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  • 99
    Thermo Fisher n2 supplement
    bax -/- bak -/- NPC cultures are able to differentiate along neural and glial lineages. NPC cultures were grown in the presence of N2 supplement, 1% FCS and FGF-2, in <t>N2</t> supplement and 1% FCS, or in the presence of 10% FCS and BDNF. After 2 weeks in differentiation medium, cells were stained with antibodies against O1 (oligodendral marker), MAP2 (neural marker), and GFAP (astrocyte marker) ( A ), and quantitation is shown in B . Culture in N2, 1% FCS, and FGF-2 resulted in small cells with relatively short blunt processes that occasionally labeled with O1 but not with GFAP or MAP2. The morphology and O1 staining indicate that some of these cells are immature oligodendrocytes. In N2 and 1% FCS media, the cells become large and elaborate extensive processes. These cells show differentiation along the lines of mature neurons (MAP2 labeling) and mature oligodendrocytes (compare the complex morphology of the O1 labeled cells with N2 alone vs those with N2 and FGF) ( B ). No astrocytes were seen with the N2 media alone. FCS and BDNF, conditions known to differentiate astrocytes from NPCs, exhibit the expected result with large flat cells showing strong GFAP immunoreactivity. Under these conditions, no neurons or oligodendrocytes were observed. For percentages of differentiated cells from wild-type mice, see Results. Scale bar, 25 μm.
    N2 Supplement, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore n acetyl l cysteine
    Involvement of ROS in inflammasome activation in  A. actinomycetemcomitans -invaded RAW264 cells.  (A)  RAW264 cells were pretreated with  N -acetyl- l -cysteine (NAC; 10 mM), ROS inhibitor, for 2 h before invasion. Untreated RAW264 cells (thin
    N Acetyl L Cysteine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cold Spring Harbor Laboratory Press n y cold spring harbor laboratory press
    Involvement of ROS in inflammasome activation in  A. actinomycetemcomitans -invaded RAW264 cells.  (A)  RAW264 cells were pretreated with  N -acetyl- l -cysteine (NAC; 10 mM), ROS inhibitor, for 2 h before invasion. Untreated RAW264 cells (thin
    N Y Cold Spring Harbor Laboratory Press, supplied by Cold Spring Harbor Laboratory Press, used in various techniques. Bioz Stars score: 92/100, based on 13865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare hybond n membrane
    Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a <t>Hybond-N+</t> membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.
    Hybond N Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 18138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc n cadherin
    Dysregulation of FGG affects the migration and invasion of HCC cells through EMT. Notes: ( A ) Western blots showed the changes in <t>E-cadherin,</t> N-cadherin, Slug, and ZEB-1 expression after artificial modification of FGG expression. siFGG means knockdown of FGG with small interfering RNA duplexes. ( B ) Quantitative analysis of EMT-associated protein expression in SK-HEP-1 cells; ( C ) Western blot results showed that Slug and ZEB-1 were knocked down in SK-HEP-1 cells with FGG overexpression. ( D ) The migration and invasion of SK-HEP-1 cells with FGG overexpression were dramatically inhibited by the knockdown of Slug and ZEB-1. In this experiment, siSlug-3 and siZEB-1–3 were used to knockdown the expression of Slug and ZEB-1, respectively. Each experiment was independently performed at least three times. Data are shown as mean ± SD. * P
    N Cadherin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 5180 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore n hydroxysuccinimide nhs
    Illustration of Gd-CDs/AFn (DOX)/FA nanocomposite preparation. Abbreviations: AFn, apoferritin; CDs, carbon dots; DEG, diethylene glycol; DOX, doxorubicin; EDC, N-(3-dimethylaminopropyl-N′-ethylcarbodiimide); FA, folic acid; Gd, gadolinium; <t>NHS,</t> <t>N-hydroxysuccinimide;</t> PEI, polyethylenimine.
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    99
    Millipore n ethylmaleimide
    Identification and characterization of liver S-palmitoylome. ( a ) A schematic overview for the site-specific identification of the S-palmitoylome by a modified alkylating RAC assay. Free thiols are first blocked with NEM. Disulfide bond was cleaved by TCEP and further blocked with IAM. Thioesters are then cleaved with neutral HA and the newly liberated thiols are purified with thiol sepharose 4B. The capture proteins on sepharoses were digested with trypsin. The supernatant of digested samples was removed and the captured peptides on sepharoses were eluted with TCEP and subjected to downstream MS analysis for S-palmitoylated site identification. Peptide alignment was applied to increase the coverage based on the retention time and m/z of identified peptides. Extracted ion chromatogram of identified peptides and the same m/z in different LC samples were used to quantify the relative abundance. The protein levels were quantified by TMT10-plex labelling. ( b ) Pie diagram illustrating the subcellular localization of identified S-palmitoylated proteins. ( c ) Pie diagram illustrating the functions of identified S-palmitoylated proteins. NEM, <t>N-ethylmaleimide;</t> IAM, Iodoacetamide; HA, hydroxylamine.
    N Ethylmaleimide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam n cadherin
    The effects of ATP-binding cassette transporter A7-knockdown on the migration of SKOV-3 cells. The data are presented as the mean ± standard deviation. (A) Images are representative of three independent experiments. The protein levels of <t>E-cadherin,</t> N-cadherin and SMAD4 were assessed by western blot analysis. All data are expressed as the mean ± standard deviation (*P
    N Cadherin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology n cadherin
    Differential <t>E-cadherin</t> expression in cranial sutures. a Microarray profile of E-cadherin gene on SAG and PF suture shows that the gene is up-regulated exclusively in the PF suture at p15. Green = Lower than mean expression; red = higher than mean expression. b Quantitative real-time RT-PCR confirmed the up-regulation of E-cadherin gene expression in PF suture at p15. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments.
    N Cadherin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nmda
    Dendritic release requires L-type Ca 2+ channels. A , Dendritic release responses to <t>AMPA</t> or <t>NMDA</t> in the presence and absence of IEM1460 (100 µM) or nimodipine (3 µM). n ≥ 5. ****p
    Nmda, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sulfo nhs ss biotin
    Internalization of truncated FcγRIIa/b is augmented by RTX treatment. Ramos cells <t>transfected</t> with FcγRIIbΔcyt or FcγRIIaΔcyt were treated with <t>sulfo-NHS-SS-biotin</t> at 4 °C to biotinylate cell surface proteins and cultured with 5 μg/ml A488-labeled RTX for 2 h at 37 °C or left untreated. Internalization of FcγRIIb/a was assessed at 0, 30, 60, and 120 min by treating cells with MESNa to remove cell surface biotin, followed by immunoprecipitation of internalized biotinylated proteins with streptavidin-coated beads. Precipitated proteins were blotted for FcγRII. A and C , representative examples of FcγRIIbΔcyt and FcγRIIaΔcyt transfectants left untreated ( left panels ) or treated with RTX ( right panels ), respectively. −, not treated ( NT ); +, MESNa-treated. B and D , densitometry demonstrating the proportion of internalized FcγRIIbΔcyt ( B ) and FcγRIIaΔcyt ( D ) in untreated cells ( left panels ) and cells treated with RTX ( right panels ), expressed as a percentage of non-MESNa-treated cells, n = 3. Horizontal bars represent the median.
    Sulfo Nhs Ss Biotin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4097 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Springer-Verlag n y springer verlag
    Internalization of truncated FcγRIIa/b is augmented by RTX treatment. Ramos cells <t>transfected</t> with FcγRIIbΔcyt or FcγRIIaΔcyt were treated with <t>sulfo-NHS-SS-biotin</t> at 4 °C to biotinylate cell surface proteins and cultured with 5 μg/ml A488-labeled RTX for 2 h at 37 °C or left untreated. Internalization of FcγRIIb/a was assessed at 0, 30, 60, and 120 min by treating cells with MESNa to remove cell surface biotin, followed by immunoprecipitation of internalized biotinylated proteins with streptavidin-coated beads. Precipitated proteins were blotted for FcγRII. A and C , representative examples of FcγRIIbΔcyt and FcγRIIaΔcyt transfectants left untreated ( left panels ) or treated with RTX ( right panels ), respectively. −, not treated ( NT ); +, MESNa-treated. B and D , densitometry demonstrating the proportion of internalized FcγRIIbΔcyt ( B ) and FcγRIIaΔcyt ( D ) in untreated cells ( left panels ) and cells treated with RTX ( right panels ), expressed as a percentage of non-MESNa-treated cells, n = 3. Horizontal bars represent the median.
    N Y Springer Verlag, supplied by Springer-Verlag, used in various techniques. Bioz Stars score: 92/100, based on 1051 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson n cadherin
    hucMSC-Ex reversed TGF-β1-induced EMT in human hepatocyte cell line HL7702 cells. (A) The morphology of HL7702 cells treated with TGF-β1 for 3 days followed by hucMSC-Ex for 3 days (200×). (a) HL7702 cells, 3 days (C3d); (b) TGF-β1+HL7702, 3 days (T3d); (c) TGF-β1+HL7702, 6 days (T6d); (d) TGF-β1+HL7702+hucMSC-Ex, 6 days (T+E 6d). (B) Quantitative RT-PCR analyses of <t>E-cadherin</t> , N-cadherin , and Twist mRNA expression. (C) Western blotting analyses of E-cadherin and N-cadherin expression in cells. (D) Density analysis of western blot bands. * P
    N Cadherin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 3028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tetramethylbenzidine
    A: Drawings depicting the connections of the ventral division of the external nucleus of the inferior colliculus (ICXv) determined from focal wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injection (filled area a) in one bat. B: Photomicrograph of diaminobenzidine (DAB)-reacted WGA-HRP-stained section (CytOx counterstained) showing the size of the injection site. In A, labeling is shown on five transverse brain sections. The rostrocaudal distance between the sections is 360 µm for the two most caudal sections, and about 600 µm between the remaining rostral sections. Large filled circles represent cell bodies labeled by retrograde transport; lines and small dots represent labeled fibers or terminals, respectively. Open circles are labeled cells in the immediate vicinity of the injection site. a: Injection site in the ICXv indicated by filled area (reconstructed from DAB-stained section shown in A). b: Retrogradely labeled cell bodies in the nucleus of the central acoustic tract (NCAT; <t>tetramethylbenzidine</t> [TMB] reaction). c: Labeled fibers coursing medial to the lateral lemniscus. d: Fibers in the deep superior colliculus (SCd). e: Fine punctate labeling in the suprageniculate nucleus (Sg) of the medial geniculate body. f: Transport of tracer to Sg was via fibers running along the ventral half of brachium of the inferior colliculus (BIC). D, dorsal; m, medial; c, caudal; v, ventral; l, lateral; r, rostral. Scale bar in B = 500 µm.
    Tetramethylbenzidine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2027 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore n 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride
    AMC can be conjugated to free Carboxyl groups on Proteins ( a ) Linear correlation between free AMC concentration, from 100nM to 1mM, and fluorescence. Here different concentrations of AMC, dissolved in proteolysis buffer, were incubated at 37°C on 96-well plates. Fluorescence was analyzed at an emission wavelength of 444nM, with excitation wavelength of 390nM. Values are means ± SE, n = 3. ( b ) Addition of increasing amounts of BSA to AMC in the presence of NaCNBH 3 progressively quenches the fluorescence of AMC. Here 0–50mg of BSA was added to 100μM AMC and 20mM NaCNBH 3 and incubated for 1hr at 37°C. Free AMC content was determined with reference to a standard curve of known AMC concentrations. Values are means ± SE, n = 3. ( c ) Here 50mg/ml of BSA was incubated with 1mM AMC in the presence or absence of 20mM NaCNBH 3 , and then run on a 12% SDS Page gel. A fluorescent BSA-AMC complex was readily observed at ≈66kDa (the approximate size of BSA), using an excitation wavelength of 365nM and an emission wavelength of 444 nm, when all three reagents were present, but could only be faintly discerned in the absence of NaCNBH 3 . A silver stain was later performed. ( d ], was incubated with 50mg of BSA for 1hr. BSA was extensively dialyzed then then prepared as in panel (c). Increasing concentrations of <t>N-(3-Dimethylamineopropyl)-N'-ethylcarbodiimide,</t> caused a progressive decrease in BSA’s electrophoretic mobility, and loss of fluorescence at 66kDa; a representative gel is shown to the left of the panel, and fluorescence is quantified in the graph to the right.
    N 3 Dimethylaminopropyl N Ethylcarbodiimide Hydrochloride, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 913 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    bax -/- bak -/- NPC cultures are able to differentiate along neural and glial lineages. NPC cultures were grown in the presence of N2 supplement, 1% FCS and FGF-2, in N2 supplement and 1% FCS, or in the presence of 10% FCS and BDNF. After 2 weeks in differentiation medium, cells were stained with antibodies against O1 (oligodendral marker), MAP2 (neural marker), and GFAP (astrocyte marker) ( A ), and quantitation is shown in B . Culture in N2, 1% FCS, and FGF-2 resulted in small cells with relatively short blunt processes that occasionally labeled with O1 but not with GFAP or MAP2. The morphology and O1 staining indicate that some of these cells are immature oligodendrocytes. In N2 and 1% FCS media, the cells become large and elaborate extensive processes. These cells show differentiation along the lines of mature neurons (MAP2 labeling) and mature oligodendrocytes (compare the complex morphology of the O1 labeled cells with N2 alone vs those with N2 and FGF) ( B ). No astrocytes were seen with the N2 media alone. FCS and BDNF, conditions known to differentiate astrocytes from NPCs, exhibit the expected result with large flat cells showing strong GFAP immunoreactivity. Under these conditions, no neurons or oligodendrocytes were observed. For percentages of differentiated cells from wild-type mice, see Results. Scale bar, 25 μm.

    Journal: The Journal of Neuroscience

    Article Title: The Proapoptotic Activities of Bax and Bak Limit the Size of the Neural Stem Cell Pool

    doi: 10.1523/JNEUROSCI.23-35-11112.2003

    Figure Lengend Snippet: bax -/- bak -/- NPC cultures are able to differentiate along neural and glial lineages. NPC cultures were grown in the presence of N2 supplement, 1% FCS and FGF-2, in N2 supplement and 1% FCS, or in the presence of 10% FCS and BDNF. After 2 weeks in differentiation medium, cells were stained with antibodies against O1 (oligodendral marker), MAP2 (neural marker), and GFAP (astrocyte marker) ( A ), and quantitation is shown in B . Culture in N2, 1% FCS, and FGF-2 resulted in small cells with relatively short blunt processes that occasionally labeled with O1 but not with GFAP or MAP2. The morphology and O1 staining indicate that some of these cells are immature oligodendrocytes. In N2 and 1% FCS media, the cells become large and elaborate extensive processes. These cells show differentiation along the lines of mature neurons (MAP2 labeling) and mature oligodendrocytes (compare the complex morphology of the O1 labeled cells with N2 alone vs those with N2 and FGF) ( B ). No astrocytes were seen with the N2 media alone. FCS and BDNF, conditions known to differentiate astrocytes from NPCs, exhibit the expected result with large flat cells showing strong GFAP immunoreactivity. Under these conditions, no neurons or oligodendrocytes were observed. For percentages of differentiated cells from wild-type mice, see Results. Scale bar, 25 μm.

    Article Snippet: Cells were resuspended in DMEM (Invitrogen), 10% FCS, 1% penicillin-streptomycin-fungizone (PSF) (Invitrogen), 1% N2 supplement (Invitrogen), and recombinant human fibroblast growth factor-2 (FGF-2) at 20 ng/ml (Promega, Madison, WI) and plated on poly- l -lysine (Sigma)-coated plates at 3 × 104 cells/cm2 .

    Techniques: Staining, Marker, Quantitation Assay, Labeling, Mouse Assay

    Effects of long-term growth factor deprivation in  bax -/- bak -/-  NPC cultures.  bax -/- bak -/-  (filled squares) and wild-type (open squares) NPC cultures were grown to confluency in standard NPC medium and then deprived of N2 supplement, 1% FCS, and FGF-2 for 31 d. At day 31, growth factors were readded. Relative cell number compared with the number of cells present at the onset of the experiment ( A ) and cell size ( B ) are shown at indicated time points. The majority of the wild-type cells died within 1 week and was not available for additional sampling. The viability of the  bax -/- bak -/-  cells was 91% at day 31. A representative experiment is shown.

    Journal: The Journal of Neuroscience

    Article Title: The Proapoptotic Activities of Bax and Bak Limit the Size of the Neural Stem Cell Pool

    doi: 10.1523/JNEUROSCI.23-35-11112.2003

    Figure Lengend Snippet: Effects of long-term growth factor deprivation in bax -/- bak -/- NPC cultures. bax -/- bak -/- (filled squares) and wild-type (open squares) NPC cultures were grown to confluency in standard NPC medium and then deprived of N2 supplement, 1% FCS, and FGF-2 for 31 d. At day 31, growth factors were readded. Relative cell number compared with the number of cells present at the onset of the experiment ( A ) and cell size ( B ) are shown at indicated time points. The majority of the wild-type cells died within 1 week and was not available for additional sampling. The viability of the bax -/- bak -/- cells was 91% at day 31. A representative experiment is shown.

    Article Snippet: Cells were resuspended in DMEM (Invitrogen), 10% FCS, 1% penicillin-streptomycin-fungizone (PSF) (Invitrogen), 1% N2 supplement (Invitrogen), and recombinant human fibroblast growth factor-2 (FGF-2) at 20 ng/ml (Promega, Madison, WI) and plated on poly- l -lysine (Sigma)-coated plates at 3 × 104 cells/cm2 .

    Techniques: Sampling

    NPC cultures from  bax -/- bak -/-  mice are resistant to apoptotic stimuli. NPC cultures from  bax -/- bak -/-  and control genotypes were grown without N2 supplement, 1% FCS, and FGF-2 for 3 d ( A ), treated with staurosporine for 24 hr ( B ), or treated with etoposide for 24 hr ( C ). Cells were harvested, and viability was determined by trypan blue exclusion. The mean ± SD of triplicate samples are shown. GF, Growth factor.

    Journal: The Journal of Neuroscience

    Article Title: The Proapoptotic Activities of Bax and Bak Limit the Size of the Neural Stem Cell Pool

    doi: 10.1523/JNEUROSCI.23-35-11112.2003

    Figure Lengend Snippet: NPC cultures from bax -/- bak -/- mice are resistant to apoptotic stimuli. NPC cultures from bax -/- bak -/- and control genotypes were grown without N2 supplement, 1% FCS, and FGF-2 for 3 d ( A ), treated with staurosporine for 24 hr ( B ), or treated with etoposide for 24 hr ( C ). Cells were harvested, and viability was determined by trypan blue exclusion. The mean ± SD of triplicate samples are shown. GF, Growth factor.

    Article Snippet: Cells were resuspended in DMEM (Invitrogen), 10% FCS, 1% penicillin-streptomycin-fungizone (PSF) (Invitrogen), 1% N2 supplement (Invitrogen), and recombinant human fibroblast growth factor-2 (FGF-2) at 20 ng/ml (Promega, Madison, WI) and plated on poly- l -lysine (Sigma)-coated plates at 3 × 104 cells/cm2 .

    Techniques: Mouse Assay

    Y88 is essential for T function Schematic illustrating the homologous recombination strategy to insert the Y88A point mutation in the endogenous  T  locus. The neomycin selection cassette was removed by transient expression of  FlpE  recombinase. Orange lines depict probes used for Southern blotting to confirm integration of the mutation. Restriction enzyme sites used for Southern blotting are depicted. Southern blot of genomic DNA from mESCs to detect homologous recombination of the Y88A mutation using  T  probes depicted in (A). A 4‐kb and 9‐kb band correspond to the wild‐type  T  allele using the 5′ and 3′  T  probe, respectively, while a 6‐kb band and 10‐kb band correspond to the mutated  T Y88A  allele containing the neomycin selection cassette. Southern blot of genomic DNA from mESCs to detect removal of the neomycin selection cassette after expression of  FlpE  recombinase using the 5′  T  probe. WISH analysis of  T Y88A  mutants shows  T  expression is normal at early stages, E7.0–7.5, and begins to decrease at E8.0. At E8.0, the left panels show a lateral view of the embryo, while the right panels show a dorsal view. The graph depicts RT‐qPCR analysis of whole embryos to confirm loss of  T  maintenance in  T Y88A  mutants compared to  T WT  controls. The mean of  n  = 2 biological replicates of pools of three to four embryos is depicted. Scale bar: 200 μm. Immunoblot from whole embryo lysates using an α‐T antibody shows a progressive loss of T protein during development in the  T Y88A  mutant. α‐LAMIN‐B1 was used as a loading control. T Y88A  mutants resemble  T null  embryos on the gross morphological level, displaying a complete loss of posterior mesoderm (arrow). Scale bar: 200 μm. Tbx6  and  Uncx4.1  expression in E9.5  Tbx6 Y137A  embryos by WISH. Black brackets indicate the PSM, and white lines indicate the plane of section shown in the lower panels. Hematoxylin and eosin staining of sections through the trunk of E9.5  Tbx6 Y137A  embryos revealed the presence of two ectopic neural tubes (arrowheads) at the expense of somite tissue, which was confirmed by immunofluorescent staining for the neural tube marker SOX2. Scale bar: 180 μm.

    Journal: EMBO Reports

    Article Title: BRACHYURY directs histone acetylation to target loci during mesoderm development

    doi: 10.15252/embr.201744201

    Figure Lengend Snippet: Y88 is essential for T function Schematic illustrating the homologous recombination strategy to insert the Y88A point mutation in the endogenous T locus. The neomycin selection cassette was removed by transient expression of FlpE recombinase. Orange lines depict probes used for Southern blotting to confirm integration of the mutation. Restriction enzyme sites used for Southern blotting are depicted. Southern blot of genomic DNA from mESCs to detect homologous recombination of the Y88A mutation using T probes depicted in (A). A 4‐kb and 9‐kb band correspond to the wild‐type T allele using the 5′ and 3′ T probe, respectively, while a 6‐kb band and 10‐kb band correspond to the mutated T Y88A allele containing the neomycin selection cassette. Southern blot of genomic DNA from mESCs to detect removal of the neomycin selection cassette after expression of FlpE recombinase using the 5′ T probe. WISH analysis of T Y88A mutants shows T expression is normal at early stages, E7.0–7.5, and begins to decrease at E8.0. At E8.0, the left panels show a lateral view of the embryo, while the right panels show a dorsal view. The graph depicts RT‐qPCR analysis of whole embryos to confirm loss of T maintenance in T Y88A mutants compared to T WT controls. The mean of n = 2 biological replicates of pools of three to four embryos is depicted. Scale bar: 200 μm. Immunoblot from whole embryo lysates using an α‐T antibody shows a progressive loss of T protein during development in the T Y88A mutant. α‐LAMIN‐B1 was used as a loading control. T Y88A mutants resemble T null embryos on the gross morphological level, displaying a complete loss of posterior mesoderm (arrow). Scale bar: 200 μm. Tbx6 and Uncx4.1 expression in E9.5 Tbx6 Y137A embryos by WISH. Black brackets indicate the PSM, and white lines indicate the plane of section shown in the lower panels. Hematoxylin and eosin staining of sections through the trunk of E9.5 Tbx6 Y137A embryos revealed the presence of two ectopic neural tubes (arrowheads) at the expense of somite tissue, which was confirmed by immunofluorescent staining for the neural tube marker SOX2. Scale bar: 180 μm.

    Article Snippet: The next day, embryoid bodies were washed thoroughly with Advanced DMEM (Gibco #12491015) and plated at a density of approximately 45 drops/cm2 onto tissue culture dishes coated with fibronectin (1:100 dilution in 1× PBS, Calbiochem #341631) in serum‐free medium [SFM, 1:1 Neurobasal:F12/DMEM (Gibco), 2 mM l ‐glutamine (Gibco), 50 U/ml penicillin, 50 μg/ml streptomycin (Lonza), 0.5× B‐27 supplement, serum‐free (Gibco), 0.5× N‐2 supplement (Gibco), 0.05% Albumin, bovine Fraction V (7.5%; Sigma‐Aldrich), 0.001% 1‐thioglycerol (Sigma‐Aldrich)] containing recombinant BMP4 (20 ng/ml, R & D Systems) for 18 h. After 18 h, SFM + rBMP4 was removed and replaced with SFM containing 40 ng/ml rWNT3A (R & D Systems) and 40 ng/ml rFGF8 (R & D Systems) for the remainder of the experiment.

    Techniques: Homologous Recombination, Mutagenesis, Selection, Expressing, Southern Blot, Quantitative RT-PCR, Staining, Marker

    T regulation of the  Lmo2  locus ChIP‐qPCR analysis of H3K27ac at the  Lmo2  −70 enhancer in  KD4‐T in vitro ‐differentiated mesodermal cells at day 4. The mean of  n  = 2 biological replicates is depicted. RT‐qPCR analysis of  T, Runx1,  and  Sox17  expression in E8.25 caudal ends of  T WT  embryos. Levels of  Runx1  and  Sox17  are significantly lower than that of  T  in E8.25 caudal ends. Error bars depict SD of  n  = 3 biological replicates. * P

    Journal: EMBO Reports

    Article Title: BRACHYURY directs histone acetylation to target loci during mesoderm development

    doi: 10.15252/embr.201744201

    Figure Lengend Snippet: T regulation of the Lmo2 locus ChIP‐qPCR analysis of H3K27ac at the Lmo2 −70 enhancer in KD4‐T in vitro ‐differentiated mesodermal cells at day 4. The mean of n = 2 biological replicates is depicted. RT‐qPCR analysis of T, Runx1, and Sox17 expression in E8.25 caudal ends of T WT embryos. Levels of Runx1 and Sox17 are significantly lower than that of T in E8.25 caudal ends. Error bars depict SD of n = 3 biological replicates. * P

    Article Snippet: The next day, embryoid bodies were washed thoroughly with Advanced DMEM (Gibco #12491015) and plated at a density of approximately 45 drops/cm2 onto tissue culture dishes coated with fibronectin (1:100 dilution in 1× PBS, Calbiochem #341631) in serum‐free medium [SFM, 1:1 Neurobasal:F12/DMEM (Gibco), 2 mM l ‐glutamine (Gibco), 50 U/ml penicillin, 50 μg/ml streptomycin (Lonza), 0.5× B‐27 supplement, serum‐free (Gibco), 0.5× N‐2 supplement (Gibco), 0.05% Albumin, bovine Fraction V (7.5%; Sigma‐Aldrich), 0.001% 1‐thioglycerol (Sigma‐Aldrich)] containing recombinant BMP4 (20 ng/ml, R & D Systems) for 18 h. After 18 h, SFM + rBMP4 was removed and replaced with SFM containing 40 ng/ml rWNT3A (R & D Systems) and 40 ng/ml rFGF8 (R & D Systems) for the remainder of the experiment.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Expressing

    Y88 is necessary for  T  maintenance T  expression in  in vitro ‐differentiated mesodermal cells from control  T WT  and  T Y88A  during the 5‐day differentiation protocol as measured by RT‐qPCR.  n  = 2 biological replicates. Schematic illustrating the RP24‐530D23  T mCherry  reporter BAC. The orange line depicts the probe used to confirm genomic integration of the reporter BAC by Southern blot. Restriction enzyme sites used for Southern blotting are depicted. Fluorescence images and WISH analysis of  T WT ‐ and  T Y88A ‐Tg ( T mCherry ) with  T  and  mCherry  probes at E9.5 reveals the co‐localization of  T  and  mCherry  expression from the  T mCherry  reporter BAC. mCHERRY protein is more stable than the  mCherry  RNA and is localized to a broader domain. Scale bars: 200 μm. T  and  mCherry  expression in  T WT ‐  and  T Y88A ‐Tg ( T mCherry ) cells differentiated into mesoderm  in vitro  during the 5‐day differentiation protocol, as measured by RT‐qPCR. The mean of  n  = 2 biological replicates is depicted. Bar graph depicting the percentage of T‐mCHERRY(+) mesodermal cells and T‐mCHERRY(−) cells, as assayed by FACS, in  T WT ‐, T Y88A ‐,  and  T null ‐Tg ( T mCherry ) cells during the 5‐day  in vitro  differentiation protocol.

    Journal: EMBO Reports

    Article Title: BRACHYURY directs histone acetylation to target loci during mesoderm development

    doi: 10.15252/embr.201744201

    Figure Lengend Snippet: Y88 is necessary for T maintenance T expression in in vitro ‐differentiated mesodermal cells from control T WT and T Y88A during the 5‐day differentiation protocol as measured by RT‐qPCR. n = 2 biological replicates. Schematic illustrating the RP24‐530D23 T mCherry reporter BAC. The orange line depicts the probe used to confirm genomic integration of the reporter BAC by Southern blot. Restriction enzyme sites used for Southern blotting are depicted. Fluorescence images and WISH analysis of T WT ‐ and T Y88A ‐Tg ( T mCherry ) with T and mCherry probes at E9.5 reveals the co‐localization of T and mCherry expression from the T mCherry reporter BAC. mCHERRY protein is more stable than the mCherry RNA and is localized to a broader domain. Scale bars: 200 μm. T and mCherry expression in T WT ‐ and T Y88A ‐Tg ( T mCherry ) cells differentiated into mesoderm in vitro during the 5‐day differentiation protocol, as measured by RT‐qPCR. The mean of n = 2 biological replicates is depicted. Bar graph depicting the percentage of T‐mCHERRY(+) mesodermal cells and T‐mCHERRY(−) cells, as assayed by FACS, in T WT ‐, T Y88A ‐, and T null ‐Tg ( T mCherry ) cells during the 5‐day in vitro differentiation protocol.

    Article Snippet: The next day, embryoid bodies were washed thoroughly with Advanced DMEM (Gibco #12491015) and plated at a density of approximately 45 drops/cm2 onto tissue culture dishes coated with fibronectin (1:100 dilution in 1× PBS, Calbiochem #341631) in serum‐free medium [SFM, 1:1 Neurobasal:F12/DMEM (Gibco), 2 mM l ‐glutamine (Gibco), 50 U/ml penicillin, 50 μg/ml streptomycin (Lonza), 0.5× B‐27 supplement, serum‐free (Gibco), 0.5× N‐2 supplement (Gibco), 0.05% Albumin, bovine Fraction V (7.5%; Sigma‐Aldrich), 0.001% 1‐thioglycerol (Sigma‐Aldrich)] containing recombinant BMP4 (20 ng/ml, R & D Systems) for 18 h. After 18 h, SFM + rBMP4 was removed and replaced with SFM containing 40 ng/ml rWNT3A (R & D Systems) and 40 ng/ml rFGF8 (R & D Systems) for the remainder of the experiment.

    Techniques: Expressing, In Vitro, Quantitative RT-PCR, BAC Assay, Southern Blot, Fluorescence, FACS

    Involvement of ROS in inflammasome activation in  A. actinomycetemcomitans -invaded RAW264 cells.  (A)  RAW264 cells were pretreated with  N -acetyl- l -cysteine (NAC; 10 mM), ROS inhibitor, for 2 h before invasion. Untreated RAW264 cells (thin

    Journal: Journal of Interferon & Cytokine Research

    Article Title: Aggregatibacter actinomycetemcomitans Invasion Induces Interleukin-1β Production Through Reactive Oxygen Species and Cathepsin B

    doi: 10.1089/jir.2014.0127

    Figure Lengend Snippet: Involvement of ROS in inflammasome activation in A. actinomycetemcomitans -invaded RAW264 cells. (A) RAW264 cells were pretreated with N -acetyl- l -cysteine (NAC; 10 mM), ROS inhibitor, for 2 h before invasion. Untreated RAW264 cells (thin

    Article Snippet: Cathepsin B inhibitor, CA-074Me (10 μM; Millipore Corporation, Billerica, MA), ROS inhibitor, N -acetyl- l -cysteine (NAC, 10 mM; Sigma-Aldrich), caspase-1 inhibitor, Z-YVAD-FMK (100 μM; Abcam), and cytochalasin D (1 μg/mL; Sigma-Aldrich) were used in this study.

    Techniques: Activation Assay

    Flow diagram of the experiment . All of the animals received the same total number of injections, as drug injections necessary for the experiment were supplemented with control solution injections. For example, group #3 received a single injection of furosemide 0.5 hours before the exposure to 3.5 hours of noise, but in addition was injected with saline over the two preceding days and over the following five days in order to make up the number of injections. NAC - N-acetyl-l-cysteine; ACE - vitamins A, C and E; IP - intraperitoneal injection; PTS - permanent threshold shift; ABR - auditory nerve brainstem evoked response; BBN - broadband noise.

    Journal: Journal of Occupational Medicine and Toxicology (London, England)

    Article Title: Uniform comparison of several drugs which provide protection from noise induced hearing loss

    doi: 10.1186/1745-6673-5-26

    Figure Lengend Snippet: Flow diagram of the experiment . All of the animals received the same total number of injections, as drug injections necessary for the experiment were supplemented with control solution injections. For example, group #3 received a single injection of furosemide 0.5 hours before the exposure to 3.5 hours of noise, but in addition was injected with saline over the two preceding days and over the following five days in order to make up the number of injections. NAC - N-acetyl-l-cysteine; ACE - vitamins A, C and E; IP - intraperitoneal injection; PTS - permanent threshold shift; ABR - auditory nerve brainstem evoked response; BBN - broadband noise.

    Article Snippet: Group 2: N-acetyl-l-cysteine (NAC), an anti-oxidant, obtained from Sigma, Israel (n = 10).

    Techniques: Flow Cytometry, Injection

    Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a Hybond-N+ membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.

    Journal: The Journal of surgical research

    Article Title: GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION

    doi: 10.1016/j.jss.2009.03.072

    Figure Lengend Snippet: Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a Hybond-N+ membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.

    Article Snippet: For Northern blot hybridization, 15 μg of total RNA was resolved on 1% agarose/formaldehyde gels by electrophoresis, transferred to Hybond=N+ membrane (Amersham Pharmacia Biotech, Buckinhamshire, England), aprobed with IL-8 cDNA labeled with [α-32 P] dATP (Perkin-Elmer Life Sciences Inc., Boston, MA) using a random-priming DNA-labeling kit (Stratagene, Cedar Creek, TX).

    Techniques: Autoradiography, Northern Blot, Isolation, Multiple Displacement Amplification, Labeling, Real-time Polymerase Chain Reaction, Negative Control

    Autoradiographs of Northern blots with B. germanica trypsin ( bgtryp -1). Total RNA was separated on 1.0% agarose gel containing 6.7% formaldehyde. RNA was transferred to Hybond-N + nylon membrane, probed with cDNA inserts of B. germanica trypsin ( bgtryp -1). Transcript size was approximately 0.9 kb.

    Journal: The Korean Journal of Parasitology

    Article Title: Cloning and expression of trypsin-encoding cDNA from Blattella germanica and its possibility as an allergen

    doi: 10.3347/kjp.2005.43.3.101

    Figure Lengend Snippet: Autoradiographs of Northern blots with B. germanica trypsin ( bgtryp -1). Total RNA was separated on 1.0% agarose gel containing 6.7% formaldehyde. RNA was transferred to Hybond-N + nylon membrane, probed with cDNA inserts of B. germanica trypsin ( bgtryp -1). Transcript size was approximately 0.9 kb.

    Article Snippet: The DNA digests were separated on 0.8% agarose gel, then transferred to Hybond-N+ nylon membranes, according to the manufacturer's instructions (Amersham, USA).

    Techniques: Northern Blot, Agarose Gel Electrophoresis

    MPP10 in subcellular fractions of HeLa cells. First, HeLa cells were fractionated into cytoplasmic and nuclear fractions. Then, the crude nuclear fraction (Nuclei I) was further fractionated into nucleoplasm (Nuclei II) and nucleoli. Fractions representing an equal number of cells were separated by SDS-PAGE, transferred to Hybond-N, and reacted with anti-MPP10 serum. Bound antibodies were subsequently reacted with horseradish peroxidase-coupled secondary antibodies and detected with ECL. Positions of migration of molecular mass markers are indicated on the right.

    Journal: Molecular Biology of the Cell

    Article Title: M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

    doi:

    Figure Lengend Snippet: MPP10 in subcellular fractions of HeLa cells. First, HeLa cells were fractionated into cytoplasmic and nuclear fractions. Then, the crude nuclear fraction (Nuclei I) was further fractionated into nucleoplasm (Nuclei II) and nucleoli. Fractions representing an equal number of cells were separated by SDS-PAGE, transferred to Hybond-N, and reacted with anti-MPP10 serum. Bound antibodies were subsequently reacted with horseradish peroxidase-coupled secondary antibodies and detected with ECL. Positions of migration of molecular mass markers are indicated on the right.

    Article Snippet: Proteins separated by SDS-PAGE were transferred to Immobilon-P or Hybond-N (Amersham, Arlington Heights, IL) and reacted with MPP10 antiserum at a dilution of 1:2000 to 1:10,000 in Tris-buffered saline containing Tween (50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 0.1% Tween-20) with or without 5% nonfat dried milk.

    Techniques: SDS Page, Migration

    Dysregulation of FGG affects the migration and invasion of HCC cells through EMT. Notes: ( A ) Western blots showed the changes in E-cadherin, N-cadherin, Slug, and ZEB-1 expression after artificial modification of FGG expression. siFGG means knockdown of FGG with small interfering RNA duplexes. ( B ) Quantitative analysis of EMT-associated protein expression in SK-HEP-1 cells; ( C ) Western blot results showed that Slug and ZEB-1 were knocked down in SK-HEP-1 cells with FGG overexpression. ( D ) The migration and invasion of SK-HEP-1 cells with FGG overexpression were dramatically inhibited by the knockdown of Slug and ZEB-1. In this experiment, siSlug-3 and siZEB-1–3 were used to knockdown the expression of Slug and ZEB-1, respectively. Each experiment was independently performed at least three times. Data are shown as mean ± SD. * P

    Journal: Cancer Management and Research

    Article Title: FGG promotes migration and invasion in hepatocellular carcinoma cells through activating epithelial to mesenchymal transition

    doi: 10.2147/CMAR.S188248

    Figure Lengend Snippet: Dysregulation of FGG affects the migration and invasion of HCC cells through EMT. Notes: ( A ) Western blots showed the changes in E-cadherin, N-cadherin, Slug, and ZEB-1 expression after artificial modification of FGG expression. siFGG means knockdown of FGG with small interfering RNA duplexes. ( B ) Quantitative analysis of EMT-associated protein expression in SK-HEP-1 cells; ( C ) Western blot results showed that Slug and ZEB-1 were knocked down in SK-HEP-1 cells with FGG overexpression. ( D ) The migration and invasion of SK-HEP-1 cells with FGG overexpression were dramatically inhibited by the knockdown of Slug and ZEB-1. In this experiment, siSlug-3 and siZEB-1–3 were used to knockdown the expression of Slug and ZEB-1, respectively. Each experiment was independently performed at least three times. Data are shown as mean ± SD. * P

    Article Snippet: During EMT, proteins involved in cell junctions, such as E-cadherin are downregulated, while mesenchymal markers, such as N-cadherin are upregulated.

    Techniques: Migration, Western Blot, Expressing, Modification, Small Interfering RNA, Over Expression

    Illustration of Gd-CDs/AFn (DOX)/FA nanocomposite preparation. Abbreviations: AFn, apoferritin; CDs, carbon dots; DEG, diethylene glycol; DOX, doxorubicin; EDC, N-(3-dimethylaminopropyl-N′-ethylcarbodiimide); FA, folic acid; Gd, gadolinium; NHS, N-hydroxysuccinimide; PEI, polyethylenimine.

    Journal: International Journal of Nanomedicine

    Article Title: Construction of magnetic-carbon-quantum-dots-probe-labeled apoferritin nanocages for bioimaging and targeted therapy

    doi: 10.2147/IJN.S108039

    Figure Lengend Snippet: Illustration of Gd-CDs/AFn (DOX)/FA nanocomposite preparation. Abbreviations: AFn, apoferritin; CDs, carbon dots; DEG, diethylene glycol; DOX, doxorubicin; EDC, N-(3-dimethylaminopropyl-N′-ethylcarbodiimide); FA, folic acid; Gd, gadolinium; NHS, N-hydroxysuccinimide; PEI, polyethylenimine.

    Article Snippet: Ferritin from equine spleen, FA, citric acid, polyethylenimine (PEI; molecular weight =2,500), diethylene glycol, ethylenediamine, formamide, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl-N′-ethylcarbodiimide) hydrochloride (EDC-HCl), and dimethyl sulfoxide were obtained from Sigma-Aldrich Co. (St Louis, MO, USA).

    Techniques:

    Synthesis scheme of magnetic nanoparticles and the final product. Notes: ( A ) The procedure for synthesizing three different types of magnetic nanoparticles: Fe 3 O 4 , Dex/Fe 3 O 4 , and Bis/Dex/Fe 3 O 4 . ( B ) The scheme of the final product Bis/Dex/Fe 3 O 4 . ( C ) Bis/Dex/Fe 3 O 4 fully dispersed in PBS at concentrations of 1, 2, and 4 mg/mL (from left to right). ( D ) High concentration (200 mg/mL) of Bis/Dex/Fe 3 O 4 magnetic nanoparticles are well dispersed in the saline and attracted by a 3,000 G magnet. Abbreviations: Fe 3 O 4 , iron (II, III) oxide; Dex, dextran; Bis, bisphosphonate; PBS, phosphate-buffered saline; DD, double-distilled; N 2 H 4 , hydrazine; NaOH, sodium hydroxide; NaH 2 PO 4 , sodium phosphate monobasic; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS, N -hydroxysuccinimide.

    Journal: International Journal of Nanomedicine

    Article Title: Synthesis of composite magnetic nanoparticles Fe3O4 with alendronate for osteoporosis treatment

    doi: 10.2147/IJN.S112415

    Figure Lengend Snippet: Synthesis scheme of magnetic nanoparticles and the final product. Notes: ( A ) The procedure for synthesizing three different types of magnetic nanoparticles: Fe 3 O 4 , Dex/Fe 3 O 4 , and Bis/Dex/Fe 3 O 4 . ( B ) The scheme of the final product Bis/Dex/Fe 3 O 4 . ( C ) Bis/Dex/Fe 3 O 4 fully dispersed in PBS at concentrations of 1, 2, and 4 mg/mL (from left to right). ( D ) High concentration (200 mg/mL) of Bis/Dex/Fe 3 O 4 magnetic nanoparticles are well dispersed in the saline and attracted by a 3,000 G magnet. Abbreviations: Fe 3 O 4 , iron (II, III) oxide; Dex, dextran; Bis, bisphosphonate; PBS, phosphate-buffered saline; DD, double-distilled; N 2 H 4 , hydrazine; NaOH, sodium hydroxide; NaH 2 PO 4 , sodium phosphate monobasic; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS, N -hydroxysuccinimide.

    Article Snippet: Ferrous chloride tetrahydrate (FeCl2 ⋅4H2 O), ferrous chloride hexahydrate (FeCl3 ⋅6H2 O), Dex ((C6 H10 O5 )n , molecular weight [MW] ~40,000 Da), hydrazine (N2 H4 ), sodium hydroxide (NaOH), sodium chloroacetate (C2 H2 ClO2 ⋅Na), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride (C8 H17 N3 ⋅HCl), N -hydroxysuccinimide (C4 H5 NO3 ; NHS), and alendronate sodium trihydrant (C4 H12 NO7 P2 Na⋅3H2 O) were all purchased from Sigma-Aldrich Co. (St Louis, MO, USA) and then used, as received, to synthesize the composite nanoparticles.

    Techniques: Concentration Assay

    Identification and characterization of liver S-palmitoylome. ( a ) A schematic overview for the site-specific identification of the S-palmitoylome by a modified alkylating RAC assay. Free thiols are first blocked with NEM. Disulfide bond was cleaved by TCEP and further blocked with IAM. Thioesters are then cleaved with neutral HA and the newly liberated thiols are purified with thiol sepharose 4B. The capture proteins on sepharoses were digested with trypsin. The supernatant of digested samples was removed and the captured peptides on sepharoses were eluted with TCEP and subjected to downstream MS analysis for S-palmitoylated site identification. Peptide alignment was applied to increase the coverage based on the retention time and m/z of identified peptides. Extracted ion chromatogram of identified peptides and the same m/z in different LC samples were used to quantify the relative abundance. The protein levels were quantified by TMT10-plex labelling. ( b ) Pie diagram illustrating the subcellular localization of identified S-palmitoylated proteins. ( c ) Pie diagram illustrating the functions of identified S-palmitoylated proteins. NEM, N-ethylmaleimide; IAM, Iodoacetamide; HA, hydroxylamine.

    Journal: Scientific Reports

    Article Title: Role of S-Palmitoylation by ZDHHC13 in Mitochondrial function and Metabolism in Liver

    doi: 10.1038/s41598-017-02159-4

    Figure Lengend Snippet: Identification and characterization of liver S-palmitoylome. ( a ) A schematic overview for the site-specific identification of the S-palmitoylome by a modified alkylating RAC assay. Free thiols are first blocked with NEM. Disulfide bond was cleaved by TCEP and further blocked with IAM. Thioesters are then cleaved with neutral HA and the newly liberated thiols are purified with thiol sepharose 4B. The capture proteins on sepharoses were digested with trypsin. The supernatant of digested samples was removed and the captured peptides on sepharoses were eluted with TCEP and subjected to downstream MS analysis for S-palmitoylated site identification. Peptide alignment was applied to increase the coverage based on the retention time and m/z of identified peptides. Extracted ion chromatogram of identified peptides and the same m/z in different LC samples were used to quantify the relative abundance. The protein levels were quantified by TMT10-plex labelling. ( b ) Pie diagram illustrating the subcellular localization of identified S-palmitoylated proteins. ( c ) Pie diagram illustrating the functions of identified S-palmitoylated proteins. NEM, N-ethylmaleimide; IAM, Iodoacetamide; HA, hydroxylamine.

    Article Snippet: Extraction of membrane proteins from mice liver Liver tissue samples from 6-week-old mice were homogenized in LB buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA; pH 7.2) with 10 mM N-ethylmaleimide (NEM, Sigma-Aldrich), 2X protease inhibitor (PI, Roche), and 2 mM PMSF for 2 min at 24,000 rpm in a Polytron PT3000 homogenizer.

    Techniques: Modification, Purification, Mass Spectrometry

    CpMet gastroprotective mechanism in an ethanol-induced gastric ulcer model in mice. (a) Protocol validation and (b) effect of CpMet . CpMet , C. procera methanolic extract 100 mg/kg; VEH, isotonic saline solution; L-NAME, N ω -nitro-L-arginine methyl ester 10 mg/kg; INDO, indomethacin 10 mg/kg; NEM, N-ethylmaleimide 1 mg/kg; GLIB, glibenclamide 5 mg/kg. Each column represents the mean ± SEM of three independent replicates ( n = 6 per group). An ANOVA was performed followed by Tukey's test. For (a), ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Gastroprotective Mechanism and Ulcer Resolution Effect of Cyrtocarpa procera Methanolic Extract on Ethanol-Induced Gastric Injury

    doi: 10.1155/2018/2862706

    Figure Lengend Snippet: CpMet gastroprotective mechanism in an ethanol-induced gastric ulcer model in mice. (a) Protocol validation and (b) effect of CpMet . CpMet , C. procera methanolic extract 100 mg/kg; VEH, isotonic saline solution; L-NAME, N ω -nitro-L-arginine methyl ester 10 mg/kg; INDO, indomethacin 10 mg/kg; NEM, N-ethylmaleimide 1 mg/kg; GLIB, glibenclamide 5 mg/kg. Each column represents the mean ± SEM of three independent replicates ( n = 6 per group). An ANOVA was performed followed by Tukey's test. For (a), ∗ P

    Article Snippet: Absolute ethanol (ETOH), Nω -nitro-L-arginine methyl ester (L-NAME), N-ethylmaleimide (NEM), glibenclamide (GLIB), indomethacin (INDO), and carbenoxolone (CAR) were purchased from Sigma-Aldrich Co. Omeprazole (OME) was obtained from Liomont®.

    Techniques: Mouse Assay

    The effects of ATP-binding cassette transporter A7-knockdown on the migration of SKOV-3 cells. The data are presented as the mean ± standard deviation. (A) Images are representative of three independent experiments. The protein levels of E-cadherin, N-cadherin and SMAD4 were assessed by western blot analysis. All data are expressed as the mean ± standard deviation (*P

    Journal: Oncology Letters

    Article Title: ATP-binding cassette transporter A7 accelerates epithelial-to-mesenchymal transition in ovarian cancer cells by upregulating the transforming growth factor-β signaling pathway

    doi: 10.3892/ol.2018.9366

    Figure Lengend Snippet: The effects of ATP-binding cassette transporter A7-knockdown on the migration of SKOV-3 cells. The data are presented as the mean ± standard deviation. (A) Images are representative of three independent experiments. The protein levels of E-cadherin, N-cadherin and SMAD4 were assessed by western blot analysis. All data are expressed as the mean ± standard deviation (*P

    Article Snippet: Following blocking in 10% bovine serum albumin at 37°C for 2 h, the membranes were incubated overnight at 4°C with antibodies specific to β-actin (cat. no. 4970S; 1:1,000; CST Biological Reagents Co., Ltd., Shanghai, China), ABCA7 (cat. no. 25339-1-AP; 1:200; ProteinTech Group, Inc., Chicago, IL, USA), N-cadherin (cat. no. ab76057; 1:1,000; Abcam), E-cadherin (cat. no. ab15148; 1:1,000; Abcam) and SMAD4 (cat. no. D3R4N; 1:1,000; CST Biological Reagents Co., Ltd.).

    Techniques: Binding Assay, Migration, Standard Deviation, Western Blot

    TGF-β1 stimulation rescues the suppression of EMT markers and migration induced by ABCA7-knockdown in SKOV-3 cell line. (A) Images are representative of three independent experiments. The protein levels of E-cadherin, N-cadherin and SMAD4 were assessed by western blot analysis. (B) All data are expressed as mean ± standard deviation (*P

    Journal: Oncology Letters

    Article Title: ATP-binding cassette transporter A7 accelerates epithelial-to-mesenchymal transition in ovarian cancer cells by upregulating the transforming growth factor-β signaling pathway

    doi: 10.3892/ol.2018.9366

    Figure Lengend Snippet: TGF-β1 stimulation rescues the suppression of EMT markers and migration induced by ABCA7-knockdown in SKOV-3 cell line. (A) Images are representative of three independent experiments. The protein levels of E-cadherin, N-cadherin and SMAD4 were assessed by western blot analysis. (B) All data are expressed as mean ± standard deviation (*P

    Article Snippet: Following blocking in 10% bovine serum albumin at 37°C for 2 h, the membranes were incubated overnight at 4°C with antibodies specific to β-actin (cat. no. 4970S; 1:1,000; CST Biological Reagents Co., Ltd., Shanghai, China), ABCA7 (cat. no. 25339-1-AP; 1:200; ProteinTech Group, Inc., Chicago, IL, USA), N-cadherin (cat. no. ab76057; 1:1,000; Abcam), E-cadherin (cat. no. ab15148; 1:1,000; Abcam) and SMAD4 (cat. no. D3R4N; 1:1,000; CST Biological Reagents Co., Ltd.).

    Techniques: Migration, Western Blot, Standard Deviation

    Differential E-cadherin expression in cranial sutures. a Microarray profile of E-cadherin gene on SAG and PF suture shows that the gene is up-regulated exclusively in the PF suture at p15. Green = Lower than mean expression; red = higher than mean expression. b Quantitative real-time RT-PCR confirmed the up-regulation of E-cadherin gene expression in PF suture at p15. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments.

    Journal: Cells, Tissues, Organs

    Article Title: Unique Modulation of Cadherin Expression Pattern during Posterior Frontal Cranial Suture Development and Closure

    doi: 10.1159/000272318

    Figure Lengend Snippet: Differential E-cadherin expression in cranial sutures. a Microarray profile of E-cadherin gene on SAG and PF suture shows that the gene is up-regulated exclusively in the PF suture at p15. Green = Lower than mean expression; red = higher than mean expression. b Quantitative real-time RT-PCR confirmed the up-regulation of E-cadherin gene expression in PF suture at p15. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments.

    Article Snippet: In vitro work demonstrated that N-cadherin is required for mesenchymal cell specification in early phases of embryonic limb bud development, and perturbation of cadherin-mediated interactions disrupts chondrogenic cell condensation [ ; ; ].

    Techniques: Expressing, Microarray, Quantitative RT-PCR

    E-cadherin expression in posterior frontal suture during the first month of life. a Quantitative real-time PCR for E-cadherin on PF suture reveals up-regulation of E-cadherin gene starting at day p9 with maximal expression at days p13–p15, a period coinciding with active bone deposition in the PF suture and suture closure. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p

    Journal: Cells, Tissues, Organs

    Article Title: Unique Modulation of Cadherin Expression Pattern during Posterior Frontal Cranial Suture Development and Closure

    doi: 10.1159/000272318

    Figure Lengend Snippet: E-cadherin expression in posterior frontal suture during the first month of life. a Quantitative real-time PCR for E-cadherin on PF suture reveals up-regulation of E-cadherin gene starting at day p9 with maximal expression at days p13–p15, a period coinciding with active bone deposition in the PF suture and suture closure. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p

    Article Snippet: In vitro work demonstrated that N-cadherin is required for mesenchymal cell specification in early phases of embryonic limb bud development, and perturbation of cadherin-mediated interactions disrupts chondrogenic cell condensation [ ; ; ].

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    a N-cadherin expression in the posterior frontal suture during the first month of life. Top panel: Quantitative real-time PCR for N-cadherin on PF suture reveals a biphasic expression profile of N-cadherin gene. N-cadherin is first up-regulated on p7, a period coinciding with a cellular condensation in the endocranial plate of the PF suture. A second phase of N-cadherin gene up-regulation is observed on p13–p15, a period marked by osteogenesis and closure of PF suture. Bottom panel: Quantitative real-time PCR for E-cadherin on SAG suture shows that the expression is steady overtime. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p

    Journal: Cells, Tissues, Organs

    Article Title: Unique Modulation of Cadherin Expression Pattern during Posterior Frontal Cranial Suture Development and Closure

    doi: 10.1159/000272318

    Figure Lengend Snippet: a N-cadherin expression in the posterior frontal suture during the first month of life. Top panel: Quantitative real-time PCR for N-cadherin on PF suture reveals a biphasic expression profile of N-cadherin gene. N-cadherin is first up-regulated on p7, a period coinciding with a cellular condensation in the endocranial plate of the PF suture. A second phase of N-cadherin gene up-regulation is observed on p13–p15, a period marked by osteogenesis and closure of PF suture. Bottom panel: Quantitative real-time PCR for E-cadherin on SAG suture shows that the expression is steady overtime. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p

    Article Snippet: In vitro work demonstrated that N-cadherin is required for mesenchymal cell specification in early phases of embryonic limb bud development, and perturbation of cadherin-mediated interactions disrupts chondrogenic cell condensation [ ; ; ].

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Expression of N- and E-cadherin transcriptional repressors Wnt7a and Snail. a Quantitative real-time PCR for Wnt7a on PF suture reveals up-regulation of Wnt7a geneas early as p7, with maximal expression on p11 and return to baseline by p13. b The inverse gene expression correlation between Wnt7a and N-cadherin. Note that Wnt7a gene expression is specifically up-regulated during the down-regulation of N-cadherin gene. c Quantitative real-time PCR for Snail on PF suture reveals a moderate up-regulation of Snail genestarting on p5, with a decrease on p9 and a return to baseline by p15. * p

    Journal: Cells, Tissues, Organs

    Article Title: Unique Modulation of Cadherin Expression Pattern during Posterior Frontal Cranial Suture Development and Closure

    doi: 10.1159/000272318

    Figure Lengend Snippet: Expression of N- and E-cadherin transcriptional repressors Wnt7a and Snail. a Quantitative real-time PCR for Wnt7a on PF suture reveals up-regulation of Wnt7a geneas early as p7, with maximal expression on p11 and return to baseline by p13. b The inverse gene expression correlation between Wnt7a and N-cadherin. Note that Wnt7a gene expression is specifically up-regulated during the down-regulation of N-cadherin gene. c Quantitative real-time PCR for Snail on PF suture reveals a moderate up-regulation of Snail genestarting on p5, with a decrease on p9 and a return to baseline by p15. * p

    Article Snippet: In vitro work demonstrated that N-cadherin is required for mesenchymal cell specification in early phases of embryonic limb bud development, and perturbation of cadherin-mediated interactions disrupts chondrogenic cell condensation [ ; ; ].

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    E-cadherin expression is not down-regulated in the PF suture of Fgf-2 null mice. a E-cadherin expression in PF suture of Fgf-2 null mice during the first month of life was assessed by quantitative real-time PCR. Up-regulation of E-cadherin gene is observed starting on p5 with maximal level on p9 and return to baseline by p11. Note that up-regulation of E-cadherin gene in Fgf-2 null mice occurs earlier than in CD-1 wild-type mice. b N-cadherin expression in PF suture of Fgf-2 null mice also shows a pattern similar to that of wild-type CD-1 mice. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments. * p

    Journal: Cells, Tissues, Organs

    Article Title: Unique Modulation of Cadherin Expression Pattern during Posterior Frontal Cranial Suture Development and Closure

    doi: 10.1159/000272318

    Figure Lengend Snippet: E-cadherin expression is not down-regulated in the PF suture of Fgf-2 null mice. a E-cadherin expression in PF suture of Fgf-2 null mice during the first month of life was assessed by quantitative real-time PCR. Up-regulation of E-cadherin gene is observed starting on p5 with maximal level on p9 and return to baseline by p11. Note that up-regulation of E-cadherin gene in Fgf-2 null mice occurs earlier than in CD-1 wild-type mice. b N-cadherin expression in PF suture of Fgf-2 null mice also shows a pattern similar to that of wild-type CD-1 mice. Quantified mRNA values were normalized by the amounts of Gapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments. * p

    Article Snippet: In vitro work demonstrated that N-cadherin is required for mesenchymal cell specification in early phases of embryonic limb bud development, and perturbation of cadherin-mediated interactions disrupts chondrogenic cell condensation [ ; ; ].

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Dendritic release requires L-type Ca 2+ channels. A , Dendritic release responses to AMPA or NMDA in the presence and absence of IEM1460 (100 µM) or nimodipine (3 µM). n ≥ 5. ****p

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

    Article Title: Action potential-independent and pharmacologically unique vesicular serotonin release from dendrites

    doi: 10.1523/JNEUROSCI.0020-12.2012

    Figure Lengend Snippet: Dendritic release requires L-type Ca 2+ channels. A , Dendritic release responses to AMPA or NMDA in the presence and absence of IEM1460 (100 µM) or nimodipine (3 µM). n ≥ 5. ****p

    Article Snippet: Parachloroamphetamine (Sigma) was also applied at 32° C. For stimulation with ionotropic glutamate receptor agonists, AMPA (Sigma, 10 µM) or NMDA (Sigma, 50 µM) were applied to slices in room temperature n-aCSF for 1 minute.

    Techniques:

    Internalization of truncated FcγRIIa/b is augmented by RTX treatment. Ramos cells transfected with FcγRIIbΔcyt or FcγRIIaΔcyt were treated with sulfo-NHS-SS-biotin at 4 °C to biotinylate cell surface proteins and cultured with 5 μg/ml A488-labeled RTX for 2 h at 37 °C or left untreated. Internalization of FcγRIIb/a was assessed at 0, 30, 60, and 120 min by treating cells with MESNa to remove cell surface biotin, followed by immunoprecipitation of internalized biotinylated proteins with streptavidin-coated beads. Precipitated proteins were blotted for FcγRII. A and C , representative examples of FcγRIIbΔcyt and FcγRIIaΔcyt transfectants left untreated ( left panels ) or treated with RTX ( right panels ), respectively. −, not treated ( NT ); +, MESNa-treated. B and D , densitometry demonstrating the proportion of internalized FcγRIIbΔcyt ( B ) and FcγRIIaΔcyt ( D ) in untreated cells ( left panels ) and cells treated with RTX ( right panels ), expressed as a percentage of non-MESNa-treated cells, n = 3. Horizontal bars represent the median.

    Journal: The Journal of Biological Chemistry

    Article Title: Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain *

    doi: 10.1074/jbc.M114.593806

    Figure Lengend Snippet: Internalization of truncated FcγRIIa/b is augmented by RTX treatment. Ramos cells transfected with FcγRIIbΔcyt or FcγRIIaΔcyt were treated with sulfo-NHS-SS-biotin at 4 °C to biotinylate cell surface proteins and cultured with 5 μg/ml A488-labeled RTX for 2 h at 37 °C or left untreated. Internalization of FcγRIIb/a was assessed at 0, 30, 60, and 120 min by treating cells with MESNa to remove cell surface biotin, followed by immunoprecipitation of internalized biotinylated proteins with streptavidin-coated beads. Precipitated proteins were blotted for FcγRII. A and C , representative examples of FcγRIIbΔcyt and FcγRIIaΔcyt transfectants left untreated ( left panels ) or treated with RTX ( right panels ), respectively. −, not treated ( NT ); +, MESNa-treated. B and D , densitometry demonstrating the proportion of internalized FcγRIIbΔcyt ( B ) and FcγRIIaΔcyt ( D ) in untreated cells ( left panels ) and cells treated with RTX ( right panels ), expressed as a percentage of non-MESNa-treated cells, n = 3. Horizontal bars represent the median.

    Article Snippet: Ramos cells transfected with WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, and FcγRIIaΔcyt were treated with sulfo-NHS-SS-biotin and then cultured in the presence of A488-labeled RTX to stimulate internalization of the FcγR.

    Techniques: Transfection, Cell Culture, Labeling, Immunoprecipitation

    Differential recycling of FcγRIIb1 and FcγRIIa is not responsible for the slower rate of internalization of CD20 mediated by FcγRIIa in response to ligation by RTX. Ramos cells transfected with WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, or FcγRIIaΔcyt were treated with sulfo-NHS-SS-biotin at 4 °C to biotinylate cell surface proteins and cultured with 5 μg/ml A488-labeled RTX for 2 h ( A and B ) or 30 min ( C and D ) to allow endocytosis. Cells were then treated with MESNa to remove cell surface biotin. Cells were either treated with MESNa for a second time or buffer lacking MESNa (0 h as indicated) or returned to 37 °C for a further 2 h to allow recycling of internalized proteins (2 h). After further MESNa treatment or treatment with buffer lacking MESNa (2 h as indicated), internalized biotinylated proteins were immunoprecipitated with streptavidin-coated beads. Precipitated proteins were blotted for FcγRII. A and C , representative examples demonstrating recycling of WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, and FcγRIIaΔcyt, respectively. −, not treated; +, MESNa-treated. B and D , densitometry demonstrating the relative density of internalized WT FcγRIIb1 ( B , left panels ), WT FcγRIIa ( B , right panels ), FcγRIIbΔcyt ( D , left panels ), or FcγRIIaΔcyt ( D , right panels ), expressed as a percentage of 0 h, non-MESNa-treated cells, n = 4. Horizontal bars represent the median. E , representative examples demonstrating recycling of CD22 in Ramos cells transfected with WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, or FcγRIIaΔcyt.

    Journal: The Journal of Biological Chemistry

    Article Title: Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain *

    doi: 10.1074/jbc.M114.593806

    Figure Lengend Snippet: Differential recycling of FcγRIIb1 and FcγRIIa is not responsible for the slower rate of internalization of CD20 mediated by FcγRIIa in response to ligation by RTX. Ramos cells transfected with WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, or FcγRIIaΔcyt were treated with sulfo-NHS-SS-biotin at 4 °C to biotinylate cell surface proteins and cultured with 5 μg/ml A488-labeled RTX for 2 h ( A and B ) or 30 min ( C and D ) to allow endocytosis. Cells were then treated with MESNa to remove cell surface biotin. Cells were either treated with MESNa for a second time or buffer lacking MESNa (0 h as indicated) or returned to 37 °C for a further 2 h to allow recycling of internalized proteins (2 h). After further MESNa treatment or treatment with buffer lacking MESNa (2 h as indicated), internalized biotinylated proteins were immunoprecipitated with streptavidin-coated beads. Precipitated proteins were blotted for FcγRII. A and C , representative examples demonstrating recycling of WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, and FcγRIIaΔcyt, respectively. −, not treated; +, MESNa-treated. B and D , densitometry demonstrating the relative density of internalized WT FcγRIIb1 ( B , left panels ), WT FcγRIIa ( B , right panels ), FcγRIIbΔcyt ( D , left panels ), or FcγRIIaΔcyt ( D , right panels ), expressed as a percentage of 0 h, non-MESNa-treated cells, n = 4. Horizontal bars represent the median. E , representative examples demonstrating recycling of CD22 in Ramos cells transfected with WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, or FcγRIIaΔcyt.

    Article Snippet: Ramos cells transfected with WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, and FcγRIIaΔcyt were treated with sulfo-NHS-SS-biotin and then cultured in the presence of A488-labeled RTX to stimulate internalization of the FcγR.

    Techniques: Ligation, Transfection, Cell Culture, Labeling, Immunoprecipitation

    Internalization of WT FcγIIb1 and FcγRIIa is augmented by RTX treatment. Ramos cells transfected with WT FcγRIIb1 or WT FcγRIIa were treated with sulfo-NHS-SS-biotin at 4 °C to biotinylate cell surface proteins and cultured with 5 μg/ml A488-labeled RTX for 6 h at 37 °C or left untreated. Internalization of FcγRIIb1/a was assessed at 0, 1, 2, and 6 h by treating cells with MESNa to remove cell surface biotin, followed by immunoprecipitation of internalized biotinylated proteins with streptavidin-coated beads. Precipitated proteins were blotted for FcγRII. A and C , representative examples of WT FcγRIIb1 and WT FcγRIIa transfectants left untreated ( left panels ) or treated with RTX ( right panels ), respectively. −, not treated ( NT ); +, MESNa-treated. B and D , densitometry demonstrating the proportion of internalized WT FcγRIIb1 ( B ) and WT FcγRIIa ( D ) in untreated cells ( left panels ) and cells treated with RTX ( right panels ), expressed as a percentage of non-MESNa-treated cells, n = 3. Horizontal bars represent the median.

    Journal: The Journal of Biological Chemistry

    Article Title: Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain *

    doi: 10.1074/jbc.M114.593806

    Figure Lengend Snippet: Internalization of WT FcγIIb1 and FcγRIIa is augmented by RTX treatment. Ramos cells transfected with WT FcγRIIb1 or WT FcγRIIa were treated with sulfo-NHS-SS-biotin at 4 °C to biotinylate cell surface proteins and cultured with 5 μg/ml A488-labeled RTX for 6 h at 37 °C or left untreated. Internalization of FcγRIIb1/a was assessed at 0, 1, 2, and 6 h by treating cells with MESNa to remove cell surface biotin, followed by immunoprecipitation of internalized biotinylated proteins with streptavidin-coated beads. Precipitated proteins were blotted for FcγRII. A and C , representative examples of WT FcγRIIb1 and WT FcγRIIa transfectants left untreated ( left panels ) or treated with RTX ( right panels ), respectively. −, not treated ( NT ); +, MESNa-treated. B and D , densitometry demonstrating the proportion of internalized WT FcγRIIb1 ( B ) and WT FcγRIIa ( D ) in untreated cells ( left panels ) and cells treated with RTX ( right panels ), expressed as a percentage of non-MESNa-treated cells, n = 3. Horizontal bars represent the median.

    Article Snippet: Ramos cells transfected with WT FcγRIIb1, WT FcγRIIa, FcγRIIbΔcyt, and FcγRIIaΔcyt were treated with sulfo-NHS-SS-biotin and then cultured in the presence of A488-labeled RTX to stimulate internalization of the FcγR.

    Techniques: Transfection, Cell Culture, Labeling, Immunoprecipitation

    Effects of mutations in the hBest1 C terminus on channel function. (A) Current amplitudes of point mutants in the C-terminal acidic region (amino acids 293–308) and the putative EF hand loop (amino acids 312–323). Each amino acid (in black) and its position in the C terminus are indicated along the axis. The corresponding mutations are colored red, green, and blue. Each data point represents four to nine cells. The solid line indicates the average current amplitude of wild type ( n = 8), and the dashed lines represent the standard error. (B) Expression of hBest1 mutants in the plasma membrane of HEK cells. Wild-type, N296L, E300Q, D301N, D302N, D303L, D304N, E306Q, and N308D mutant-transfected and nontransfected cells were exposed to membrane-impermeable Sulfo-NHS-LC biotin. Biotinylated membrane proteins (20 μl; left) purified with streptavidin resin and total proteins (5 μl; right) were probed with antibodies to the myc tag on hBest1 (68-kD band; top) and with antibodies to intracellular protein GAPDH (37-kD band; bottom).

    Journal: The Journal of General Physiology

    Article Title: Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

    doi: 10.1085/jgp.200810056

    Figure Lengend Snippet: Effects of mutations in the hBest1 C terminus on channel function. (A) Current amplitudes of point mutants in the C-terminal acidic region (amino acids 293–308) and the putative EF hand loop (amino acids 312–323). Each amino acid (in black) and its position in the C terminus are indicated along the axis. The corresponding mutations are colored red, green, and blue. Each data point represents four to nine cells. The solid line indicates the average current amplitude of wild type ( n = 8), and the dashed lines represent the standard error. (B) Expression of hBest1 mutants in the plasma membrane of HEK cells. Wild-type, N296L, E300Q, D301N, D302N, D303L, D304N, E306Q, and N308D mutant-transfected and nontransfected cells were exposed to membrane-impermeable Sulfo-NHS-LC biotin. Biotinylated membrane proteins (20 μl; left) purified with streptavidin resin and total proteins (5 μl; right) were probed with antibodies to the myc tag on hBest1 (68-kD band; top) and with antibodies to intracellular protein GAPDH (37-kD band; bottom).

    Article Snippet: In brief, cells transfected with wild-type and mutant hBest1 and nontransfected cells were placed on ice, washed three times with PBS, and biotinylated with 0.5 mg/ml Sulfo-NHS-LC Biotin (Thermo Fisher Scientific) in PBS for 30 min.

    Techniques: Expressing, Mutagenesis, Transfection, Purification

    Stabilizing beta-catenin signaling suppresses EC fenestrae and reduces vascular permeability. ( A–F ) Transmission electron micrographs from control mice (left) and from age-matched mice with EC-specific deletion of Ctnnb1 exon 3 (right) showing representative sections of choriocapillaris ( A,B ), posterior pituitary ( C,D ), and choroid plexus ( E,F ). RPE, retinal pigment epithelium. Vertical arrows mark fenestrae. Neurohormone secretory granules are seen in the posterior pituitary parenchyma. Scale bar, 500 nm. ( G ) Quantification of the density of capillary EC fenestrae in age-matched and 4HT-treated control ( Ctnnb1 flex3/+ ) vs. Ctnnb1 exon 3 stabilized ( Ctnnb1 flex3/+ ;Pdgfb-CreER ) mice for the choriocapillaris, posterior pituitary, choroid plexus, and ciliary body. Each data-point represents all of the vascular wall length within a single 10 μm x 10 μm TEM image. Bars show mean ± S.D. Each cluster of data points represents a different mouse. For each location and genotype, 3–5 mice were analyzed. For each of the four anatomic locations, the p-values are calculated for all of the Ctnnb1 flex3/+ vs. all of the Ctnnb1 flex3/+ ;Pdgfb-CreER data-points. ( H,I ) Sagittal brain sections show vascular markers GLUT1 and PLVAP and perivascular accumulation of Sulfo-NHS-biotin (following IP injection) in the SFO and adjacent choroid plexus ( H ) and in the area postrema and adjacent choroid plexus ( I ) from ~P30 WT vs. Ctnnb1 flex3/+ ;Pdgfb-CreER mice. AP, area postrema; CP, choroid plexus. A, anterior; P, posterior. Scale bars in ( H ) and ( I ), 200 μm.

    Journal: eLife

    Article Title: Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures

    doi: 10.7554/eLife.43257

    Figure Lengend Snippet: Stabilizing beta-catenin signaling suppresses EC fenestrae and reduces vascular permeability. ( A–F ) Transmission electron micrographs from control mice (left) and from age-matched mice with EC-specific deletion of Ctnnb1 exon 3 (right) showing representative sections of choriocapillaris ( A,B ), posterior pituitary ( C,D ), and choroid plexus ( E,F ). RPE, retinal pigment epithelium. Vertical arrows mark fenestrae. Neurohormone secretory granules are seen in the posterior pituitary parenchyma. Scale bar, 500 nm. ( G ) Quantification of the density of capillary EC fenestrae in age-matched and 4HT-treated control ( Ctnnb1 flex3/+ ) vs. Ctnnb1 exon 3 stabilized ( Ctnnb1 flex3/+ ;Pdgfb-CreER ) mice for the choriocapillaris, posterior pituitary, choroid plexus, and ciliary body. Each data-point represents all of the vascular wall length within a single 10 μm x 10 μm TEM image. Bars show mean ± S.D. Each cluster of data points represents a different mouse. For each location and genotype, 3–5 mice were analyzed. For each of the four anatomic locations, the p-values are calculated for all of the Ctnnb1 flex3/+ vs. all of the Ctnnb1 flex3/+ ;Pdgfb-CreER data-points. ( H,I ) Sagittal brain sections show vascular markers GLUT1 and PLVAP and perivascular accumulation of Sulfo-NHS-biotin (following IP injection) in the SFO and adjacent choroid plexus ( H ) and in the area postrema and adjacent choroid plexus ( I ) from ~P30 WT vs. Ctnnb1 flex3/+ ;Pdgfb-CreER mice. AP, area postrema; CP, choroid plexus. A, anterior; P, posterior. Scale bars in ( H ) and ( I ), 200 μm.

    Article Snippet: We have performed a permeability analysis using Sulfo-NHS-biotin and the results are shown in panels H and I in the newly expanded Figure 5.

    Techniques: Permeability, Transmission Assay, Mouse Assay, Transmission Electron Microscopy, Injection

    hucMSC-Ex reversed TGF-β1-induced EMT in human hepatocyte cell line HL7702 cells. (A) The morphology of HL7702 cells treated with TGF-β1 for 3 days followed by hucMSC-Ex for 3 days (200×). (a) HL7702 cells, 3 days (C3d); (b) TGF-β1+HL7702, 3 days (T3d); (c) TGF-β1+HL7702, 6 days (T6d); (d) TGF-β1+HL7702+hucMSC-Ex, 6 days (T+E 6d). (B) Quantitative RT-PCR analyses of E-cadherin , N-cadherin , and Twist mRNA expression. (C) Western blotting analyses of E-cadherin and N-cadherin expression in cells. (D) Density analysis of western blot bands. * P

    Journal: Stem Cells and Development

    Article Title: Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Alleviate Liver Fibrosis

    doi: 10.1089/scd.2012.0395

    Figure Lengend Snippet: hucMSC-Ex reversed TGF-β1-induced EMT in human hepatocyte cell line HL7702 cells. (A) The morphology of HL7702 cells treated with TGF-β1 for 3 days followed by hucMSC-Ex for 3 days (200×). (a) HL7702 cells, 3 days (C3d); (b) TGF-β1+HL7702, 3 days (T3d); (c) TGF-β1+HL7702, 6 days (T6d); (d) TGF-β1+HL7702+hucMSC-Ex, 6 days (T+E 6d). (B) Quantitative RT-PCR analyses of E-cadherin , N-cadherin , and Twist mRNA expression. (C) Western blotting analyses of E-cadherin and N-cadherin expression in cells. (D) Density analysis of western blot bands. * P

    Article Snippet: After TGF-β1 induction for 3 days, the expression of E-cadherin mRNA decreased, while N- cadherin and Twist mRNA expression increased in HL7702 cells by quantitative RT-PCR.

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    hucMSC-Ex inhibited epithelial-to-mesenchymal transition (EMT). (A) Immunohistochemistry analysis of E-cadherin, N-cadherin, and vimentin 3 weeks after hucMSC-Ex transplantation (200×). (a, d, g) normal group; (b, e, h) PBS group; (c, f, i) hucMSC-Ex group. (B) Positive cells analysis of immunohistochemical sections. ( n =3). * P

    Journal: Stem Cells and Development

    Article Title: Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Alleviate Liver Fibrosis

    doi: 10.1089/scd.2012.0395

    Figure Lengend Snippet: hucMSC-Ex inhibited epithelial-to-mesenchymal transition (EMT). (A) Immunohistochemistry analysis of E-cadherin, N-cadherin, and vimentin 3 weeks after hucMSC-Ex transplantation (200×). (a, d, g) normal group; (b, e, h) PBS group; (c, f, i) hucMSC-Ex group. (B) Positive cells analysis of immunohistochemical sections. ( n =3). * P

    Article Snippet: After TGF-β1 induction for 3 days, the expression of E-cadherin mRNA decreased, while N- cadherin and Twist mRNA expression increased in HL7702 cells by quantitative RT-PCR.

    Techniques: Immunohistochemistry, Transplantation Assay

    A: Drawings depicting the connections of the ventral division of the external nucleus of the inferior colliculus (ICXv) determined from focal wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injection (filled area a) in one bat. B: Photomicrograph of diaminobenzidine (DAB)-reacted WGA-HRP-stained section (CytOx counterstained) showing the size of the injection site. In A, labeling is shown on five transverse brain sections. The rostrocaudal distance between the sections is 360 µm for the two most caudal sections, and about 600 µm between the remaining rostral sections. Large filled circles represent cell bodies labeled by retrograde transport; lines and small dots represent labeled fibers or terminals, respectively. Open circles are labeled cells in the immediate vicinity of the injection site. a: Injection site in the ICXv indicated by filled area (reconstructed from DAB-stained section shown in A). b: Retrogradely labeled cell bodies in the nucleus of the central acoustic tract (NCAT; tetramethylbenzidine [TMB] reaction). c: Labeled fibers coursing medial to the lateral lemniscus. d: Fibers in the deep superior colliculus (SCd). e: Fine punctate labeling in the suprageniculate nucleus (Sg) of the medial geniculate body. f: Transport of tracer to Sg was via fibers running along the ventral half of brachium of the inferior colliculus (BIC). D, dorsal; m, medial; c, caudal; v, ventral; l, lateral; r, rostral. Scale bar in B = 500 µm.

    Journal: The Journal of comparative neurology

    Article Title: An Extralemniscal Component of the Mustached Bat Inferior Colliculus Selective for Direction and Rate of Linear Frequency Modulations

    doi:

    Figure Lengend Snippet: A: Drawings depicting the connections of the ventral division of the external nucleus of the inferior colliculus (ICXv) determined from focal wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injection (filled area a) in one bat. B: Photomicrograph of diaminobenzidine (DAB)-reacted WGA-HRP-stained section (CytOx counterstained) showing the size of the injection site. In A, labeling is shown on five transverse brain sections. The rostrocaudal distance between the sections is 360 µm for the two most caudal sections, and about 600 µm between the remaining rostral sections. Large filled circles represent cell bodies labeled by retrograde transport; lines and small dots represent labeled fibers or terminals, respectively. Open circles are labeled cells in the immediate vicinity of the injection site. a: Injection site in the ICXv indicated by filled area (reconstructed from DAB-stained section shown in A). b: Retrogradely labeled cell bodies in the nucleus of the central acoustic tract (NCAT; tetramethylbenzidine [TMB] reaction). c: Labeled fibers coursing medial to the lateral lemniscus. d: Fibers in the deep superior colliculus (SCd). e: Fine punctate labeling in the suprageniculate nucleus (Sg) of the medial geniculate body. f: Transport of tracer to Sg was via fibers running along the ventral half of brachium of the inferior colliculus (BIC). D, dorsal; m, medial; c, caudal; v, ventral; l, lateral; r, rostral. Scale bar in B = 500 µm.

    Article Snippet: For the other two bats, two interleaved sets of brain sections were collected; one set was reacted with tetramethylbenzidine (TMB, Sigma-Aldrich), the other with DAB.

    Techniques: Whole Genome Amplification, Injection, Staining, Labeling

    AMC can be conjugated to free Carboxyl groups on Proteins ( a ) Linear correlation between free AMC concentration, from 100nM to 1mM, and fluorescence. Here different concentrations of AMC, dissolved in proteolysis buffer, were incubated at 37°C on 96-well plates. Fluorescence was analyzed at an emission wavelength of 444nM, with excitation wavelength of 390nM. Values are means ± SE, n = 3. ( b ) Addition of increasing amounts of BSA to AMC in the presence of NaCNBH 3 progressively quenches the fluorescence of AMC. Here 0–50mg of BSA was added to 100μM AMC and 20mM NaCNBH 3 and incubated for 1hr at 37°C. Free AMC content was determined with reference to a standard curve of known AMC concentrations. Values are means ± SE, n = 3. ( c ) Here 50mg/ml of BSA was incubated with 1mM AMC in the presence or absence of 20mM NaCNBH 3 , and then run on a 12% SDS Page gel. A fluorescent BSA-AMC complex was readily observed at ≈66kDa (the approximate size of BSA), using an excitation wavelength of 365nM and an emission wavelength of 444 nm, when all three reagents were present, but could only be faintly discerned in the absence of NaCNBH 3 . A silver stain was later performed. ( d ], was incubated with 50mg of BSA for 1hr. BSA was extensively dialyzed then then prepared as in panel (c). Increasing concentrations of N-(3-Dimethylamineopropyl)-N'-ethylcarbodiimide, caused a progressive decrease in BSA’s electrophoretic mobility, and loss of fluorescence at 66kDa; a representative gel is shown to the left of the panel, and fluorescence is quantified in the graph to the right.

    Journal: Free radical biology & medicine

    Article Title: A SIMPLE FLUORESCENT LABELING METHOD FOR STUDIES OF PROTEIN OXIDATION, PROTEIN MODIFICATION, AND PROTEOLYSIS

    doi: 10.1016/j.freeradbiomed.2011.08.018

    Figure Lengend Snippet: AMC can be conjugated to free Carboxyl groups on Proteins ( a ) Linear correlation between free AMC concentration, from 100nM to 1mM, and fluorescence. Here different concentrations of AMC, dissolved in proteolysis buffer, were incubated at 37°C on 96-well plates. Fluorescence was analyzed at an emission wavelength of 444nM, with excitation wavelength of 390nM. Values are means ± SE, n = 3. ( b ) Addition of increasing amounts of BSA to AMC in the presence of NaCNBH 3 progressively quenches the fluorescence of AMC. Here 0–50mg of BSA was added to 100μM AMC and 20mM NaCNBH 3 and incubated for 1hr at 37°C. Free AMC content was determined with reference to a standard curve of known AMC concentrations. Values are means ± SE, n = 3. ( c ) Here 50mg/ml of BSA was incubated with 1mM AMC in the presence or absence of 20mM NaCNBH 3 , and then run on a 12% SDS Page gel. A fluorescent BSA-AMC complex was readily observed at ≈66kDa (the approximate size of BSA), using an excitation wavelength of 365nM and an emission wavelength of 444 nm, when all three reagents were present, but could only be faintly discerned in the absence of NaCNBH 3 . A silver stain was later performed. ( d ], was incubated with 50mg of BSA for 1hr. BSA was extensively dialyzed then then prepared as in panel (c). Increasing concentrations of N-(3-Dimethylamineopropyl)-N'-ethylcarbodiimide, caused a progressive decrease in BSA’s electrophoretic mobility, and loss of fluorescence at 66kDa; a representative gel is shown to the left of the panel, and fluorescence is quantified in the graph to the right.

    Article Snippet: In some experiments samples were pre-treated with either N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma-Aldrich, MO, USA, catalogue# E6383-1G) to block free protein carboxyl groups, with sulfo- N -hydroxysulfosuccinimide-acetate (Pierce, Rockford, IL, USA, catalogue #26777) to block free protein amino groups, or with tryptamine (Sigma-Aldrich, MO, USA, catalogue# 193747-10G) to disrupt potential non-covalent interactions in protein hydrophobic pockets.

    Techniques: Concentration Assay, Fluorescence, Incubation, SDS Page, Silver Staining