( b ) Representative blots showing phospho-p38 (upper panel), p38 (middle panel), and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. ( c ) Representative blots showing phospho-JNK (upper panel) and JNK (lower panel) immunoreactivity levels in MKR and WT mice. ( d ) Representative blots showing phospho-Akt (upper panel) and Akt (lower panel) immunoreactivity levels in MKR and WT mice. Each blot represents three independent experiments, using five animals in each group.

    Techniques Used: Mouse Assay, Western Blot

    Differential expression of p21 and Twist in MKR mice. Hindlimb muscles were removed from WT and MKR mice at the ages of 0–3 weeks, 5 weeks, and 8 weeks, and subjected to Northern or Western blot analysis, as indicated below. ( a ) Northern blot analysis of Twist levels in MKR and WT mice (upper panel). 18S RNA levels were similar in all the groups (lower panel). ( b ) Western blot analysis of Twist (upper panel) and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. ( c ) Western blot analysis of p21 (upper panel) and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. Data are expressed as the mean ± SEM of three independent experiments, using five animals in each group.
    Figure Legend Snippet: Differential expression of p21 and Twist in MKR mice. Hindlimb muscles were removed from WT and MKR mice at the ages of 0–3 weeks, 5 weeks, and 8 weeks, and subjected to Northern or Western blot analysis, as indicated below. ( a ) Northern blot analysis of Twist levels in MKR and WT mice (upper panel). 18S RNA levels were similar in all the groups (lower panel). ( b ) Western blot analysis of Twist (upper panel) and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. ( c ) Western blot analysis of p21 (upper panel) and β-actin (lower panel) immunoreactivity levels in MKR and WT mice. Data are expressed as the mean ± SEM of three independent experiments, using five animals in each group.

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

    12) Product Images from "Endothelial Cells Support Persistent Gammaherpesvirus 68 Infection"

    Article Title: Endothelial Cells Support Persistent Gammaherpesvirus 68 Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000152

    Optimal viability of endothelial cells after γHV68 infection requires the viral cyclin. (A) MB114 cells contained the γHV68 v-cyclin transcript. RT-PCR analysis of the γHV68 v-cyclin transcript. Total RNA was isolated from infected MB114 cells and 3T3 cells at the indicated times post-infection, as well as from latently infected S11 B cells. 100 ng of RNA from each sample was subjected to RT-PCR analysis with primers specific for the v-cyclin transcript. As a loading control, we also amplified the cellular transcript β-actin. No RNA could be isolated from cultured 3T3 cells at 12 days post-infection. No RT and no template controls are indicated. (B C) Viability and persistent viral replication in the presence or absence of the v-cyclin. MB114 cells were infected with wildtype (black) or v-cyclin.STOP γHV68 (grey) at an MOI of 5 PFU/cell. Cells were harvested at six days post-infection by centrifugation from supernatant, and cultured in complete RPMI. Every six days cells were centrifuged to remove supernatant for titer (C) and stained with propidium iodine (PI) every three days (B). Viability of post-infection cultures was determined as percent PI negative cells by flow cytometry. Significant differences in viability were observed between wildtype γHV68 and v-cyclin.STOP infections at day six (p
    Figure Legend Snippet: Optimal viability of endothelial cells after γHV68 infection requires the viral cyclin. (A) MB114 cells contained the γHV68 v-cyclin transcript. RT-PCR analysis of the γHV68 v-cyclin transcript. Total RNA was isolated from infected MB114 cells and 3T3 cells at the indicated times post-infection, as well as from latently infected S11 B cells. 100 ng of RNA from each sample was subjected to RT-PCR analysis with primers specific for the v-cyclin transcript. As a loading control, we also amplified the cellular transcript β-actin. No RNA could be isolated from cultured 3T3 cells at 12 days post-infection. No RT and no template controls are indicated. (B C) Viability and persistent viral replication in the presence or absence of the v-cyclin. MB114 cells were infected with wildtype (black) or v-cyclin.STOP γHV68 (grey) at an MOI of 5 PFU/cell. Cells were harvested at six days post-infection by centrifugation from supernatant, and cultured in complete RPMI. Every six days cells were centrifuged to remove supernatant for titer (C) and stained with propidium iodine (PI) every three days (B). Viability of post-infection cultures was determined as percent PI negative cells by flow cytometry. Significant differences in viability were observed between wildtype γHV68 and v-cyclin.STOP infections at day six (p

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Cell Culture, Centrifugation, Staining, Flow Cytometry, Cytometry

    Primary endothelial cells were infected in vivo , and demonstrated prolonged viability while supporting γHV68 growth ex vivo . Multi-step growth assay of γHV68 in primary C57/BL6 lung endothelial cells (A) and corresponding cell viability (B). Primary cells were infected at an MOI of 0.05 PFU/cell with either wildtype γHV68 or v-cyclin.STOP γHV68, harvested at the indicated times, and trypan blue exclusion counts performed (B). (A) Samples were multiply freeze-thawed prior to quantification by plaque assay on NIH 3T12 cells. n = 3. (B) At 0, 48, 96, and 120 hours post-infection, trypan blue exclusion counts were performed on primary lung endothelial cells and primary MEFs infected with either wildtype or v-cyclin.STOP γHV68 at an MOI of 0.05 PFU/cell. n = 3. Limiting dilution-PCR of viral DNA (C) and RT-PCR (D) analysis of endothelial enriched (E) and depleted (D) lung cells from CD8-alpha knockout mice at six days post-intranasal infection. (C) Frequency of viral genome-positive cells was determined by LD-PCR. Percentage of positive PCR reactions are indicated on the y axis and the number of cells analyzed is indicated on the x axis. For each cell dilution, 12 PCR reactions were analyzed. The frequency of viral genome positive cells was determined by Poisson distribution indicated by the dashed line at 63.2%. Data represent the averages of three separate infected animals. Error bars represent standard errors of the mean. (D) Total RNA was isolated from D and E populations and 100 ng of RNA subjected to RT-PCR analysis with primers specific for the γHV68 pol III-1 transcript and the cellular transcripts CD31 and β-actin. Results are shown from three infected and one mock infected mouse.
    Figure Legend Snippet: Primary endothelial cells were infected in vivo , and demonstrated prolonged viability while supporting γHV68 growth ex vivo . Multi-step growth assay of γHV68 in primary C57/BL6 lung endothelial cells (A) and corresponding cell viability (B). Primary cells were infected at an MOI of 0.05 PFU/cell with either wildtype γHV68 or v-cyclin.STOP γHV68, harvested at the indicated times, and trypan blue exclusion counts performed (B). (A) Samples were multiply freeze-thawed prior to quantification by plaque assay on NIH 3T12 cells. n = 3. (B) At 0, 48, 96, and 120 hours post-infection, trypan blue exclusion counts were performed on primary lung endothelial cells and primary MEFs infected with either wildtype or v-cyclin.STOP γHV68 at an MOI of 0.05 PFU/cell. n = 3. Limiting dilution-PCR of viral DNA (C) and RT-PCR (D) analysis of endothelial enriched (E) and depleted (D) lung cells from CD8-alpha knockout mice at six days post-intranasal infection. (C) Frequency of viral genome-positive cells was determined by LD-PCR. Percentage of positive PCR reactions are indicated on the y axis and the number of cells analyzed is indicated on the x axis. For each cell dilution, 12 PCR reactions were analyzed. The frequency of viral genome positive cells was determined by Poisson distribution indicated by the dashed line at 63.2%. Data represent the averages of three separate infected animals. Error bars represent standard errors of the mean. (D) Total RNA was isolated from D and E populations and 100 ng of RNA subjected to RT-PCR analysis with primers specific for the γHV68 pol III-1 transcript and the cellular transcripts CD31 and β-actin. Results are shown from three infected and one mock infected mouse.

    Techniques Used: Infection, In Vivo, Ex Vivo, Growth Assay, Plaque Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Mouse Assay, Isolation

    Endothelial cells which were viable after γHV68 infection were productively infected. (A) MB114 cells contained viral gene transcripts as far as 12 days post-infection. RT-PCR analysis of viral gene transcripts. 100 ng of total RNA from mock infected and infected MB114 and 3T3 cells and from S11 cells was added to each of the RT reactions along with primers specific for the viral transcripts polymerase III-1 and M2, and the cellular transcripts β-actin and 18S. No RT and no template controls are indicated. (B) MB114 cells contained viral proteins as far as 12 days post-infection. Immunoblot of lytic viral protein expression. 10 µg of total protein from mock infected and infected MB114 and 3T3 cells and from S11 cells were loaded per lane and blots probed with antibodies to the lytic γHV68 proteins M3 (top) and gB (middle), and mouse β-actin (bottom) as a loading control. Mock infected cells were collected at 24 hours. Latent S11 cells do not express lytic viral proteins and served as a negative control. (C) At the indicated times post-infection, MB114 cells were analyzed for gp150 surface expression by flow cytometry. Fluorescence was determined relative to unstained cells (grey). Histograms are representative of two independent infections. (D) Six days post-infection with GFP-γHV68, MB114 cells were collected and analyzed by flow cytometry for GFP expression. Cells were cultured and brightfield (top) and fluorescent (bottom) images taken at 20 days post-infection, 100X magnification. Fluorescence was determined relative to cells infected with wildtype γHV68 (grey), and histogram is representative of two independent infections.
    Figure Legend Snippet: Endothelial cells which were viable after γHV68 infection were productively infected. (A) MB114 cells contained viral gene transcripts as far as 12 days post-infection. RT-PCR analysis of viral gene transcripts. 100 ng of total RNA from mock infected and infected MB114 and 3T3 cells and from S11 cells was added to each of the RT reactions along with primers specific for the viral transcripts polymerase III-1 and M2, and the cellular transcripts β-actin and 18S. No RT and no template controls are indicated. (B) MB114 cells contained viral proteins as far as 12 days post-infection. Immunoblot of lytic viral protein expression. 10 µg of total protein from mock infected and infected MB114 and 3T3 cells and from S11 cells were loaded per lane and blots probed with antibodies to the lytic γHV68 proteins M3 (top) and gB (middle), and mouse β-actin (bottom) as a loading control. Mock infected cells were collected at 24 hours. Latent S11 cells do not express lytic viral proteins and served as a negative control. (C) At the indicated times post-infection, MB114 cells were analyzed for gp150 surface expression by flow cytometry. Fluorescence was determined relative to unstained cells (grey). Histograms are representative of two independent infections. (D) Six days post-infection with GFP-γHV68, MB114 cells were collected and analyzed by flow cytometry for GFP expression. Cells were cultured and brightfield (top) and fluorescent (bottom) images taken at 20 days post-infection, 100X magnification. Fluorescence was determined relative to cells infected with wildtype γHV68 (grey), and histogram is representative of two independent infections.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Flow Cytometry, Cytometry, Fluorescence, Cell Culture

    13) Product Images from "Induction of Hemeoxygenase-1 Reduces Renal Oxidative Stress and Inflammation in Diabetic Spontaneously Hypertensive Rats"

    Article Title: Induction of Hemeoxygenase-1 Reduces Renal Oxidative Stress and Inflammation in Diabetic Spontaneously Hypertensive Rats

    Journal: International Journal of Hypertension

    doi: 10.1155/2012/957235

    Renal cortical HO-1 expression relative to β -actin (a), HO-1 activity (b), and HO-2 expression relative to β -actin (c) in control and diabetic (D) SHR with or without CoPP treatment ( n = 6, *indicates significant difference from control SHR and # indicates significant difference from diabetic SHR).
    Figure Legend Snippet: Renal cortical HO-1 expression relative to β -actin (a), HO-1 activity (b), and HO-2 expression relative to β -actin (c) in control and diabetic (D) SHR with or without CoPP treatment ( n = 6, *indicates significant difference from control SHR and # indicates significant difference from diabetic SHR).

    Techniques Used: Expressing, Activity Assay

    14) Product Images from "Induction of hemeoxygenase-1 reduces glomerular injury and apoptosis in diabetic spontaneously hypertensive rats"

    Article Title: Induction of hemeoxygenase-1 reduces glomerular injury and apoptosis in diabetic spontaneously hypertensive rats

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00472.2011

    Renal cortical hemeoxygenase (HO)-1 ( A ) and HO-2 ( B ) expression relative to β-actin in control and diabetic (D) spontaneously hypertensive rats (SHR) with or without cobalt protoporphyrin (CoPP) treatment ( n = 5). *Significant difference from
    Figure Legend Snippet: Renal cortical hemeoxygenase (HO)-1 ( A ) and HO-2 ( B ) expression relative to β-actin in control and diabetic (D) spontaneously hypertensive rats (SHR) with or without cobalt protoporphyrin (CoPP) treatment ( n = 5). *Significant difference from

    Techniques Used: Expressing

    Glomerular α 3 - and β 1 -integrin protein expression relative to β-actin ( A and B ) and glomerular nephrin staining and protein expression ( C ) in control and diabetic SHR with or without CoPP treatment ( n = 5). Also shown is urinary
    Figure Legend Snippet: Glomerular α 3 - and β 1 -integrin protein expression relative to β-actin ( A and B ) and glomerular nephrin staining and protein expression ( C ) in control and diabetic SHR with or without CoPP treatment ( n = 5). Also shown is urinary

    Techniques Used: Expressing, Staining

    Assessment of ICAM-1 expression relative to β-actin ( A ) and superoxide using dihydroethidine (DHE) staining and lucigenin method ( B and C , respectively) in glomeruli isolated from control and diabetic SHR and incubated with or without CoPP and/or
    Figure Legend Snippet: Assessment of ICAM-1 expression relative to β-actin ( A ) and superoxide using dihydroethidine (DHE) staining and lucigenin method ( B and C , respectively) in glomeruli isolated from control and diabetic SHR and incubated with or without CoPP and/or

    Techniques Used: Expressing, Staining, Isolation, Incubation

    15) Product Images from "The involvement of ATF4 and S-opsin in retinal photoreceptor cell damage induced by blue LED light"

    Article Title: The involvement of ATF4 and S-opsin in retinal photoreceptor cell damage induced by blue LED light

    Journal: Molecular Vision

    doi:

    Effects of S-opsin siRNA on exposure to blue LED light in murine photoreceptor cells. A : Representative immunoblotting images of short-wavelength opsin (S-opsin), ATF4, and β-actin. B : Expression level of S-opsin. C : Expression level of ATF4. Data are presented as the mean ± standard error of the mean (SEM; n=7 to 9). **p
    Figure Legend Snippet: Effects of S-opsin siRNA on exposure to blue LED light in murine photoreceptor cells. A : Representative immunoblotting images of short-wavelength opsin (S-opsin), ATF4, and β-actin. B : Expression level of S-opsin. C : Expression level of ATF4. Data are presented as the mean ± standard error of the mean (SEM; n=7 to 9). **p

    Techniques Used: Expressing

    16) Product Images from "Interplay between SIN3A and STAT3 Mediates Chromatin Conformational Changes and GFAP Expression during Cellular Differentiation"

    Article Title: Interplay between SIN3A and STAT3 Mediates Chromatin Conformational Changes and GFAP Expression during Cellular Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022018

    Differentiation of NTera-2 cells toward an astrocytic lineage. NTera-2 cells were induced to cellular differentiation by infection with lentiviruses engineered to express pFUGW- Oct-4 -RNAi at a multiplicity of infection of 25. A. Construction map of pFUGW- Oct-4 -RNAi. B. Immunofluorescence (red) and Western blots of Oct4 expression in undifferentiated NTera-2 cells and cells 21 days after differentiation. β-Actin was used as an internal control in the Western blots. Note the substantial reduction in Oct-4 expression after differentiation. C. Phase-contrast micrographs for the morphological analysis (upper panel) and immunofluorescence staining for nestin expression (lower panel, red) of NTera-2 cells 3 and 21 days after differentiation, respectively. Magnification 400×. D. Immunofluorescence (red) and Western blots for GFAP expression in undifferentiated NTera-2 cells and NTera-2 cells 21 days after differentiation. E. Immunofluorescence staining and Western blot analyses of glutamine synthetase (GS) expression in NTera-2 cells 21 days after differentiation. The red staining for GS was compared with Hoechst dye (blue) for nuclei. β-actin was used as an internal control in the Western blots.
    Figure Legend Snippet: Differentiation of NTera-2 cells toward an astrocytic lineage. NTera-2 cells were induced to cellular differentiation by infection with lentiviruses engineered to express pFUGW- Oct-4 -RNAi at a multiplicity of infection of 25. A. Construction map of pFUGW- Oct-4 -RNAi. B. Immunofluorescence (red) and Western blots of Oct4 expression in undifferentiated NTera-2 cells and cells 21 days after differentiation. β-Actin was used as an internal control in the Western blots. Note the substantial reduction in Oct-4 expression after differentiation. C. Phase-contrast micrographs for the morphological analysis (upper panel) and immunofluorescence staining for nestin expression (lower panel, red) of NTera-2 cells 3 and 21 days after differentiation, respectively. Magnification 400×. D. Immunofluorescence (red) and Western blots for GFAP expression in undifferentiated NTera-2 cells and NTera-2 cells 21 days after differentiation. E. Immunofluorescence staining and Western blot analyses of glutamine synthetase (GS) expression in NTera-2 cells 21 days after differentiation. The red staining for GS was compared with Hoechst dye (blue) for nuclei. β-actin was used as an internal control in the Western blots.

    Techniques Used: Cell Differentiation, Infection, Immunofluorescence, Western Blot, Expressing, Staining

    Phosphoproteomic signature in cellular differentiation revealed by a label-free quantitation strategy. A. Experiment workflow for quantitation of astrocyte phosphoproteomics. B. Functional annotation chart of the differentially phosphorylated proteins analyzed by DAVID [47] , [48] ; the highly represented categories are shown. Ontology terms are shown on the y axis; p-values for the significance of enrichment are graphed along the x axis. C. Analysis of the regulation of genes through label-free quantitation in NTera-2 cells 3, 7, 14 and 21 days during cell differentiation compared to undifferentiated NTera-2 cells. Data are expressed as log2 of fold change. D. Western blots showing levels of Sin3A expression in undifferentiated NTera-2 cells and NTera-2 cells 14 and 21 days after differentiation. β-actin was used as an internal control in the Western blots.
    Figure Legend Snippet: Phosphoproteomic signature in cellular differentiation revealed by a label-free quantitation strategy. A. Experiment workflow for quantitation of astrocyte phosphoproteomics. B. Functional annotation chart of the differentially phosphorylated proteins analyzed by DAVID [47] , [48] ; the highly represented categories are shown. Ontology terms are shown on the y axis; p-values for the significance of enrichment are graphed along the x axis. C. Analysis of the regulation of genes through label-free quantitation in NTera-2 cells 3, 7, 14 and 21 days during cell differentiation compared to undifferentiated NTera-2 cells. Data are expressed as log2 of fold change. D. Western blots showing levels of Sin3A expression in undifferentiated NTera-2 cells and NTera-2 cells 14 and 21 days after differentiation. β-actin was used as an internal control in the Western blots.

    Techniques Used: Cell Differentiation, Quantitation Assay, Functional Assay, Western Blot, Expressing

    17) Product Images from "Respiratory distress and perinatal lethality in Nedd4-2-deficient mice"

    Article Title: Respiratory distress and perinatal lethality in Nedd4-2-deficient mice

    Journal: Nature Communications

    doi: 10.1038/ncomms1284

    Increased ENaC expression in Nedd4-2 −/− mice. ( a ) α and βENaC expression in E18.5 lung and kidney. ( b ) Quantification of ENaC expression from ( a ) normalized to β-actin and relative to wild type. ( c ) βENaC expression (green) in E18.5 lung and kidney. Scale bars, 50 μm.
    Figure Legend Snippet: Increased ENaC expression in Nedd4-2 −/− mice. ( a ) α and βENaC expression in E18.5 lung and kidney. ( b ) Quantification of ENaC expression from ( a ) normalized to β-actin and relative to wild type. ( c ) βENaC expression (green) in E18.5 lung and kidney. Scale bars, 50 μm.

    Techniques Used: Expressing, Mouse Assay

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    Western Blot:

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    Article Snippet: .. ZEB-1 antibody was from Santa Cruz Biotechnology, Inc. Antibodies against β-actin, and vimentin purchased from Sigma (Saint Louis, MO, USA) and pFAK (Tyr397), total FAK, and E-cadherin from BD Biosciences were used for Western blots according to standard protocol. .. Bound primary antibodies were detected with peroxidase-coupled secondary antibodies (Southern BioTech; Birmingham, AL, USA) and developed by enhanced chemiluminescence (Luminata Classico Western HRP substrate; EMD Millipore Corp.; Billerica, MA, USA).

    Incubation:

    Article Title: Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells
    Article Snippet: .. After incubation with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, the membrane was incubated with primary antibodies (mouse anti-β-catenin (Santa Cruz Biotechnology), mouse anti-EN1 (Abcam, Cambridge, UK), and mouse anti-β-actin (Sigma-Aldrich)) for 1 h at room temperature or overnight at 4 °C. .. The membrane was washed three times with TBS-T and then incubated in a 1:3000 dilution of horseradish peroxidase-conjugated anti-mouse secondary antibody for 30 min at room temperature.

    Article Title: SPARCL1 suppresses metastasis in prostate cancer
    Article Snippet: .. Membranes were incubated with chicken polyclonal antibody specific for human SPARCL1 (Abcam, Cambridge, United Kingdom) at a 1:2000 dilution, and then the membranes were re‐probed using mouse monoclonal antibody specific for human anti‐β‐actin (Sigma Chemical Company, Saint Louis, MO, USA). .. The human gene SPARCL1 ORF (RC207583, OriGene Technologies, Inc, Rockville, MD) was subcloned into pBMN‐I‐GFP (Addgene Inc., Cambridge, MA, USA) to obtain a pBMN‐SPARCL1‐I‐GFP plasmid.

    Article Title: Photodynamic therapy inhibits p-glycoprotein mediated multidrug resistance via JNK activation in human hepatocellular carcinoma using the photosensitizer pheophorbide a
    Article Snippet: .. The membrane was blocked with 10% non-fat milk in Tris buffered saline containing Tween-20 (TBS-T) (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween-20) and then incubated with primary human antibodies against β-actin (Sigma), Bcl-2, JNK, p-JNK, p-ERK, procaspase-3 (Santa Cruz), caspase-9 (Stressgen), and P-glycoprotein (Merck) in TBS-T. After incubation with the secondary antibody conjugated with horseradish peroxidase, immunodetected proteins were visualized by using an enhanced chemiluminescence assay kit (Amersham Life Science). ..

    Article Title: ORP150/HSP12A Regulates Purkinje Cell Survival: A Role for Endoplasmic Reticulum Stress in Cerebellar Development
    Article Snippet: .. Tissue extracts were prepared in PBS containing NP-40 (1%), and proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride paper, followed by incubation with either anti-human ORP150 antibody (1 μg/ml) or anti-human β-actin antibody (1000× dilution; Sigma), the latter as an internal control. .. Where indicated, Western blotting using anti-KDEL monoclonal antibody (Stressgen Biotechnologies Co.; 0.2 μg/ml) was used to assess levels of GRP78 (78 kDa glucose-regulated protein; ).

    Chemiluminescence Immunoassay:

    Article Title: Photodynamic therapy inhibits p-glycoprotein mediated multidrug resistance via JNK activation in human hepatocellular carcinoma using the photosensitizer pheophorbide a
    Article Snippet: .. The membrane was blocked with 10% non-fat milk in Tris buffered saline containing Tween-20 (TBS-T) (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween-20) and then incubated with primary human antibodies against β-actin (Sigma), Bcl-2, JNK, p-JNK, p-ERK, procaspase-3 (Santa Cruz), caspase-9 (Stressgen), and P-glycoprotein (Merck) in TBS-T. After incubation with the secondary antibody conjugated with horseradish peroxidase, immunodetected proteins were visualized by using an enhanced chemiluminescence assay kit (Amersham Life Science). ..

    SDS Page:

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    FLAG-tag:

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    Millipore mouse anti human β actin monoclonal antibody
    Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with <t>anti-β-actin</t> antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.
    Mouse Anti Human β Actin Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of <t>GSK-3β</t> inhibition on the expression, phosphorylation, subcellular localization and co-transcriptional activity of β-catenin in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis was used to compare the expression and phosphorylation of β-catenin between cells treated with DMSO and either GSK-3β inhibitor. Expression of β-actin was monitored as a loading control. ( B ) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of β-catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 μm. The number shown below each panel indicates the percentage of nuclear β-catenin-positive cells among the total number of cells. The bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of β-catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear β-catenin-positive cells in 3 microscopic fields was evaluated with standard deviation. ( C ) Relative co-transcriptional activity of β-catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3β inhibitors.
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    Molecular interactions involving IQGAPs, <t>β-catenin,</t> and E-cadherin. (A) Immunoprecipitations were completed using liver lysates (20 mg of total protein per sample) from 12-month-old wild-type (WT), Iqgap1 −/− , or Iqgap2 −/− mice and a protein G agarose-conjugated anti-β-catenin antibody (5 μg per sample). Complexes were fractionated by 4 to 15% gradient SDS-PAGE and immunodetected using antibodies specific to IQGAP1, IQGAP2, β-catenin, and E-cadherin. Results represent one complete set of experiments reproduced on two different occasions. (B) Immunoprecipitation (IP) experiments using COS1 lysates (20 mg of total protein per sample) transiently expressing enhanced green fluorescent protein (EGFP) as a control or human IQGAP1 or IQGAP2 (24 h posttransfection) were completed using protein G agarose-conjugated antibodies (5 μg per sample) directed against IQGAP1 or IQGAP2. Immunodetection was completed using the same antibodies. An immunoblot of corresponding lysates is shown to document protein expression. Note the high level of endogenous IQGAP1 expression in COS1 cells transfected with enhanced green fluorescent protein, with no evidence of IQGAP1-IQGAP2 complexes. Results represent one complete set of experiments reproduced on three different occasions.
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    Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.

    Journal: PLoS ONE

    Article Title: Ser276 Phosphorylation of NF-kB p65 by MSK1 Controls SCF Expression in Inflammation

    doi: 10.1371/journal.pone.0004393

    Figure Lengend Snippet: Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.

    Article Snippet: Immunoblotting used the following antibodies: rabbit anti-human IκB-α polyclonal antibody (1/1000, Calbiochem, La Jolla, CA), mouse anti-human phospho- IκB-α monoclonal antibody, (1/1000, Ab-1, Oncogene Research Product, Boston, MA), rabbit anti-human phospho-Ser276 p65 antibody (1/1000, 3037, Cell Signaling Technology, Danvers MA), rabbit anti-human phospho-Ser536 p65 antibody (1/1000, 3031, Cell Signaling Technology), rabbit anti-human p65 polyclonal antibody (1/200, sc-109, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human CBP polyclonal antibody (1/200, sc-369, Santa Cruz Biotechnology), mouse anti-human β-actin monoclonal antibody (1/5000, Ab-1, Oncogene Research Product), goat anti-human MSK1 (1/200, sc-9392, Santa Cruz Biotechnology.

    Techniques: Expressing, Transfection, Luciferase, Construct, Incubation, Activity Assay, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

    Effect of GSK-3β inhibition on the expression, phosphorylation, subcellular localization and co-transcriptional activity of β-catenin in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis was used to compare the expression and phosphorylation of β-catenin between cells treated with DMSO and either GSK-3β inhibitor. Expression of β-actin was monitored as a loading control. ( B ) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of β-catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 μm. The number shown below each panel indicates the percentage of nuclear β-catenin-positive cells among the total number of cells. The bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of β-catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear β-catenin-positive cells in 3 microscopic fields was evaluated with standard deviation. ( C ) Relative co-transcriptional activity of β-catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3β inhibitors.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Effect of GSK-3β inhibition on the expression, phosphorylation, subcellular localization and co-transcriptional activity of β-catenin in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis was used to compare the expression and phosphorylation of β-catenin between cells treated with DMSO and either GSK-3β inhibitor. Expression of β-actin was monitored as a loading control. ( B ) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of β-catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 μm. The number shown below each panel indicates the percentage of nuclear β-catenin-positive cells among the total number of cells. The bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of β-catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear β-catenin-positive cells in 3 microscopic fields was evaluated with standard deviation. ( C ) Relative co-transcriptional activity of β-catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3β inhibitors.

    Article Snippet: The effect of siRNA on GSK-3β expression was observed by Western blotting with an antibody that recognizes both GSK-3α and GSK-3β (Millipore) as described above.

    Techniques: Inhibition, Expressing, Activity Assay, Western Blot, Immunofluorescence, Standard Deviation

    Effect of small-molecule GSK-3β inhibitors on the survival of osteosarcoma cells The osteosarcoma cells were treated with DMSO or the indicated concentrations of AR-A014418 or SB-216763 for the designated times. The relative number of viable cells at each time point was measured by the WST-8 assay. Values shown are the means ± SD of six separate experiments.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Effect of small-molecule GSK-3β inhibitors on the survival of osteosarcoma cells The osteosarcoma cells were treated with DMSO or the indicated concentrations of AR-A014418 or SB-216763 for the designated times. The relative number of viable cells at each time point was measured by the WST-8 assay. Values shown are the means ± SD of six separate experiments.

    Article Snippet: The effect of siRNA on GSK-3β expression was observed by Western blotting with an antibody that recognizes both GSK-3α and GSK-3β (Millipore) as described above.

    Techniques:

    Effects of GSK-3β inhibitors on the proliferation and apoptosis of osteosarcoma cells and osteoblasts ( A ) The indicated cells were treated with DMSO or 25 μmol/L each of either AR-A014418 or SB-216763 for 72 hours. The relative number of proliferating cells was evaluated by measuring the amount of BrdU incorporation. ( B ) Relative numbers of TUNEL-positive apoptotic cells were scored for the indicated cells at 12 hours after treatment with DMSO or 25 μmol/L each of either GSK-3β inhibitor. (A, B) Values shown are the means ± SD of six separate experiments. Asterisks denote a statistically-significant difference ( p

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Effects of GSK-3β inhibitors on the proliferation and apoptosis of osteosarcoma cells and osteoblasts ( A ) The indicated cells were treated with DMSO or 25 μmol/L each of either AR-A014418 or SB-216763 for 72 hours. The relative number of proliferating cells was evaluated by measuring the amount of BrdU incorporation. ( B ) Relative numbers of TUNEL-positive apoptotic cells were scored for the indicated cells at 12 hours after treatment with DMSO or 25 μmol/L each of either GSK-3β inhibitor. (A, B) Values shown are the means ± SD of six separate experiments. Asterisks denote a statistically-significant difference ( p

    Article Snippet: The effect of siRNA on GSK-3β expression was observed by Western blotting with an antibody that recognizes both GSK-3α and GSK-3β (Millipore) as described above.

    Techniques: BrdU Incorporation Assay, TUNEL Assay

    Effect of RNA interference on the expression of GSK-3β, cell viability, proliferation and apoptosis in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis compared the level of expression of GSK-3α and GSK-3β between cells treated with non-specific (N) and GSK-3β-specific (S) siRNA (20 nmol/L each), respectively. Expression of β-actin was monitored as a loading control. ( B – D ) Relative number of surviving cells, BrdU-positive proliferating cells and TUNEL-positive apoptotic cells were counted and compared between cell types 96 hours after transfection of non-specific and GSK3β-specific siRNA. Values shown are the mean ± SD of six separate experiments. Asterisks denote a statistically-significant difference between cells transfected with non-specific and GSK- 3β-specific siRNA.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Effect of RNA interference on the expression of GSK-3β, cell viability, proliferation and apoptosis in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis compared the level of expression of GSK-3α and GSK-3β between cells treated with non-specific (N) and GSK-3β-specific (S) siRNA (20 nmol/L each), respectively. Expression of β-actin was monitored as a loading control. ( B – D ) Relative number of surviving cells, BrdU-positive proliferating cells and TUNEL-positive apoptotic cells were counted and compared between cell types 96 hours after transfection of non-specific and GSK3β-specific siRNA. Values shown are the mean ± SD of six separate experiments. Asterisks denote a statistically-significant difference between cells transfected with non-specific and GSK- 3β-specific siRNA.

    Article Snippet: The effect of siRNA on GSK-3β expression was observed by Western blotting with an antibody that recognizes both GSK-3α and GSK-3β (Millipore) as described above.

    Techniques: Expressing, Western Blot, TUNEL Assay, Transfection

    ( A, B ) Efficacy of GSK-3β inhibitors on the size and weight of orthotopic 143B tumors (A) Tumor size was measured weekly and the volume calculated. (B) Mean weight of the tumors removed at necropsy. Asterisks denote a statistically-significant difference in tumor volume and weight compared to mice treated with DMSO. The scatter plots corresponding to the data in (A) and (B) are shown in Supplementary Figure S7 . ( C ) Histological and immunohistochemical findings for orthotopic tumors in mice treated with DMSO or with GSK-3β inhibitors. Representative paraffin-embedded sections of tumors were stained with H E or immunostained for β-catenin. Magnified images of the sections (upper three panels) are shown in Supplementary Figure S8 . Higher resolution versions of the lower six panels are shown in Supplementary Figure S9 . Scale bar in each of the upper three panels indicates 5 mm and that of the middle and lower six panels indicates 100 μm.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: ( A, B ) Efficacy of GSK-3β inhibitors on the size and weight of orthotopic 143B tumors (A) Tumor size was measured weekly and the volume calculated. (B) Mean weight of the tumors removed at necropsy. Asterisks denote a statistically-significant difference in tumor volume and weight compared to mice treated with DMSO. The scatter plots corresponding to the data in (A) and (B) are shown in Supplementary Figure S7 . ( C ) Histological and immunohistochemical findings for orthotopic tumors in mice treated with DMSO or with GSK-3β inhibitors. Representative paraffin-embedded sections of tumors were stained with H E or immunostained for β-catenin. Magnified images of the sections (upper three panels) are shown in Supplementary Figure S8 . Higher resolution versions of the lower six panels are shown in Supplementary Figure S9 . Scale bar in each of the upper three panels indicates 5 mm and that of the middle and lower six panels indicates 100 μm.

    Article Snippet: The effect of siRNA on GSK-3β expression was observed by Western blotting with an antibody that recognizes both GSK-3α and GSK-3β (Millipore) as described above.

    Techniques: Mouse Assay, Immunohistochemistry, Staining

    Expression and phosphorylation of GSK3β in osteosarcoma cells and in untransformed osteoblasts Fractions of phosphorylated GSK-3β (pGSK-3β Y216 : active form; pGSK-3β S9 : inactive form) and total GSK-3β were detected in protein extracts from osteosarcoma and hFOB1.19 cells by Western immunoblotting analysis. The amount of protein extract from each sample was compared to the expression of β-actin.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Expression and phosphorylation of GSK3β in osteosarcoma cells and in untransformed osteoblasts Fractions of phosphorylated GSK-3β (pGSK-3β Y216 : active form; pGSK-3β S9 : inactive form) and total GSK-3β were detected in protein extracts from osteosarcoma and hFOB1.19 cells by Western immunoblotting analysis. The amount of protein extract from each sample was compared to the expression of β-actin.

    Article Snippet: The effect of siRNA on GSK-3β expression was observed by Western blotting with an antibody that recognizes both GSK-3α and GSK-3β (Millipore) as described above.

    Techniques: Expressing, Western Blot

    Molecular interactions involving IQGAPs, β-catenin, and E-cadherin. (A) Immunoprecipitations were completed using liver lysates (20 mg of total protein per sample) from 12-month-old wild-type (WT), Iqgap1 −/− , or Iqgap2 −/− mice and a protein G agarose-conjugated anti-β-catenin antibody (5 μg per sample). Complexes were fractionated by 4 to 15% gradient SDS-PAGE and immunodetected using antibodies specific to IQGAP1, IQGAP2, β-catenin, and E-cadherin. Results represent one complete set of experiments reproduced on two different occasions. (B) Immunoprecipitation (IP) experiments using COS1 lysates (20 mg of total protein per sample) transiently expressing enhanced green fluorescent protein (EGFP) as a control or human IQGAP1 or IQGAP2 (24 h posttransfection) were completed using protein G agarose-conjugated antibodies (5 μg per sample) directed against IQGAP1 or IQGAP2. Immunodetection was completed using the same antibodies. An immunoblot of corresponding lysates is shown to document protein expression. Note the high level of endogenous IQGAP1 expression in COS1 cells transfected with enhanced green fluorescent protein, with no evidence of IQGAP1-IQGAP2 complexes. Results represent one complete set of experiments reproduced on three different occasions.

    Journal: Molecular and Cellular Biology

    Article Title: Development of Hepatocellular Carcinoma in Iqgap2-Deficient Mice Is IQGAP1 Dependent ▿

    doi: 10.1128/MCB.01090-07

    Figure Lengend Snippet: Molecular interactions involving IQGAPs, β-catenin, and E-cadherin. (A) Immunoprecipitations were completed using liver lysates (20 mg of total protein per sample) from 12-month-old wild-type (WT), Iqgap1 −/− , or Iqgap2 −/− mice and a protein G agarose-conjugated anti-β-catenin antibody (5 μg per sample). Complexes were fractionated by 4 to 15% gradient SDS-PAGE and immunodetected using antibodies specific to IQGAP1, IQGAP2, β-catenin, and E-cadherin. Results represent one complete set of experiments reproduced on two different occasions. (B) Immunoprecipitation (IP) experiments using COS1 lysates (20 mg of total protein per sample) transiently expressing enhanced green fluorescent protein (EGFP) as a control or human IQGAP1 or IQGAP2 (24 h posttransfection) were completed using protein G agarose-conjugated antibodies (5 μg per sample) directed against IQGAP1 or IQGAP2. Immunodetection was completed using the same antibodies. An immunoblot of corresponding lysates is shown to document protein expression. Note the high level of endogenous IQGAP1 expression in COS1 cells transfected with enhanced green fluorescent protein, with no evidence of IQGAP1-IQGAP2 complexes. Results represent one complete set of experiments reproduced on three different occasions.

    Article Snippet: To further investigate the age-dependent activation of the Wnt/β-catenin pathway in Iqgap2 −/− livers, liver lysates from 4-month-old wild-type and Iqgap2 −/− mice, as well as those from 2-year-old Iqgap2 −/− mice affected with HCC, were probed with an antibody specifically recognizing the active form of β-catenin, dephoshorylated on Ser37 or Thr41 (Upstate Biotechnology/Millipore).

    Techniques: Mouse Assay, SDS Page, Immunoprecipitation, Expressing, Immunodetection, Transfection

    Molecular analysis of HCC in Iqgap2 −/− mice. (A) Immunohistochemical detection of IQGAP1, β-catenin, and E-cadherin in livers from 4-month-old wild-type or Iqgap2 −/− mice without HCC or 2-year-old Iqgap2 −/− mice with HCC. Note the loss of E-cadherin from cell membranes, along with the enhanced cytoplasmic expression and translocation of both IQGAP1 and β-catenin, occurring only with HCC. Scale bars correspond to 50 μm. (B) Immunoblot analysis was completed using an anti-β-catenin monoclonal antibody and liver lysates (10 μg/lane) from mice (2 years old) with HCC or age-matched controls; the arrow indicates the 92-kDa β-catenin band, with clear evidence for truncated β-catenin mutant forms in the case of HCC. (C) Immunoblot (10 μg of lysate/lane) demonstrating the presence of active (dephosphorylated) β-catenin mutant forms in HCC liver tissue. Results for livers from two 4-month-old wild-type (WT) and Iqgap2 −/− (−/−) mice are also shown. Cell lysate from the A431 cell line (Upstate Biotechnology/Millipore) was used as a positive control. (D) Immunoblot analysis (10 μg of lysate/lane) to assess E-cadherin, cyclin D1, and IQGAP1 expression in Iqgap2 −/− liver lobes without HCC or affected by HCC; paired normal (N) and tumor (T) samples were collected from the same animals. (E) Immunoblot densitometric analysis of E-cadherin, cyclin D1, and IQGAP1 expression was conducted using liver lysates (10 μg/lane) from lobes affected by HCC ( n = 4), those of 4- and 12-month-old Iqgap2 −/− mice without HCC ( n = 3), and those of 4-month-old wild-type mice ( n = 2). Data are presented as mean ratios ± standard deviations of the expression of individual proteins relative to that of proteins in the wild-type samples. (F) IQGAP1 expression was assessed by immunoblotting of liver lysates (10 μg/lane) from wild-type (WT) and Iqgap2 −/− (−/−) mice of various ages (6 weeks and 4, 12, and 24 months). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific antibody served as a control for the loading of equal quantities. A representative immunoblot is shown. (G) Immunoblot densitometric analysis of hepatic IQGAP1 expression in wild-type and Iqgap2 −/− mice of various ages. Data are presented as means ± SEM of the integrated optical densities of bands. n = 3 for each age group.

    Journal: Molecular and Cellular Biology

    Article Title: Development of Hepatocellular Carcinoma in Iqgap2-Deficient Mice Is IQGAP1 Dependent ▿

    doi: 10.1128/MCB.01090-07

    Figure Lengend Snippet: Molecular analysis of HCC in Iqgap2 −/− mice. (A) Immunohistochemical detection of IQGAP1, β-catenin, and E-cadherin in livers from 4-month-old wild-type or Iqgap2 −/− mice without HCC or 2-year-old Iqgap2 −/− mice with HCC. Note the loss of E-cadherin from cell membranes, along with the enhanced cytoplasmic expression and translocation of both IQGAP1 and β-catenin, occurring only with HCC. Scale bars correspond to 50 μm. (B) Immunoblot analysis was completed using an anti-β-catenin monoclonal antibody and liver lysates (10 μg/lane) from mice (2 years old) with HCC or age-matched controls; the arrow indicates the 92-kDa β-catenin band, with clear evidence for truncated β-catenin mutant forms in the case of HCC. (C) Immunoblot (10 μg of lysate/lane) demonstrating the presence of active (dephosphorylated) β-catenin mutant forms in HCC liver tissue. Results for livers from two 4-month-old wild-type (WT) and Iqgap2 −/− (−/−) mice are also shown. Cell lysate from the A431 cell line (Upstate Biotechnology/Millipore) was used as a positive control. (D) Immunoblot analysis (10 μg of lysate/lane) to assess E-cadherin, cyclin D1, and IQGAP1 expression in Iqgap2 −/− liver lobes without HCC or affected by HCC; paired normal (N) and tumor (T) samples were collected from the same animals. (E) Immunoblot densitometric analysis of E-cadherin, cyclin D1, and IQGAP1 expression was conducted using liver lysates (10 μg/lane) from lobes affected by HCC ( n = 4), those of 4- and 12-month-old Iqgap2 −/− mice without HCC ( n = 3), and those of 4-month-old wild-type mice ( n = 2). Data are presented as mean ratios ± standard deviations of the expression of individual proteins relative to that of proteins in the wild-type samples. (F) IQGAP1 expression was assessed by immunoblotting of liver lysates (10 μg/lane) from wild-type (WT) and Iqgap2 −/− (−/−) mice of various ages (6 weeks and 4, 12, and 24 months). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific antibody served as a control for the loading of equal quantities. A representative immunoblot is shown. (G) Immunoblot densitometric analysis of hepatic IQGAP1 expression in wild-type and Iqgap2 −/− mice of various ages. Data are presented as means ± SEM of the integrated optical densities of bands. n = 3 for each age group.

    Article Snippet: To further investigate the age-dependent activation of the Wnt/β-catenin pathway in Iqgap2 −/− livers, liver lysates from 4-month-old wild-type and Iqgap2 −/− mice, as well as those from 2-year-old Iqgap2 −/− mice affected with HCC, were probed with an antibody specifically recognizing the active form of β-catenin, dephoshorylated on Ser37 or Thr41 (Upstate Biotechnology/Millipore).

    Techniques: Mouse Assay, Immunohistochemistry, Expressing, Translocation Assay, Mutagenesis, Positive Control