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  • 99
    Vector Laboratories biotinylated secondary antibodies
    Avastin attenuates astrogliosis and blood vessel formation in the injured cortex. a, c Cortical sections were obtained from mice in which Avastin or PBS was infused into the ventricle 14 d post-ATP injection. GFAP and CoL1A1 were visualized with Alexa-488 and Alexa-594 conjugated secondary antibodies ( a ) or <t>biotinylated</t> secondary antibodies and DAB-based color reaction ( c ). b Relative fluorescence intensity of GFAP and CoL1A1 were measured using ZEN software and plotted. Values are means ± SEMs for animals treated with saline ( n = 4) or Avastin ( n = 3) (*, p
    Biotinylated Secondary Antibodies, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 10949 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher goat anti mouse igg h l cross adsorbed secondary antibody
    In vivo expression of non-tenocyte markers PPARγ, collagen type II, and osteocalcin in rats implanted with hTSCs treated with various concentrations of PGE 2 and their respective hematoxylin and eosin (H E) stained tissue sections. hTSCs cultured with three concentrations (0.1, 10, and 100 ng/ml) of PGE 2 were implanted subcutaneously into rats; later, immunohistochemical and histological analyses were performed on tissue sections. For the immunohistochemical staining, fixed tissue sections were incubated with mouse anti-human PPARγ antibody, mouse anti-collagen type II (Collagen II) antibody, or mouse anti-human osteocalcin antibody. Cy3-conjugated goat anti-mouse <t>IgG</t> was then used to detect primary binding. Nuclei were stained with Hoechst (blue). Expression levels of PPARγ, collagen type II, and osteocalcin (red) were lower in cells treated with 0.1 ng/ml PGE 2 ( A–C ) than those treated with the higher concentrations (10 and 100 ng/ml) of PGE 2 ( E–G, I–K ). H E staining was also performed on tissue sections ( D, H, L ). More cells (black dots; see insets in D , H , and L ) were observed in tissues implanted with hTSCs that had been treated with high concentrations of PGE 2 in culture ( H, L ). Specifically, at 100 ng/ml ( L ), cells were concentrated in a specific region (triangle). Additionally, semi-quantification of the stained cells was performed by counting immuno-positive cells and calculating percentage staining ( M ) (*p
    Goat Anti Mouse Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti flag m2 antibody
    Cysteine trapping of the complex between ACKR3 and CXCL12. ( a ) CRS2 interactions detected using SDS–PAGE of purified ACKR3:CXCL12 complexes in the absence of reducing agent. Upper bands correspond to the crosslinked complex and lower bands to free receptor. Crosslinking of thermostabilized apocytochrome b562 (bRIL)-R197C-ACKR3 with Cys mutants in the N terminus of CXCL12. No crosslinking is seen for control samples N45C-CXCL12 or WT-CXCL12. ( b ) Crosslinking of Y7C-CXCL12 with ECL2 mutants of ACKR3. R197C-ACKR3 efficiently crosslinks with the chemokine, while no crosslinking is detected for F199C-ACKR3 or WT-ACKR3. ( c ) Schematic summary of disulfide trapping experiments involving ECL2 of ACKR3 and the N terminus of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Crosslinked sites are connected with solid lines, where the line thickness is proportional to crosslinking efficiency. Dashed lines indicate unsuccessful cross-links. ( d ) Cysteine trapping of CRS1 interactions detected by western blot of ACKR3:CXCL12 complexes under non-reducing conditions. <t>ACKR3-FLAG</t> and CXCL12-HA were detected with primary <t>anti-FLAG</t> and anti-HA antibodies and fluorescent secondary antibodies; 16, 17 and 20 refer to the residue in CXCL12 mutated to Cys. ( e ) Schematic representation of CRS1 disulfide trapping experiments. Mutation of C21 or C26 to Ser gives a free Cys side chain that can be crosslinked to residue 20 of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Full, uncropped versions of the gels and blots are shown in Supplementary Fig. 7 .
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 37956 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti beta actin antibody
    Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. <t>Beta-actin</t> was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti alpha tubulin antibody
    Roles of individual MARTX toxin effector domains. (A) Schematic representation of the Δeffector strain library. DUF1 = domain of unknown function in the first position; RID = Rho Inactivation Domain; ABH = <t>Alpha/Beta</t> Hydrolase domain; MCF = Makes Caterpillars Floppy-like domain; RRSP = Ras/Rap1 Specific Protease domain; CPD = Cysteine Protease domain; striped boxes = repeat regions; (B) Lysis of T84 monolayers exposed to the indicated bacterial strains for 180 minutes, expressed relative to Triton X-100 treated monolayers. (C) Western blot using lysates from HeLa cells exposed to the indicated strains for 60 minutes at MOI = 100, probed with anti-Ras10 antibody (bottom) and <t>anti-tubulin</t> (top) (D) Percent of rounded HeLa cells observed upon exposure to the indicated bacterial strains for 120 minutes at MOI = 10. (E-F) TER of polarized monolayers measured at 60 mins (E) or 120 mins (F) minutes following apical exposure to the indicated strains of V . vulnificus at MOI = 2.6. Data analyzed by one-way ANOVA followed by multiple comparison’s test to determine p valules; **** p
    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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    94
    Millipore mouse monoclonal
    Roles of individual MARTX toxin effector domains. (A) Schematic representation of the Δeffector strain library. DUF1 = domain of unknown function in the first position; RID = Rho Inactivation Domain; ABH = <t>Alpha/Beta</t> Hydrolase domain; MCF = Makes Caterpillars Floppy-like domain; RRSP = Ras/Rap1 Specific Protease domain; CPD = Cysteine Protease domain; striped boxes = repeat regions; (B) Lysis of T84 monolayers exposed to the indicated bacterial strains for 180 minutes, expressed relative to Triton X-100 treated monolayers. (C) Western blot using lysates from HeLa cells exposed to the indicated strains for 60 minutes at MOI = 100, probed with anti-Ras10 antibody (bottom) and <t>anti-tubulin</t> (top) (D) Percent of rounded HeLa cells observed upon exposure to the indicated bacterial strains for 120 minutes at MOI = 10. (E-F) TER of polarized monolayers measured at 60 mins (E) or 120 mins (F) minutes following apical exposure to the indicated strains of V . vulnificus at MOI = 2.6. Data analyzed by one-way ANOVA followed by multiple comparison’s test to determine p valules; **** p
    Mouse Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 915 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti neun
    Progression of neurogenesis and gliogenesis in the tuberal hypothalamus of CD1 wildtype mice. a P0 brain sections of <t>BrdU</t> birthdating studies indicating the neurons, marked by <t>NeuN,</t> that were born at E11.5, E13.5 and E15.5 embryonic time points during neurogenesis in the tuberal hypothalamus. Yellow arrows indicate examples of NeuN+/BrdU+ co-labeled neurons, third ventricle location is highlighted with a white dotted line. b Sox9+ glioblasts in the tuberal hypothalamus at E13.5, E15.5 and E17.5. Scale bars equal 200 μm
    Mouse Anti Neun, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti beta actin antibody mouse monoclonal
    BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and <t>beta-actin</t> (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.
    Anti Beta Actin Antibody Mouse Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher donkey anti mouse igg h l highly cross adsorbed secondary antibody
    BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and <t>beta-actin</t> (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.
    Donkey Anti Mouse Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6700 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse anti α tubulin
    PP2A activity does not substantially affect progression beyond anaphase Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into three sets that received either vehicle as control (CTRL), OA at 0.5 µM (OA), or LB-100. Cells were then taken at the indicated time points of further incubation and spun onto microscopy slides and processed for immunofluorescence staining for <t>α-tubulin</t> and the centromere marker CREST, DNA was stained with Hoechst. Upper graphs: post-anaphase cells were visually scored through microscopy. Error bars refer to variation within three independent experiments performed under identical conditions. Lower photographs: indicative images of HeLa and hTERT-RPE1 cells taken at 60 or 40 min, respectively, of incubation. Scale bars, 10 μm.
    Mouse Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti mouse igg
    In vitro and in vivo binding of HSF1 to the COX-2 promoter. ( A ) Nucleotide sequence of the 100 bp (from −2579 to −2480) DNA fragment of the COX-2 promoter region including binding sites for the GATA and HSF1 transcription factors. The 100 bp fragment was amplified by PCR, 32 P-labeled and used as probe for gel-shift analysis shown in B and C . ( B ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), whole-cell extracts were analyzed for HSF DNA-binding activity by EMSA in a 3% polyacrylamide gel using the probe described in ( A ). Position of HSF-DNA binding complex (HSF), constitutive HSE binding activity (CHBA) and non specific protein-DNA interactions (NS) are shown (upper panel). In parallel samples whole-cell extracts were analyzed for levels of HSF1 and <t>β-actin</t> proteins by Western blot (lower panels). Arrow indicates the position of the low-mobility phosphorylated HSF1 isoform. ( C ) Specificity of HSF1-DNA binding complexes. Whole-cell extracts from HUVECs subjected to heat shock at 43°C for 40 min were preincubated with different dilutions of anti-HSF1 polyclonal antibodies for 15 min before supershift assay. Position of HSF, CHBA and NS are indicated as in B . ( D,E ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), recruitment of HSF1 to the COX-2 and HSP70 promoters was analyzed by ChIP assay. ( D ) ChIP-enriched DNAs using preimmune serum (IP NS <t>IgG)</t> or anti-HSF1 serum (IP anti-HSF1), as well as input DNAs (INPUT) were prepared, and DNA fragments of the COX-2 gene (−2629 to −2420) and HSP70 gene (−262 to −70) were amplified by PCR. ( E ) Quantification of ChIP assay shown in ( D ). Samples from at least three independent experiments were analyzed by real time PCR. Relative promoter occupancy is expressed as fold induction of control arbitrarily set to a value of 1. Error bars indicate ± S.D. * = P
    Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher goat anti mouse igg2a
    β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10 9 PFU of AdβGal (Ad) or 5 × 10 4 AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and <t>IgG2a-specific</t> antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.
    Goat Anti Mouse Igg2a, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti mouse igg whole molecule peroxidase antibody
    Binding of <t>IgG</t> and <t>C3b</t> with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.
    Anti Mouse Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti gfap
    Ultrastructural immunolocalization of <t>GABA</t> in astrocytes of hippocampal CA1, CA3, and dentate gyrus regions. Combined pre-embedding immunogold and immunoperoxidase methods for electron microscopy. Astrocytes <t>(GFAP+)</t> and GABA profiles are labeled by DAB and silver-intensified gold particles, respectively. GABA immunoparticles are localized in synaptic terminals (Ter) with symmetric synapses (arrows) as well as in astrocytes (Ast, white arrowheads) in CA1 (A) and CA3 (B) stratum radiatum and in the dentate gyrus (C) . In A, a GAFP+ astrocytic portion containing GABA is delineated.
    Mouse Anti Gfap, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4780 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse monoclonal antibodies
    Ultrastructural immunolocalization of <t>GABA</t> in astrocytes of hippocampal CA1, CA3, and dentate gyrus regions. Combined pre-embedding immunogold and immunoperoxidase methods for electron microscopy. Astrocytes <t>(GFAP+)</t> and GABA profiles are labeled by DAB and silver-intensified gold particles, respectively. GABA immunoparticles are localized in synaptic terminals (Ter) with symmetric synapses (arrows) as well as in astrocytes (Ast, white arrowheads) in CA1 (A) and CA3 (B) stratum radiatum and in the dentate gyrus (C) . In A, a GAFP+ astrocytic portion containing GABA is delineated.
    Mouse Monoclonal Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4917 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher alexa fluor 488 goat anti mouse igg
    FANCJ-R707C is a separation-of-function mutant that partially rescues sensitivity to the replication stress inducing agents aphidicolin or telomestatin but fails to restore cisplatin resistance. ( A ) Schematic diagram of green fluorescence protein (GFP)-FANCJ recombinant protein expressed in fancj −/− cells for genetic complementation assays. ( B ) Western blot analysis of whole cell lysate protein (40 μg) from fancj −/− DT40 cells transfected with plasmid encoding (pEGFP-Vector), pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C), or pEGFP-FANCJ-H396D (FANCJ-H396D). Protein was detected with antibody against FANCJ or actin (as a loading control, 10% loaded). (C-E) Cell survival assays, as measured by methyl cellulose colony formation, for the indicated fancj −/− cells expressing mutant or wild-type FANCJ proteins. Cells were exposed to the indicated concentrations of CisPt ( C ), APH ( D ) or TMS ( E ). Filled square, fancj −/− cells transfected with FANCJ-WT; open square, fancj −/− cells transfected with pEGFP-Vector; filled triangle, fancj −/− cells transfected with FANCJ-R707C; open cross- fancj −/− cells transfected with FANCJ-H396D. (F, G) FANCJ-R707C restores normal replication restart after APH exposure. Schematic representation of the protocol used to track DNA replication fibers is shown in Panel F. Cells were pulse-chased with CldU ( red label ), and then labeled with IdU ( green label ) for the indicated times. ( G ) Representative images of fluorescently-labeled DNA fibers from the indicated cell lines treated with DMSO or APH. ( H ) Box and whiskers graphs indicating the 10–90 percentile of the IdU tract length (μm) for ongoing forks. The data, presented as mean ± standard error of the mean (s.e.m.), are based on the measurement of at least 100 DNA fibers from two independent experiments. ( I – N ), DNA damage, as marked by immunofluorescent detection of γ-H2AX foci, in the indicated fancj −/− transfected cell lines. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to: CisPt (1 μM) (I, J); APH (200 nM) (K, L); TMS (5 μM) (M, N). Immunofluorescence detection of γ-H2AX foci by <t>Alexa</t> <t>fluor</t> 488 is shown along with DAPI, or merged (DAPI and Alexa fluor 488). Quantitative analyses of γ-H2AX foci are shown with S.D. (ns-not significant; ** P
    Alexa Fluor 488 Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 6906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti vinculin antibody
    Low correlation of colocalization of pacsin2 and the VE-cadherin complex at super-resolution level. ( a ) Representative deconvolved images of stimulated emission depletion (STED) and confocal microscopy performed on IF labelled HUVECs stained for VE-cadherin (red) and catenins, <t>vinculin</t> or pacsin2 (all green). ( b ) Pearson correlation analysis of pixel intensities between VE-cadherin and indicated proteins at FAJ sections. Graph shows the average Pearson correlation coefficient ( R )±s.e.m. of indicated protein pairs, which was determined on junctional region of interests (ROIs) from deconvolved STED images. n ≥17 FAJs per correlation analysis. An R of 1 indicates perfect colocalization between two proteins, whereas an R of 0 indicates no correlation. ** P ≤0.01 (Student's t -test).
    Monoclonal Anti Vinculin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology goat anti mouse igg hrp
    Schematic representation of DNA hα-Syn vaccine constructs and expression in transfected CHO cells. (A ) Strategy for cloning genes encoding several hα-Syn B cell epitope/s fused with MultiTEP into the pVAX1 vector and schematic representation of PV-1947D, PV-1948D, PV-1949D, and PV-1950D constructs. ( B–D ) The expression of PV-1947D ( B ), PV-1948D ( C ), PV-1949D ( D ), and PV-1950D ( B–D ) and secretion of protein was demonstrated by WB of conditioned media of transiently transfected CHO cells. Proteins were visualized by staining with anti-hα-Syn 85–99 Abs ( A ), anti-α-hSyn 109–126 Abs ( B ), and anti-α-hSyn 126–140 Abs ( C ) followed by <t>HRP</t> conjugated anti-mouse <t>IgG.</t>
    Goat Anti Mouse Igg Hrp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 4245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti ha antibody
    HrpF stabilizes HrpA, and the 67th lysine residue of HrpF is critical for its interaction with HrpA. (A) Immunoblots of immunoprecipitated proteins probed with anti‐HrpA (top) and anti‐haemagglutinin <t>(anti‐HA)</t> (bottom) antibodies to show in vitro protein interactions. Escherichia coli lysates expressing HrpF‐HA27 (pNCHU1867), HrpF K67A ‐HA27 (pNCHU1868), HrpA (pNCHU1869) (‘+’) or vector alone (‘−’) were mixed with anti‐HA agarose beads for immunoprecipitation and detected by anti‐HrpA serum as described in Experimental procedures. Gel‐bound, immunoprecipitated proteins; lysate, total proteins as a control to detect the expression of recombinant proteins. (B) Far western analysis shows that HrpF‐HA27 and HrpF K67A ‐HA27 bind to HrpA‐His in vitro . Escherichia coli ‐expressed HrpA‐His (pNCHU1944) or EV1 (pETite) was probed with bacterial lysates expressing EV2 (pET29a), HrpF‐HA27 (pNCHU1867) or HrpF K67A ‐HA27 (pNCHU1868) as described in Experimental procedures. HrpA‐His‐bound HrpF‐HA was further detected with HA monoclonal antibody. (C) Immunodetection of HrpA stability in the Pseudomonas syringae pv. averrhoi (Pav) hrpF mutant. PavHL1, wild‐type harbouring pRK415 and pNCHU2046; Δ hrpF , HL1‐N1589 harbouring pRK415 and pNCHU2046; Δ hrpF/hrpF , HL1‐N1589 harbouring pNCHU1590 and pNCHU2046, Δ hrpF/hrpF K67A , HL1‐N1589 harbouring pNCHU2068 and pNCHU2046. Total cell cultures were harvested at 0, 2 and 4 h post‐chloramphenicol treatment for immunodetection with anti‐HrpZ and anti‐HrpA sera. The stability of HrpA is shown by the fold changes in HrpA banding intensity, which was first normalized by the HrpZ bands and calculated using the value at 0 h post‐chloramphenicol treatment as the denominator.
    Monoclonal Anti Ha Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti mouse igg2b
    PA0833 stimulates protective immunity in a P. aeruginosa sepsis model. (A) Comparison of total <t>IgG</t> and IgG subgroups <t>(IgG1,</t> <t>IgG2a,</t> and <t>IgG2b)</t> in the mice ( n = 5) immunized with PA0833. Serum was obtained at 7 days after the final immunization, and the levels of total IgG and IgG subgroups were expressed as the means of log 2 titers. Multiple comparisons among different groups were analyzed using one-way ANOVA. Data are shown as the means ± SD. (B) BALB/c mice ( n = 10) were immunized with PA0833 plus an Al(OH) 3 adjuvant and challenged with PAO1 at 7.0 × 10 7 CFUs/mouse by intravenous injection. The survival rate was monitored for 14 days. The P -values were calculated using the Mantel–Cox log-rank test. (C,D) Efficacy of immunization with PA0833 on the spread of P. aeruginosa . The number of viable bacteria in the blood, liver, and spleen of mice ( n = 10) at 1 and 3 days post-infection are shown. Data are presented in box and whisker plots, and the medians are shown. Differences were compared to determine their significance using Student’s t -test.
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    Image Search Results


    Avastin attenuates astrogliosis and blood vessel formation in the injured cortex. a, c Cortical sections were obtained from mice in which Avastin or PBS was infused into the ventricle 14 d post-ATP injection. GFAP and CoL1A1 were visualized with Alexa-488 and Alexa-594 conjugated secondary antibodies ( a ) or biotinylated secondary antibodies and DAB-based color reaction ( c ). b Relative fluorescence intensity of GFAP and CoL1A1 were measured using ZEN software and plotted. Values are means ± SEMs for animals treated with saline ( n = 4) or Avastin ( n = 3) (*, p

    Journal: Molecular Brain

    Article Title: Region-specific astrogliosis: differential vessel formation contributes to different patterns of astrogliosis in the cortex and striatum

    doi: 10.1186/s13041-020-00642-0

    Figure Lengend Snippet: Avastin attenuates astrogliosis and blood vessel formation in the injured cortex. a, c Cortical sections were obtained from mice in which Avastin or PBS was infused into the ventricle 14 d post-ATP injection. GFAP and CoL1A1 were visualized with Alexa-488 and Alexa-594 conjugated secondary antibodies ( a ) or biotinylated secondary antibodies and DAB-based color reaction ( c ). b Relative fluorescence intensity of GFAP and CoL1A1 were measured using ZEN software and plotted. Values are means ± SEMs for animals treated with saline ( n = 4) or Avastin ( n = 3) (*, p

    Article Snippet: The sections were rinsed three times with PBS and incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

    Techniques: Mouse Assay, Injection, Fluorescence, Software

    In vivo expression of non-tenocyte markers PPARγ, collagen type II, and osteocalcin in rats implanted with hTSCs treated with various concentrations of PGE 2 and their respective hematoxylin and eosin (H E) stained tissue sections. hTSCs cultured with three concentrations (0.1, 10, and 100 ng/ml) of PGE 2 were implanted subcutaneously into rats; later, immunohistochemical and histological analyses were performed on tissue sections. For the immunohistochemical staining, fixed tissue sections were incubated with mouse anti-human PPARγ antibody, mouse anti-collagen type II (Collagen II) antibody, or mouse anti-human osteocalcin antibody. Cy3-conjugated goat anti-mouse IgG was then used to detect primary binding. Nuclei were stained with Hoechst (blue). Expression levels of PPARγ, collagen type II, and osteocalcin (red) were lower in cells treated with 0.1 ng/ml PGE 2 ( A–C ) than those treated with the higher concentrations (10 and 100 ng/ml) of PGE 2 ( E–G, I–K ). H E staining was also performed on tissue sections ( D, H, L ). More cells (black dots; see insets in D , H , and L ) were observed in tissues implanted with hTSCs that had been treated with high concentrations of PGE 2 in culture ( H, L ). Specifically, at 100 ng/ml ( L ), cells were concentrated in a specific region (triangle). Additionally, semi-quantification of the stained cells was performed by counting immuno-positive cells and calculating percentage staining ( M ) (*p

    Journal: PLoS ONE

    Article Title: Prostaglandin E2 (PGE2) Exerts Biphasic Effects on Human Tendon Stem Cells

    doi: 10.1371/journal.pone.0087706

    Figure Lengend Snippet: In vivo expression of non-tenocyte markers PPARγ, collagen type II, and osteocalcin in rats implanted with hTSCs treated with various concentrations of PGE 2 and their respective hematoxylin and eosin (H E) stained tissue sections. hTSCs cultured with three concentrations (0.1, 10, and 100 ng/ml) of PGE 2 were implanted subcutaneously into rats; later, immunohistochemical and histological analyses were performed on tissue sections. For the immunohistochemical staining, fixed tissue sections were incubated with mouse anti-human PPARγ antibody, mouse anti-collagen type II (Collagen II) antibody, or mouse anti-human osteocalcin antibody. Cy3-conjugated goat anti-mouse IgG was then used to detect primary binding. Nuclei were stained with Hoechst (blue). Expression levels of PPARγ, collagen type II, and osteocalcin (red) were lower in cells treated with 0.1 ng/ml PGE 2 ( A–C ) than those treated with the higher concentrations (10 and 100 ng/ml) of PGE 2 ( E–G, I–K ). H E staining was also performed on tissue sections ( D, H, L ). More cells (black dots; see insets in D , H , and L ) were observed in tissues implanted with hTSCs that had been treated with high concentrations of PGE 2 in culture ( H, L ). Specifically, at 100 ng/ml ( L ), cells were concentrated in a specific region (triangle). Additionally, semi-quantification of the stained cells was performed by counting immuno-positive cells and calculating percentage staining ( M ) (*p

    Article Snippet: The cells were then washed three times with PBS, followed by incubation with Cy3-conjugated goat anti-mouse IgG (1∶500; Invitrogen, Cat. # A10521) secondary antibody at room temperature for 2 hrs.

    Techniques: In Vivo, Expressing, Staining, Cell Culture, Immunohistochemistry, Incubation, Binding Assay

    Cysteine trapping of the complex between ACKR3 and CXCL12. ( a ) CRS2 interactions detected using SDS–PAGE of purified ACKR3:CXCL12 complexes in the absence of reducing agent. Upper bands correspond to the crosslinked complex and lower bands to free receptor. Crosslinking of thermostabilized apocytochrome b562 (bRIL)-R197C-ACKR3 with Cys mutants in the N terminus of CXCL12. No crosslinking is seen for control samples N45C-CXCL12 or WT-CXCL12. ( b ) Crosslinking of Y7C-CXCL12 with ECL2 mutants of ACKR3. R197C-ACKR3 efficiently crosslinks with the chemokine, while no crosslinking is detected for F199C-ACKR3 or WT-ACKR3. ( c ) Schematic summary of disulfide trapping experiments involving ECL2 of ACKR3 and the N terminus of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Crosslinked sites are connected with solid lines, where the line thickness is proportional to crosslinking efficiency. Dashed lines indicate unsuccessful cross-links. ( d ) Cysteine trapping of CRS1 interactions detected by western blot of ACKR3:CXCL12 complexes under non-reducing conditions. ACKR3-FLAG and CXCL12-HA were detected with primary anti-FLAG and anti-HA antibodies and fluorescent secondary antibodies; 16, 17 and 20 refer to the residue in CXCL12 mutated to Cys. ( e ) Schematic representation of CRS1 disulfide trapping experiments. Mutation of C21 or C26 to Ser gives a free Cys side chain that can be crosslinked to residue 20 of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Full, uncropped versions of the gels and blots are shown in Supplementary Fig. 7 .

    Journal: Nature Communications

    Article Title: Structural basis of ligand interaction with atypical chemokine receptor 3

    doi: 10.1038/ncomms14135

    Figure Lengend Snippet: Cysteine trapping of the complex between ACKR3 and CXCL12. ( a ) CRS2 interactions detected using SDS–PAGE of purified ACKR3:CXCL12 complexes in the absence of reducing agent. Upper bands correspond to the crosslinked complex and lower bands to free receptor. Crosslinking of thermostabilized apocytochrome b562 (bRIL)-R197C-ACKR3 with Cys mutants in the N terminus of CXCL12. No crosslinking is seen for control samples N45C-CXCL12 or WT-CXCL12. ( b ) Crosslinking of Y7C-CXCL12 with ECL2 mutants of ACKR3. R197C-ACKR3 efficiently crosslinks with the chemokine, while no crosslinking is detected for F199C-ACKR3 or WT-ACKR3. ( c ) Schematic summary of disulfide trapping experiments involving ECL2 of ACKR3 and the N terminus of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Crosslinked sites are connected with solid lines, where the line thickness is proportional to crosslinking efficiency. Dashed lines indicate unsuccessful cross-links. ( d ) Cysteine trapping of CRS1 interactions detected by western blot of ACKR3:CXCL12 complexes under non-reducing conditions. ACKR3-FLAG and CXCL12-HA were detected with primary anti-FLAG and anti-HA antibodies and fluorescent secondary antibodies; 16, 17 and 20 refer to the residue in CXCL12 mutated to Cys. ( e ) Schematic representation of CRS1 disulfide trapping experiments. Mutation of C21 or C26 to Ser gives a free Cys side chain that can be crosslinked to residue 20 of CXCL12. Residues are coloured based on radiolytic footprinting results (see Figs 3 and 4 ). Full, uncropped versions of the gels and blots are shown in Supplementary Fig. 7 .

    Article Snippet: Mouse anti-Flag M2 primary antibody (1:5,000 dilution, F3165; Sigma Aldrich) and IRDye 680-conjugated donkey anti-mouse IgG (1:20,000 dilution; LI-COR Biosciences) secondary antibody were used to detect the FLAG-tagged receptor.

    Techniques: SDS Page, Purification, Footprinting, Western Blot, Mutagenesis

    Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using anti‐HA/Strep/FLAG antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).

    Journal: Molecular Microbiology

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

    doi: 10.1111/mmi.14357

    Figure Lengend Snippet: Detection of the T. gondii mitochondrial ribosome. A. Protein immunoblot analysis of endogenously tagged TgmS35, TgbL12m and TguL3m from total cell lysate separated by SDS‐PAGE and detected using anti‐HA/Strep/FLAG antibodies. B. Total cell lysate separated by blue‐native PAGE and immunoblotted to detect TgmS35, TgbL12m and TguL3m with anti‐HA/Strep/FLAG antibodies. C. Validation of the promoter integration in the Tg mS15 locus via PCR analysis using primers 1, 2, 3, and 4, represented in Fig. S4 D. Western blot (top panel) of TgmS35‐3xHA in lines where Tg mS35 is under its native promoter (TgmS35‐3HA) or where Tg mS35 or Tg uS15m are under regulatable promoters (r Tg mS35‐3HA and Tg mS35‐3HA/r Tg uS15m respectively). Low panel shows instant blue staining for loading control. E. comparison of results from RT‐PCR performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (total), from IP of TgTom22 (Tom22) or from IP of TgmS35 (TgmS35). F. An example RT‐PCR experiment performed with primers for a mitochondrial rRNA sequence (Fig. S5 ) (mito‐rRNA), for an apicoplast rRNA sequence (api‐rRNA) and for a cytosolic mRNA (actin). Template is RNA extracted from total cell lysate of TATi∆ku80 (Parental – total), from IP of TgTom22 (Tom22‐IP) or from IP of TgmS35 (TgmS35‐IP).

    Article Snippet: Cells were then labelled with different sets of primary and secondary antibodies: mouse anti‐HA antibody (1:1000, Sigma), rabbit anti‐TgMys (1:1000) (Ovciarikova et al. , ), mouse anti‐Myc (1:1000, Cell Signalling), rabbit anti‐TgTOM40 (1:2000)(van Dooren et al. , ); mouse anti‐Strep (1:1000, StrepMAB‐Classic, IBA), mouse anti‐FLAG (1:1000, Monoclonal ANTI‐FLAG® M2, Sigma‐Aldrich) coupled with goat anti‐mouse or anti‐rabbit fluorescent antibody (AlexaFluor 594 or 488 1:1000 (Invitrogen)).

    Techniques: Western Blot, SDS Page, Blue Native PAGE, Polymerase Chain Reaction, Staining, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Mitochondrial localisation of putative mitochondrial ribosomal proteins by epitope tagging. A. Localisation through fluorescence microscopy analysis of 4 gene‐products predicted to encode for mitoribosomal proteins identified in our search. B. Localisation of 2 additional gene‐products predicted to be mitochondrial ribosomal proteins not found in our search. For both panels, mitochondria are marked with anti‐TgMys or anti‐TOM40; the mentioned GOI (genes of interest) are marked with anti‐HA, anti‐FLAG or anti‐Strep. Scale bar 1 μm. Images in A also appear in Fig. S2 . [Colour figure can be viewed at https://www.wileyonlinelibrary.com ]

    Journal: Molecular Microbiology

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

    doi: 10.1111/mmi.14357

    Figure Lengend Snippet: Mitochondrial localisation of putative mitochondrial ribosomal proteins by epitope tagging. A. Localisation through fluorescence microscopy analysis of 4 gene‐products predicted to encode for mitoribosomal proteins identified in our search. B. Localisation of 2 additional gene‐products predicted to be mitochondrial ribosomal proteins not found in our search. For both panels, mitochondria are marked with anti‐TgMys or anti‐TOM40; the mentioned GOI (genes of interest) are marked with anti‐HA, anti‐FLAG or anti‐Strep. Scale bar 1 μm. Images in A also appear in Fig. S2 . [Colour figure can be viewed at https://www.wileyonlinelibrary.com ]

    Article Snippet: Cells were then labelled with different sets of primary and secondary antibodies: mouse anti‐HA antibody (1:1000, Sigma), rabbit anti‐TgMys (1:1000) (Ovciarikova et al. , ), mouse anti‐Myc (1:1000, Cell Signalling), rabbit anti‐TgTOM40 (1:2000)(van Dooren et al. , ); mouse anti‐Strep (1:1000, StrepMAB‐Classic, IBA), mouse anti‐FLAG (1:1000, Monoclonal ANTI‐FLAG® M2, Sigma‐Aldrich) coupled with goat anti‐mouse or anti‐rabbit fluorescent antibody (AlexaFluor 594 or 488 1:1000 (Invitrogen)).

    Techniques: Fluorescence, Microscopy

    Transient expression of Cas9 results in mitochondria morphology defects. A. Representative immunofluorescence micrographs of parasites transiently expressing CAS9 and sgRNA for the mentioned GOI. Mitochondria in red are marked by anti‐TgMys. CAS9‐FLAG in green is marked with anti‐FLAG. Merge shows DAPI, FLAG and TgMys. Green boxes highlight parasites with wild‐type looking mitochondria. Red boxes highlight parasites with mitochondria that appear to have a morphological defect herein named ‘abnormal mitochondria.’ B. Quantification of vacuole with abnormal mitochondria morphologies for each condition. Bars represent the mean ± SEM ( n = 3), * p

    Journal: Molecular Microbiology

    Article Title: Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

    doi: 10.1111/mmi.14357

    Figure Lengend Snippet: Transient expression of Cas9 results in mitochondria morphology defects. A. Representative immunofluorescence micrographs of parasites transiently expressing CAS9 and sgRNA for the mentioned GOI. Mitochondria in red are marked by anti‐TgMys. CAS9‐FLAG in green is marked with anti‐FLAG. Merge shows DAPI, FLAG and TgMys. Green boxes highlight parasites with wild‐type looking mitochondria. Red boxes highlight parasites with mitochondria that appear to have a morphological defect herein named ‘abnormal mitochondria.’ B. Quantification of vacuole with abnormal mitochondria morphologies for each condition. Bars represent the mean ± SEM ( n = 3), * p

    Article Snippet: Cells were then labelled with different sets of primary and secondary antibodies: mouse anti‐HA antibody (1:1000, Sigma), rabbit anti‐TgMys (1:1000) (Ovciarikova et al. , ), mouse anti‐Myc (1:1000, Cell Signalling), rabbit anti‐TgTOM40 (1:2000)(van Dooren et al. , ); mouse anti‐Strep (1:1000, StrepMAB‐Classic, IBA), mouse anti‐FLAG (1:1000, Monoclonal ANTI‐FLAG® M2, Sigma‐Aldrich) coupled with goat anti‐mouse or anti‐rabbit fluorescent antibody (AlexaFluor 594 or 488 1:1000 (Invitrogen)).

    Techniques: Expressing, Immunofluorescence

    Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. Beta-actin was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p

    Journal: Scientific Reports

    Article Title: Parkin truncating variants result in a loss-of-function phenotype

    doi: 10.1038/s41598-019-52534-6

    Figure Lengend Snippet: Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. Beta-actin was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p

    Article Snippet: Antibodies Primary antibodies: mouse monoclonal anti-EGFP antibody (Abnova, MAB1765), mouse monoclonal anti-GFP antibody (Rockland, 600-301-215), mouse monoclonal anti-beta-actin (Sigma-Aldrich, A5441), mouse monoclonal anti-GM130 (BD Biosciences, 610822), mouse monoclonal anti-caspase 3 (Cell Signaling, 9668), mouse monoclonal anti-HDAC2 (Santa Cruz Biotechnology, sc-9959), rabbit polyclonal anti-histone H3 (Abcam, ab1791), mouse monoclonal anti-p62 (Proteintech, 66184-1-Ig), mouse monoclonal anti-TOM20 (BD Biosciences, 612278).

    Techniques: Expressing, Functional Assay, Clone Assay

    Roles of individual MARTX toxin effector domains. (A) Schematic representation of the Δeffector strain library. DUF1 = domain of unknown function in the first position; RID = Rho Inactivation Domain; ABH = Alpha/Beta Hydrolase domain; MCF = Makes Caterpillars Floppy-like domain; RRSP = Ras/Rap1 Specific Protease domain; CPD = Cysteine Protease domain; striped boxes = repeat regions; (B) Lysis of T84 monolayers exposed to the indicated bacterial strains for 180 minutes, expressed relative to Triton X-100 treated monolayers. (C) Western blot using lysates from HeLa cells exposed to the indicated strains for 60 minutes at MOI = 100, probed with anti-Ras10 antibody (bottom) and anti-tubulin (top) (D) Percent of rounded HeLa cells observed upon exposure to the indicated bacterial strains for 120 minutes at MOI = 10. (E-F) TER of polarized monolayers measured at 60 mins (E) or 120 mins (F) minutes following apical exposure to the indicated strains of V . vulnificus at MOI = 2.6. Data analyzed by one-way ANOVA followed by multiple comparison’s test to determine p valules; **** p

    Journal: PLoS Pathogens

    Article Title: The Effector Domain Region of the Vibrio vulnificus MARTX Toxin Confers Biphasic Epithelial Barrier Disruption and Is Essential for Systemic Spread from the Intestine

    doi: 10.1371/journal.ppat.1006119

    Figure Lengend Snippet: Roles of individual MARTX toxin effector domains. (A) Schematic representation of the Δeffector strain library. DUF1 = domain of unknown function in the first position; RID = Rho Inactivation Domain; ABH = Alpha/Beta Hydrolase domain; MCF = Makes Caterpillars Floppy-like domain; RRSP = Ras/Rap1 Specific Protease domain; CPD = Cysteine Protease domain; striped boxes = repeat regions; (B) Lysis of T84 monolayers exposed to the indicated bacterial strains for 180 minutes, expressed relative to Triton X-100 treated monolayers. (C) Western blot using lysates from HeLa cells exposed to the indicated strains for 60 minutes at MOI = 100, probed with anti-Ras10 antibody (bottom) and anti-tubulin (top) (D) Percent of rounded HeLa cells observed upon exposure to the indicated bacterial strains for 120 minutes at MOI = 10. (E-F) TER of polarized monolayers measured at 60 mins (E) or 120 mins (F) minutes following apical exposure to the indicated strains of V . vulnificus at MOI = 2.6. Data analyzed by one-way ANOVA followed by multiple comparison’s test to determine p valules; **** p

    Article Snippet: Strains were validated by assessment of LDH release as described below, by western blotting for Ras proteolysis using the pan-Ras RAS10 (EMD Millipore, 05–516, 1:500) and tubulin (Sigma-Alrich, T6074, (1:10,000) monoclonal antibodies as previously described [ ], and by assessment and quantification of HeLa cell rounding as previously described [ ].

    Techniques: Lysis, Western Blot

    Progression of neurogenesis and gliogenesis in the tuberal hypothalamus of CD1 wildtype mice. a P0 brain sections of BrdU birthdating studies indicating the neurons, marked by NeuN, that were born at E11.5, E13.5 and E15.5 embryonic time points during neurogenesis in the tuberal hypothalamus. Yellow arrows indicate examples of NeuN+/BrdU+ co-labeled neurons, third ventricle location is highlighted with a white dotted line. b Sox9+ glioblasts in the tuberal hypothalamus at E13.5, E15.5 and E17.5. Scale bars equal 200 μm

    Journal: Neural Development

    Article Title: Oligodendrocyte development in the embryonic tuberal hypothalamus and the influence of Ascl1

    doi: 10.1186/s13064-016-0075-9

    Figure Lengend Snippet: Progression of neurogenesis and gliogenesis in the tuberal hypothalamus of CD1 wildtype mice. a P0 brain sections of BrdU birthdating studies indicating the neurons, marked by NeuN, that were born at E11.5, E13.5 and E15.5 embryonic time points during neurogenesis in the tuberal hypothalamus. Yellow arrows indicate examples of NeuN+/BrdU+ co-labeled neurons, third ventricle location is highlighted with a white dotted line. b Sox9+ glioblasts in the tuberal hypothalamus at E13.5, E15.5 and E17.5. Scale bars equal 200 μm

    Article Snippet: Primary antibodies were as follows: Mouse anti-NeuN (Millipore; 1:400), Rat anti-BrdU (Cedar Lane; 1:300), Goat anti-Ki67 (Santa Cruz; 1:300), Rabbit anti-Ki67 (Abcam; 1:100), Goat anti-Sox9 (R & D systems; 1:40); Rabbit anti-Olig2 (Millipore; 1:500); Mouse anti-Olig2 (Millipore; 1:300), Goat anti-PdgfRα (R & D Systems; 1:150); Rat anti-SF-1 (graciously provided by Dr Taro Tachibana, Osaka City University JAPAN, 1:800); Rabbit anti-TTF-1 (alternatively Nkx2.1; Santa Cruz; 1:500), Goat anti-Sox10 (Santa Cruz, 1:500), Rabbit anti-pHH3 (Millipore; 1:500), Rabbit anti-Cyclin E (Santa Cruz; 1:200), Rabbit anti-Cyclin B1 (Santa Cruz; 1:200), Mouse anti-Cyclin D2 (ThermoFisher Scientific; 1:200), Rabbit anti-p57kip (Sigma; 1:200), Rabbit anti-Aldh1L1 (Abcam; 1:500), Rat anti-MBP (Millipore; 1:50), and Rabbit anti-Cleaved Caspase 3 (Abcam; 1:800).

    Techniques: Mouse Assay, Labeling

    BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and beta-actin (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.

    Journal: Current Alzheimer research

    Article Title: BACE1 Levels by APOE Genotype in Non-Demented and Alzheimer\u2019s Post-Mortem Brains

    doi:

    Figure Lengend Snippet: BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and beta-actin (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.

    Article Snippet: The membranes were then stripped and reprobed with mouse anti-β actin (#A1978; Sigma-Aldrich, St. Louis, MO).

    Techniques: Western Blot

    Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

    Journal: Experimental & Molecular Medicine

    Article Title: Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells

    doi: 10.1038/s12276-018-0044-y

    Figure Lengend Snippet: Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

    Article Snippet: After incubation with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, the membrane was incubated with primary antibodies (mouse anti-β-catenin (Santa Cruz Biotechnology), mouse anti-EN1 (Abcam, Cambridge, UK), and mouse anti-β-actin (Sigma-Aldrich)) for 1 h at room temperature or overnight at 4 °C.

    Techniques: Activation Assay, Derivative Assay, Expressing, ALP Assay, shRNA

    dnj-14 (tm3223) worms are delayed in ubiquitylated protein degradation (A) Representative image of Western blot detection of GFP-UbV accumulation in PP563 and PP545 strains after overnight incubation with or without 10 μM bortezomib. There was no GFP detected in untreated PP563 worms but a signal was detectable after bortezomib treatment. In contrast GFP was already accumulated in PP545 worms before treatment. Quantitation using ImageJ showed that the average GFP signal from PP545 worm lysates after bortezomib treatment was 94% +/− 0.005% (+/−SEM) compared to untreated PP545 worms (n = 3). ( B ) Representative image of Western blot detection of GFP-UbV accumulation in N2 and dnj-14 (tm3223) crossed with the pSur5::UbV-GFP expressing strain, PP563, generating the strains, dnj-14 (wt) ; PP563 and dnj-14 (tm3223) ; PP563. There was an average intensity increase of 354 +/−109% (+/−SEM) in dnj-14 (tm3223) ; PP563 compared to dnj-14 (wt) ; PP563 (n = 3). Worms were harvested and lysed at day 1 of adulthood. 50 worms were lysed in each SDS-PAGE sample and anti-beta-actin was used as a loading control. ( B ) GFP fluorescence was imaged in dnj-14 (wt) ; PP563 and dnj-14 (tm3223) ; PP563 worms after overnight incubation with increasing concentrations of bortezomib. ( C ) Western blot detection of GFP-UbV accumulation, using an anti-GFP antibody, after overnight treatment of dnj-14 (wt) ; PP563 and dnj-14 (tm3223) ; PP563 with increasing concentrations of bortezomib. ( D ) Quantitation of anti-GFP signal detected in ( C ). Band intensity for 0 mM was assumed to be background and was subtracted from the band intensity for drug treated samples for the corresponding strain. All data was then normalised against the signal for dnj-14 (wt) ; PP563 at 0.5 mM. Error bars represent the standard error of the mean. Use of ANOVAR showed that data from the dnj-14 (tm3223) worms was statistically significantly different from the control worm data (n = 3, p

    Journal: Scientific Reports

    Article Title: Expression profile of a Caenorhabditis elegans model of adult neuronal ceroid lipofuscinosis reveals down regulation of ubiquitin E3 ligase components

    doi: 10.1038/srep14392

    Figure Lengend Snippet: dnj-14 (tm3223) worms are delayed in ubiquitylated protein degradation (A) Representative image of Western blot detection of GFP-UbV accumulation in PP563 and PP545 strains after overnight incubation with or without 10 μM bortezomib. There was no GFP detected in untreated PP563 worms but a signal was detectable after bortezomib treatment. In contrast GFP was already accumulated in PP545 worms before treatment. Quantitation using ImageJ showed that the average GFP signal from PP545 worm lysates after bortezomib treatment was 94% +/− 0.005% (+/−SEM) compared to untreated PP545 worms (n = 3). ( B ) Representative image of Western blot detection of GFP-UbV accumulation in N2 and dnj-14 (tm3223) crossed with the pSur5::UbV-GFP expressing strain, PP563, generating the strains, dnj-14 (wt) ; PP563 and dnj-14 (tm3223) ; PP563. There was an average intensity increase of 354 +/−109% (+/−SEM) in dnj-14 (tm3223) ; PP563 compared to dnj-14 (wt) ; PP563 (n = 3). Worms were harvested and lysed at day 1 of adulthood. 50 worms were lysed in each SDS-PAGE sample and anti-beta-actin was used as a loading control. ( B ) GFP fluorescence was imaged in dnj-14 (wt) ; PP563 and dnj-14 (tm3223) ; PP563 worms after overnight incubation with increasing concentrations of bortezomib. ( C ) Western blot detection of GFP-UbV accumulation, using an anti-GFP antibody, after overnight treatment of dnj-14 (wt) ; PP563 and dnj-14 (tm3223) ; PP563 with increasing concentrations of bortezomib. ( D ) Quantitation of anti-GFP signal detected in ( C ). Band intensity for 0 mM was assumed to be background and was subtracted from the band intensity for drug treated samples for the corresponding strain. All data was then normalised against the signal for dnj-14 (wt) ; PP563 at 0.5 mM. Error bars represent the standard error of the mean. Use of ANOVAR showed that data from the dnj-14 (tm3223) worms was statistically significantly different from the control worm data (n = 3, p

    Article Snippet: Membranes were subsequently incubated with mouse anti-beta-actin (sigma) for 1 hour at room temperature as a loading control.

    Techniques: Western Blot, Incubation, Quantitation Assay, Expressing, SDS Page, Fluorescence

    PP2A activity does not substantially affect progression beyond anaphase Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into three sets that received either vehicle as control (CTRL), OA at 0.5 µM (OA), or LB-100. Cells were then taken at the indicated time points of further incubation and spun onto microscopy slides and processed for immunofluorescence staining for α-tubulin and the centromere marker CREST, DNA was stained with Hoechst. Upper graphs: post-anaphase cells were visually scored through microscopy. Error bars refer to variation within three independent experiments performed under identical conditions. Lower photographs: indicative images of HeLa and hTERT-RPE1 cells taken at 60 or 40 min, respectively, of incubation. Scale bars, 10 μm.

    Journal: Oncotarget

    Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1

    doi: 10.18632/oncotarget.23329

    Figure Lengend Snippet: PP2A activity does not substantially affect progression beyond anaphase Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into three sets that received either vehicle as control (CTRL), OA at 0.5 µM (OA), or LB-100. Cells were then taken at the indicated time points of further incubation and spun onto microscopy slides and processed for immunofluorescence staining for α-tubulin and the centromere marker CREST, DNA was stained with Hoechst. Upper graphs: post-anaphase cells were visually scored through microscopy. Error bars refer to variation within three independent experiments performed under identical conditions. Lower photographs: indicative images of HeLa and hTERT-RPE1 cells taken at 60 or 40 min, respectively, of incubation. Scale bars, 10 μm.

    Article Snippet: After blocking with 3% BSA (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 hour, samples were incubated for 3 hours with mouse anti α-tubulin (1:500; Sigma-Aldrich, St. Louis, MO, USA) and human anti CREST (1:100; Fitzgerald Industries International, North Acton, MA, USA) primary antibodies, both in PBS + 1% BSA.

    Techniques: Activity Assay, Incubation, Microscopy, Immunofluorescence, Staining, Marker

    Vemurafenib hyperactivates the Raf/MEK/ERK pathway while inhibiting various cellular signaling cascades. (A) Analysis of the effects of Vemurafenib on IAV-induced cellular signaling pathways by western blot. A549 cells were infected with FPV (MOI 5) and subsequently treated with Vemurafenib (25 μM) or DMSO, respectively. Total cell lysates were harvested at the times indicated. Activity of the Raf/MEK/ERK pathway was analyzed by detection of phosphorylated kinases MEK1/2 and ERK1/2. JNK and p38 MAPK pathway activities were elucidated by analysis of JNK1/2 and p38 phosphorylation as well as by detection of phosphorylation of upstream kinases MKK3/6 and downstream target ATF2. Activity of the PI3K/Akt pathway was determined by detection of Akt phosphorylation. Alpha-tubulin served as loading control. (B,C) Stimulation of Vemurafenib-treated A549 cells with EGF (30 ng/ml) for 5 min (B) or TNFα (5 ng/ml) for 30 min (C) . Activity of different cellular signaling pathways was analyzed by western blot. ERK2 and alpha-tubulin served as loading controls. (A–C) Blots are representative of three independent experiments.

    Journal: Frontiers in Microbiology

    Article Title: Vemurafenib Limits Influenza A Virus Propagation by Targeting Multiple Signaling Pathways

    doi: 10.3389/fmicb.2017.02426

    Figure Lengend Snippet: Vemurafenib hyperactivates the Raf/MEK/ERK pathway while inhibiting various cellular signaling cascades. (A) Analysis of the effects of Vemurafenib on IAV-induced cellular signaling pathways by western blot. A549 cells were infected with FPV (MOI 5) and subsequently treated with Vemurafenib (25 μM) or DMSO, respectively. Total cell lysates were harvested at the times indicated. Activity of the Raf/MEK/ERK pathway was analyzed by detection of phosphorylated kinases MEK1/2 and ERK1/2. JNK and p38 MAPK pathway activities were elucidated by analysis of JNK1/2 and p38 phosphorylation as well as by detection of phosphorylation of upstream kinases MKK3/6 and downstream target ATF2. Activity of the PI3K/Akt pathway was determined by detection of Akt phosphorylation. Alpha-tubulin served as loading control. (B,C) Stimulation of Vemurafenib-treated A549 cells with EGF (30 ng/ml) for 5 min (B) or TNFα (5 ng/ml) for 30 min (C) . Activity of different cellular signaling pathways was analyzed by western blot. ERK2 and alpha-tubulin served as loading controls. (A–C) Blots are representative of three independent experiments.

    Article Snippet: Antisera directed against ERK2 (C-14; #sc-154) and IAV PB1 (vK-20; #sc-17601) were purchased from Santa Cruz Biotechnology and α-Tubulin antibodies (#T6199) from Sigma-Aldrich.

    Techniques: Western Blot, Infection, Activity Assay

    Vemurafenib inhibits IAV-induced apoptosis by interfering with cytokine expression. (A) A549 cells were treated with Vemurafenib (25 μM), DMSO or left untreated and were stimulated with Staurosporine (1 μM) for 5 h. Cleavage of caspases 8, 9, and 3 as well as PARP was visualized by western blot of total cell lysates. ERK2 and alpha-tubulin served as loading controls. Blots are representative of three independent experiments. (B) Expression levels of TRAIL mRNA in FPV-infected (MOI 5) A549 cells which were subsequently treated with Vemurafenib (25 μM) or DMSO, respectively. Untreated samples served as negative (uninfected) and positive control (infected). Results are depicted as mean n -fold expression (±SEM) of three independent experiments normalized to negative control. Data were analyzed for statistical significance by Kruskal–Wallis test followed by Dunn's multiple comparisons test. (C) A549 cells were pre-incubated with U0126 (50 μM) for 90 min and Vemurafenib (25 μM) for 60 min or DMSO before transfection of 500 ng total RNA isolated from infected A549 cells (vRNA; FPV, MOI 5, 8 h). Total RNA from uninfected cells (cRNA) was used as control. Phosphorylation of ERK1/2 was confirmed by immunostaining with phospho-specific antibodies. Apoptosis induction was analyzed by detection of cleaved PARP. Equal loading was confirmed by staining of ERK2. Blots are representative of three independent experiments. (D) A549 cells were infected with H7N7 (SC35M) and afterwards treated with Vemurafenib (25 μM) or DMSO, respectively. Cells were stimulated with human TRAIL (50 ng/ml) (left panel) or human TNFα (5 ng/ml) (right panel) 4 hpi. Infectious virus particles in the supernatant were measured 8 hpi by standard plaque assay and are depicted as mean (± SD ) of three independent experiments. Statistical significance was analyzed by one way ANOVA followed by Sidak's multiple comparisons test.

    Journal: Frontiers in Microbiology

    Article Title: Vemurafenib Limits Influenza A Virus Propagation by Targeting Multiple Signaling Pathways

    doi: 10.3389/fmicb.2017.02426

    Figure Lengend Snippet: Vemurafenib inhibits IAV-induced apoptosis by interfering with cytokine expression. (A) A549 cells were treated with Vemurafenib (25 μM), DMSO or left untreated and were stimulated with Staurosporine (1 μM) for 5 h. Cleavage of caspases 8, 9, and 3 as well as PARP was visualized by western blot of total cell lysates. ERK2 and alpha-tubulin served as loading controls. Blots are representative of three independent experiments. (B) Expression levels of TRAIL mRNA in FPV-infected (MOI 5) A549 cells which were subsequently treated with Vemurafenib (25 μM) or DMSO, respectively. Untreated samples served as negative (uninfected) and positive control (infected). Results are depicted as mean n -fold expression (±SEM) of three independent experiments normalized to negative control. Data were analyzed for statistical significance by Kruskal–Wallis test followed by Dunn's multiple comparisons test. (C) A549 cells were pre-incubated with U0126 (50 μM) for 90 min and Vemurafenib (25 μM) for 60 min or DMSO before transfection of 500 ng total RNA isolated from infected A549 cells (vRNA; FPV, MOI 5, 8 h). Total RNA from uninfected cells (cRNA) was used as control. Phosphorylation of ERK1/2 was confirmed by immunostaining with phospho-specific antibodies. Apoptosis induction was analyzed by detection of cleaved PARP. Equal loading was confirmed by staining of ERK2. Blots are representative of three independent experiments. (D) A549 cells were infected with H7N7 (SC35M) and afterwards treated with Vemurafenib (25 μM) or DMSO, respectively. Cells were stimulated with human TRAIL (50 ng/ml) (left panel) or human TNFα (5 ng/ml) (right panel) 4 hpi. Infectious virus particles in the supernatant were measured 8 hpi by standard plaque assay and are depicted as mean (± SD ) of three independent experiments. Statistical significance was analyzed by one way ANOVA followed by Sidak's multiple comparisons test.

    Article Snippet: Antisera directed against ERK2 (C-14; #sc-154) and IAV PB1 (vK-20; #sc-17601) were purchased from Santa Cruz Biotechnology and α-Tubulin antibodies (#T6199) from Sigma-Aldrich.

    Techniques: Expressing, Western Blot, Infection, Positive Control, Negative Control, Incubation, Transfection, Isolation, Immunostaining, Staining, Plaque Assay

    Vemurafenib strongly limits influenza A virus replication by a multifactorial mode of action. (A,B) Immunofluorescence of Vemurafenib (25 μM)- or DMSO-treated A549 cells infected with FPV (MOI 5) for the time points indicated. Viral NP localization was detected using IAV NP primary antibodies and nuclei were counterstained with DAPI. (A) NP positive cells (open bars) and cells with nuclear NP localization (checkered bars) were quantified and related to the number of DAPI positive cells. FIJI software was used for counting. Statistical significance of % NP positive cells and % nuclear NP was evaluated separately by one-way ANOVA followed by Sidak's multiple comparisons test ( * p = 0.01–0.05). (B) Exemplary sections of one of three independent experiments. Scale represents 50 μm. (C,D) A549 cells were infected with FPV (MOI 5) and afterwards treated with Vemurafenib (25 μM) or DMSO, respectively. (C) Expression of viral proteins was analyzed by western blot assay at the times indicated. ERK2 and alpha-tubulin served as loading controls. (D) Samples were further analyzed for phosphorylation levels of MK2, 4E-BP1, p70S6K, and pS6 Rib. Prot. by western blot. Alpha-Tubulin served as loading control. (C,D) Blots are representative of three independent experiments. (E) A549 cells were infected with 0.01 MOI SC35M and subsequently treated with 10 μM SB202190, 10 μM SP600125 or both inhibitors in presence of Vemurafenib (25 μM) or DMSO, respectively. Amounts of progeny virions were measured by standard plaque assay. Shown are means (± SD ) of three independent experiments. Statistical significance was calculated by two-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test relative to respective DMSO controls ( **** p

    Journal: Frontiers in Microbiology

    Article Title: Vemurafenib Limits Influenza A Virus Propagation by Targeting Multiple Signaling Pathways

    doi: 10.3389/fmicb.2017.02426

    Figure Lengend Snippet: Vemurafenib strongly limits influenza A virus replication by a multifactorial mode of action. (A,B) Immunofluorescence of Vemurafenib (25 μM)- or DMSO-treated A549 cells infected with FPV (MOI 5) for the time points indicated. Viral NP localization was detected using IAV NP primary antibodies and nuclei were counterstained with DAPI. (A) NP positive cells (open bars) and cells with nuclear NP localization (checkered bars) were quantified and related to the number of DAPI positive cells. FIJI software was used for counting. Statistical significance of % NP positive cells and % nuclear NP was evaluated separately by one-way ANOVA followed by Sidak's multiple comparisons test ( * p = 0.01–0.05). (B) Exemplary sections of one of three independent experiments. Scale represents 50 μm. (C,D) A549 cells were infected with FPV (MOI 5) and afterwards treated with Vemurafenib (25 μM) or DMSO, respectively. (C) Expression of viral proteins was analyzed by western blot assay at the times indicated. ERK2 and alpha-tubulin served as loading controls. (D) Samples were further analyzed for phosphorylation levels of MK2, 4E-BP1, p70S6K, and pS6 Rib. Prot. by western blot. Alpha-Tubulin served as loading control. (C,D) Blots are representative of three independent experiments. (E) A549 cells were infected with 0.01 MOI SC35M and subsequently treated with 10 μM SB202190, 10 μM SP600125 or both inhibitors in presence of Vemurafenib (25 μM) or DMSO, respectively. Amounts of progeny virions were measured by standard plaque assay. Shown are means (± SD ) of three independent experiments. Statistical significance was calculated by two-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test relative to respective DMSO controls ( **** p

    Article Snippet: Antisera directed against ERK2 (C-14; #sc-154) and IAV PB1 (vK-20; #sc-17601) were purchased from Santa Cruz Biotechnology and α-Tubulin antibodies (#T6199) from Sigma-Aldrich.

    Techniques: Immunofluorescence, Infection, Software, Expressing, Western Blot, Plaque Assay

    In vitro and in vivo binding of HSF1 to the COX-2 promoter. ( A ) Nucleotide sequence of the 100 bp (from −2579 to −2480) DNA fragment of the COX-2 promoter region including binding sites for the GATA and HSF1 transcription factors. The 100 bp fragment was amplified by PCR, 32 P-labeled and used as probe for gel-shift analysis shown in B and C . ( B ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), whole-cell extracts were analyzed for HSF DNA-binding activity by EMSA in a 3% polyacrylamide gel using the probe described in ( A ). Position of HSF-DNA binding complex (HSF), constitutive HSE binding activity (CHBA) and non specific protein-DNA interactions (NS) are shown (upper panel). In parallel samples whole-cell extracts were analyzed for levels of HSF1 and β-actin proteins by Western blot (lower panels). Arrow indicates the position of the low-mobility phosphorylated HSF1 isoform. ( C ) Specificity of HSF1-DNA binding complexes. Whole-cell extracts from HUVECs subjected to heat shock at 43°C for 40 min were preincubated with different dilutions of anti-HSF1 polyclonal antibodies for 15 min before supershift assay. Position of HSF, CHBA and NS are indicated as in B . ( D,E ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), recruitment of HSF1 to the COX-2 and HSP70 promoters was analyzed by ChIP assay. ( D ) ChIP-enriched DNAs using preimmune serum (IP NS IgG) or anti-HSF1 serum (IP anti-HSF1), as well as input DNAs (INPUT) were prepared, and DNA fragments of the COX-2 gene (−2629 to −2420) and HSP70 gene (−262 to −70) were amplified by PCR. ( E ) Quantification of ChIP assay shown in ( D ). Samples from at least three independent experiments were analyzed by real time PCR. Relative promoter occupancy is expressed as fold induction of control arbitrarily set to a value of 1. Error bars indicate ± S.D. * = P

    Journal: PLoS ONE

    Article Title: Regulation of Cyclooxygenase-2 Expression by Heat: A Novel Aspect of Heat Shock Factor 1 Function in Human Cells

    doi: 10.1371/journal.pone.0031304

    Figure Lengend Snippet: In vitro and in vivo binding of HSF1 to the COX-2 promoter. ( A ) Nucleotide sequence of the 100 bp (from −2579 to −2480) DNA fragment of the COX-2 promoter region including binding sites for the GATA and HSF1 transcription factors. The 100 bp fragment was amplified by PCR, 32 P-labeled and used as probe for gel-shift analysis shown in B and C . ( B ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), whole-cell extracts were analyzed for HSF DNA-binding activity by EMSA in a 3% polyacrylamide gel using the probe described in ( A ). Position of HSF-DNA binding complex (HSF), constitutive HSE binding activity (CHBA) and non specific protein-DNA interactions (NS) are shown (upper panel). In parallel samples whole-cell extracts were analyzed for levels of HSF1 and β-actin proteins by Western blot (lower panels). Arrow indicates the position of the low-mobility phosphorylated HSF1 isoform. ( C ) Specificity of HSF1-DNA binding complexes. Whole-cell extracts from HUVECs subjected to heat shock at 43°C for 40 min were preincubated with different dilutions of anti-HSF1 polyclonal antibodies for 15 min before supershift assay. Position of HSF, CHBA and NS are indicated as in B . ( D,E ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), recruitment of HSF1 to the COX-2 and HSP70 promoters was analyzed by ChIP assay. ( D ) ChIP-enriched DNAs using preimmune serum (IP NS IgG) or anti-HSF1 serum (IP anti-HSF1), as well as input DNAs (INPUT) were prepared, and DNA fragments of the COX-2 gene (−2629 to −2420) and HSP70 gene (−262 to −70) were amplified by PCR. ( E ) Quantification of ChIP assay shown in ( D ). Samples from at least three independent experiments were analyzed by real time PCR. Relative promoter occupancy is expressed as fold induction of control arbitrarily set to a value of 1. Error bars indicate ± S.D. * = P

    Article Snippet: After blocking with 5% skim milk solution, membranes were incubated with rabbit polyclonal anti-HSF1, (Santa Cruz Biotechnology), antibodies, or monoclonal anti-COX-2 (SC-19999, Santa Cruz Biotechnology), anti-HSP70 (Stressgene), and anti-β-actin (Sigma) antibodies followed by decoration with peroxidase-labeled anti-rabbit or anti-mouse IgG respectively (Super Signal detection kit, Pierce).

    Techniques: In Vitro, In Vivo, Binding Assay, Sequencing, Amplification, Polymerase Chain Reaction, Labeling, Electrophoretic Mobility Shift Assay, Activity Assay, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10 9 PFU of AdβGal (Ad) or 5 × 10 4 AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and IgG2a-specific antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.

    Journal: Journal of Virology

    Article Title: Distinct Roles of Adenovirus Vector-Transduced Dendritic Cells, Myoblasts, and Endothelial Cells in Mediating an Immune Response against a Transgene Product

    doi: 10.1128/JVI.76.6.2899-2911.2002

    Figure Lengend Snippet: β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10 9 PFU of AdβGal (Ad) or 5 × 10 4 AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and IgG2a-specific antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.

    Article Snippet: Different anti-mouse Ig's were used: goat-anti-mouse IgG (Amersham Pharmacia), goat anti-mouse IgG1 (Caltag), and goat anti-mouse IgG2a (Caltag).

    Techniques: Mouse Assay, Injection, Expressing, Enzyme-linked Immunosorbent Assay

    Binding of IgG and C3b with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.

    Journal: Frontiers in Immunology

    Article Title: Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation

    doi: 10.3389/fimmu.2020.00462

    Figure Lengend Snippet: Binding of IgG and C3b with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.

    Article Snippet: C3b was detected in bacterial lysates ran on a 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) without heating under non-reducing conditions (without adding 2-mercaptoethanol), transferred to nitrocellulose membrane (MDI), and probed with mouse anti-human C3b monoclonal antibodies (Thermo Fisher, Cat. No. MA1-70054) followed with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (Sigma, Cat. No. A4416).

    Techniques: Binding Assay, Incubation, Purification

    Ultrastructural immunolocalization of GABA in astrocytes of hippocampal CA1, CA3, and dentate gyrus regions. Combined pre-embedding immunogold and immunoperoxidase methods for electron microscopy. Astrocytes (GFAP+) and GABA profiles are labeled by DAB and silver-intensified gold particles, respectively. GABA immunoparticles are localized in synaptic terminals (Ter) with symmetric synapses (arrows) as well as in astrocytes (Ast, white arrowheads) in CA1 (A) and CA3 (B) stratum radiatum and in the dentate gyrus (C) . In A, a GAFP+ astrocytic portion containing GABA is delineated.

    Journal: Frontiers in Computational Neuroscience

    Article Title: GABA release by hippocampal astrocytes

    doi: 10.3389/fncom.2012.00059

    Figure Lengend Snippet: Ultrastructural immunolocalization of GABA in astrocytes of hippocampal CA1, CA3, and dentate gyrus regions. Combined pre-embedding immunogold and immunoperoxidase methods for electron microscopy. Astrocytes (GFAP+) and GABA profiles are labeled by DAB and silver-intensified gold particles, respectively. GABA immunoparticles are localized in synaptic terminals (Ter) with symmetric synapses (arrows) as well as in astrocytes (Ast, white arrowheads) in CA1 (A) and CA3 (B) stratum radiatum and in the dentate gyrus (C) . In A, a GAFP+ astrocytic portion containing GABA is delineated.

    Article Snippet: They were incubated in primary rabbit anti-GABA (1:1000; Somogyi and Hodgson, ) and mouse anti-GFAP (1:1000; Sigma Chemical Company St. Louis, MO, USA) antibodies both in 10% BSA/TBS containing 0.1% sodium azide and 0.004% saponin on a shaker for 2 days at 4°C.

    Techniques: Electron Microscopy, Labeling, AST Assay

    FANCJ-R707C is a separation-of-function mutant that partially rescues sensitivity to the replication stress inducing agents aphidicolin or telomestatin but fails to restore cisplatin resistance. ( A ) Schematic diagram of green fluorescence protein (GFP)-FANCJ recombinant protein expressed in fancj −/− cells for genetic complementation assays. ( B ) Western blot analysis of whole cell lysate protein (40 μg) from fancj −/− DT40 cells transfected with plasmid encoding (pEGFP-Vector), pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C), or pEGFP-FANCJ-H396D (FANCJ-H396D). Protein was detected with antibody against FANCJ or actin (as a loading control, 10% loaded). (C-E) Cell survival assays, as measured by methyl cellulose colony formation, for the indicated fancj −/− cells expressing mutant or wild-type FANCJ proteins. Cells were exposed to the indicated concentrations of CisPt ( C ), APH ( D ) or TMS ( E ). Filled square, fancj −/− cells transfected with FANCJ-WT; open square, fancj −/− cells transfected with pEGFP-Vector; filled triangle, fancj −/− cells transfected with FANCJ-R707C; open cross- fancj −/− cells transfected with FANCJ-H396D. (F, G) FANCJ-R707C restores normal replication restart after APH exposure. Schematic representation of the protocol used to track DNA replication fibers is shown in Panel F. Cells were pulse-chased with CldU ( red label ), and then labeled with IdU ( green label ) for the indicated times. ( G ) Representative images of fluorescently-labeled DNA fibers from the indicated cell lines treated with DMSO or APH. ( H ) Box and whiskers graphs indicating the 10–90 percentile of the IdU tract length (μm) for ongoing forks. The data, presented as mean ± standard error of the mean (s.e.m.), are based on the measurement of at least 100 DNA fibers from two independent experiments. ( I – N ), DNA damage, as marked by immunofluorescent detection of γ-H2AX foci, in the indicated fancj −/− transfected cell lines. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to: CisPt (1 μM) (I, J); APH (200 nM) (K, L); TMS (5 μM) (M, N). Immunofluorescence detection of γ-H2AX foci by Alexa fluor 488 is shown along with DAPI, or merged (DAPI and Alexa fluor 488). Quantitative analyses of γ-H2AX foci are shown with S.D. (ns-not significant; ** P

    Journal: Nucleic Acids Research

    Article Title: A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair

    doi: 10.1093/nar/gky403

    Figure Lengend Snippet: FANCJ-R707C is a separation-of-function mutant that partially rescues sensitivity to the replication stress inducing agents aphidicolin or telomestatin but fails to restore cisplatin resistance. ( A ) Schematic diagram of green fluorescence protein (GFP)-FANCJ recombinant protein expressed in fancj −/− cells for genetic complementation assays. ( B ) Western blot analysis of whole cell lysate protein (40 μg) from fancj −/− DT40 cells transfected with plasmid encoding (pEGFP-Vector), pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C), or pEGFP-FANCJ-H396D (FANCJ-H396D). Protein was detected with antibody against FANCJ or actin (as a loading control, 10% loaded). (C-E) Cell survival assays, as measured by methyl cellulose colony formation, for the indicated fancj −/− cells expressing mutant or wild-type FANCJ proteins. Cells were exposed to the indicated concentrations of CisPt ( C ), APH ( D ) or TMS ( E ). Filled square, fancj −/− cells transfected with FANCJ-WT; open square, fancj −/− cells transfected with pEGFP-Vector; filled triangle, fancj −/− cells transfected with FANCJ-R707C; open cross- fancj −/− cells transfected with FANCJ-H396D. (F, G) FANCJ-R707C restores normal replication restart after APH exposure. Schematic representation of the protocol used to track DNA replication fibers is shown in Panel F. Cells were pulse-chased with CldU ( red label ), and then labeled with IdU ( green label ) for the indicated times. ( G ) Representative images of fluorescently-labeled DNA fibers from the indicated cell lines treated with DMSO or APH. ( H ) Box and whiskers graphs indicating the 10–90 percentile of the IdU tract length (μm) for ongoing forks. The data, presented as mean ± standard error of the mean (s.e.m.), are based on the measurement of at least 100 DNA fibers from two independent experiments. ( I – N ), DNA damage, as marked by immunofluorescent detection of γ-H2AX foci, in the indicated fancj −/− transfected cell lines. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to: CisPt (1 μM) (I, J); APH (200 nM) (K, L); TMS (5 μM) (M, N). Immunofluorescence detection of γ-H2AX foci by Alexa fluor 488 is shown along with DAPI, or merged (DAPI and Alexa fluor 488). Quantitative analyses of γ-H2AX foci are shown with S.D. (ns-not significant; ** P

    Article Snippet: After four washes in PBS with 0.1% Tween 20, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:500, Invitrogen) or Alexa Fluor 633 goat anti-mouse IgG (1:500, Invitrogen) for 1 h at room temperature.

    Techniques: Mutagenesis, Fluorescence, Recombinant, Western Blot, Transfection, Plasmid Preparation, Expressing, Labeling, Immunofluorescence

    FANCJ-R707C fails to suppress single-stranded DNA or Rad51 persistence in cells exposed to CisPt. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to CisPt (1 μM) ( A, B ) or APH (200 nM) ( C, D ). Note that for BrdU staining, cells were pre-labeled with BrdU prior to drug exposure as described in Materials and Methods. (A, C) Immunofluorescent BrdU foci (A) or Rad51 foci (C) were detected by Alexa fluor 488 and are shown along with DAPI, or merged (DAPI and Alexa fluor 488). (B, D) Quantitative analyses of BrdU foci (B) or Rad51 foci (D) are shown with S.D. indicated by error bars.

    Journal: Nucleic Acids Research

    Article Title: A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair

    doi: 10.1093/nar/gky403

    Figure Lengend Snippet: FANCJ-R707C fails to suppress single-stranded DNA or Rad51 persistence in cells exposed to CisPt. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to CisPt (1 μM) ( A, B ) or APH (200 nM) ( C, D ). Note that for BrdU staining, cells were pre-labeled with BrdU prior to drug exposure as described in Materials and Methods. (A, C) Immunofluorescent BrdU foci (A) or Rad51 foci (C) were detected by Alexa fluor 488 and are shown along with DAPI, or merged (DAPI and Alexa fluor 488). (B, D) Quantitative analyses of BrdU foci (B) or Rad51 foci (D) are shown with S.D. indicated by error bars.

    Article Snippet: After four washes in PBS with 0.1% Tween 20, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:500, Invitrogen) or Alexa Fluor 633 goat anti-mouse IgG (1:500, Invitrogen) for 1 h at room temperature.

    Techniques: Transfection, Plasmid Preparation, BrdU Staining, Labeling

    Low correlation of colocalization of pacsin2 and the VE-cadherin complex at super-resolution level. ( a ) Representative deconvolved images of stimulated emission depletion (STED) and confocal microscopy performed on IF labelled HUVECs stained for VE-cadherin (red) and catenins, vinculin or pacsin2 (all green). ( b ) Pearson correlation analysis of pixel intensities between VE-cadherin and indicated proteins at FAJ sections. Graph shows the average Pearson correlation coefficient ( R )±s.e.m. of indicated protein pairs, which was determined on junctional region of interests (ROIs) from deconvolved STED images. n ≥17 FAJs per correlation analysis. An R of 1 indicates perfect colocalization between two proteins, whereas an R of 0 indicates no correlation. ** P ≤0.01 (Student's t -test).

    Journal: Nature Communications

    Article Title: The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions

    doi: 10.1038/ncomms12210

    Figure Lengend Snippet: Low correlation of colocalization of pacsin2 and the VE-cadherin complex at super-resolution level. ( a ) Representative deconvolved images of stimulated emission depletion (STED) and confocal microscopy performed on IF labelled HUVECs stained for VE-cadherin (red) and catenins, vinculin or pacsin2 (all green). ( b ) Pearson correlation analysis of pixel intensities between VE-cadherin and indicated proteins at FAJ sections. Graph shows the average Pearson correlation coefficient ( R )±s.e.m. of indicated protein pairs, which was determined on junctional region of interests (ROIs) from deconvolved STED images. n ≥17 FAJs per correlation analysis. An R of 1 indicates perfect colocalization between two proteins, whereas an R of 0 indicates no correlation. ** P ≤0.01 (Student's t -test).

    Article Snippet: Antibodies and reagents Mouse monoclonal anti-actin (clone AC-40, Cat # A3853, diluted 1/3,000 for western) anti-vinculin antibody hVIN-1 (Cat # V9131, diluted 1/100 for immunofluorescence (IF)), rabbit anti-α-catenin serum (Cat # C2081, diluted 1/400 for IF) and anti-β-catenin serum (Cat # C2206, diluted 1/400 for IF) were obtained from Sigma-Aldrich.

    Techniques: Confocal Microscopy, Staining

    F-BAR protein recruitment to force-remodelling adherens junctions. ( a ) Widefield immunofluorescence (IF) images of HUVECs stained for pacsin2 (green), F-actin (red) and VE-cadherin (blue). Magnified images of regions of interest at focal adherens junctions (FAJs) and linear adherens junctions (LAJs). Arrows indicate pacsin2-positive FAJs. Graph shows the average ratio±s.e. of the mean (s.e.m.) between pacsin2 and VE-cadherin fluorescence integrated intensities along junctional linescans. n =44 FAJs; n =36 LAJs from three independent experiments. ( b ) Widefield IF images of FAJs in HMEC-1 and BMEC-28 monolayers stained for pacsin2 (green), F-actin (red) and VE-cadherin (blue). ( c ) Graph shows a quantification of the percentage±s.e.m. of FAJs that are pacsin2-positive in IF images of HUVEC ( n =22 images), HMEC-1 ( n =16 images) or BMEC-28 cells ( n =30 images) from n ≥2 independent experiments. ( d ) Graph shows the average distribution±s.e.m. of VE-cadherin, pacsin2 and vinculin intensities along pacsin2-positive FAJs. A ratio of 1 represents symmetric distribution along FAJs. For VE-cadherin and pacsin2, the analysis was from n =40 FAJs, and for vinculin n =27 FAJs from n ≥2 independent experiments. ( e ) Widefield IF images of FAJs in mesenchymal stromal cells (MSC) stained for pacsin2 (green), F-actin (red) and N-cadherin (blue); and prostate epithelial cells (DU145) stained for pacsin2 (green), F-actin (red) and β-catenin (blue). ( f ) En face confocal IF images of human mesenteric artery stained for VE-cadherin (red) and pacsin2 (green). ( g ) Widefield IF images of FAJs in HUVEC monolayer stained for nostrin (green), F-actin (red) and VE-cadherin (blue). First graph shows the average ratio±s.e.m. between nostrin and VE-cadherin fluorescence integrated intensities along junctional linescans. n =33 FAJs; n =31 LAJs from three independent experiments. The second graph shows the average percentage±s.e.m. of FAJs that are nostrin positive ( n =12 HUVEC images from three independent experiments). ( h ) Widefield IF images of FAJs in HUVECs stained for pacsin2 (green), vinculin (red) and VE-cadherin (blue). Linescan analysis along the white dotted line shows the fluorescence intensities of VE-cadherin (right y axis), vinculin and pacsin2 signals (left y axis). ** P ≤0.01 (Student's t -test).

    Journal: Nature Communications

    Article Title: The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions

    doi: 10.1038/ncomms12210

    Figure Lengend Snippet: F-BAR protein recruitment to force-remodelling adherens junctions. ( a ) Widefield immunofluorescence (IF) images of HUVECs stained for pacsin2 (green), F-actin (red) and VE-cadherin (blue). Magnified images of regions of interest at focal adherens junctions (FAJs) and linear adherens junctions (LAJs). Arrows indicate pacsin2-positive FAJs. Graph shows the average ratio±s.e. of the mean (s.e.m.) between pacsin2 and VE-cadherin fluorescence integrated intensities along junctional linescans. n =44 FAJs; n =36 LAJs from three independent experiments. ( b ) Widefield IF images of FAJs in HMEC-1 and BMEC-28 monolayers stained for pacsin2 (green), F-actin (red) and VE-cadherin (blue). ( c ) Graph shows a quantification of the percentage±s.e.m. of FAJs that are pacsin2-positive in IF images of HUVEC ( n =22 images), HMEC-1 ( n =16 images) or BMEC-28 cells ( n =30 images) from n ≥2 independent experiments. ( d ) Graph shows the average distribution±s.e.m. of VE-cadherin, pacsin2 and vinculin intensities along pacsin2-positive FAJs. A ratio of 1 represents symmetric distribution along FAJs. For VE-cadherin and pacsin2, the analysis was from n =40 FAJs, and for vinculin n =27 FAJs from n ≥2 independent experiments. ( e ) Widefield IF images of FAJs in mesenchymal stromal cells (MSC) stained for pacsin2 (green), F-actin (red) and N-cadherin (blue); and prostate epithelial cells (DU145) stained for pacsin2 (green), F-actin (red) and β-catenin (blue). ( f ) En face confocal IF images of human mesenteric artery stained for VE-cadherin (red) and pacsin2 (green). ( g ) Widefield IF images of FAJs in HUVEC monolayer stained for nostrin (green), F-actin (red) and VE-cadherin (blue). First graph shows the average ratio±s.e.m. between nostrin and VE-cadherin fluorescence integrated intensities along junctional linescans. n =33 FAJs; n =31 LAJs from three independent experiments. The second graph shows the average percentage±s.e.m. of FAJs that are nostrin positive ( n =12 HUVEC images from three independent experiments). ( h ) Widefield IF images of FAJs in HUVECs stained for pacsin2 (green), vinculin (red) and VE-cadherin (blue). Linescan analysis along the white dotted line shows the fluorescence intensities of VE-cadherin (right y axis), vinculin and pacsin2 signals (left y axis). ** P ≤0.01 (Student's t -test).

    Article Snippet: Antibodies and reagents Mouse monoclonal anti-actin (clone AC-40, Cat # A3853, diluted 1/3,000 for western) anti-vinculin antibody hVIN-1 (Cat # V9131, diluted 1/100 for immunofluorescence (IF)), rabbit anti-α-catenin serum (Cat # C2081, diluted 1/400 for IF) and anti-β-catenin serum (Cat # C2206, diluted 1/400 for IF) were obtained from Sigma-Aldrich.

    Techniques: Immunofluorescence, Staining, Fluorescence

    Schematic representation of DNA hα-Syn vaccine constructs and expression in transfected CHO cells. (A ) Strategy for cloning genes encoding several hα-Syn B cell epitope/s fused with MultiTEP into the pVAX1 vector and schematic representation of PV-1947D, PV-1948D, PV-1949D, and PV-1950D constructs. ( B–D ) The expression of PV-1947D ( B ), PV-1948D ( C ), PV-1949D ( D ), and PV-1950D ( B–D ) and secretion of protein was demonstrated by WB of conditioned media of transiently transfected CHO cells. Proteins were visualized by staining with anti-hα-Syn 85–99 Abs ( A ), anti-α-hSyn 109–126 Abs ( B ), and anti-α-hSyn 126–140 Abs ( C ) followed by HRP conjugated anti-mouse IgG.

    Journal: Neurobiology of aging

    Article Title: MultiTEP Platform-based DNA Vaccines for alpha-Synucleinopathies: Pre-clinical Evaluation of Immunogenicity and Therapeutic Potency

    doi: 10.1016/j.neurobiolaging.2017.08.006

    Figure Lengend Snippet: Schematic representation of DNA hα-Syn vaccine constructs and expression in transfected CHO cells. (A ) Strategy for cloning genes encoding several hα-Syn B cell epitope/s fused with MultiTEP into the pVAX1 vector and schematic representation of PV-1947D, PV-1948D, PV-1949D, and PV-1950D constructs. ( B–D ) The expression of PV-1947D ( B ), PV-1948D ( C ), PV-1949D ( D ), and PV-1950D ( B–D ) and secretion of protein was demonstrated by WB of conditioned media of transiently transfected CHO cells. Proteins were visualized by staining with anti-hα-Syn 85–99 Abs ( A ), anti-α-hSyn 109–126 Abs ( B ), and anti-α-hSyn 126–140 Abs ( C ) followed by HRP conjugated anti-mouse IgG.

    Article Snippet: Membranes were stained with purified anti-hα-Syn85–99 , anti-hα-Syn109–126 and anti-hα-Syn126–140 antibodies at concentration 1µg/ml and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology).

    Techniques: Construct, Expressing, Transfection, Clone Assay, Plasmid Preparation, Western Blot, Staining

    HrpF stabilizes HrpA, and the 67th lysine residue of HrpF is critical for its interaction with HrpA. (A) Immunoblots of immunoprecipitated proteins probed with anti‐HrpA (top) and anti‐haemagglutinin (anti‐HA) (bottom) antibodies to show in vitro protein interactions. Escherichia coli lysates expressing HrpF‐HA27 (pNCHU1867), HrpF K67A ‐HA27 (pNCHU1868), HrpA (pNCHU1869) (‘+’) or vector alone (‘−’) were mixed with anti‐HA agarose beads for immunoprecipitation and detected by anti‐HrpA serum as described in Experimental procedures. Gel‐bound, immunoprecipitated proteins; lysate, total proteins as a control to detect the expression of recombinant proteins. (B) Far western analysis shows that HrpF‐HA27 and HrpF K67A ‐HA27 bind to HrpA‐His in vitro . Escherichia coli ‐expressed HrpA‐His (pNCHU1944) or EV1 (pETite) was probed with bacterial lysates expressing EV2 (pET29a), HrpF‐HA27 (pNCHU1867) or HrpF K67A ‐HA27 (pNCHU1868) as described in Experimental procedures. HrpA‐His‐bound HrpF‐HA was further detected with HA monoclonal antibody. (C) Immunodetection of HrpA stability in the Pseudomonas syringae pv. averrhoi (Pav) hrpF mutant. PavHL1, wild‐type harbouring pRK415 and pNCHU2046; Δ hrpF , HL1‐N1589 harbouring pRK415 and pNCHU2046; Δ hrpF/hrpF , HL1‐N1589 harbouring pNCHU1590 and pNCHU2046, Δ hrpF/hrpF K67A , HL1‐N1589 harbouring pNCHU2068 and pNCHU2046. Total cell cultures were harvested at 0, 2 and 4 h post‐chloramphenicol treatment for immunodetection with anti‐HrpZ and anti‐HrpA sera. The stability of HrpA is shown by the fold changes in HrpA banding intensity, which was first normalized by the HrpZ bands and calculated using the value at 0 h post‐chloramphenicol treatment as the denominator.

    Journal: Molecular Plant Pathology

    Article Title: The pathogenicity factor HrpF interacts with HrpA and HrpG to modulate type III secretion system (T3SS) function and t3ss expression in Pseudomonas syringae pv. averrhoi

    doi: 10.1111/mpp.12349

    Figure Lengend Snippet: HrpF stabilizes HrpA, and the 67th lysine residue of HrpF is critical for its interaction with HrpA. (A) Immunoblots of immunoprecipitated proteins probed with anti‐HrpA (top) and anti‐haemagglutinin (anti‐HA) (bottom) antibodies to show in vitro protein interactions. Escherichia coli lysates expressing HrpF‐HA27 (pNCHU1867), HrpF K67A ‐HA27 (pNCHU1868), HrpA (pNCHU1869) (‘+’) or vector alone (‘−’) were mixed with anti‐HA agarose beads for immunoprecipitation and detected by anti‐HrpA serum as described in Experimental procedures. Gel‐bound, immunoprecipitated proteins; lysate, total proteins as a control to detect the expression of recombinant proteins. (B) Far western analysis shows that HrpF‐HA27 and HrpF K67A ‐HA27 bind to HrpA‐His in vitro . Escherichia coli ‐expressed HrpA‐His (pNCHU1944) or EV1 (pETite) was probed with bacterial lysates expressing EV2 (pET29a), HrpF‐HA27 (pNCHU1867) or HrpF K67A ‐HA27 (pNCHU1868) as described in Experimental procedures. HrpA‐His‐bound HrpF‐HA was further detected with HA monoclonal antibody. (C) Immunodetection of HrpA stability in the Pseudomonas syringae pv. averrhoi (Pav) hrpF mutant. PavHL1, wild‐type harbouring pRK415 and pNCHU2046; Δ hrpF , HL1‐N1589 harbouring pRK415 and pNCHU2046; Δ hrpF/hrpF , HL1‐N1589 harbouring pNCHU1590 and pNCHU2046, Δ hrpF/hrpF K67A , HL1‐N1589 harbouring pNCHU2068 and pNCHU2046. Total cell cultures were harvested at 0, 2 and 4 h post‐chloramphenicol treatment for immunodetection with anti‐HrpZ and anti‐HrpA sera. The stability of HrpA is shown by the fold changes in HrpA banding intensity, which was first normalized by the HrpZ bands and calculated using the value at 0 h post‐chloramphenicol treatment as the denominator.

    Article Snippet: Immunodetection was performed using monoclonal anti‐HA antibody (Sigma).

    Techniques: Western Blot, Immunoprecipitation, In Vitro, Expressing, Plasmid Preparation, Recombinant, Immunodetection, Mutagenesis

    PA0833 stimulates protective immunity in a P. aeruginosa sepsis model. (A) Comparison of total IgG and IgG subgroups (IgG1, IgG2a, and IgG2b) in the mice ( n = 5) immunized with PA0833. Serum was obtained at 7 days after the final immunization, and the levels of total IgG and IgG subgroups were expressed as the means of log 2 titers. Multiple comparisons among different groups were analyzed using one-way ANOVA. Data are shown as the means ± SD. (B) BALB/c mice ( n = 10) were immunized with PA0833 plus an Al(OH) 3 adjuvant and challenged with PAO1 at 7.0 × 10 7 CFUs/mouse by intravenous injection. The survival rate was monitored for 14 days. The P -values were calculated using the Mantel–Cox log-rank test. (C,D) Efficacy of immunization with PA0833 on the spread of P. aeruginosa . The number of viable bacteria in the blood, liver, and spleen of mice ( n = 10) at 1 and 3 days post-infection are shown. Data are presented in box and whisker plots, and the medians are shown. Differences were compared to determine their significance using Student’s t -test.

    Journal: Frontiers in Microbiology

    Article Title: PA0833 Is an OmpA C-Like Protein That Confers Protection Against Pseudomonas aeruginosa Infection

    doi: 10.3389/fmicb.2018.01062

    Figure Lengend Snippet: PA0833 stimulates protective immunity in a P. aeruginosa sepsis model. (A) Comparison of total IgG and IgG subgroups (IgG1, IgG2a, and IgG2b) in the mice ( n = 5) immunized with PA0833. Serum was obtained at 7 days after the final immunization, and the levels of total IgG and IgG subgroups were expressed as the means of log 2 titers. Multiple comparisons among different groups were analyzed using one-way ANOVA. Data are shown as the means ± SD. (B) BALB/c mice ( n = 10) were immunized with PA0833 plus an Al(OH) 3 adjuvant and challenged with PAO1 at 7.0 × 10 7 CFUs/mouse by intravenous injection. The survival rate was monitored for 14 days. The P -values were calculated using the Mantel–Cox log-rank test. (C,D) Efficacy of immunization with PA0833 on the spread of P. aeruginosa . The number of viable bacteria in the blood, liver, and spleen of mice ( n = 10) at 1 and 3 days post-infection are shown. Data are presented in box and whisker plots, and the medians are shown. Differences were compared to determine their significance using Student’s t -test.

    Article Snippet: Diluted serum samples were used as the primary antibodies, and the secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a or anti-mouse IgG2b (Sigma).

    Techniques: Mouse Assay, Injection, Infection, Whisker Assay