tween 20 Millipore Search Results


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  • 99
    Millipore immobilon p membrane
    pGEM and pEXI reacted with surface biotinylated PMN and HL-60 cells. PMN or HL-60 cells were lysed with Triton X-100 and mixed with bacteria. After incubation the bacteria were recovered by centrifugation, lysed, the lysates subjected to SDS/PAGE and transferred to <t>Immobilon-P</t> membrane. Biotinylated proteins were detected by staining with extravidin-conjugated peroxidase and chemiluminescence. PMN total lysate (lane 1) and HL-60 cell total lysate (lane 2). Proteins recovered from PMN lysate with pEXI (lane 3) and pGEM (lane 4). Proteins recovered from HL-60 cells lysate with pEXI (lane 5) and pGEM (lane 6). pEXI bind a 30-kDa band specifically from PMN lysates (lane 3). The 23-kDa protein recognized by extravidin is probably the biotin carboxy carrier protein found in E. coli ).
    Immobilon P Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14510 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pbs
    a : Logarithmic conversion of <t>PSA</t> values [ln(1 +PSA)] from intraosseous tumor models originating from C4-2B cells treated invitro with match TEI, mismatch TEI, or <t>PBS</t> control. There is no statistical difference between mismatch TEI, and PBS treated groups ( P -value =1.000). Match TEI treated mice had significantly lower PSA values than PBS, and mismatch TEI combined groups ( P -value 0.0055). Panel b reveals all the three groups, saline control (left), mismatch TEI, and match TEI(right), with SEM bars.
    Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bsa
    SEM images of random <t>PCL</t> nanofibers (A) before and (B–;D) after plasma treatment for 2 min, followed by soaking in (B) PBS, (C) 0.1% <t>BSA</t> solution, and (D) 0.5% BSA solution, respectively, for 1 h. The morphology and texture of the nanofibers are essentially preserved during the treatments.
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 58255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore polysorbate 80
    In vitro  release of plumbagin from oleic acid-based nanoemulsion (polysorbate 80 (3.5%) as a function of time. SGF, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) at 37°C was used as release media.
    Polysorbate 80, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore immobilon p pvdf membranes
    Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to <t>PVDF</t> membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.
    Immobilon P Pvdf Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tris buffered saline
    Analysis of MSNP protein coronas by SDS-PAGE. Notes: MSNPs of different sizes were incubated with cell-culture medium with 5% or 10% serum at <t>37°C</t> for 24 hours. After centrifugation to remove unbound serum proteins, pellets were washed three times and then resuspended in 30 µL water and analyzed by SDS-PAGE on a 12% <t>bis-Tris-glycine</t> gel (Thermo Fisher Scientific), followed by EZ blue staining. Representative gels are shown. ( A ) Protein coronas of MSNPs after incubation in cell-culture medium with 5% serum. Lane 1, aliquot of medium; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, medium alone submitted to centrifugation as negative control; lane 5, molecular weight marker. ( B ) Protein coronas of MSNPs after incubation in cell-culture medium with 10% serum. Lane 1, medium alone submitted to centrifugation as negative control; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, an aliquot of medium; lane 5, molecular weight marker. Abbreviations: MSNP, mesoporous silica nanoparticle; SDS-PAGE, sodium dodecyl sulfate polyacrylamide-gel electrophoresis.
    Tris Buffered Saline, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore triton x 100
    Li AS-A expression and localization in  L .  infantum . A)  AS-A expression in different stages of  L .  infantum  life cycle. Promastigote forms: logarithmic phase (Log), early stationary phase (ES), late stationary phase (LS); axenic amastigote forms (Am). Twenty μg of total extract were analysed by Western-blot and probed with rabbit polyclonal anti- Li AS-A. α-tubulin (mouse monoclonal antibody) was used as loading control. These results are representative of 3 independent experiments.  B)  Immunofluorescence analysis showing AS-A (red upper panel; green lower panel) localization in  L .  infantum  promastigote form. Nucleus and kinetoplast DNA, cytosol and mitochondria were stained with DAPI (blue), sheep anti- Li TDR1 (thiol-dependent reductase in green) and Mitotracker Orange CMTMROS (red), respectively. Images were acquired with a 100x objective, using a Zeiss AxioImager Z1. The scale bar corresponds to 5 μm. Data is representative of 4 independent experiments.  C)  Digitonin fractionation of mid-log  L .  infantum  promastigotes. Pellet (P) and supernatant (S) fractions obtained using increasing concentrations of digitonin or positive control with 1% of Triton X-100 (TR–Total Release) were subjected to Western-blot analysis and probed with antibodies against  Li Enolase (cytosolic marker) and hypoxanthine guanine phosphoribosyltransferase  Li HGPRT (glycosomal marker). Data is representative of 5 independent experiments.
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nitrocellulose membrane
    Li AS-A expression and localization in  L .  infantum . A)  AS-A expression in different stages of  L .  infantum  life cycle. Promastigote forms: logarithmic phase (Log), early stationary phase (ES), late stationary phase (LS); axenic amastigote forms (Am). Twenty μg of total extract were analysed by Western-blot and probed with rabbit polyclonal anti- Li AS-A. α-tubulin (mouse monoclonal antibody) was used as loading control. These results are representative of 3 independent experiments.  B)  Immunofluorescence analysis showing AS-A (red upper panel; green lower panel) localization in  L .  infantum  promastigote form. Nucleus and kinetoplast DNA, cytosol and mitochondria were stained with DAPI (blue), sheep anti- Li TDR1 (thiol-dependent reductase in green) and Mitotracker Orange CMTMROS (red), respectively. Images were acquired with a 100x objective, using a Zeiss AxioImager Z1. The scale bar corresponds to 5 μm. Data is representative of 4 independent experiments.  C)  Digitonin fractionation of mid-log  L .  infantum  promastigotes. Pellet (P) and supernatant (S) fractions obtained using increasing concentrations of digitonin or positive control with 1% of Triton X-100 (TR–Total Release) were subjected to Western-blot analysis and probed with antibodies against  Li Enolase (cytosolic marker) and hypoxanthine guanine phosphoribosyltransferase  Li HGPRT (glycosomal marker). Data is representative of 5 independent experiments.
    Nitrocellulose Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β actin
    KIF14 activates Akt signaling and promotes CRC tumorigenesis in vivo . Alterations in Akt phosphorylation activity due to (A) attenuated or (B) upregulated KIF14 expression in CRC cells were determined by western blotting. <t>β-actin</t> served as an internal control. (C) Transduction efficiency of recombinant lentiviruses KIF14 shRNA was determined by RT-qPCR and western blotting. All values were presented as the mean ± standard deviation (n=3). (D) Resected subcutaneous tumors generated by SW480 cells stably transduced with KIF14 shRNA T460 or the control (n=5). (E) KIF14 expression in xenografts at day 32 was measured by RT-qPCR. (F) Tumor volume was recorded seven times at equal intervals from the 14th day following injection. (G) Weight of resected xenografts was measured. The results from the xenograft tumor model were presented as the mean ± standard deviation (n=5). ** P
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 67284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti beta actin antibody
    A. Invasion of Clone #8 through matrigel, laminin and fibronectin and motility assay . B. Adhesion assay of Clone #8 to matrigel, laminin and fibronectin. C. Anoikis assay. Experiments were performed 48 hours post-transfection with two different exon targeted siRNA integrin <t>Beta</t> 1. Student's t -test; p ≤ 0.05*, 0.01**, 0.005***.
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33089 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sodium dodecyl sulfate polyacrylamide gel electrophoresis
    Effect of NPs on the expression of muscle specific markers in myotubes. Notes: ( A ) Fluorescence images of myotubes after incubation with and without NPs. Red, staining of alpha-actinin characteristic of the sarcomeric organization of myofibers; blue, Hoechst stained-nuclei. Scale bar, 1 μm. ( B ) Expression of muscle-specific markers. After myoblast treatment with NPs, proteins were extracted from myotubes at day 7 of differentiation and subjected to <t>sodium</t> <t>dodecyl</t> <t>sulfate</t> <t>polyacrylamide</t> <t>gel</t> <t>electrophoresis.</t> Immunodetection was carried out using specific antibodies against alpha-actinin, myosin heavy chain, and myogenin. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. ( C ) The level of the different proteins was quantified and expressed as a percentage of the level obtained in the untreated control cells. Bars on the graph represent the standard error of the mean. *Significantly different from the control ( P ≤0.05). Abbreviation: NP, silica nanoparticle.
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15742 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dapi
    Surface coatings: (a) representative CLSM images, taken at different focal planes, of the coated Alg + (i) and Alg c (ii) particles cocultured with the HepG2 cells for 72 h (cell-to-particle ratio 25/1). The scale bars are 50 μm [blue: 6-diamidino-2-phenylindole <t>(DAPI)-stained</t> nuclei; green: fluorescently labeled Alg; and red: <t>phalloidin-stained</t> cytoskeleton]. (b) Cell viability: the viability of the HepG2 cells in the cocultures in comparison to that of a HepG2 monoculture is shown by assessing the activity of cellular dehydrogenase (culture time: 72 h, cell-to-Alg-based particle ratio: 25/1, and n = 3).
    Dapi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 87206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ethylenediaminetetraacetic acid
    Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with <t>ethylenediaminetetraacetic</t> acid shows absence of activity. (C)
    Ethylenediaminetetraacetic Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4351 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sds polyacrylamide gel
    Western blot analyses of freestanding and operon-encoded BioH production. Equal volumes of the freestanding and operon-encoded protein eluates were loaded into each lane of an <t>SDS-polyacrylamide</t> gel. After electrophoresis the proteins were transferred to <t>Immobilon-P</t> and the membranes were subjected to immunoblotting with anti-His 6 tag antibody. Lane 1, the P. aeruginosa bioD::Gm strain expressing chromosomal His 6 -tagged BioH under limited biotin conditions (1.6 nM); Lane 2, the P. aeruginosa bioD::Gm strain expressing chromosomal His 6 -tagged operon-encoded BioH under excess biotin conditions (40 μM); Lane 3, negative control, P. aeruginosa bioD::Gm strain expressing operon-encoded native BioH lacking a His 6 tag under limited biotin conditions (1.6 nM); Lane 4, positive control, purified P. aeruginosa BioH protein with a C-terminal His 6 -tag; L, ladder; Lane 5, positive control, purified E. coli BioH protein with a C-terminal His 6 -tag; Lane 6, negative control, the E. coli ∆bioD strain expressing the native freestanding chromosomal BioH lacking a His 6 tag under limited biotin conditions (1.6 nM); Lane 7, the E. coli ∆bioD strain expressing a chromosomal His 6 -tagged bioH under excess biotin (40 μM) conditions and Lane 8, the E. coli ∆bioD expressing the chromosomal His 6 -tagged BioH under limited biotin conditions (1.6 nM).
    Sds Polyacrylamide Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12897 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Merck KGaA pvdf membrane
    Western blot analyses of freestanding and operon-encoded BioH production. Equal volumes of the freestanding and operon-encoded protein eluates were loaded into each lane of an <t>SDS-polyacrylamide</t> gel. After electrophoresis the proteins were transferred to <t>Immobilon-P</t> and the membranes were subjected to immunoblotting with anti-His 6 tag antibody. Lane 1, the P. aeruginosa bioD::Gm strain expressing chromosomal His 6 -tagged BioH under limited biotin conditions (1.6 nM); Lane 2, the P. aeruginosa bioD::Gm strain expressing chromosomal His 6 -tagged operon-encoded BioH under excess biotin conditions (40 μM); Lane 3, negative control, P. aeruginosa bioD::Gm strain expressing operon-encoded native BioH lacking a His 6 tag under limited biotin conditions (1.6 nM); Lane 4, positive control, purified P. aeruginosa BioH protein with a C-terminal His 6 -tag; L, ladder; Lane 5, positive control, purified E. coli BioH protein with a C-terminal His 6 -tag; Lane 6, negative control, the E. coli ∆bioD strain expressing the native freestanding chromosomal BioH lacking a His 6 tag under limited biotin conditions (1.6 nM); Lane 7, the E. coli ∆bioD strain expressing a chromosomal His 6 -tagged bioH under excess biotin (40 μM) conditions and Lane 8, the E. coli ∆bioD expressing the chromosomal His 6 -tagged BioH under limited biotin conditions (1.6 nM).
    Pvdf Membrane, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 94/100, based on 3071 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tween20
    SWCNT interactions with live cells probed by NIR photoluminescence. ( a ) Bright-field and NIR photoluminescence imaging (inserts) of live cells incubated for 24 h with <t>Tween20-,</t> F108-, or PLPEG-coated SWCNTs and further rinsed before imaging. PLPEG- and F108-coated SWCNTs displayed lower non-specific interactions with live cells compared to Tween20-coated SWCNTs. Scale bars: 25 µm for the bright field images and 10 µm for the magnified NIR photoluminescence images of SWCNTs; ( b ) Corresponding median (red), 25–75th percentile (blue), and 0–100th percentile (black) of the number of SWCNT PL spots observed on live cells for Tween20-, F108- or PLPEG-coated SWCNTs ( N = 70, 53, and 86 cells respectively, n.s.: not significant, ** p
    Tween20, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3363 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti alpha tubulin antibody
    The endosome to lysosome pathway and Lrsam1 mutations in mice. (A) Schematic of the endosome to lysosome pathway. Proteins are endocytosed from the plasma membrane and either recycled back to the membrane or transited through the endosomal pathway. From the late endosome, multivesicular bodies are targeted to the lysosome for degradation. The formation of multivesicular bodies requires the ESCRT complexes for vesicle scission and the proper membrane composition of PI3,5P2. Key proteins that function in this pathway and also cause CMT disease when mutated are shown in bold. (B) Representation of the protein domains of LRSAM1. From the N-terminus to the C-terminus, LRSAM1 contains four leucine-rich repeats (LR), two coil-coiled domains (CC), the <t>sterile-alpha</t> motif (SAM), two PTAP domains and a zinc-finger motif of the RING type (ZnF). (C) Gene-trap alleles of the mouse Lrsam1. Representation of the Lrsam1 gene structure and localization of the RRK239 and RRK461 gene-trap insertions. Exons are represented by black boxes. The diagram is not to scale. The intron sizes and positions of the insertions within the introns are shown. (D) Anti-LRSAM1 western blots on mouse spinal cord lysates and COS7 cells transfected with the Lrsam1 cDNA. Samples were prepared from wild-type, heterozygous and homozygous mice for the RRK461 gene trap insertion. A specific band of the expected size for LRSAM1 (84 kDa) was detected in wild-type samples and this size was consistent with recombinant protein expressed in COS7 cells (rLRSAM1). This band was reduced in heterozygotes and below detection in homozygotes. Blots were probed with either mouse anti-LRSAM1 (left panels) or rabbit anti-LRSAM1 (right panels) with similar results. The anti-mouse secondary antibody also recognized nonspecific immunoglobulin bands from the tissue whereas the rabbit anti-LRSAM1 antibody recognized a number of nonspecific background bands. With longer exposures, higher molecular weight bands were also detected in transfected COS7 cells (right panel, right lane), but not in untransfected cells. GAPDH (left lane) and <t>tubulin</t> were used as loading controls. (E) qRT-PCR on spinal cord extracts with primer pairs recognizing amplicons either upstream (5′) or downstream (3′) of the insertions. Relative transcript abundance in the heterozygotes (HET) and homozygote mutants. (MUT) for Lrsam1 RRK461 compared with wild-type mice (set at 1.0) is indicated. Error bars represent the confidence interval of the fold-change. n =7 for HET and 5 for MUT.
    Monoclonal Anti Alpha Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    pGEM and pEXI reacted with surface biotinylated PMN and HL-60 cells. PMN or HL-60 cells were lysed with Triton X-100 and mixed with bacteria. After incubation the bacteria were recovered by centrifugation, lysed, the lysates subjected to SDS/PAGE and transferred to Immobilon-P membrane. Biotinylated proteins were detected by staining with extravidin-conjugated peroxidase and chemiluminescence. PMN total lysate (lane 1) and HL-60 cell total lysate (lane 2). Proteins recovered from PMN lysate with pEXI (lane 3) and pGEM (lane 4). Proteins recovered from HL-60 cells lysate with pEXI (lane 5) and pGEM (lane 6). pEXI bind a 30-kDa band specifically from PMN lysates (lane 3). The 23-kDa protein recognized by extravidin is probably the biotin carboxy carrier protein found in E. coli ).

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

    Article Title: CGM1a antigen of neutrophils, a receptor of gonococcal opacity proteins

    doi:

    Figure Lengend Snippet: pGEM and pEXI reacted with surface biotinylated PMN and HL-60 cells. PMN or HL-60 cells were lysed with Triton X-100 and mixed with bacteria. After incubation the bacteria were recovered by centrifugation, lysed, the lysates subjected to SDS/PAGE and transferred to Immobilon-P membrane. Biotinylated proteins were detected by staining with extravidin-conjugated peroxidase and chemiluminescence. PMN total lysate (lane 1) and HL-60 cell total lysate (lane 2). Proteins recovered from PMN lysate with pEXI (lane 3) and pGEM (lane 4). Proteins recovered from HL-60 cells lysate with pEXI (lane 5) and pGEM (lane 6). pEXI bind a 30-kDa band specifically from PMN lysates (lane 3). The 23-kDa protein recognized by extravidin is probably the biotin carboxy carrier protein found in E. coli ).

    Article Snippet: The Immobilon-P membrane (Millipore) with transferred biotinylated bacterial–PMN lysates was incubated in 0.05 M Tris at pH 7.4, 150 mM NaCl buffer containing 5% vitamin free-casein (Sigma) for 4 h at room temperature or overnight at 4°C.

    Techniques: Incubation, Centrifugation, SDS Page, Staining

    a : Logarithmic conversion of PSA values [ln(1 +PSA)] from intraosseous tumor models originating from C4-2B cells treated invitro with match TEI, mismatch TEI, or PBS control. There is no statistical difference between mismatch TEI, and PBS treated groups ( P -value =1.000). Match TEI treated mice had significantly lower PSA values than PBS, and mismatch TEI combined groups ( P -value 0.0055). Panel b reveals all the three groups, saline control (left), mismatch TEI, and match TEI(right), with SEM bars.

    Journal: The Prostate

    Article Title: Telomerase Enzyme Inhibition (TEI) and Cytolytic Therapy in the Management of Androgen Independent Osseous Metastatic Prostate Cancer

    doi: 10.1002/pros.21096

    Figure Lengend Snippet: a : Logarithmic conversion of PSA values [ln(1 +PSA)] from intraosseous tumor models originating from C4-2B cells treated invitro with match TEI, mismatch TEI, or PBS control. There is no statistical difference between mismatch TEI, and PBS treated groups ( P -value =1.000). Match TEI treated mice had significantly lower PSA values than PBS, and mismatch TEI combined groups ( P -value 0.0055). Panel b reveals all the three groups, saline control (left), mismatch TEI, and match TEI(right), with SEM bars.

    Article Snippet: Tissue sections with either primary PSA antibody or PBS alone were inoculated at 4°C overnight in a humidified chamber, then washed with 0.05% Tween 20 (Sigma–Aldrich) in PBS 3 times and incubated with biotin-conjugated goat anti-rabbit IgG (Sigma–Aldrich) at room temperature for 60 min. After washing 3 times with 0.05% PBS-Tween 20, VECTOR-STAIN Elite ABC reagent (Vector Laboratories, Burlin-game, CA) was applied on tissues for 30 min at room temperature and brown color positive staining was developed with the Sigma FAST 3-3′-diaminobenzi-dine tablet set at room temperature for 10 min.

    Techniques: Mouse Assay

    Femurs from PBS treated (control), mismatch telomerase inhibitor, which also is another control, and match telomerase inhibitor. Control PBS is the far left column. Mismatch telomerase inhibitor is the middle column, and match telomerase inhibitor is the far right column. All photos were taken at 200× magnification. Row 1 constituting A , B , and C is hematoxylin and eosin stain. Row 2 consisting of D , E , and F is PSA immunohistochemistry stain. Row 3 consisting of G , H , and I is trichrome stain. BM, bone marrow; Tum, tumor. In A and B control treated mice panels, there is obvious bone destruction and C4-2B cells are seen through out the marrow cavity and invading into the bone (arrow). However, panel C, match treated mouse reveals normal bone marrow and normal calcified osseous bone structures. In panels D and E, on the PSA immunohistochemistry stain and adjacent slices, the brown PSA staining is seen throughout the marrow space and within the bone(arrow), in both the control treated PBS and mismatch telomerase in panels D and E, respectively. However, once again, in panel F, no PSA immunostain is seen in the match treated mouse. Lastly, in panels G and H, adjacent sections, control treated animals reveal bone destruction, whereas match treated animals in panel I reveal a normal architecture. This pattern in these panels was consistent in all the respective mice treatment groups.

    Journal: The Prostate

    Article Title: Telomerase Enzyme Inhibition (TEI) and Cytolytic Therapy in the Management of Androgen Independent Osseous Metastatic Prostate Cancer

    doi: 10.1002/pros.21096

    Figure Lengend Snippet: Femurs from PBS treated (control), mismatch telomerase inhibitor, which also is another control, and match telomerase inhibitor. Control PBS is the far left column. Mismatch telomerase inhibitor is the middle column, and match telomerase inhibitor is the far right column. All photos were taken at 200× magnification. Row 1 constituting A , B , and C is hematoxylin and eosin stain. Row 2 consisting of D , E , and F is PSA immunohistochemistry stain. Row 3 consisting of G , H , and I is trichrome stain. BM, bone marrow; Tum, tumor. In A and B control treated mice panels, there is obvious bone destruction and C4-2B cells are seen through out the marrow cavity and invading into the bone (arrow). However, panel C, match treated mouse reveals normal bone marrow and normal calcified osseous bone structures. In panels D and E, on the PSA immunohistochemistry stain and adjacent slices, the brown PSA staining is seen throughout the marrow space and within the bone(arrow), in both the control treated PBS and mismatch telomerase in panels D and E, respectively. However, once again, in panel F, no PSA immunostain is seen in the match treated mouse. Lastly, in panels G and H, adjacent sections, control treated animals reveal bone destruction, whereas match treated animals in panel I reveal a normal architecture. This pattern in these panels was consistent in all the respective mice treatment groups.

    Article Snippet: Tissue sections with either primary PSA antibody or PBS alone were inoculated at 4°C overnight in a humidified chamber, then washed with 0.05% Tween 20 (Sigma–Aldrich) in PBS 3 times and incubated with biotin-conjugated goat anti-rabbit IgG (Sigma–Aldrich) at room temperature for 60 min. After washing 3 times with 0.05% PBS-Tween 20, VECTOR-STAIN Elite ABC reagent (Vector Laboratories, Burlin-game, CA) was applied on tissues for 30 min at room temperature and brown color positive staining was developed with the Sigma FAST 3-3′-diaminobenzi-dine tablet set at room temperature for 10 min.

    Techniques: H&E Stain, Immunohistochemistry, Staining, Mouse Assay

    LNA wing modifications of PS-ONs strongly suppress platelet activation and binding to GPVI and PF4. Increase in platelet activation marker (A) PAC-1 (Glycoprotein IIb/IIIa) and (B) P-selectin, after 10 min incubation with indicated controls (20 μM ADP, 20 μM TRAP) or 10 μM ONs with different length (16mer (AC)8, 18 mer (AC)9 and 20 mer (AC)10) without and with 3 LNA modifications in the flanks. (A) and (B) are box-plots of n = 5 or n = 10 individual data points as indicated with the bars showing the median value. Dotted lines represent the 95% ULN confidence interval of unstimulated platelets. (C) Binding ONs of different length ((AC)8-10 without and with 3 LNA modifications in the flanks to His -tagged GPVI protein captured on SPR chip via anti-His antibodies. Data are means with error bars demonstrating data range from two replicates. (D) Activity of ONs of different length ((AC)8-10 without and with 3 LNA modifications in the flanks in the PF4 ELISA expressed as % maximal heparin activity. Data represent means ±SD (n = 4). (E) Changes in platelet activation markers PAC-1 and P-selectin induced by controls (ADP, TRAP and negative control (PBS)) or AC11 (white bars) and in the presence of 5 μM Bay 61–3606 (grey bars) or 5 μM Src inhibitor PP2 (black bars). Data are means ±SD (n = 5).

    Journal: PLoS ONE

    Article Title: Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia

    doi: 10.1371/journal.pone.0187574

    Figure Lengend Snippet: LNA wing modifications of PS-ONs strongly suppress platelet activation and binding to GPVI and PF4. Increase in platelet activation marker (A) PAC-1 (Glycoprotein IIb/IIIa) and (B) P-selectin, after 10 min incubation with indicated controls (20 μM ADP, 20 μM TRAP) or 10 μM ONs with different length (16mer (AC)8, 18 mer (AC)9 and 20 mer (AC)10) without and with 3 LNA modifications in the flanks. (A) and (B) are box-plots of n = 5 or n = 10 individual data points as indicated with the bars showing the median value. Dotted lines represent the 95% ULN confidence interval of unstimulated platelets. (C) Binding ONs of different length ((AC)8-10 without and with 3 LNA modifications in the flanks to His -tagged GPVI protein captured on SPR chip via anti-His antibodies. Data are means with error bars demonstrating data range from two replicates. (D) Activity of ONs of different length ((AC)8-10 without and with 3 LNA modifications in the flanks in the PF4 ELISA expressed as % maximal heparin activity. Data represent means ±SD (n = 4). (E) Changes in platelet activation markers PAC-1 and P-selectin induced by controls (ADP, TRAP and negative control (PBS)) or AC11 (white bars) and in the presence of 5 μM Bay 61–3606 (grey bars) or 5 μM Src inhibitor PP2 (black bars). Data are means ±SD (n = 5).

    Article Snippet: Briefly, microtiter plates (Maxisorp 96well, Nunc #442404) were coated with human recombinant PF4 (10 μg/ml, ProSpec-Tany TechnoGene, Rehovot, Israel) in phosphate buffered saline (PBS) for 2h followed by the addition of either vehicle, heparin (0.002–200 μg/ml, Sigma-Aldrich, Darmstadt, Germany) or ONs (0.001–3 μM).

    Techniques: Activation Assay, Binding Assay, Marker, Incubation, SPR Assay, Chromatin Immunoprecipitation, Activity Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    SEM images of random PCL nanofibers (A) before and (B–;D) after plasma treatment for 2 min, followed by soaking in (B) PBS, (C) 0.1% BSA solution, and (D) 0.5% BSA solution, respectively, for 1 h. The morphology and texture of the nanofibers are essentially preserved during the treatments.

    Journal: Journal of materials chemistry. B, Materials for biology and medicine

    Article Title: A General Strategy for Generating Gradients of Bioactive Proteins on Electrospun Nanofiber Mats by Masking with Bovine Serum Albumin

    doi: 10.1039/C7TB00974G

    Figure Lengend Snippet: SEM images of random PCL nanofibers (A) before and (B–;D) after plasma treatment for 2 min, followed by soaking in (B) PBS, (C) 0.1% BSA solution, and (D) 0.5% BSA solution, respectively, for 1 h. The morphology and texture of the nanofibers are essentially preserved during the treatments.

    Article Snippet: BSA, BSA labeled with fluorescein (BSA-FITC), polycaprolactone (PCL, 80,000 g/mol in average molecular weight), formaldehyde, pyridine, formic acid, dimethylformamide (DMF), dichloromethane (DCM), phosphate-buffered saline (PBS), Tween 20, and anti-neurofilament 200 were all purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques:

    (A) Plots of the relative fluorescence intensities for the BSA-FITC adsorbed on the nanofibers as a function of soaking time in the 0.1% and 0.5% BSA solutions, respectively. The corresponding representative fluorescence micrographs of the nanofibers are also included (upper row: 0.1% BSA, and lower row: 0.5%). The extent of BSA adsorbed on PCL nanofibers is dependent on the duration of exposure to BSA solution (n=3 for each time point). The BSA concentration also affects the rate at which BSA adsorbs onto the nanofibers. (B) Representative fluorescence micrographs and their corresponding relative fluorescence intensities at different positions of nanofiber strips by varying the duration of contact time with a 0.1% BSA solution over a distance of 35 mm (n=9 at each position). A gradient in BSA can be clearly seen across the strip of nanofibers.

    Journal: Journal of materials chemistry. B, Materials for biology and medicine

    Article Title: A General Strategy for Generating Gradients of Bioactive Proteins on Electrospun Nanofiber Mats by Masking with Bovine Serum Albumin

    doi: 10.1039/C7TB00974G

    Figure Lengend Snippet: (A) Plots of the relative fluorescence intensities for the BSA-FITC adsorbed on the nanofibers as a function of soaking time in the 0.1% and 0.5% BSA solutions, respectively. The corresponding representative fluorescence micrographs of the nanofibers are also included (upper row: 0.1% BSA, and lower row: 0.5%). The extent of BSA adsorbed on PCL nanofibers is dependent on the duration of exposure to BSA solution (n=3 for each time point). The BSA concentration also affects the rate at which BSA adsorbs onto the nanofibers. (B) Representative fluorescence micrographs and their corresponding relative fluorescence intensities at different positions of nanofiber strips by varying the duration of contact time with a 0.1% BSA solution over a distance of 35 mm (n=9 at each position). A gradient in BSA can be clearly seen across the strip of nanofibers.

    Article Snippet: BSA, BSA labeled with fluorescein (BSA-FITC), polycaprolactone (PCL, 80,000 g/mol in average molecular weight), formaldehyde, pyridine, formic acid, dimethylformamide (DMF), dichloromethane (DCM), phosphate-buffered saline (PBS), Tween 20, and anti-neurofilament 200 were all purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Fluorescence, Concentration Assay, Stripping Membranes

    Inhibitory effects of peptides on the SARS-CoV S protein and ACE2 interaction by competitive biotinylated ELISA. Biotin-labeled S protein (1 nmol) was mixed with 10 nmol of synthetic peptides or BSA and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were added to wells, which were coated with 1 ng of ACE2 and incubated at 37 °C for 1 h. Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. The results are expressed as inhibition described in Section 2 . Values are mean ± standard error of six independent assays. ** p

    Journal: Antiviral Research

    Article Title: Design and biological activities of novel inhibitory peptides for SARS-CoV spike protein and angiotensin-converting enzyme 2 interaction

    doi: 10.1016/j.antiviral.2005.10.005

    Figure Lengend Snippet: Inhibitory effects of peptides on the SARS-CoV S protein and ACE2 interaction by competitive biotinylated ELISA. Biotin-labeled S protein (1 nmol) was mixed with 10 nmol of synthetic peptides or BSA and incubated at 37 °C with shaking. After a 2-h incubation, the mixtures were added to wells, which were coated with 1 ng of ACE2 and incubated at 37 °C for 1 h. Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. The results are expressed as inhibition described in Section 2 . Values are mean ± standard error of six independent assays. ** p

    Article Snippet: 2.4 Biotinylated enzyme-linked immunosorbent assay (ELISA) Microtiter plates (MaxiSorp Nunc-Immum™ plates, Nunc, Denmark) were coated at 4 °C overnight with 50 μl of spike, ACE2 (R & D Systems, Minneapolis, MN, USA), or bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA), which was diluted in 0.05 M carbonate buffer (16 mM Na2 CO2 , 34 mM NaHCO3 , pH 9.6).

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Incubation, Avidin-Biotin Assay, Inhibition

    Analysis of SARS-CoV S protein and ACE2 interaction by biotinylated ELISA. The wells were coated with 1 ng of ACE2 and challenged with various amounts of biotin-labeled S protein (●), biotin-labeled BSA (○) or biotin (▴). Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. Values are mean ± standard error of four independent assays.

    Journal: Antiviral Research

    Article Title: Design and biological activities of novel inhibitory peptides for SARS-CoV spike protein and angiotensin-converting enzyme 2 interaction

    doi: 10.1016/j.antiviral.2005.10.005

    Figure Lengend Snippet: Analysis of SARS-CoV S protein and ACE2 interaction by biotinylated ELISA. The wells were coated with 1 ng of ACE2 and challenged with various amounts of biotin-labeled S protein (●), biotin-labeled BSA (○) or biotin (▴). Following three washes, peroxidase-conjugated avidin and chromatic substrate were sequentially added. The absorbance was read at 405 nm in an ELISA plate reader. Values are mean ± standard error of four independent assays.

    Article Snippet: 2.4 Biotinylated enzyme-linked immunosorbent assay (ELISA) Microtiter plates (MaxiSorp Nunc-Immum™ plates, Nunc, Denmark) were coated at 4 °C overnight with 50 μl of spike, ACE2 (R & D Systems, Minneapolis, MN, USA), or bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA), which was diluted in 0.05 M carbonate buffer (16 mM Na2 CO2 , 34 mM NaHCO3 , pH 9.6).

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Avidin-Biotin Assay

    In vitro  release of plumbagin from oleic acid-based nanoemulsion (polysorbate 80 (3.5%) as a function of time. SGF, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) at 37°C was used as release media.

    Journal: BioMed Research International

    Article Title: Plumbagin-Loaded Nanoemulsion Drug Delivery Formulation and Evaluation of Antiproliferative Effect on Prostate Cancer Cells

    doi: 10.1155/2018/9035452

    Figure Lengend Snippet: In vitro release of plumbagin from oleic acid-based nanoemulsion (polysorbate 80 (3.5%) as a function of time. SGF, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) at 37°C was used as release media.

    Article Snippet: Plumbagin, polysorbate 80, oleic acid and all other chemicals were supplied by Sigma (St. Louis, MO, USA) unless otherwise stated.

    Techniques: In Vitro

    Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae *

    doi: 10.1074/jbc.M114.633131

    Figure Lengend Snippet: Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP. Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting. Mitochondria (25 μg of protein) ( M ) and 2.5% of the first anti-PC elution ( E1 ) were analyzed for each of the two strains.

    Article Snippet: Protein samples incubated with SDS sample buffer (50 m m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 1.33% β-mercaptoethanol) were separated on 12% Tris-glycine SDS-polyacrylamide gels by electrophoresis ( ) followed by transfer to Immobilon-P PVDF membranes (Millipore) at 100 V for 1.5 h. Membranes were then blocked overnight in 3% nonfat milk, phosphate-buffered saline (140.7 m m NaCl, 9.3 m m Na2 HPO4 , pH 7.4), 0.1% Tween 20.

    Techniques: Purification, Affinity Purification, SDS Page

    CNAP-tagged Coq proteins co-precipitate several other Coq proteins. W303, CNAP3, CNAP6, and CNAP9 purified mitochondria (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification with Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting with antisera to the designated yeast polypeptides. 25 μg of mitochondria protein were analyzed for each strain ( M ), and 2.5% of the first anti-PC elution ( E1 ) volume was loaded per strain (25 μl). Arrows denote each tagged protein in their respective blots. The predominant band in the Coq3 blot detected in the mitochondrial samples represents a background protein and not Coq3, accounting for its presence in CNAP3 mitochondria.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae *

    doi: 10.1074/jbc.M114.633131

    Figure Lengend Snippet: CNAP-tagged Coq proteins co-precipitate several other Coq proteins. W303, CNAP3, CNAP6, and CNAP9 purified mitochondria (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification with Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting with antisera to the designated yeast polypeptides. 25 μg of mitochondria protein were analyzed for each strain ( M ), and 2.5% of the first anti-PC elution ( E1 ) volume was loaded per strain (25 μl). Arrows denote each tagged protein in their respective blots. The predominant band in the Coq3 blot detected in the mitochondrial samples represents a background protein and not Coq3, accounting for its presence in CNAP3 mitochondria.

    Article Snippet: Protein samples incubated with SDS sample buffer (50 m m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 1.33% β-mercaptoethanol) were separated on 12% Tris-glycine SDS-polyacrylamide gels by electrophoresis ( ) followed by transfer to Immobilon-P PVDF membranes (Millipore) at 100 V for 1.5 h. Membranes were then blocked overnight in 3% nonfat milk, phosphate-buffered saline (140.7 m m NaCl, 9.3 m m Na2 HPO4 , pH 7.4), 0.1% Tween 20.

    Techniques: Purification, Affinity Purification, SDS Page

    Pilot scale lunasin purification. A) Flow diagram of the optimized lunasin purification method. (B) Coomassie-stained SDS-PAGE gel of protein samples representing each stage of the pilot-scale purification. SDS-PAGE using a 15% Tris-glycine gel and diluted samples of clarified extract (1∶20), Q anion-exchange fraction (1∶40), UF permeate (1∶20), and RPC fraction (1∶40). Synthetic lunasin (500 ng) was loaded as a positive control. Molecular weight standards (M) are shown in the first lane. (C) Immunoblot analysis of protein samples representing each stage of pilot-scale purification. Proteins separated by SDS-PAGE as described for (B) were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody. Lunasin was detected in all the samples as a band with an apparent molecular weight of ∼5 kDa. (D) Coomassie-stained SDS-PAGE gel of final RPC-purified lunasin product. SDS-PAGE was performed on a 15% gel using 10 µg of RPC-purified lunasin. Molecular weight standards (M) are shown in the first lane.

    Journal: PLoS ONE

    Article Title: Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean

    doi: 10.1371/journal.pone.0035409

    Figure Lengend Snippet: Pilot scale lunasin purification. A) Flow diagram of the optimized lunasin purification method. (B) Coomassie-stained SDS-PAGE gel of protein samples representing each stage of the pilot-scale purification. SDS-PAGE using a 15% Tris-glycine gel and diluted samples of clarified extract (1∶20), Q anion-exchange fraction (1∶40), UF permeate (1∶20), and RPC fraction (1∶40). Synthetic lunasin (500 ng) was loaded as a positive control. Molecular weight standards (M) are shown in the first lane. (C) Immunoblot analysis of protein samples representing each stage of pilot-scale purification. Proteins separated by SDS-PAGE as described for (B) were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody. Lunasin was detected in all the samples as a band with an apparent molecular weight of ∼5 kDa. (D) Coomassie-stained SDS-PAGE gel of final RPC-purified lunasin product. SDS-PAGE was performed on a 15% gel using 10 µg of RPC-purified lunasin. Molecular weight standards (M) are shown in the first lane.

    Article Snippet: Proteins were transferred to Immobilon-P 0.45 um PVDF membranes (Millipore, Billerica, MA, USA) at 20 V for 90 min at 4°C.

    Techniques: Purification, Flow Cytometry, Staining, SDS Page, Positive Control, Molecular Weight

    Reversed-phase chromatography (RPC) method development. Bench-scale RPC was performed using a 1.6×8.0 cm Source15RPC column that was sanitized with 1 N NaOH and equilibrated with ten CV of Buffer A (75.5 mM sodium phosphate/68.4 mM NaCl, pH 7.4) prior to sample load. The ultrafiltration (UF) permeate was brought to a final concentration of 1 M ammonium sulfate and then applied to the column followed by a five CV wash with Buffer A. Bound proteins were eluted using a five-step gradient consisting of 20%, 40%, 60%, 80%, and 100% Buffer B (64.2 mM sodium phosphate/58 mM NaCl/15% n-propanol (v/v)). Each gradient step was approximately five CV except the 100% B step which was ten CV. The column was then stripped using 65% n-propanol. (A) RPC of the UF permeate. Letters with arrows represent beginning of (a) sample load, (b) column wash, and (c) column strip. The presence of lunasin in each sample was determined by ELISA and SDS-PAGE. Lunasin was detected primarily within the 100% B elution fraction. Chromatogram shows the A 280 (solid line ______ ), the A 215 ( .__. __. ), percent Buffer B ( _ _ _ _ _ ), and the percent maximum lunasin content as determined by ELISA (-------). (B) Coomassie-stained SDS-PAGE gel of RPC fractions. SDS-PAGE using a 15% Tris-glycine gel was performed on 1∶10 dilution and 1∶4 dilutions of the UF permeate and column strip, respectively, and undiluted samples from the column flow through and Buffer B step gradient fractions. Molecular weight standards (MW Std) are shown in the first lane. The majority of lunasin ( > 95%) was detected in the 100% B eluate, with minor amounts detected in the 80% B eluate and in the column strip. The major contaminating ∼9 kDa protein was detected exclusively in the column strip. (C) Immunoblot analysis of the UF permeate, column flow through, step gradient fractions, and column strip. Proteins separated by SDS-PAGE were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody. For SDS-PAGE, dilutions of the UF permeate (1∶10), 80% B eluate (1∶4), 100% B eluate (1∶4), and column strip (1∶4) were made. All other samples were undiluted. The position of the 4 kDa and 6 kDa molecular weight standards (MW Std) are shown in the first lane.

    Journal: PLoS ONE

    Article Title: Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean

    doi: 10.1371/journal.pone.0035409

    Figure Lengend Snippet: Reversed-phase chromatography (RPC) method development. Bench-scale RPC was performed using a 1.6×8.0 cm Source15RPC column that was sanitized with 1 N NaOH and equilibrated with ten CV of Buffer A (75.5 mM sodium phosphate/68.4 mM NaCl, pH 7.4) prior to sample load. The ultrafiltration (UF) permeate was brought to a final concentration of 1 M ammonium sulfate and then applied to the column followed by a five CV wash with Buffer A. Bound proteins were eluted using a five-step gradient consisting of 20%, 40%, 60%, 80%, and 100% Buffer B (64.2 mM sodium phosphate/58 mM NaCl/15% n-propanol (v/v)). Each gradient step was approximately five CV except the 100% B step which was ten CV. The column was then stripped using 65% n-propanol. (A) RPC of the UF permeate. Letters with arrows represent beginning of (a) sample load, (b) column wash, and (c) column strip. The presence of lunasin in each sample was determined by ELISA and SDS-PAGE. Lunasin was detected primarily within the 100% B elution fraction. Chromatogram shows the A 280 (solid line ______ ), the A 215 ( .__. __. ), percent Buffer B ( _ _ _ _ _ ), and the percent maximum lunasin content as determined by ELISA (-------). (B) Coomassie-stained SDS-PAGE gel of RPC fractions. SDS-PAGE using a 15% Tris-glycine gel was performed on 1∶10 dilution and 1∶4 dilutions of the UF permeate and column strip, respectively, and undiluted samples from the column flow through and Buffer B step gradient fractions. Molecular weight standards (MW Std) are shown in the first lane. The majority of lunasin ( > 95%) was detected in the 100% B eluate, with minor amounts detected in the 80% B eluate and in the column strip. The major contaminating ∼9 kDa protein was detected exclusively in the column strip. (C) Immunoblot analysis of the UF permeate, column flow through, step gradient fractions, and column strip. Proteins separated by SDS-PAGE were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody. For SDS-PAGE, dilutions of the UF permeate (1∶10), 80% B eluate (1∶4), 100% B eluate (1∶4), and column strip (1∶4) were made. All other samples were undiluted. The position of the 4 kDa and 6 kDa molecular weight standards (MW Std) are shown in the first lane.

    Article Snippet: Proteins were transferred to Immobilon-P 0.45 um PVDF membranes (Millipore, Billerica, MA, USA) at 20 V for 90 min at 4°C.

    Techniques: Reversed-phase Chromatography, Concentration Assay, Stripping Membranes, Enzyme-linked Immunosorbent Assay, SDS Page, Staining, Flow Cytometry, Molecular Weight

    SDS-PAGE and immunoblot analysis of anion-exchanged purified lunasin. Aliquots of samples corresponding to the bench-scale anion-exchange chromatography method where lunasin was eluted using a step gradient ( Figure 1C ) were subjected to SDS-PAGE and immunoblot analysis. (A) SDS-PAGE of the clarified extract, column flow through (Q flow through), and the 30% Buffer B elution (Q 30% B Fraction). Clarified extract, Q flow through, and Q-30%B fraction were prepared at dilutions of 1∶8, 1∶8, and 1∶10, respectively, and electrophoresed using 15% Tris-glycine gels. Molecular weight standards (MW Std) are shown in the first lane. (B) Immunoblot analysis of the clarified extract, Q flow through, and the Q 30% B Fraction. Proteins were separated by SDS-PAGE as described in (A), transferred to a PVDF membrane, and probed with a lunasin-specific mouse monoclonal antibody. For SDS-PAGE, clarified extract, Q flow through, and Q-30% B fraction were prepared at dilutions of 1∶20, 1∶20, and 1∶40, respectively. Molecular weight standards (MW Std) are shown in the first lane.

    Journal: PLoS ONE

    Article Title: Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean

    doi: 10.1371/journal.pone.0035409

    Figure Lengend Snippet: SDS-PAGE and immunoblot analysis of anion-exchanged purified lunasin. Aliquots of samples corresponding to the bench-scale anion-exchange chromatography method where lunasin was eluted using a step gradient ( Figure 1C ) were subjected to SDS-PAGE and immunoblot analysis. (A) SDS-PAGE of the clarified extract, column flow through (Q flow through), and the 30% Buffer B elution (Q 30% B Fraction). Clarified extract, Q flow through, and Q-30%B fraction were prepared at dilutions of 1∶8, 1∶8, and 1∶10, respectively, and electrophoresed using 15% Tris-glycine gels. Molecular weight standards (MW Std) are shown in the first lane. (B) Immunoblot analysis of the clarified extract, Q flow through, and the Q 30% B Fraction. Proteins were separated by SDS-PAGE as described in (A), transferred to a PVDF membrane, and probed with a lunasin-specific mouse monoclonal antibody. For SDS-PAGE, clarified extract, Q flow through, and Q-30% B fraction were prepared at dilutions of 1∶20, 1∶20, and 1∶40, respectively. Molecular weight standards (MW Std) are shown in the first lane.

    Article Snippet: Proteins were transferred to Immobilon-P 0.45 um PVDF membranes (Millipore, Billerica, MA, USA) at 20 V for 90 min at 4°C.

    Techniques: SDS Page, Purification, Chromatography, Flow Cytometry, Molecular Weight

    Quantitation of lunasin and lunasin complex in clarified extracts. White flake (120 g) was extracted with 1.5 L of 75.5 mM sodium phosphate/150 mM NaCl/20 mM ascorbic acid/10 mM sodium metabisulfite, pH 7.4 for one hour. A clarified extract was produced by treating the initial extract with Celpure P100 and filtration using one micron M-503 filter pads. (A) SDS-PAGE analysis of reduced and non-reduced clarified extract. The clarified extract was diluted 1∶10 and analyzed by SDS-PAGE using a 15% Tris-glycine gel under standard reducing and non-reducing (without BME in the sample buffer) conditions. Molecular weight standards (MW Std) are shown in the first lane. The arrow indicates a ∼5 kDa band that corresponds to lunasin. The lack of a clear lunasin band in the sample analyzed under non-reducing conditions indicates that most of the lunasin present in the clarified extract is in protein complexes stabilized by disulfide bridges. (B) Immunoblot analysis of reduced and non-reduced clarified extract. Clarified extract was diluted 1∶10 and subjected to SDS-PAGE along with a series of synthetic lunasin standards as described in (A). The separated proteins were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody diluted 1∶100,000. The amount of lunasin present was determined by image analysis using the synthetic lunasin band intensities to generate a standard curve. This analysis demonstrated that

    Journal: PLoS ONE

    Article Title: Scalable Purification and Characterization of the Anticancer Lunasin Peptide from Soybean

    doi: 10.1371/journal.pone.0035409

    Figure Lengend Snippet: Quantitation of lunasin and lunasin complex in clarified extracts. White flake (120 g) was extracted with 1.5 L of 75.5 mM sodium phosphate/150 mM NaCl/20 mM ascorbic acid/10 mM sodium metabisulfite, pH 7.4 for one hour. A clarified extract was produced by treating the initial extract with Celpure P100 and filtration using one micron M-503 filter pads. (A) SDS-PAGE analysis of reduced and non-reduced clarified extract. The clarified extract was diluted 1∶10 and analyzed by SDS-PAGE using a 15% Tris-glycine gel under standard reducing and non-reducing (without BME in the sample buffer) conditions. Molecular weight standards (MW Std) are shown in the first lane. The arrow indicates a ∼5 kDa band that corresponds to lunasin. The lack of a clear lunasin band in the sample analyzed under non-reducing conditions indicates that most of the lunasin present in the clarified extract is in protein complexes stabilized by disulfide bridges. (B) Immunoblot analysis of reduced and non-reduced clarified extract. Clarified extract was diluted 1∶10 and subjected to SDS-PAGE along with a series of synthetic lunasin standards as described in (A). The separated proteins were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody diluted 1∶100,000. The amount of lunasin present was determined by image analysis using the synthetic lunasin band intensities to generate a standard curve. This analysis demonstrated that

    Article Snippet: Proteins were transferred to Immobilon-P 0.45 um PVDF membranes (Millipore, Billerica, MA, USA) at 20 V for 90 min at 4°C.

    Techniques: Quantitation Assay, Produced, Filtration, SDS Page, Molecular Weight

    Analysis of MSNP protein coronas by SDS-PAGE. Notes: MSNPs of different sizes were incubated with cell-culture medium with 5% or 10% serum at 37°C for 24 hours. After centrifugation to remove unbound serum proteins, pellets were washed three times and then resuspended in 30 µL water and analyzed by SDS-PAGE on a 12% bis-Tris-glycine gel (Thermo Fisher Scientific), followed by EZ blue staining. Representative gels are shown. ( A ) Protein coronas of MSNPs after incubation in cell-culture medium with 5% serum. Lane 1, aliquot of medium; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, medium alone submitted to centrifugation as negative control; lane 5, molecular weight marker. ( B ) Protein coronas of MSNPs after incubation in cell-culture medium with 10% serum. Lane 1, medium alone submitted to centrifugation as negative control; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, an aliquot of medium; lane 5, molecular weight marker. Abbreviations: MSNP, mesoporous silica nanoparticle; SDS-PAGE, sodium dodecyl sulfate polyacrylamide-gel electrophoresis.

    Journal: International Journal of Nanomedicine

    Article Title: Mesoporous silica nanoparticles trigger mitophagy in endothelial cells and perturb neuronal network activity in a size- and time-dependent manner

    doi: 10.2147/IJN.S127663

    Figure Lengend Snippet: Analysis of MSNP protein coronas by SDS-PAGE. Notes: MSNPs of different sizes were incubated with cell-culture medium with 5% or 10% serum at 37°C for 24 hours. After centrifugation to remove unbound serum proteins, pellets were washed three times and then resuspended in 30 µL water and analyzed by SDS-PAGE on a 12% bis-Tris-glycine gel (Thermo Fisher Scientific), followed by EZ blue staining. Representative gels are shown. ( A ) Protein coronas of MSNPs after incubation in cell-culture medium with 5% serum. Lane 1, aliquot of medium; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, medium alone submitted to centrifugation as negative control; lane 5, molecular weight marker. ( B ) Protein coronas of MSNPs after incubation in cell-culture medium with 10% serum. Lane 1, medium alone submitted to centrifugation as negative control; lane 2, protein coronas of 30 nm MSNPs; lane 3, protein coronas of 250 nm MSNPs; lane 4, an aliquot of medium; lane 5, molecular weight marker. Abbreviations: MSNP, mesoporous silica nanoparticle; SDS-PAGE, sodium dodecyl sulfate polyacrylamide-gel electrophoresis.

    Article Snippet: To detect β-actin and caspase 3, the membrane was blocked for 30 minutes at 37°C in Tris-buffered saline containing 0.1% Tween and 5% nonfat milk and incubated overnight at 4°C with anti-β-actin (1:1,500; Sigma-Aldrich) or anti-caspase 3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA).

    Techniques: SDS Page, Incubation, Cell Culture, Centrifugation, Staining, Negative Control, Molecular Weight, Marker, Polyacrylamide Gel Electrophoresis

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

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0004365

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

    Article Snippet: Chemicals and reagents L-asparagine, L-aspartic acid sodium salt monohydrate, L-glutamine, L-glutamatic acid salt hydrate, ATP disodium salt hydrate, AMP disodium salt, sodium pyrophosphate decahydrate, ninhydrin, dNTPs, ammonium chloride, magnesium chloride, tween-20, tris-base, urea, thiourea, DTT, triton X-100 and IPTG (isopropyl-β-D- thiogalactopyranoside) were purchased from Sigma.

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

    KIF14 activates Akt signaling and promotes CRC tumorigenesis in vivo . Alterations in Akt phosphorylation activity due to (A) attenuated or (B) upregulated KIF14 expression in CRC cells were determined by western blotting. β-actin served as an internal control. (C) Transduction efficiency of recombinant lentiviruses KIF14 shRNA was determined by RT-qPCR and western blotting. All values were presented as the mean ± standard deviation (n=3). (D) Resected subcutaneous tumors generated by SW480 cells stably transduced with KIF14 shRNA T460 or the control (n=5). (E) KIF14 expression in xenografts at day 32 was measured by RT-qPCR. (F) Tumor volume was recorded seven times at equal intervals from the 14th day following injection. (G) Weight of resected xenografts was measured. The results from the xenograft tumor model were presented as the mean ± standard deviation (n=5). ** P

    Journal: International Journal of Oncology

    Article Title: KIF14 promotes cell proliferation via activation of Akt and is directly targeted by miR-200c in colorectal cancer

    doi: 10.3892/ijo.2018.4546

    Figure Lengend Snippet: KIF14 activates Akt signaling and promotes CRC tumorigenesis in vivo . Alterations in Akt phosphorylation activity due to (A) attenuated or (B) upregulated KIF14 expression in CRC cells were determined by western blotting. β-actin served as an internal control. (C) Transduction efficiency of recombinant lentiviruses KIF14 shRNA was determined by RT-qPCR and western blotting. All values were presented as the mean ± standard deviation (n=3). (D) Resected subcutaneous tumors generated by SW480 cells stably transduced with KIF14 shRNA T460 or the control (n=5). (E) KIF14 expression in xenografts at day 32 was measured by RT-qPCR. (F) Tumor volume was recorded seven times at equal intervals from the 14th day following injection. (G) Weight of resected xenografts was measured. The results from the xenograft tumor model were presented as the mean ± standard deviation (n=5). ** P

    Article Snippet: Following blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against KIF14 (polyclonal, rabbit anti-human IgG; 1:1,000; cat. no. ab3746; Abcam, Cambridge, MA, USA), protein kinase B (Akt; polyclonal, rabbit anti-human IgG; 1:1,000; cat. no. 9272s; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated-Akt (polyclonal, rabbit anti-human IgG; 1:200; cat. no. 9271s; Cell Signaling Technology, Inc.) or β-actin (monoclonal, mouse anti-human IgG; 1:3,000; cat. no. AC-15; Sigma- Aldrich; Merck KGaA, Darmstadt, Germany).

    Techniques: In Vivo, Activity Assay, Expressing, Western Blot, Transduction, Recombinant, shRNA, Quantitative RT-PCR, Standard Deviation, Generated, Stable Transfection, Injection

    miR-200c modulates KIF14 expression most potently. (A) miR-200c, miR-200b and miR-429 expression levels were detected in five colorectal cancer cell lines by RT-qPCR. (B) Expression of miR-200c, miR-200b and miR-429 was measured by RT-qPCR in SW480 and RKO cells transfected with KIF14 siRNA or the control. (C) miR-200c mimics or inhibitor was transfected into SW480, RKO or LoVo cells respectively, and the transfection efficiency was determined by RT-qPCR. Effects of miR-200c (D) mimics or (E) inhibitor on KIF14 protein expression levels and the phosphorylation levels of Akt were examined by western blotting. β-actin served as an internal control. RT-qPCR was used to detect the alteration of KIF14 mRNA expression. (F) Schematic diagram of miR-200c binding sites in the 3′UTR of KIF14 from microRNA.org. (G) Left panel, construction of KIF14 luciferase reporter plasmids harboring the wild-type, single mutated or double mutated miR-200c response element. Right panel, the relative luciferase activity was measured in HEK293 cells co-transfected with reporter plasmids containing the wild-type or mutant KIF14 3′UTR sequences and miR-200c mimics/mi-NC. All data were expressed as the mean ± standard deviation (n=3). * P

    Journal: International Journal of Oncology

    Article Title: KIF14 promotes cell proliferation via activation of Akt and is directly targeted by miR-200c in colorectal cancer

    doi: 10.3892/ijo.2018.4546

    Figure Lengend Snippet: miR-200c modulates KIF14 expression most potently. (A) miR-200c, miR-200b and miR-429 expression levels were detected in five colorectal cancer cell lines by RT-qPCR. (B) Expression of miR-200c, miR-200b and miR-429 was measured by RT-qPCR in SW480 and RKO cells transfected with KIF14 siRNA or the control. (C) miR-200c mimics or inhibitor was transfected into SW480, RKO or LoVo cells respectively, and the transfection efficiency was determined by RT-qPCR. Effects of miR-200c (D) mimics or (E) inhibitor on KIF14 protein expression levels and the phosphorylation levels of Akt were examined by western blotting. β-actin served as an internal control. RT-qPCR was used to detect the alteration of KIF14 mRNA expression. (F) Schematic diagram of miR-200c binding sites in the 3′UTR of KIF14 from microRNA.org. (G) Left panel, construction of KIF14 luciferase reporter plasmids harboring the wild-type, single mutated or double mutated miR-200c response element. Right panel, the relative luciferase activity was measured in HEK293 cells co-transfected with reporter plasmids containing the wild-type or mutant KIF14 3′UTR sequences and miR-200c mimics/mi-NC. All data were expressed as the mean ± standard deviation (n=3). * P

    Article Snippet: Following blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against KIF14 (polyclonal, rabbit anti-human IgG; 1:1,000; cat. no. ab3746; Abcam, Cambridge, MA, USA), protein kinase B (Akt; polyclonal, rabbit anti-human IgG; 1:1,000; cat. no. 9272s; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated-Akt (polyclonal, rabbit anti-human IgG; 1:200; cat. no. 9271s; Cell Signaling Technology, Inc.) or β-actin (monoclonal, mouse anti-human IgG; 1:3,000; cat. no. AC-15; Sigma- Aldrich; Merck KGaA, Darmstadt, Germany).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Binding Assay, Luciferase, Activity Assay, Mutagenesis, Standard Deviation

    A. Invasion of Clone #8 through matrigel, laminin and fibronectin and motility assay . B. Adhesion assay of Clone #8 to matrigel, laminin and fibronectin. C. Anoikis assay. Experiments were performed 48 hours post-transfection with two different exon targeted siRNA integrin Beta 1. Student's t -test; p ≤ 0.05*, 0.01**, 0.005***.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Alterations in integrin expression modulates invasion of pancreatic cancer cells

    doi: 10.1186/1756-9966-28-140

    Figure Lengend Snippet: A. Invasion of Clone #8 through matrigel, laminin and fibronectin and motility assay . B. Adhesion assay of Clone #8 to matrigel, laminin and fibronectin. C. Anoikis assay. Experiments were performed 48 hours post-transfection with two different exon targeted siRNA integrin Beta 1. Student's t -test; p ≤ 0.05*, 0.01**, 0.005***.

    Article Snippet: Beta-actin was used as loading control (Sigma, A5441).

    Techniques: Motility Assay, Cell Adhesion Assay, Transfection

    Immunoblot of A. Integrin β1 B. Integrin α5 C. Integrin α6 and D. β-actin used as loading control in MiaPaCa-2, Clone #3 and Clone #8 . E. Knockdown of integrin β1 in Clone #8 cells 48 hours post transfection (siRNAs ITGβ1 #1 and #2). F. Knockdown of integrin α5 in Clone #8 cells 48 hours post transfection (siRNAs ITGα5 #1, #2). G. Knockdown of integrin α6 in Clone #8 cells 48 hours post transfection (siRNAs ITGα6 #1 and #2). H. Beta-actin used as loading control.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Alterations in integrin expression modulates invasion of pancreatic cancer cells

    doi: 10.1186/1756-9966-28-140

    Figure Lengend Snippet: Immunoblot of A. Integrin β1 B. Integrin α5 C. Integrin α6 and D. β-actin used as loading control in MiaPaCa-2, Clone #3 and Clone #8 . E. Knockdown of integrin β1 in Clone #8 cells 48 hours post transfection (siRNAs ITGβ1 #1 and #2). F. Knockdown of integrin α5 in Clone #8 cells 48 hours post transfection (siRNAs ITGα5 #1, #2). G. Knockdown of integrin α6 in Clone #8 cells 48 hours post transfection (siRNAs ITGα6 #1 and #2). H. Beta-actin used as loading control.

    Article Snippet: Beta-actin was used as loading control (Sigma, A5441).

    Techniques: Transfection

    TXNIP is highly expressed in DTC cell lines and low or undetectable in ATC cell lines, and glucose uptake is inversely proportional to TXNIP expression levels. (A) HTh74 cells were transduced with lentivirus expressing a PPARγ-specific shRNA or scrambled control, and stable pools were generated under antibiotic selection. Western blot analysis of nuclear lysates (top immunoblot) reveals PPARγ expression levels in the transduced cells. Using Western blot analysis of whole cell lysates, TXNIP and β-actin (loading control) were detected using specific antibodies. Nonspecific band is indicated by “ns”. (B) Western blot analysis for TXNIP, Trx-1, and β-actin (loading control) were performed on whole cell lysates prepared from a panel of DTC and ATC cell lines grown under standard conditions. (C) Glucose uptake assays were performed. Each condition was performed in triplicate per experiment, and each experiment was performed at least three times. Nonspecific glucose uptake as determined by parallel treatment of a subset with cytochalasin B was subtracted from measurements. Data from all experiments were combined, and glucose uptake from each cell line was normalized to levels of HTh74 (average set at 1). Normalized averages are plotted, and error bars indicate SEM. ***p

    Journal: Molecular Cancer

    Article Title: Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer

    doi: 10.1186/1476-4598-13-62

    Figure Lengend Snippet: TXNIP is highly expressed in DTC cell lines and low or undetectable in ATC cell lines, and glucose uptake is inversely proportional to TXNIP expression levels. (A) HTh74 cells were transduced with lentivirus expressing a PPARγ-specific shRNA or scrambled control, and stable pools were generated under antibiotic selection. Western blot analysis of nuclear lysates (top immunoblot) reveals PPARγ expression levels in the transduced cells. Using Western blot analysis of whole cell lysates, TXNIP and β-actin (loading control) were detected using specific antibodies. Nonspecific band is indicated by “ns”. (B) Western blot analysis for TXNIP, Trx-1, and β-actin (loading control) were performed on whole cell lysates prepared from a panel of DTC and ATC cell lines grown under standard conditions. (C) Glucose uptake assays were performed. Each condition was performed in triplicate per experiment, and each experiment was performed at least three times. Nonspecific glucose uptake as determined by parallel treatment of a subset with cytochalasin B was subtracted from measurements. Data from all experiments were combined, and glucose uptake from each cell line was normalized to levels of HTh74 (average set at 1). Normalized averages are plotted, and error bars indicate SEM. ***p

    Article Snippet: Primary antibodies used for the current studies include anti-VDUP1 (TXNIP) at 1:500 (rabbit polyclonal; Invitrogen, catalog # 403700), anti-PPARγ at 1:500 (rabbit polyclonal; Santa Cruz Biotechnology, catalog # sc-7196), anti-thioredoxin-1 at 1:1,000 (C63C6 rabbit monoclonal; Cell Signaling Technology, catalog #2429), and anti-β-actin at 1:5,000 (mouse monoclonal; Sigma-Aldrich, catalog # A5441).

    Techniques: Expressing, Transduction, shRNA, Generated, Selection, Western Blot

    TXNIP overexpression in ATC HTh74 and T238 cells attenuates glucose uptake. HTh74 and T238 cells were transduced with retrovirus encoding human TXNIP or vector control as well as a selectable antibiotic resistance marker, and stable pools were generated under antibiotic selection. Western blot analysis of whole cell lysates with TXNIP- and β-actin-specific antibodies is shown for HTh74 (A) and T238 (B) . Glucose uptake assays were performed as described in Figure 1 using the HTh74 stable cell lines (C) and T238 stable cell lines (D) . Data from all experiments were combined, and glucose uptake from each cell line was normalized to vector control levels (average set at 1). **p = 0.001, ***p

    Journal: Molecular Cancer

    Article Title: Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer

    doi: 10.1186/1476-4598-13-62

    Figure Lengend Snippet: TXNIP overexpression in ATC HTh74 and T238 cells attenuates glucose uptake. HTh74 and T238 cells were transduced with retrovirus encoding human TXNIP or vector control as well as a selectable antibiotic resistance marker, and stable pools were generated under antibiotic selection. Western blot analysis of whole cell lysates with TXNIP- and β-actin-specific antibodies is shown for HTh74 (A) and T238 (B) . Glucose uptake assays were performed as described in Figure 1 using the HTh74 stable cell lines (C) and T238 stable cell lines (D) . Data from all experiments were combined, and glucose uptake from each cell line was normalized to vector control levels (average set at 1). **p = 0.001, ***p

    Article Snippet: Primary antibodies used for the current studies include anti-VDUP1 (TXNIP) at 1:500 (rabbit polyclonal; Invitrogen, catalog # 403700), anti-PPARγ at 1:500 (rabbit polyclonal; Santa Cruz Biotechnology, catalog # sc-7196), anti-thioredoxin-1 at 1:1,000 (C63C6 rabbit monoclonal; Cell Signaling Technology, catalog #2429), and anti-β-actin at 1:5,000 (mouse monoclonal; Sigma-Aldrich, catalog # A5441).

    Techniques: Over Expression, Transduction, Plasmid Preparation, Marker, Generated, Selection, Western Blot, Stable Transfection

    Effect of NPs on the expression of muscle specific markers in myotubes. Notes: ( A ) Fluorescence images of myotubes after incubation with and without NPs. Red, staining of alpha-actinin characteristic of the sarcomeric organization of myofibers; blue, Hoechst stained-nuclei. Scale bar, 1 μm. ( B ) Expression of muscle-specific markers. After myoblast treatment with NPs, proteins were extracted from myotubes at day 7 of differentiation and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunodetection was carried out using specific antibodies against alpha-actinin, myosin heavy chain, and myogenin. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. ( C ) The level of the different proteins was quantified and expressed as a percentage of the level obtained in the untreated control cells. Bars on the graph represent the standard error of the mean. *Significantly different from the control ( P ≤0.05). Abbreviation: NP, silica nanoparticle.

    Journal: International Journal of Nanomedicine

    Article Title: Internalization and fate of silica nanoparticles in C2C12 skeletal muscle cells: evidence of a beneficial effect on myoblast fusion

    doi: 10.2147/IJN.S74158

    Figure Lengend Snippet: Effect of NPs on the expression of muscle specific markers in myotubes. Notes: ( A ) Fluorescence images of myotubes after incubation with and without NPs. Red, staining of alpha-actinin characteristic of the sarcomeric organization of myofibers; blue, Hoechst stained-nuclei. Scale bar, 1 μm. ( B ) Expression of muscle-specific markers. After myoblast treatment with NPs, proteins were extracted from myotubes at day 7 of differentiation and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunodetection was carried out using specific antibodies against alpha-actinin, myosin heavy chain, and myogenin. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. ( C ) The level of the different proteins was quantified and expressed as a percentage of the level obtained in the untreated control cells. Bars on the graph represent the standard error of the mean. *Significantly different from the control ( P ≤0.05). Abbreviation: NP, silica nanoparticle.

    Article Snippet: After treatment with 5× sodium dodecyl sulfate loading buffer, 50 μg protein was resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore) by the semidry transfer method.

    Techniques: Expressing, Fluorescence, Incubation, Staining, Polyacrylamide Gel Electrophoresis, Immunodetection

    Cdk1 phosphorylates sororin. ( A ). Each of these nine sites has a serine or threonine followed by proline (minimal Cdk consensus), whereas sites marked with an asterisk conform to the full Cdk consensus ([S/T]Px[K/R]). RDLEM is the potential CIM. Below the wild-type sororin are the various truncation and multi-site mutants of sororin that were generated. In addition, a single mutant at each of the nine sites was generated (not shown). ( B ) Phosphorylation of sororin by Cdk1 in vitro. Recombinant Cdk1–cyclin B1 was mixed with GST–sororin and a phosphorylation reaction was carried out in vitro with [ 32 P]ATP. Reactions were separated by SDS-PAGE and analyzed by autoradiography. ( C ) Effect of serine to alanine mutations on sororin phosphorylation. Recombinant sororin WT or sororin 9A with an N-terminal GST tag was phosphorylated in an in vitro reaction with Cdk1–cyclin B1 and [ 32 P]ATP. Reactions were analyzed by autoradiography with histone H1 serving as a positive control. CBB, Coomassie-Brilliant-Blue-stained gels.

    Journal: Journal of Cell Science

    Article Title: Regulation of sororin by Cdk1-mediated phosphorylation

    doi: 10.1242/jcs.085431

    Figure Lengend Snippet: Cdk1 phosphorylates sororin. ( A ). Each of these nine sites has a serine or threonine followed by proline (minimal Cdk consensus), whereas sites marked with an asterisk conform to the full Cdk consensus ([S/T]Px[K/R]). RDLEM is the potential CIM. Below the wild-type sororin are the various truncation and multi-site mutants of sororin that were generated. In addition, a single mutant at each of the nine sites was generated (not shown). ( B ) Phosphorylation of sororin by Cdk1 in vitro. Recombinant Cdk1–cyclin B1 was mixed with GST–sororin and a phosphorylation reaction was carried out in vitro with [ 32 P]ATP. Reactions were separated by SDS-PAGE and analyzed by autoradiography. ( C ) Effect of serine to alanine mutations on sororin phosphorylation. Recombinant sororin WT or sororin 9A with an N-terminal GST tag was phosphorylated in an in vitro reaction with Cdk1–cyclin B1 and [ 32 P]ATP. Reactions were analyzed by autoradiography with histone H1 serving as a positive control. CBB, Coomassie-Brilliant-Blue-stained gels.

    Article Snippet: Proteins were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the gels were transferred to polyvinylidene difluoride membranes (Millipore).

    Techniques: Generated, Mutagenesis, In Vitro, Recombinant, SDS Page, Autoradiography, Positive Control, Staining

    Surface coatings: (a) representative CLSM images, taken at different focal planes, of the coated Alg + (i) and Alg c (ii) particles cocultured with the HepG2 cells for 72 h (cell-to-particle ratio 25/1). The scale bars are 50 μm [blue: 6-diamidino-2-phenylindole (DAPI)-stained nuclei; green: fluorescently labeled Alg; and red: phalloidin-stained cytoskeleton]. (b) Cell viability: the viability of the HepG2 cells in the cocultures in comparison to that of a HepG2 monoculture is shown by assessing the activity of cellular dehydrogenase (culture time: 72 h, cell-to-Alg-based particle ratio: 25/1, and n = 3).

    Journal: ACS Omega

    Article Title: Planar and Cell Aggregate-Like Assemblies Consisting of Microreactors and HepG2 Cells

    doi: 10.1021/acsomega.7b01234

    Figure Lengend Snippet: Surface coatings: (a) representative CLSM images, taken at different focal planes, of the coated Alg + (i) and Alg c (ii) particles cocultured with the HepG2 cells for 72 h (cell-to-particle ratio 25/1). The scale bars are 50 μm [blue: 6-diamidino-2-phenylindole (DAPI)-stained nuclei; green: fluorescently labeled Alg; and red: phalloidin-stained cytoskeleton]. (b) Cell viability: the viability of the HepG2 cells in the cocultures in comparison to that of a HepG2 monoculture is shown by assessing the activity of cellular dehydrogenase (culture time: 72 h, cell-to-Alg-based particle ratio: 25/1, and n = 3).

    Article Snippet: Materials Sodium Alg, PLL (molecular weight of 40–60 kDa), sodium chloride (NaCl), calcium chloride (CaCl2 ), tris(hydroxymethyl)aminomethane (TRIS), HEPES, sodium bicarbonate (NaHCO3 ), Triton X-100, Span 80, TWEEN 80, 1 H,1 H,2 H,2 H-perfluorooctyltriethoxysilane (PFOTES), acetic acid, ethanol, chloroform (purity of ≥99.5%), phalloidin tetramethylrhodamine B isothiocyanate (phalloidin), DAPI, primary antibody monoclonal antivinculin, FDA and propidium iodide (PI), catalase from bovine liver (10 000 units mg–1 solid, 240 kDa), from horseradish peroxidase (HRP, 250–330 units mg–1 solid), cell counting kit-8 (CCK-8), and hydrogen peroxide (H2 O2 , 30 w/w %) were purchased from Sigma-Aldrich.

    Techniques: Confocal Laser Scanning Microscopy, Staining, Labeling, Activity Assay

    (a) Representative CLSM images, taken at different focal planes, of Alg c cocultured with the HepG2 cells for 7 d (i) and 10 d (ii) (cell-to-Alg c ratio 25/1). The scale bars are 50 μm (blue: DAPI-stained nuclei; green: fluorescently labeled Alg; and red: phalloidin-stained cytoskeleton). (b) Viability of the HepG2 cells in the cocultures in comparison to that of a HepG2 monoculture is shown by assessing the activity of cellular dehydrogenase (culture time: 7 and 10 d, cell-to-Alg c ratio: 25/1, and n = 3). (c) dsDNA quantification of HepG2 cell/Alg c cocultures (cell-to-Alg c ratios: 25/1 and 10/1) in comparison to cells only after 3 d, 5 d, 7 d, 10 d, and 13 d ( n = 3).

    Journal: ACS Omega

    Article Title: Planar and Cell Aggregate-Like Assemblies Consisting of Microreactors and HepG2 Cells

    doi: 10.1021/acsomega.7b01234

    Figure Lengend Snippet: (a) Representative CLSM images, taken at different focal planes, of Alg c cocultured with the HepG2 cells for 7 d (i) and 10 d (ii) (cell-to-Alg c ratio 25/1). The scale bars are 50 μm (blue: DAPI-stained nuclei; green: fluorescently labeled Alg; and red: phalloidin-stained cytoskeleton). (b) Viability of the HepG2 cells in the cocultures in comparison to that of a HepG2 monoculture is shown by assessing the activity of cellular dehydrogenase (culture time: 7 and 10 d, cell-to-Alg c ratio: 25/1, and n = 3). (c) dsDNA quantification of HepG2 cell/Alg c cocultures (cell-to-Alg c ratios: 25/1 and 10/1) in comparison to cells only after 3 d, 5 d, 7 d, 10 d, and 13 d ( n = 3).

    Article Snippet: Materials Sodium Alg, PLL (molecular weight of 40–60 kDa), sodium chloride (NaCl), calcium chloride (CaCl2 ), tris(hydroxymethyl)aminomethane (TRIS), HEPES, sodium bicarbonate (NaHCO3 ), Triton X-100, Span 80, TWEEN 80, 1 H,1 H,2 H,2 H-perfluorooctyltriethoxysilane (PFOTES), acetic acid, ethanol, chloroform (purity of ≥99.5%), phalloidin tetramethylrhodamine B isothiocyanate (phalloidin), DAPI, primary antibody monoclonal antivinculin, FDA and propidium iodide (PI), catalase from bovine liver (10 000 units mg–1 solid, 240 kDa), from horseradish peroxidase (HRP, 250–330 units mg–1 solid), cell counting kit-8 (CCK-8), and hydrogen peroxide (H2 O2 , 30 w/w %) were purchased from Sigma-Aldrich.

    Techniques: Confocal Laser Scanning Microscopy, Staining, Labeling, Activity Assay

    Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with ethylenediaminetetraacetic acid shows absence of activity. (C)

    Journal: Tissue Engineering. Part A

    Article Title: Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue

    doi: 10.1089/ten.tea.2018.0036

    Figure Lengend Snippet: Matrix metalloproteinase (MMP) gene expression and protein activity. (A) RNA-seq showed MMP2 was not differentially regulated but MMP14 was significantly upregulated. (B) On zymography, bands corresponding to pro MMP2 and active MMP2 were seen. The Std lane corresponds to recombinant MMP2. Inhibition with ethylenediaminetetraacetic acid shows absence of activity. (C)

    Article Snippet: Ethylenediaminetetraacetic acid (15 mM) was used for positive control of MMP inhibition and recombinant MMP2 (MilliporeSigma, Burlington, MA) was used as a positive control.

    Techniques: Expressing, Activity Assay, RNA Sequencing Assay, Zymography, Recombinant, Inhibition

    Western blot analyses of freestanding and operon-encoded BioH production. Equal volumes of the freestanding and operon-encoded protein eluates were loaded into each lane of an SDS-polyacrylamide gel. After electrophoresis the proteins were transferred to Immobilon-P and the membranes were subjected to immunoblotting with anti-His 6 tag antibody. Lane 1, the P. aeruginosa bioD::Gm strain expressing chromosomal His 6 -tagged BioH under limited biotin conditions (1.6 nM); Lane 2, the P. aeruginosa bioD::Gm strain expressing chromosomal His 6 -tagged operon-encoded BioH under excess biotin conditions (40 μM); Lane 3, negative control, P. aeruginosa bioD::Gm strain expressing operon-encoded native BioH lacking a His 6 tag under limited biotin conditions (1.6 nM); Lane 4, positive control, purified P. aeruginosa BioH protein with a C-terminal His 6 -tag; L, ladder; Lane 5, positive control, purified E. coli BioH protein with a C-terminal His 6 -tag; Lane 6, negative control, the E. coli ∆bioD strain expressing the native freestanding chromosomal BioH lacking a His 6 tag under limited biotin conditions (1.6 nM); Lane 7, the E. coli ∆bioD strain expressing a chromosomal His 6 -tagged bioH under excess biotin (40 μM) conditions and Lane 8, the E. coli ∆bioD expressing the chromosomal His 6 -tagged BioH under limited biotin conditions (1.6 nM).

    Journal: Scientific Reports

    Article Title: Expression and Activity of the BioH Esterase of Biotin Synthesis is Independent of Genome Context

    doi: 10.1038/s41598-017-01490-0

    Figure Lengend Snippet: Western blot analyses of freestanding and operon-encoded BioH production. Equal volumes of the freestanding and operon-encoded protein eluates were loaded into each lane of an SDS-polyacrylamide gel. After electrophoresis the proteins were transferred to Immobilon-P and the membranes were subjected to immunoblotting with anti-His 6 tag antibody. Lane 1, the P. aeruginosa bioD::Gm strain expressing chromosomal His 6 -tagged BioH under limited biotin conditions (1.6 nM); Lane 2, the P. aeruginosa bioD::Gm strain expressing chromosomal His 6 -tagged operon-encoded BioH under excess biotin conditions (40 μM); Lane 3, negative control, P. aeruginosa bioD::Gm strain expressing operon-encoded native BioH lacking a His 6 tag under limited biotin conditions (1.6 nM); Lane 4, positive control, purified P. aeruginosa BioH protein with a C-terminal His 6 -tag; L, ladder; Lane 5, positive control, purified E. coli BioH protein with a C-terminal His 6 -tag; Lane 6, negative control, the E. coli ∆bioD strain expressing the native freestanding chromosomal BioH lacking a His 6 tag under limited biotin conditions (1.6 nM); Lane 7, the E. coli ∆bioD strain expressing a chromosomal His 6 -tagged bioH under excess biotin (40 μM) conditions and Lane 8, the E. coli ∆bioD expressing the chromosomal His 6 -tagged BioH under limited biotin conditions (1.6 nM).

    Article Snippet: Equal volumes of soluble proteins were loaded and separated on a 12% SDS-polyacrylamide gel and transferred by electrophoresis to Immobilon-P membranes (Millipore) for 15 min at 15 V. The membranes were preblocked with TBS buffer (100 mM Tris base and 0.9% NaCl, pH 7.5) containing 0.1% Tween 20 and 5% nonfat milk powder.

    Techniques: Western Blot, Electrophoresis, Expressing, Negative Control, Positive Control, Purification

    Upregulation of cellular RNR expression and activity by MCMV. (A) Cellular R1 and R2 levels during MCMV infection. NIH 3T3 cells were growth-arrested in 0.5% calf serum and then either infected with active or UV-irradiated MCMV (MOI, 5 PFU/cell) or mock-infected. Whole-cell extracts were prepared at various times after infection, separated by SDS-PAGE, and analyzed by immunoblotting with the anti-R1, anti-R2, and anti-actin antibodies. A sample from quiescent cells stimulated with 10% calf serum for 24 h was also included. Lanes: 1, mock infection; 2, 6 hpi; 3, 12 hpi; 4, 24 hpi; 5, 36 hpi; 6, 48 hpi; 7, 60 hpi; 8, UV-inactivated MCMV 48 hpi; 9, noninfected cells grown in the presence of 10% serum. (B) Effect of MCMV infection on dNTP pool sizes in resting cells. NIH 3T3 cells were growth arrested in 0.5% calf serum and then infected with active or UV-inactivated MCMV (MOI, 5 PFU/cell). After virus adsorption, cells were either treated with 0.5 mM HU or left untreated. The nucleotides were extracted at 48 hpi, and their levels were measured by high-performance liquid chromatography. Levels of nucleotide pools are expressed as percentages of the total nucleoside triphosphate pool (CTP + UTP + ATP + GTP + dCTP + dTTP + dATP + dGTP) to minimize variations due to small differences in cell number in the samples.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: Upregulation of cellular RNR expression and activity by MCMV. (A) Cellular R1 and R2 levels during MCMV infection. NIH 3T3 cells were growth-arrested in 0.5% calf serum and then either infected with active or UV-irradiated MCMV (MOI, 5 PFU/cell) or mock-infected. Whole-cell extracts were prepared at various times after infection, separated by SDS-PAGE, and analyzed by immunoblotting with the anti-R1, anti-R2, and anti-actin antibodies. A sample from quiescent cells stimulated with 10% calf serum for 24 h was also included. Lanes: 1, mock infection; 2, 6 hpi; 3, 12 hpi; 4, 24 hpi; 5, 36 hpi; 6, 48 hpi; 7, 60 hpi; 8, UV-inactivated MCMV 48 hpi; 9, noninfected cells grown in the presence of 10% serum. (B) Effect of MCMV infection on dNTP pool sizes in resting cells. NIH 3T3 cells were growth arrested in 0.5% calf serum and then infected with active or UV-inactivated MCMV (MOI, 5 PFU/cell). After virus adsorption, cells were either treated with 0.5 mM HU or left untreated. The nucleotides were extracted at 48 hpi, and their levels were measured by high-performance liquid chromatography. Levels of nucleotide pools are expressed as percentages of the total nucleoside triphosphate pool (CTP + UTP + ATP + GTP + dCTP + dTTP + dATP + dGTP) to minimize variations due to small differences in cell number in the samples.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: Expressing, Activity Assay, Infection, Irradiation, SDS Page, Adsorption, High Performance Liquid Chromatography

    (A) Time course of M45 expression during MCMV replication as determined by immunoblotting. Whole-cell lysates of mock-infected and MCMV-infected NIH 3T3 cells at various times after infection were separated by SDS-PAGE, transferred to a membrane, and probed with the anti-M45 antiserum or with the anti-actin monoclonal antibody. Lanes: 1, mock-infected cells; 2, 6 hpi; 3, 9 hpi; 4, 12 hpi; 5, 18 hpi; 6, 24 hpi; 7, 36 hpi; 8, 48 hpi. (B) Effect of PFA on M45 expression. Whole-cell extracts were prepared at 18 hpi (lanes 1 and 2), 24 hpi (lanes 3 and 4), or 48 hpi (lanes 5 and 6) from MCMV-infected NIH 3T3 cells treated with PFA (250 μg/ml) after virus adsorption (lanes 2, 4, and 6) or left untreated (lanes 1, 3, and 5). Protein expression was analyzed by immunoblotting with the anti-M45 antiserum or with the anti-actin monoclonal antibody. (C) Subcellular localization of M45 in MCMV-infected NIH 3T3 cells at 48 hpi, detected by immunofluorescence and confocal microscopy. Cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. (D) Subcellular localization of transiently expressed M45 protein in NIH 3T3 cells transfected with the pcDNA3-45 vector at 24 h posttransfection. For immunofluorescence and confocal microscopy analysis, cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. The merged pictures are shown.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: (A) Time course of M45 expression during MCMV replication as determined by immunoblotting. Whole-cell lysates of mock-infected and MCMV-infected NIH 3T3 cells at various times after infection were separated by SDS-PAGE, transferred to a membrane, and probed with the anti-M45 antiserum or with the anti-actin monoclonal antibody. Lanes: 1, mock-infected cells; 2, 6 hpi; 3, 9 hpi; 4, 12 hpi; 5, 18 hpi; 6, 24 hpi; 7, 36 hpi; 8, 48 hpi. (B) Effect of PFA on M45 expression. Whole-cell extracts were prepared at 18 hpi (lanes 1 and 2), 24 hpi (lanes 3 and 4), or 48 hpi (lanes 5 and 6) from MCMV-infected NIH 3T3 cells treated with PFA (250 μg/ml) after virus adsorption (lanes 2, 4, and 6) or left untreated (lanes 1, 3, and 5). Protein expression was analyzed by immunoblotting with the anti-M45 antiserum or with the anti-actin monoclonal antibody. (C) Subcellular localization of M45 in MCMV-infected NIH 3T3 cells at 48 hpi, detected by immunofluorescence and confocal microscopy. Cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. (D) Subcellular localization of transiently expressed M45 protein in NIH 3T3 cells transfected with the pcDNA3-45 vector at 24 h posttransfection. For immunofluorescence and confocal microscopy analysis, cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. The merged pictures are shown.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: Expressing, Infection, SDS Page, Adsorption, Immunofluorescence, Confocal Microscopy, Incubation, Transfection, Plasmid Preparation

    Identification of the MCMV M45 protein by the specific antiserum. (A) Immunoblot analysis of native or recombinant M45 expression in MCMV-infected NIH 3T3 cells, in cells transfected with an M45 expression vector, or in E. coli . Proteins of whole-cell lysates were separated by SDS-PAGE (5 to 15% acrylamide), transferred to a membrane, and probed with the M45 antiserum. Lanes: 1, extracts from mock-infected NIH 3T3 cells; 2, extracts from NIH 3T3 cells 48 h after infection with MCMV; 3, extracts from NIH 3T3 cells transiently transfected with the control vector pcDNA3; 4, extracts from NIH 3T3 cells transiently transfected with the M45 expression vector pcDNA3-M45; 5, extracts from IPTG-induced E. coli containing the M45 expression vector pET30-M45. (B) Immunoprecipitation of M45 by the specific antiserum. M45 was immunoprecipitated from cell extracts of MCMV-infected NIH 3T3 cells prepared at 48 hpi and was analyzed by immunoblotting with the M45 antiserum. Lanes: 1, cell extract from infected cells; 2, cell extract immunoprecipitated with the M45 antiserum; 3, cell extract immunoprecipitated with preimmune serum. Sizes of the molecular mass markers are shown on the left of each panel. Asterisk indicates the Ig heavy chains recognized by the secondary antibody.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: Identification of the MCMV M45 protein by the specific antiserum. (A) Immunoblot analysis of native or recombinant M45 expression in MCMV-infected NIH 3T3 cells, in cells transfected with an M45 expression vector, or in E. coli . Proteins of whole-cell lysates were separated by SDS-PAGE (5 to 15% acrylamide), transferred to a membrane, and probed with the M45 antiserum. Lanes: 1, extracts from mock-infected NIH 3T3 cells; 2, extracts from NIH 3T3 cells 48 h after infection with MCMV; 3, extracts from NIH 3T3 cells transiently transfected with the control vector pcDNA3; 4, extracts from NIH 3T3 cells transiently transfected with the M45 expression vector pcDNA3-M45; 5, extracts from IPTG-induced E. coli containing the M45 expression vector pET30-M45. (B) Immunoprecipitation of M45 by the specific antiserum. M45 was immunoprecipitated from cell extracts of MCMV-infected NIH 3T3 cells prepared at 48 hpi and was analyzed by immunoblotting with the M45 antiserum. Lanes: 1, cell extract from infected cells; 2, cell extract immunoprecipitated with the M45 antiserum; 3, cell extract immunoprecipitated with preimmune serum. Sizes of the molecular mass markers are shown on the left of each panel. Asterisk indicates the Ig heavy chains recognized by the secondary antibody.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: Recombinant, Expressing, Infection, Transfection, Plasmid Preparation, SDS Page, Immunoprecipitation

    Detection of M45 in purified MCMV particles by immunoblotting. Proteins from a whole-cell lysate of MCMV-infected NIH 3T3 cells at 48 hpi (lane 1) or from virus particles purified by two rounds of centrifugation through a 15% sucrose cushion (lane 2) or via density gradient centrifugation (lane 3) were separated by SDS-PAGE, blotted onto a membrane, and probed with antibodies against the viral proteins M45 and M44 and the cellular proteins R2 and actin.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: Detection of M45 in purified MCMV particles by immunoblotting. Proteins from a whole-cell lysate of MCMV-infected NIH 3T3 cells at 48 hpi (lane 1) or from virus particles purified by two rounds of centrifugation through a 15% sucrose cushion (lane 2) or via density gradient centrifugation (lane 3) were separated by SDS-PAGE, blotted onto a membrane, and probed with antibodies against the viral proteins M45 and M44 and the cellular proteins R2 and actin.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: Purification, Infection, Centrifugation, Gradient Centrifugation, SDS Page

    RNR assays. (A) SDS-PAGE analyses of purified recombinant M45. Lane 1, molecular mass markers at 203, 120, 90, and 51 kDa; lane 2, recombinant His-tagged M45 purified by chromatography on a nickel-agarose column followed by chromatography on a Superdex 200 column. (B) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 protein (10 μg) before and after the addition of a constant amount of mouse R1 protein (2 μg). The increasing amounts of purified recombinant M45 are 0, 7, 14, and 28 μg. (C) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 (10 μg) together with a constant amount of mouse R1 (2 μg) or of the catalytically inactive R1 C429A protein (9 μg). The increasing amounts of purified recombinant M45 are 0, 7, and 14 μg. (D) Allosteric inhibition of ATP-stimulated CDP reduction by dATP. Catalytic activity of the mouse complex R1-R2 alone or together with recombinant M45 protein was assayed in the presence of 0, 20, 80, or 400 μM dATP.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: RNR assays. (A) SDS-PAGE analyses of purified recombinant M45. Lane 1, molecular mass markers at 203, 120, 90, and 51 kDa; lane 2, recombinant His-tagged M45 purified by chromatography on a nickel-agarose column followed by chromatography on a Superdex 200 column. (B) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 protein (10 μg) before and after the addition of a constant amount of mouse R1 protein (2 μg). The increasing amounts of purified recombinant M45 are 0, 7, 14, and 28 μg. (C) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 (10 μg) together with a constant amount of mouse R1 (2 μg) or of the catalytically inactive R1 C429A protein (9 μg). The increasing amounts of purified recombinant M45 are 0, 7, and 14 μg. (D) Allosteric inhibition of ATP-stimulated CDP reduction by dATP. Catalytic activity of the mouse complex R1-R2 alone or together with recombinant M45 protein was assayed in the presence of 0, 20, 80, or 400 μM dATP.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: SDS Page, Purification, Recombinant, Chromatography, Activity Assay, Inhibition

    Analysis of heparin–Sepharose purified GH3NEs by western blotting. Equal amounts (25 µg) of each heparin–Sepharose fraction, along with 25 µg of GH3NE for the GABP westerns, and 50 µg of GH3NE for the Ets-1 and Ets-2 westerns, were resolved on 10% SDS–polyacrylamide gels and transferred to Immobilon-P membranes. Blots were probed with antibodies against GABPα, GABPβ or Ets-1, where indicated. Protein was detected using ECL, as described in the Materials and Methods.

    Journal: Nucleic Acids Research

    Article Title: Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of the rat prolactin promoter

    doi:

    Figure Lengend Snippet: Analysis of heparin–Sepharose purified GH3NEs by western blotting. Equal amounts (25 µg) of each heparin–Sepharose fraction, along with 25 µg of GH3NE for the GABP westerns, and 50 µg of GH3NE for the Ets-1 and Ets-2 westerns, were resolved on 10% SDS–polyacrylamide gels and transferred to Immobilon-P membranes. Blots were probed with antibodies against GABPα, GABPβ or Ets-1, where indicated. Protein was detected using ECL, as described in the Materials and Methods.

    Article Snippet: Equal amounts (25 µg) of the peak heparin–Sepharose fractions, along with 25 or 50 µg of GH3NE, where indicated, were resolved on 10% SDS–polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA), according to the manufacturer’s protocol.

    Techniques: Purification, Western Blot

    SWCNT interactions with live cells probed by NIR photoluminescence. ( a ) Bright-field and NIR photoluminescence imaging (inserts) of live cells incubated for 24 h with Tween20-, F108-, or PLPEG-coated SWCNTs and further rinsed before imaging. PLPEG- and F108-coated SWCNTs displayed lower non-specific interactions with live cells compared to Tween20-coated SWCNTs. Scale bars: 25 µm for the bright field images and 10 µm for the magnified NIR photoluminescence images of SWCNTs; ( b ) Corresponding median (red), 25–75th percentile (blue), and 0–100th percentile (black) of the number of SWCNT PL spots observed on live cells for Tween20-, F108- or PLPEG-coated SWCNTs ( N = 70, 53, and 86 cells respectively, n.s.: not significant, ** p

    Journal: Nanomaterials

    Article Title: Evaluation of Different Single-Walled Carbon Nanotube Surface Coatings for Single-Particle Tracking Applications in Biological Environments

    doi: 10.3390/nano7110393

    Figure Lengend Snippet: SWCNT interactions with live cells probed by NIR photoluminescence. ( a ) Bright-field and NIR photoluminescence imaging (inserts) of live cells incubated for 24 h with Tween20-, F108-, or PLPEG-coated SWCNTs and further rinsed before imaging. PLPEG- and F108-coated SWCNTs displayed lower non-specific interactions with live cells compared to Tween20-coated SWCNTs. Scale bars: 25 µm for the bright field images and 10 µm for the magnified NIR photoluminescence images of SWCNTs; ( b ) Corresponding median (red), 25–75th percentile (blue), and 0–100th percentile (black) of the number of SWCNT PL spots observed on live cells for Tween20-, F108- or PLPEG-coated SWCNTs ( N = 70, 53, and 86 cells respectively, n.s.: not significant, ** p

    Article Snippet: Preparation of SWCNT Dispersions Pluoronic (F108-), Tween20-, and Brij35-coated SWCNTs: All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) if not stated otherwise.

    Techniques: Imaging, Incubation

    Live cell biocompatibility of SWCNTs encapsulated with different coatings. Top ( a ): Bright-field images of COS-7 incubated with PLPEG-, F108-, Tween20-, Brij35-, and ISPVP-coated SWCNTs for one day and four days. Scale bar: 30 µm. Bottom: Corresponding comparisons of cellular ( b ) proliferation and ( c ) viability. Starting concentration of COS-7 cells: 1 × 10 5 cells/mL, SWCNT: 1 μg/mL, ce ll cultured at 37 °C with 5% CO 2 ; Three independent experiments were performed to obtain standard variations. Cell viability was evaluated using trypan blue dye staining.

    Journal: Nanomaterials

    Article Title: Evaluation of Different Single-Walled Carbon Nanotube Surface Coatings for Single-Particle Tracking Applications in Biological Environments

    doi: 10.3390/nano7110393

    Figure Lengend Snippet: Live cell biocompatibility of SWCNTs encapsulated with different coatings. Top ( a ): Bright-field images of COS-7 incubated with PLPEG-, F108-, Tween20-, Brij35-, and ISPVP-coated SWCNTs for one day and four days. Scale bar: 30 µm. Bottom: Corresponding comparisons of cellular ( b ) proliferation and ( c ) viability. Starting concentration of COS-7 cells: 1 × 10 5 cells/mL, SWCNT: 1 μg/mL, ce ll cultured at 37 °C with 5% CO 2 ; Three independent experiments were performed to obtain standard variations. Cell viability was evaluated using trypan blue dye staining.

    Article Snippet: Preparation of SWCNT Dispersions Pluoronic (F108-), Tween20-, and Brij35-coated SWCNTs: All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) if not stated otherwise.

    Techniques: Incubation, Concentration Assay, Cell Culture, Staining

    The endosome to lysosome pathway and Lrsam1 mutations in mice. (A) Schematic of the endosome to lysosome pathway. Proteins are endocytosed from the plasma membrane and either recycled back to the membrane or transited through the endosomal pathway. From the late endosome, multivesicular bodies are targeted to the lysosome for degradation. The formation of multivesicular bodies requires the ESCRT complexes for vesicle scission and the proper membrane composition of PI3,5P2. Key proteins that function in this pathway and also cause CMT disease when mutated are shown in bold. (B) Representation of the protein domains of LRSAM1. From the N-terminus to the C-terminus, LRSAM1 contains four leucine-rich repeats (LR), two coil-coiled domains (CC), the sterile-alpha motif (SAM), two PTAP domains and a zinc-finger motif of the RING type (ZnF). (C) Gene-trap alleles of the mouse Lrsam1. Representation of the Lrsam1 gene structure and localization of the RRK239 and RRK461 gene-trap insertions. Exons are represented by black boxes. The diagram is not to scale. The intron sizes and positions of the insertions within the introns are shown. (D) Anti-LRSAM1 western blots on mouse spinal cord lysates and COS7 cells transfected with the Lrsam1 cDNA. Samples were prepared from wild-type, heterozygous and homozygous mice for the RRK461 gene trap insertion. A specific band of the expected size for LRSAM1 (84 kDa) was detected in wild-type samples and this size was consistent with recombinant protein expressed in COS7 cells (rLRSAM1). This band was reduced in heterozygotes and below detection in homozygotes. Blots were probed with either mouse anti-LRSAM1 (left panels) or rabbit anti-LRSAM1 (right panels) with similar results. The anti-mouse secondary antibody also recognized nonspecific immunoglobulin bands from the tissue whereas the rabbit anti-LRSAM1 antibody recognized a number of nonspecific background bands. With longer exposures, higher molecular weight bands were also detected in transfected COS7 cells (right panel, right lane), but not in untransfected cells. GAPDH (left lane) and tubulin were used as loading controls. (E) qRT-PCR on spinal cord extracts with primer pairs recognizing amplicons either upstream (5′) or downstream (3′) of the insertions. Relative transcript abundance in the heterozygotes (HET) and homozygote mutants. (MUT) for Lrsam1 RRK461 compared with wild-type mice (set at 1.0) is indicated. Error bars represent the confidence interval of the fold-change. n =7 for HET and 5 for MUT.

    Journal: Disease Models & Mechanisms

    Article Title: Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease

    doi: 10.1242/dmm.010942

    Figure Lengend Snippet: The endosome to lysosome pathway and Lrsam1 mutations in mice. (A) Schematic of the endosome to lysosome pathway. Proteins are endocytosed from the plasma membrane and either recycled back to the membrane or transited through the endosomal pathway. From the late endosome, multivesicular bodies are targeted to the lysosome for degradation. The formation of multivesicular bodies requires the ESCRT complexes for vesicle scission and the proper membrane composition of PI3,5P2. Key proteins that function in this pathway and also cause CMT disease when mutated are shown in bold. (B) Representation of the protein domains of LRSAM1. From the N-terminus to the C-terminus, LRSAM1 contains four leucine-rich repeats (LR), two coil-coiled domains (CC), the sterile-alpha motif (SAM), two PTAP domains and a zinc-finger motif of the RING type (ZnF). (C) Gene-trap alleles of the mouse Lrsam1. Representation of the Lrsam1 gene structure and localization of the RRK239 and RRK461 gene-trap insertions. Exons are represented by black boxes. The diagram is not to scale. The intron sizes and positions of the insertions within the introns are shown. (D) Anti-LRSAM1 western blots on mouse spinal cord lysates and COS7 cells transfected with the Lrsam1 cDNA. Samples were prepared from wild-type, heterozygous and homozygous mice for the RRK461 gene trap insertion. A specific band of the expected size for LRSAM1 (84 kDa) was detected in wild-type samples and this size was consistent with recombinant protein expressed in COS7 cells (rLRSAM1). This band was reduced in heterozygotes and below detection in homozygotes. Blots were probed with either mouse anti-LRSAM1 (left panels) or rabbit anti-LRSAM1 (right panels) with similar results. The anti-mouse secondary antibody also recognized nonspecific immunoglobulin bands from the tissue whereas the rabbit anti-LRSAM1 antibody recognized a number of nonspecific background bands. With longer exposures, higher molecular weight bands were also detected in transfected COS7 cells (right panel, right lane), but not in untransfected cells. GAPDH (left lane) and tubulin were used as loading controls. (E) qRT-PCR on spinal cord extracts with primer pairs recognizing amplicons either upstream (5′) or downstream (3′) of the insertions. Relative transcript abundance in the heterozygotes (HET) and homozygote mutants. (MUT) for Lrsam1 RRK461 compared with wild-type mice (set at 1.0) is indicated. Error bars represent the confidence interval of the fold-change. n =7 for HET and 5 for MUT.

    Article Snippet: The membranes were subsequently blocked in 2% milk in TBS-0.1% Tween20 and incubated overnight at 4°C with the primary antibody (mouse anti-LRSAM1, Abcam ab73113; rabbit anti-LRSAM1, Sigma-Aldrich HPA021403; rabbit anti-GAPDH, Abcam ab9385; or mouse anti-tubulin, clone DM1A, Sigma-Aldrich T9026) diluted in the blocking solution.

    Techniques: Mouse Assay, Western Blot, Transfection, Recombinant, Molecular Weight, Quantitative RT-PCR