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  • 99
    Thermo Fisher alexa fluor
    AChR clustering in wt and ColQ-deficient muscle cells. AChR clusters in wt ( a ) and ColQ −/− ( b ) myotubes (2 d old) labeled with <t>Alexa</t> 488 <t>α-BTX.</t> Projections of confocal stacks. Scale bar, 20 μm.
    Alexa Fluor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher alexa fluor 647
    DENV does not induce the SREBP-2 pathway or upreglation of HMGCR early after infection. 2fTGH cells were mock-treated (A) or treated with DTT (B) for 6 h. Cells were then fixed and stained intracellularly for cellular proteins using antibodies against SREBP-2 (green) and HMGCR (red) followed by secondary antibodies conjugated to <t>Alexa</t> Fluor® 488 and Alexa Fluor® 546, respectively. 2fTGH cells were infected with DENV-2 for 12 h and stained intracellularly for (C) SREBP-2 (magenta) and (D) HMGCR (magenta) followed by secondary antibodies conjugated to Alexa Fluor® 647; mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively, were used to detect DENV proteins. Nuclear staining (blue) was performed using DAPI stain. The multiple-exposure images were captured using a 40×/1.4 Plan-Apochromat DIC oil objective on a Zeiss 710 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 and were processed as previously described in Figure 1 . (E) SREBP-2 proteolyic processing by immunoblot analysis. Actin was used as a loading control. In (F), 2fTGH cells were infected with DENV-2 over a 3–12 h time-course. Lysates were collected and analyzed for SREBP-2 and HMGCR expression by immunoblot analysis. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection. A time-matched mock-infected control was included at each time-point. Total magnification 600×.
    Alexa Fluor 647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9558 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher alexa 647 fluor
    Seldegs increase the accumulation of antigen-specific antibodies in human endothelial (HMEC-1) cells expressing FcRn-GFP. ( a ) HMEC-1 cells were incubated with 100 nM Alexa 647-labelled 8-18C5 (MOG-specific) or TZB (HER2-specific) in complex with 400 nM MOG-Seldeg/MOG-WT or HER2-Seldeg/HER2-WT for 30 min and chased for 0 (30' P) or 60 min (30' P, 60' C). Mean fluorescence intensities (MFI) of Alexa 647-labelled 8-18C5 or TZB for triplicate samples were determined by flow cytometry. Error bars indicate s.d. ( b , c ) HMEC-1 cells were plated on coverslips and treated as in a , except that Seldegs or control WT proteins were labelled with Alexa 555 and cells were fixed for microscopy. Images of representative cells from multiple cells analysed are shown with GFP, Alexa 555 and <t>Alexa</t> 647 in overlays pseudocoloured green, red and blue, respectively. Representative endosomes in the insets are cropped and expanded. ( d ) HMEC-1 cells were pre-pulsed with Alexa 555-labelled dextran for 2 h, washed and pulsed with 8-18C5 in complex with MOG-Seldeg, MOG-WT and HER2-Seldeg (concentrations and labels as for a ) for 30 min, followed by an 8 h chase. Cells were washed, fixed and imaged, and images for a representative cell from multiple cells analysed are presented. Representative lysosomes in the insets are cropped and expanded. For the overlay, GFP, Alexa 555 and Alexa 647 are pseudocoloured as in b . For b – d , scale bars=5 μm, and for insets, scale bars=0.25 μm. Data shown are representative of at least two independent experiments.
    Alexa 647 Fluor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher alexa fluo 488 green
    Mouse primary and immortalized osteoblast cell proliferation. Proliferation of primary and immortalized cells was immunostained using a mouse monoclonal anti-BrdU antibody (1:100 dilution) after a 4-h BrdU incorporation (30 μM), followed by a 1:1,000 dilution of the secondary antibody (goat-anti-mouse) with <t>Alexa</t> Fluo® 488 green. For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst. Images were obtained by a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei. BrdU positive staining of the primary cells acts as 100% and asterisk (*) shows significant difference between the primary and immortalized cells ( p
    Alexa Fluo 488 Green, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher alex fluor 488
    Mouse primary and immortalized osteoblast cell proliferation. Proliferation of primary and immortalized cells was immunostained using a mouse monoclonal anti-BrdU antibody (1:100 dilution) after a 4-h BrdU incorporation (30 μM), followed by a 1:1,000 dilution of the secondary antibody (goat-anti-mouse) with <t>Alexa</t> Fluo® 488 green. For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst. Images were obtained by a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei. BrdU positive staining of the primary cells acts as 100% and asterisk (*) shows significant difference between the primary and immortalized cells ( p
    Alex Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher alexa 488 fluor
    Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for <t>Alexa</t> 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.
    Alexa 488 Fluor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher alexa fluor 405
    Neuronal calcium sensor (NCS1) in the Golgi apparatus recognizes Ca 2+ signal for focal release of mannosidase-II vesicles. A, mCherrry-MAN2A-expressing U937 macrophages were incubated with latex beads (30 min) or infected with PKH67-labeled H37Rv (1 h). Cells were fixed and stained with anti-NCS1 antibody followed by <t>Alexa</t> Fluor 405-labeled secondary antibody. For the upper panel, latex beads were given green pseudo-color using Imaris ( scale bar, 4 μm). B, upper panel , in U937 macrophages, incubated with beads for 30 min or 1 h, samples were stained with anti-NCS1 antibody. Presence of NCS1 at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments. For lower panel , mCherry-MAN2A ( red )-expressing U937-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-tagged secondary antibody. The images are representative of the 1-h time point. Percent co-localization of H37Rv with mannosidase-II, NCS1, or both mannosidase-II and NCS1 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.). DIC , differential interference contrast. C, co-localization of mannosidase-II and NCS1 in U937 macrophages. U937 macrophages expressing mCherry-MAN2A were stained with anti-NCS1 antibody followed by secondary antibody (pseudo-colored green ) ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. D, siRNA-mediated knockdown of NCS1 was confirmed by Western blottings on the whole cell lysates from the transfected cells. Knockdown was monitored at 48 h post-transfection. E, THP-1-derived macrophages treated with NCS1 siRNA were incubated with GFP expressing E. coli for 5 and 15 min. Cells were stained with anti-mannosidase-II antibody to assess the recruitment of mannosidase-II at the phagosomes in NCS1-depleted cells. Percent co-localization of E. coli with mannosidase-II was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.E., *, p value
    Alexa Fluor 405, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 705 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher alexa fluor 647 dye
    Characterization of VCAM-1 + macrophages in the CHT. a, Transgenic Tg(kdrl:eGFP) embryos, stained with an anti-VCAM-1 antibody (magenta, arrows), show that the VCAM-1 + macrophage first appeared in the CHT at 32 h.p.f. b, Tg(kdrl:eGFP) embryos in the vcam1 cas011 , itga4 cas005 or runx1 w84x mutant background are stained with an anti-VCAM-1 antibody (magenta, white arrows) at 54 h.p.f. Signals in itga4 cas005 and runx1 w84x are similar to that in wild-type siblings, whereas there is almost no detectable signal in vcam1 cas011 mutants. c, Schematic diagrams (left) and confocal imaging (right) of VCAM-1 + macrophages (labelled with <t>Alexa</t> <t>Fluor</t> 647 dye-conjugated anti-VCAM-1 antibody by intravascular injection) that patrol the CHT of wild-type embryos. VCAM-1 + macrophages were mainly located intravascularly ( > 91%) with round or unpolarized cell morphology ( > 84%). Cross indicates the original position of VCAM-1 + . DA, dorsal aorta; VC, venous capillaries. Scale bars, 50 μm ( a, b ) and 20 μm ( c ).
    Alexa Fluor 647 Dye, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher alexa fluor conjugated
    Characterization of VCAM-1 + macrophages in the CHT. a, Transgenic Tg(kdrl:eGFP) embryos, stained with an anti-VCAM-1 antibody (magenta, arrows), show that the VCAM-1 + macrophage first appeared in the CHT at 32 h.p.f. b, Tg(kdrl:eGFP) embryos in the vcam1 cas011 , itga4 cas005 or runx1 w84x mutant background are stained with an anti-VCAM-1 antibody (magenta, white arrows) at 54 h.p.f. Signals in itga4 cas005 and runx1 w84x are similar to that in wild-type siblings, whereas there is almost no detectable signal in vcam1 cas011 mutants. c, Schematic diagrams (left) and confocal imaging (right) of VCAM-1 + macrophages (labelled with <t>Alexa</t> <t>Fluor</t> 647 dye-conjugated anti-VCAM-1 antibody by intravascular injection) that patrol the CHT of wild-type embryos. VCAM-1 + macrophages were mainly located intravascularly ( > 91%) with round or unpolarized cell morphology ( > 84%). Cross indicates the original position of VCAM-1 + . DA, dorsal aorta; VC, venous capillaries. Scale bars, 50 μm ( a, b ) and 20 μm ( c ).
    Alexa Fluor Conjugated, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 472 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    AChR clustering in wt and ColQ-deficient muscle cells. AChR clusters in wt ( a ) and ColQ −/− ( b ) myotubes (2 d old) labeled with Alexa 488 α-BTX. Projections of confocal stacks. Scale bar, 20 μm.

    Journal: The Journal of Neuroscience

    Article Title: ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

    doi: 10.1523/JNEUROSCI.4374-09.2010

    Figure Lengend Snippet: AChR clustering in wt and ColQ-deficient muscle cells. AChR clusters in wt ( a ) and ColQ −/− ( b ) myotubes (2 d old) labeled with Alexa 488 α-BTX. Projections of confocal stacks. Scale bar, 20 μm.

    Article Snippet: α-Bungarotoxin (α-BTX) Alexa Fluor 488 or 594 conjugate, rabbit monoclonal or polyclonal anti-GFP, Alexa Fluor 488- or 594-conjugated goat anti-rabbit or anti-mouse and mouse monoclonal anti-transferrin receptor (TfR) were purchased from Invitrogen.

    Techniques: Labeling

    ColQ regulates AChR and rapsyn mRNA levels and MuSK membrane insertion at neonatal NMJs. A , Quantification of AChR subunits, MuSK, and rapsyn mRNAs on wt and ColQ −/− P7 gastrocnemius muscles by real-time RT-PCR. Levels of mRNAs are represented as relative expression (2 −Δ C t versus reference gene × 100). β-AChR subunit and rapsyn mRNA levels relative to GAPDH mRNAs were higher in ColQ −/− mice compared to wt mice whereas other AChR subunits and MuSK mRNAs were not significantly increased. B , AChR, MuSK and rapsyn clusters labeled respectively with Alexa 488 α-BTX, MuSK and rapsyn antibodies on cross-sections of STM muscle from wt and ColQ −/− P7 mice. C , D , Quantification of MuSK and rapsyn fluorescence intensities from projection of confocal stacks. MuSK fluorescence intensity per AChR cluster was decreased in the absence of ColQ whereas rapsyn was increased ( C ). STM and Sol synapses from ColQ −/− mice displayed lower MuSK-to-α-AChR fluorescence ratio but higher rapsyn-to-α-AChR ratio compared to wt synapses ( D ). Data are presented as percentage of wt control ± SEM from five mice for mRNA analysis ( A ) and from three mice for immunofluorescence analysis ( n ≥32 NMJs for C , D ). * p

    Journal: The Journal of Neuroscience

    Article Title: ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

    doi: 10.1523/JNEUROSCI.4374-09.2010

    Figure Lengend Snippet: ColQ regulates AChR and rapsyn mRNA levels and MuSK membrane insertion at neonatal NMJs. A , Quantification of AChR subunits, MuSK, and rapsyn mRNAs on wt and ColQ −/− P7 gastrocnemius muscles by real-time RT-PCR. Levels of mRNAs are represented as relative expression (2 −Δ C t versus reference gene × 100). β-AChR subunit and rapsyn mRNA levels relative to GAPDH mRNAs were higher in ColQ −/− mice compared to wt mice whereas other AChR subunits and MuSK mRNAs were not significantly increased. B , AChR, MuSK and rapsyn clusters labeled respectively with Alexa 488 α-BTX, MuSK and rapsyn antibodies on cross-sections of STM muscle from wt and ColQ −/− P7 mice. C , D , Quantification of MuSK and rapsyn fluorescence intensities from projection of confocal stacks. MuSK fluorescence intensity per AChR cluster was decreased in the absence of ColQ whereas rapsyn was increased ( C ). STM and Sol synapses from ColQ −/− mice displayed lower MuSK-to-α-AChR fluorescence ratio but higher rapsyn-to-α-AChR ratio compared to wt synapses ( D ). Data are presented as percentage of wt control ± SEM from five mice for mRNA analysis ( A ) and from three mice for immunofluorescence analysis ( n ≥32 NMJs for C , D ). * p

    Article Snippet: α-Bungarotoxin (α-BTX) Alexa Fluor 488 or 594 conjugate, rabbit monoclonal or polyclonal anti-GFP, Alexa Fluor 488- or 594-conjugated goat anti-rabbit or anti-mouse and mouse monoclonal anti-transferrin receptor (TfR) were purchased from Invitrogen.

    Techniques: Quantitative RT-PCR, Expressing, Mouse Assay, Labeling, Fluorescence, Immunofluorescence

    ColQ deficiency decreases cluster size and increases AChR packing in vivo . A , AChR clusters labeled with Alexa 488 α-BTX on cross-sections of STM ( a , b ) and Sol ( c , d ) muscles from wt ( a , c ) and ColQ −/− ( b , d ) P7 mice. B–E , Area and fluorescence intensity of individual NMJs measured from projection of confocal stacks. AChR cluster area ( B ) and AChR cluster area normalized to fiber diameter ( C ) were decreased in ColQ-deficient STM and Sol muscles. AChR cluster fluorescence intensity was increased in STM muscle but not in Sol muscle from ColQ −/− mice ( D ). The fluorescence intensity per μm 2 was significantly increased both in STM and Sol muscles from ColQ −/− mice ( E ). Data are presented as means ± SEM or percentage of wt control ± SEM ( n ≥ 32 NMJs from 3 mice per group). * p

    Journal: The Journal of Neuroscience

    Article Title: ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

    doi: 10.1523/JNEUROSCI.4374-09.2010

    Figure Lengend Snippet: ColQ deficiency decreases cluster size and increases AChR packing in vivo . A , AChR clusters labeled with Alexa 488 α-BTX on cross-sections of STM ( a , b ) and Sol ( c , d ) muscles from wt ( a , c ) and ColQ −/− ( b , d ) P7 mice. B–E , Area and fluorescence intensity of individual NMJs measured from projection of confocal stacks. AChR cluster area ( B ) and AChR cluster area normalized to fiber diameter ( C ) were decreased in ColQ-deficient STM and Sol muscles. AChR cluster fluorescence intensity was increased in STM muscle but not in Sol muscle from ColQ −/− mice ( D ). The fluorescence intensity per μm 2 was significantly increased both in STM and Sol muscles from ColQ −/− mice ( E ). Data are presented as means ± SEM or percentage of wt control ± SEM ( n ≥ 32 NMJs from 3 mice per group). * p

    Article Snippet: α-Bungarotoxin (α-BTX) Alexa Fluor 488 or 594 conjugate, rabbit monoclonal or polyclonal anti-GFP, Alexa Fluor 488- or 594-conjugated goat anti-rabbit or anti-mouse and mouse monoclonal anti-transferrin receptor (TfR) were purchased from Invitrogen.

    Techniques: In Vivo, Labeling, Mouse Assay, Fluorescence

    Restoring ColQ reverses the effects of ColQ deficiency on AChR clustering. A , AChR clusters labeled with Alexa 594 α-BTX in ColQ −/− , wt, and C2C12 myotubes overexpressing GFP-ColQ or GFP-ColQ-ΔColΔCt. ColQ −/− cells ( a , b ) as well as wt ( c , d ) and C2C12 cells ( e , f ) overexpressing GFP-ColQ (white arrows) were nearly devoid of AChR clusters, whereas clusters were observed in adjacent untransfected myotubes (white stars). ColQ −/− myotubes overexpressing GFP-ColQ-ΔColΔCt ( g , h ; white arrows) had similar number of AChR clusters compared to ColQ −/− myotubes. B , Relative frequency distribution for the number of AChR clusters per myotube (expressed as bins) observed in ColQ −/− (white bars) and ColQ −/− cells transfected with GFP-ColQ (black bars). A significant decrease in AChR cluster number was observed in cells transfected with GFP-ColQ. C , Non permeabilized ColQ −/− cells overexpressing GFP-ColQ ( a ) and stained with anti-GFP antibody ( b ), indicating that GFP-ColQ was secreted. D , Western blot of cell surface GFP from ColQ −/− cells transfected with GFP-ColQ showing that the GFP tagged ColQ was secreted. Cell surface TfR was used as a loading control. E , AChR clusters labeled with Alexa 594 α-BTX in ColQ −/− myotubes transfected with GFP-ColQ. In transfected ColQ −/− cells overexpressing GFP-ColQ, remaining AChR clusters colocalized with GFP-ColQ ( a , b and white arrows). In ColQ −/− myotubes overexpressing GFP-ColQ (white arrows), agrin treatment was able to stimulate AChR clustering ( c , d ). Data are presented as means ± SEM from three independent experiments ( n ≥36 myotubes). ** p

    Journal: The Journal of Neuroscience

    Article Title: ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

    doi: 10.1523/JNEUROSCI.4374-09.2010

    Figure Lengend Snippet: Restoring ColQ reverses the effects of ColQ deficiency on AChR clustering. A , AChR clusters labeled with Alexa 594 α-BTX in ColQ −/− , wt, and C2C12 myotubes overexpressing GFP-ColQ or GFP-ColQ-ΔColΔCt. ColQ −/− cells ( a , b ) as well as wt ( c , d ) and C2C12 cells ( e , f ) overexpressing GFP-ColQ (white arrows) were nearly devoid of AChR clusters, whereas clusters were observed in adjacent untransfected myotubes (white stars). ColQ −/− myotubes overexpressing GFP-ColQ-ΔColΔCt ( g , h ; white arrows) had similar number of AChR clusters compared to ColQ −/− myotubes. B , Relative frequency distribution for the number of AChR clusters per myotube (expressed as bins) observed in ColQ −/− (white bars) and ColQ −/− cells transfected with GFP-ColQ (black bars). A significant decrease in AChR cluster number was observed in cells transfected with GFP-ColQ. C , Non permeabilized ColQ −/− cells overexpressing GFP-ColQ ( a ) and stained with anti-GFP antibody ( b ), indicating that GFP-ColQ was secreted. D , Western blot of cell surface GFP from ColQ −/− cells transfected with GFP-ColQ showing that the GFP tagged ColQ was secreted. Cell surface TfR was used as a loading control. E , AChR clusters labeled with Alexa 594 α-BTX in ColQ −/− myotubes transfected with GFP-ColQ. In transfected ColQ −/− cells overexpressing GFP-ColQ, remaining AChR clusters colocalized with GFP-ColQ ( a , b and white arrows). In ColQ −/− myotubes overexpressing GFP-ColQ (white arrows), agrin treatment was able to stimulate AChR clustering ( c , d ). Data are presented as means ± SEM from three independent experiments ( n ≥36 myotubes). ** p

    Article Snippet: α-Bungarotoxin (α-BTX) Alexa Fluor 488 or 594 conjugate, rabbit monoclonal or polyclonal anti-GFP, Alexa Fluor 488- or 594-conjugated goat anti-rabbit or anti-mouse and mouse monoclonal anti-transferrin receptor (TfR) were purchased from Invitrogen.

    Techniques: Labeling, Transfection, Staining, Western Blot

    ColQ deficiency decreases membrane-bound MuSK in vitro and alters the activity of MuSK signaling pathway. A , Western blot of total transferrin receptor proteins (TfR) in wt and ColQ −/− myotubes. GAPDH was used as a loading control. Total TfR protein level relative to GAPDH was not significantly different between wt and ColQ −/− myotubes. B , Western blot of membrane-bound MuSK and α-AChR subunit in wt and ColQ −/− myotubes. Membrane-bound proteins were labeled with NHS-biotin and isolated with streptavidin. TfR was used as a loading control. MuSK surface protein level relative to TfR was decreased by 35% in the absence of ColQ. Conversely, loss of ColQ resulted in a 178% increase of α-AChR subunit surface protein level relative to TfR. C , Fluorescence analyses of AChR cluster number in wt and ColQ −/− myotubes treated with agrin and/or transfected with GFP-ColQ. Compared to untransfected cells (white bars), AChR cluster number was drastically decreased both in wt and ColQ −/− cells overexpressing GFP-ColQ (light gray bars). Agrin treatment had less effect on the number of AChR clusters in ColQ −/− cells compared to wt cells (dark gray bars), whereas GFP-ColQ transfection restored agrin capacity to induce normal AChR clustering (black bars). D , Western blot (up panel) and quantification (bottom panel) of membrane-bound β-AChR subunit and β-AChR subunit phosphorylated on Y390 residue (pβ-AChR). Agrin treatment increased β-AChR subunit phosphorylation in both cell lines but was threefold less efficient in ColQ −/− cells as shown by the pβ-AChR-to-β-AChR ratio. E , AChR clusters labeled with Alexa 488 α-BTX and pAChRβ1 antibody on cross-sections of STM muscle from wt and ColQ −/− P7 mice (left panel). Quantification of pβ-AChR fluorescence intensity from projection of confocal stacks showed that STM and Sol synapses from ColQ −/− mice displayed lower pβ-AChR-to-α-AChR fluorescence ratio than muscles from wt mice (right panel). Data are presented as means ± SEM or percentage of wt or ColQ −/− control ± SEM from three to five independent experiments ( n ≥ 35 NMJs for E ). * p

    Journal: The Journal of Neuroscience

    Article Title: ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

    doi: 10.1523/JNEUROSCI.4374-09.2010

    Figure Lengend Snippet: ColQ deficiency decreases membrane-bound MuSK in vitro and alters the activity of MuSK signaling pathway. A , Western blot of total transferrin receptor proteins (TfR) in wt and ColQ −/− myotubes. GAPDH was used as a loading control. Total TfR protein level relative to GAPDH was not significantly different between wt and ColQ −/− myotubes. B , Western blot of membrane-bound MuSK and α-AChR subunit in wt and ColQ −/− myotubes. Membrane-bound proteins were labeled with NHS-biotin and isolated with streptavidin. TfR was used as a loading control. MuSK surface protein level relative to TfR was decreased by 35% in the absence of ColQ. Conversely, loss of ColQ resulted in a 178% increase of α-AChR subunit surface protein level relative to TfR. C , Fluorescence analyses of AChR cluster number in wt and ColQ −/− myotubes treated with agrin and/or transfected with GFP-ColQ. Compared to untransfected cells (white bars), AChR cluster number was drastically decreased both in wt and ColQ −/− cells overexpressing GFP-ColQ (light gray bars). Agrin treatment had less effect on the number of AChR clusters in ColQ −/− cells compared to wt cells (dark gray bars), whereas GFP-ColQ transfection restored agrin capacity to induce normal AChR clustering (black bars). D , Western blot (up panel) and quantification (bottom panel) of membrane-bound β-AChR subunit and β-AChR subunit phosphorylated on Y390 residue (pβ-AChR). Agrin treatment increased β-AChR subunit phosphorylation in both cell lines but was threefold less efficient in ColQ −/− cells as shown by the pβ-AChR-to-β-AChR ratio. E , AChR clusters labeled with Alexa 488 α-BTX and pAChRβ1 antibody on cross-sections of STM muscle from wt and ColQ −/− P7 mice (left panel). Quantification of pβ-AChR fluorescence intensity from projection of confocal stacks showed that STM and Sol synapses from ColQ −/− mice displayed lower pβ-AChR-to-α-AChR fluorescence ratio than muscles from wt mice (right panel). Data are presented as means ± SEM or percentage of wt or ColQ −/− control ± SEM from three to five independent experiments ( n ≥ 35 NMJs for E ). * p

    Article Snippet: α-Bungarotoxin (α-BTX) Alexa Fluor 488 or 594 conjugate, rabbit monoclonal or polyclonal anti-GFP, Alexa Fluor 488- or 594-conjugated goat anti-rabbit or anti-mouse and mouse monoclonal anti-transferrin receptor (TfR) were purchased from Invitrogen.

    Techniques: In Vitro, Activity Assay, Western Blot, Labeling, Isolation, Fluorescence, Transfection, Mouse Assay

    NDRG1 directly interacts with PCNA. (A)Schematic procedure for purification and identification of NDRG1 binding proteins via TAP assay (left). Plasmid expressing Strep-Flag-tagged NDRG1 was stable transfected into KSHV positive iSLK.RGB cells. The equivalent empty vector was stable transfected as a control. Cell lysates were subjected to affinity purification with streptavidin beads, followed by IP with flag M2 beads. The purified elutes were resolved by SDS-PAGE and visualized with silver staining (right), and were also analyzed by MS. (B) Pathway pie chart showing classified and predicted functions of MS identified proteins. (C) Co-IP of NDRG1 and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-NDRG1 and anti-PCNA antibodies. (D) In vitro interaction between NDRG1 and PCNA via GST pull down assay. Purified GST, and GST-fused full length NDRG1 were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (E) NDRG1 colocalized with PCNA in the nucleus. Cells were fixed and probed with rabbit antibody against NDRG1 and mouse antibody against PCNA, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 555 (red), goat anti-mouse IgG conjugated with Alexa Fluor 488 (green), DAPI (blue). Scale bars represent 5μm.

    Journal: PLoS Pathogens

    Article Title: NDRG1 facilitates the replication and persistence of Kaposi’s sarcoma-associated herpesvirus by interacting with the DNA polymerase clamp PCNA

    doi: 10.1371/journal.ppat.1007628

    Figure Lengend Snippet: NDRG1 directly interacts with PCNA. (A)Schematic procedure for purification and identification of NDRG1 binding proteins via TAP assay (left). Plasmid expressing Strep-Flag-tagged NDRG1 was stable transfected into KSHV positive iSLK.RGB cells. The equivalent empty vector was stable transfected as a control. Cell lysates were subjected to affinity purification with streptavidin beads, followed by IP with flag M2 beads. The purified elutes were resolved by SDS-PAGE and visualized with silver staining (right), and were also analyzed by MS. (B) Pathway pie chart showing classified and predicted functions of MS identified proteins. (C) Co-IP of NDRG1 and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-NDRG1 and anti-PCNA antibodies. (D) In vitro interaction between NDRG1 and PCNA via GST pull down assay. Purified GST, and GST-fused full length NDRG1 were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (E) NDRG1 colocalized with PCNA in the nucleus. Cells were fixed and probed with rabbit antibody against NDRG1 and mouse antibody against PCNA, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 555 (red), goat anti-mouse IgG conjugated with Alexa Fluor 488 (green), DAPI (blue). Scale bars represent 5μm.

    Article Snippet: The secondary antibodies used for western blotting and the IF assay were goat anti-mouse IRDye 800CW (Li-Cor, 926–32210); goat anti-rabbit IRDye 680RD (Li-Cor, 926–68071); goat anti-rabbit antibodies conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, A-11094), 555 (Thermo Fisher Scientific, A27017), and 680 (Thermo Fisher Scientific, A27020); goat anti-mouse antibodies conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, A-11001) and 555 (Thermo Fisher Scientific, A-21422); and goat anti-rat antibodies conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, A-11006).

    Techniques: Purification, Binding Assay, Plasmid Preparation, Expressing, Transfection, Affinity Purification, SDS Page, Silver Staining, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, In Vitro, Pull Down Assay, Staining, Incubation

    NDRG1 forms a complex with LANA and PCNA. (A) LANA colocalized with NDRG1 and PCNA in the nucleus. Cells were fixed and probed with rat antibody against LANA, mouse antibody against PCNA, and rabbit antibody against NDRG1, followed by incubation with goat anti-rat IgG conjugated with Alexa Fluor 488 (green), goat anti-mouse IgG conjugated with Alexa Fluor 555 (red), goat anti-rabbit IgG conjugated with Alexa Fluor 680 (purple). Scale bars represent 5μm. (B) Co-IP of endogenous LANA, NDRG1, and PCNA in BCBL1 cells. Cell lysates were subjected to IP with anti-LANA mouse monoclonal antibody(1B5) or mouse IgG controls. Purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1, and anti-PCNA antibodies. (C) Co-IP of LANA, NDRG1, and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA and HA-LANA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1 and anti-PCNA antibodies. (D) Co-IP of LANA, NDRG1, and PCNA in HEK293T-Strep-Flag-LANA cells. pCDNA3.1-NDRG1 or pCDNA3.1-vector was transfected into cells. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1 and anti-PCNA antibodies. Tubulin was performed as the loading control of input samples, and IgG was used as the loading control of M2-Flag beads. (E) In vitro interaction between LANA and PCNA via GST pull down assay. Purified GST, and GST-fused LANA-N (1–340 aa) and GST-fused LANA-C (1022–1162 aa) beads were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (F) In vitro interaction between LANA and NDRG1 via GST pull down assay. Purified GST, and GST-fused LANA-N and GST-fused LANA-C beads were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent IVT–NDRG1, and pulled down proteins were subjected to western blotting detection (upper panel). (G) In vitro interaction between LANA and PCNA in the presence of NDRG1 via GST pull down assay. Purified GST, and GST-fused LANA-N and GST-fused LANA-C beads were incubated with equivalent IVT-PCNA and IVT–NDRG1, and pulled down proteins were subjected to western blotting detection.

    Journal: PLoS Pathogens

    Article Title: NDRG1 facilitates the replication and persistence of Kaposi’s sarcoma-associated herpesvirus by interacting with the DNA polymerase clamp PCNA

    doi: 10.1371/journal.ppat.1007628

    Figure Lengend Snippet: NDRG1 forms a complex with LANA and PCNA. (A) LANA colocalized with NDRG1 and PCNA in the nucleus. Cells were fixed and probed with rat antibody against LANA, mouse antibody against PCNA, and rabbit antibody against NDRG1, followed by incubation with goat anti-rat IgG conjugated with Alexa Fluor 488 (green), goat anti-mouse IgG conjugated with Alexa Fluor 555 (red), goat anti-rabbit IgG conjugated with Alexa Fluor 680 (purple). Scale bars represent 5μm. (B) Co-IP of endogenous LANA, NDRG1, and PCNA in BCBL1 cells. Cell lysates were subjected to IP with anti-LANA mouse monoclonal antibody(1B5) or mouse IgG controls. Purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1, and anti-PCNA antibodies. (C) Co-IP of LANA, NDRG1, and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA and HA-LANA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1 and anti-PCNA antibodies. (D) Co-IP of LANA, NDRG1, and PCNA in HEK293T-Strep-Flag-LANA cells. pCDNA3.1-NDRG1 or pCDNA3.1-vector was transfected into cells. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1 and anti-PCNA antibodies. Tubulin was performed as the loading control of input samples, and IgG was used as the loading control of M2-Flag beads. (E) In vitro interaction between LANA and PCNA via GST pull down assay. Purified GST, and GST-fused LANA-N (1–340 aa) and GST-fused LANA-C (1022–1162 aa) beads were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (F) In vitro interaction between LANA and NDRG1 via GST pull down assay. Purified GST, and GST-fused LANA-N and GST-fused LANA-C beads were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent IVT–NDRG1, and pulled down proteins were subjected to western blotting detection (upper panel). (G) In vitro interaction between LANA and PCNA in the presence of NDRG1 via GST pull down assay. Purified GST, and GST-fused LANA-N and GST-fused LANA-C beads were incubated with equivalent IVT-PCNA and IVT–NDRG1, and pulled down proteins were subjected to western blotting detection.

    Article Snippet: The secondary antibodies used for western blotting and the IF assay were goat anti-mouse IRDye 800CW (Li-Cor, 926–32210); goat anti-rabbit IRDye 680RD (Li-Cor, 926–68071); goat anti-rabbit antibodies conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, A-11094), 555 (Thermo Fisher Scientific, A27017), and 680 (Thermo Fisher Scientific, A27020); goat anti-mouse antibodies conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, A-11001) and 555 (Thermo Fisher Scientific, A-21422); and goat anti-rat antibodies conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, A-11006).

    Techniques: Incubation, Co-Immunoprecipitation Assay, Purification, Western Blot, Transfection, Plasmid Preparation, Affinity Purification, In Vitro, Pull Down Assay, SDS Page, Staining

    Reelin does not affect cell morphology and cytoskeletal content. Patient and control cells were fixed in 4% paraformaldehyde (PFA) stained for cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) and cytoplasm with CellMask Deep Red stain. Specific probes were used to visualize cytoskeletal proteins in actin and microtubules. Images were captured on Operetta High Content Imaging System (PerkinElmer, Llantrisant, UK) and analyzed by Harmony software. Age-matched patient cell lines ( N =9) were compared with control cell lines ( N =9). For all individual cell lines, 120 single cells were quantified and grouped into subsequent disease or non-diseased dataset. ( a ) “Find nuclei” algorithm was used to locate all cell nuclei stained with DAPI to identify all cells within the imaged field of views. ( b ) “Select population” algorithm was used to filter for cells that are fully within the field of view (green highlight) and omit cells partially in the field (red highlight). ( c ) Representative image of cells within a random field of view co-stained with DAPI (blue; nuclei), CellMask (red; cell cytoplasm), Alexa Fluor 488-conjugated phalloidin (green; filamentous actin), and anti-acetylated α-tubulin antibody followed by Alexa Fluor 546 secondary antibody (yellow; stable microtubules). ( d ) CellMask staining allows measurement of area covered by cell cytoplasm and cell roundness. ( e ) Filamentous actin expression levels were quantified based on mean fluorescence intensity of Alexa Fluor 488 conjugated to phalloidin within cell cytoplasm stained with CellMask. ( f ) Microtubule stability was measured based on expression levels of acetylated α-tubulin within cell cytoplasm stained with CellMask. ( g ) Quantitation of cell area, which correlates to the size of the cell. ( h ) Cell roundness was quantified as a relative roundness index where an index of “1” represents a perfect circle and “0” represents a straight line. ( i ) Actin expression graphs measured as Alexa Fluor 488 fluorescence intensities. ( j ) Stable microtubule expression measured as Alexa Fluor 546 fluorescence intensities. All plotted data represents mean of all cells measured per group±s.e.m. Open bars represent control cell dataset; closed gray bars represent patient cell dataset. Scale bar=50 μm. * P

    Journal: NPJ Schizophrenia

    Article Title: Schizophrenia patient-derived olfactory neurosphere-derived cells do not respond to extracellular reelin

    doi: 10.1038/npjschz.2016.27

    Figure Lengend Snippet: Reelin does not affect cell morphology and cytoskeletal content. Patient and control cells were fixed in 4% paraformaldehyde (PFA) stained for cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) and cytoplasm with CellMask Deep Red stain. Specific probes were used to visualize cytoskeletal proteins in actin and microtubules. Images were captured on Operetta High Content Imaging System (PerkinElmer, Llantrisant, UK) and analyzed by Harmony software. Age-matched patient cell lines ( N =9) were compared with control cell lines ( N =9). For all individual cell lines, 120 single cells were quantified and grouped into subsequent disease or non-diseased dataset. ( a ) “Find nuclei” algorithm was used to locate all cell nuclei stained with DAPI to identify all cells within the imaged field of views. ( b ) “Select population” algorithm was used to filter for cells that are fully within the field of view (green highlight) and omit cells partially in the field (red highlight). ( c ) Representative image of cells within a random field of view co-stained with DAPI (blue; nuclei), CellMask (red; cell cytoplasm), Alexa Fluor 488-conjugated phalloidin (green; filamentous actin), and anti-acetylated α-tubulin antibody followed by Alexa Fluor 546 secondary antibody (yellow; stable microtubules). ( d ) CellMask staining allows measurement of area covered by cell cytoplasm and cell roundness. ( e ) Filamentous actin expression levels were quantified based on mean fluorescence intensity of Alexa Fluor 488 conjugated to phalloidin within cell cytoplasm stained with CellMask. ( f ) Microtubule stability was measured based on expression levels of acetylated α-tubulin within cell cytoplasm stained with CellMask. ( g ) Quantitation of cell area, which correlates to the size of the cell. ( h ) Cell roundness was quantified as a relative roundness index where an index of “1” represents a perfect circle and “0” represents a straight line. ( i ) Actin expression graphs measured as Alexa Fluor 488 fluorescence intensities. ( j ) Stable microtubule expression measured as Alexa Fluor 546 fluorescence intensities. All plotted data represents mean of all cells measured per group±s.e.m. Open bars represent control cell dataset; closed gray bars represent patient cell dataset. Scale bar=50 μm. * P

    Article Snippet: Fluorophore-conjugated secondary antibodies used were: Alexa Fluor 546 (1:400; Life Technologies; for acetylated α-tubulin) and Alexa Fluor 488 (1:400; Life Technologies; for phospho-FAK Tyr397).

    Techniques: Staining, Imaging, Software, Expressing, Fluorescence, Quantitation Assay

    Double-labeling of the CNS of third instar larvae, using staining with a mouse monoclonal antibody against Drosophila synapsin (red fluorescence) and a rabbit antiserum against CCHamide-2 (green fluorescence). A. Dorsal view of the ventral plane of the CNS, showing the central neuropil in each hemisphere and in the anterior part of the ventral nerve cord. The excitation and emission wavelengths used were selective for Alexa Fluor 594 fluorescence (red). B. The same focal plane as in A, but now using excitation and emission wavelengths selective for Alexa Fluor 488 fluorescence (green). C. Merge of A and B. The CCHamide-2 immunoreactive neuropil is located in the anterior part of the central neuropil in each hemisphere. The arrow points to a group of CCHamide-2 immunoreactive perikarya (somewhat out of focus) belonging to the group 4 perikarya of Figure 5 . Scale bar = 20 µm.

    Journal: PLoS ONE

    Article Title: Expression Patterns of the Drosophila Neuropeptide CCHamide-2 and Its Receptor May Suggest Hormonal Signaling from the Gut to the Brain

    doi: 10.1371/journal.pone.0076131

    Figure Lengend Snippet: Double-labeling of the CNS of third instar larvae, using staining with a mouse monoclonal antibody against Drosophila synapsin (red fluorescence) and a rabbit antiserum against CCHamide-2 (green fluorescence). A. Dorsal view of the ventral plane of the CNS, showing the central neuropil in each hemisphere and in the anterior part of the ventral nerve cord. The excitation and emission wavelengths used were selective for Alexa Fluor 594 fluorescence (red). B. The same focal plane as in A, but now using excitation and emission wavelengths selective for Alexa Fluor 488 fluorescence (green). C. Merge of A and B. The CCHamide-2 immunoreactive neuropil is located in the anterior part of the central neuropil in each hemisphere. The arrow points to a group of CCHamide-2 immunoreactive perikarya (somewhat out of focus) belonging to the group 4 perikarya of Figure 5 . Scale bar = 20 µm.

    Article Snippet: After the incubation and washing procedures as described above, the specimen were incubated with a mixture of donkey anti-rabbit IgG coupled to Alexa Fluor 488 diluted 1:1500 as described above and goat anti-mouse IgG coupled to Alexa Fluor 594 (Invitrogen) diluted 1:1000.

    Techniques: Labeling, Staining, Fluorescence

    DENV does not induce the SREBP-2 pathway or upreglation of HMGCR early after infection. 2fTGH cells were mock-treated (A) or treated with DTT (B) for 6 h. Cells were then fixed and stained intracellularly for cellular proteins using antibodies against SREBP-2 (green) and HMGCR (red) followed by secondary antibodies conjugated to Alexa Fluor® 488 and Alexa Fluor® 546, respectively. 2fTGH cells were infected with DENV-2 for 12 h and stained intracellularly for (C) SREBP-2 (magenta) and (D) HMGCR (magenta) followed by secondary antibodies conjugated to Alexa Fluor® 647; mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively, were used to detect DENV proteins. Nuclear staining (blue) was performed using DAPI stain. The multiple-exposure images were captured using a 40×/1.4 Plan-Apochromat DIC oil objective on a Zeiss 710 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 and were processed as previously described in Figure 1 . (E) SREBP-2 proteolyic processing by immunoblot analysis. Actin was used as a loading control. In (F), 2fTGH cells were infected with DENV-2 over a 3–12 h time-course. Lysates were collected and analyzed for SREBP-2 and HMGCR expression by immunoblot analysis. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection. A time-matched mock-infected control was included at each time-point. Total magnification 600×.

    Journal: PLoS ONE

    Article Title: Early Dengue Virus Protein Synthesis Induces Extensive Rearrangement of the Endoplasmic Reticulum Independent of the UPR and SREBP-2 Pathway

    doi: 10.1371/journal.pone.0038202

    Figure Lengend Snippet: DENV does not induce the SREBP-2 pathway or upreglation of HMGCR early after infection. 2fTGH cells were mock-treated (A) or treated with DTT (B) for 6 h. Cells were then fixed and stained intracellularly for cellular proteins using antibodies against SREBP-2 (green) and HMGCR (red) followed by secondary antibodies conjugated to Alexa Fluor® 488 and Alexa Fluor® 546, respectively. 2fTGH cells were infected with DENV-2 for 12 h and stained intracellularly for (C) SREBP-2 (magenta) and (D) HMGCR (magenta) followed by secondary antibodies conjugated to Alexa Fluor® 647; mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively, were used to detect DENV proteins. Nuclear staining (blue) was performed using DAPI stain. The multiple-exposure images were captured using a 40×/1.4 Plan-Apochromat DIC oil objective on a Zeiss 710 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 and were processed as previously described in Figure 1 . (E) SREBP-2 proteolyic processing by immunoblot analysis. Actin was used as a loading control. In (F), 2fTGH cells were infected with DENV-2 over a 3–12 h time-course. Lysates were collected and analyzed for SREBP-2 and HMGCR expression by immunoblot analysis. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection. A time-matched mock-infected control was included at each time-point. Total magnification 600×.

    Article Snippet: Alexa Fluor™ 488 (goat anti-rabbit), Alexa Fluor™ 546 (donkey anti-goat), and Alexa Fluor™ 647 (goat anti-rabbit) were diluted 1∶200 (Molecular Probes, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Infection, Staining, Expressing

    DENV-induced ER rearrangement and expansion is independent of the ATF6 pathway. ATF6 +/+ (A–C) and ATF6 −/− (D–F) MEF cells were infected with DENV-2 for 12 h and intracellularly stained for cellular proteins (A and D) GRP78, (B and E) GRP94, and (C and F) XBP1 (magenta), followed by secondary antibodies conjugated to Alexa Fluor® 647. DENV proteins were detected with mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Images were acquired and processed as described in Fig. 1. Total Magnification 630×.

    Journal: PLoS ONE

    Article Title: Early Dengue Virus Protein Synthesis Induces Extensive Rearrangement of the Endoplasmic Reticulum Independent of the UPR and SREBP-2 Pathway

    doi: 10.1371/journal.pone.0038202

    Figure Lengend Snippet: DENV-induced ER rearrangement and expansion is independent of the ATF6 pathway. ATF6 +/+ (A–C) and ATF6 −/− (D–F) MEF cells were infected with DENV-2 for 12 h and intracellularly stained for cellular proteins (A and D) GRP78, (B and E) GRP94, and (C and F) XBP1 (magenta), followed by secondary antibodies conjugated to Alexa Fluor® 647. DENV proteins were detected with mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Images were acquired and processed as described in Fig. 1. Total Magnification 630×.

    Article Snippet: Alexa Fluor™ 488 (goat anti-rabbit), Alexa Fluor™ 546 (donkey anti-goat), and Alexa Fluor™ 647 (goat anti-rabbit) were diluted 1∶200 (Molecular Probes, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Infection, Staining

    DENV induces rearrangement of the ER early after infection. 2fTGH cells were (A) mock-infected or (B–E) infected with DENV-2 for 12 h and stained intracellularly for UPR markers and viral proteins E and NS3. (A) Mock-infected 2fTGH cells were stained with mouse MAbs against DENV E (4G2; green) and NS3 (E1D8; red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Intracellular staining of DENV-infected cells was carried out as in (A); in addition primary antibodies specific for cellular proteins (B) GRP78, (C) GRP94, (D) XBP1 and (E) ATF6 (magenta) were used, followed by secondary antibodies conjugated to Alexa Fluor® 647. The multiple-exposure images were captured using 63X/1.25 Plan-Neofluar DIC oil objective on Zeiss 510 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647. For individual images, the additional channels were turned OFF post-acquisition using the Zeiss operating software. Co-localization is observed in the “Merged” image as a yellow hue. Merged and DIC images represent a single image with all detector channels ON. Total magnification 630×. (F) Western blot analysis of GRP78 over a 12-h course of DENV infection. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection.

    Journal: PLoS ONE

    Article Title: Early Dengue Virus Protein Synthesis Induces Extensive Rearrangement of the Endoplasmic Reticulum Independent of the UPR and SREBP-2 Pathway

    doi: 10.1371/journal.pone.0038202

    Figure Lengend Snippet: DENV induces rearrangement of the ER early after infection. 2fTGH cells were (A) mock-infected or (B–E) infected with DENV-2 for 12 h and stained intracellularly for UPR markers and viral proteins E and NS3. (A) Mock-infected 2fTGH cells were stained with mouse MAbs against DENV E (4G2; green) and NS3 (E1D8; red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Intracellular staining of DENV-infected cells was carried out as in (A); in addition primary antibodies specific for cellular proteins (B) GRP78, (C) GRP94, (D) XBP1 and (E) ATF6 (magenta) were used, followed by secondary antibodies conjugated to Alexa Fluor® 647. The multiple-exposure images were captured using 63X/1.25 Plan-Neofluar DIC oil objective on Zeiss 510 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647. For individual images, the additional channels were turned OFF post-acquisition using the Zeiss operating software. Co-localization is observed in the “Merged” image as a yellow hue. Merged and DIC images represent a single image with all detector channels ON. Total magnification 630×. (F) Western blot analysis of GRP78 over a 12-h course of DENV infection. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection.

    Article Snippet: Alexa Fluor™ 488 (goat anti-rabbit), Alexa Fluor™ 546 (donkey anti-goat), and Alexa Fluor™ 647 (goat anti-rabbit) were diluted 1∶200 (Molecular Probes, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Infection, Staining, Software, Western Blot, Expressing

    DENV-induced ER rearrangement and expansion is independent of the XBP-1 pathway. XBP1 +/+ (A–D) and XBP1 −/− (E–H) MEF cells were infected with DENV-2 for 12 h and stained intracellularly for cellular proteins using antibodies against (A and E) GRP78, (B and F) GRP94, (C) XBP1, and (G) ATF6 (magenta), followed by secondary antibodies conjugated to Alexa Fluor® 647. DENV proteins were detected with mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Images were acquired and processed as described in Fig. 1. Total magnification 630×.

    Journal: PLoS ONE

    Article Title: Early Dengue Virus Protein Synthesis Induces Extensive Rearrangement of the Endoplasmic Reticulum Independent of the UPR and SREBP-2 Pathway

    doi: 10.1371/journal.pone.0038202

    Figure Lengend Snippet: DENV-induced ER rearrangement and expansion is independent of the XBP-1 pathway. XBP1 +/+ (A–D) and XBP1 −/− (E–H) MEF cells were infected with DENV-2 for 12 h and stained intracellularly for cellular proteins using antibodies against (A and E) GRP78, (B and F) GRP94, (C) XBP1, and (G) ATF6 (magenta), followed by secondary antibodies conjugated to Alexa Fluor® 647. DENV proteins were detected with mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Images were acquired and processed as described in Fig. 1. Total magnification 630×.

    Article Snippet: Alexa Fluor™ 488 (goat anti-rabbit), Alexa Fluor™ 546 (donkey anti-goat), and Alexa Fluor™ 647 (goat anti-rabbit) were diluted 1∶200 (Molecular Probes, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Infection, Staining

    Pulse labelling of nascent DNA in E. coli cells with EdU. ( a ) Pulse labelling experiment in a fast-growing E. coli batch culture (LB medium, 32 °C). Growth curves of cultures with and without EdU are shown in the upper left panel. Culture aliquots were fixed after 2, 3.5 and 5 min and labelled with Alexa Fluor 647 via click-chemistry. Parallel multi-colour imaging was performed as described in the Methods section. Nucleoids were probed using JF 503 ). Increasing EdU incubation time leads to an increase of nucleoid coverage (ratio of the area populated by pulse-labelled DNA and the entire nucleoid) (upper right panel). The lower panel shows representative cells for the indicated EdU pulse durations, labelled for nascent DNA (red hot), the entire nucleoid (cyan hot) and the membrane (green). Outlines were determined in the membrane PAINT image and are shown by dashed lines in the DNA d STORM and PAINT images. ( b ) Pulse labelling during metabolic arrest. MG1655 cells were grown in LB medium at 32 °C. Growth curves of cultures with and without SHX are shown in the upper left panel. The treated culture was split into 3 cultures and EdU was added for the last 30 min of SHX treatment, finally resulting in cells arrested for 30 min, 60 min and 90 min. Parallel multi-colour imaging was performed as described in the Methods section. The nucleoid coverage of nascent DNA decreases with the time of SHX treatment (upper right panel). The lower panel shows representative cells for the different time points during metabolic arrest, the colour-coding is kept identical to ( a ). Bars represent mean values and error bars the respecting standard deviation (scale bars: 1 µm).

    Journal: Scientific Reports

    Article Title: A toolbox for multiplexed super-resolution imaging of the E. coli nucleoid and membrane using novel PAINT labels

    doi: 10.1038/s41598-018-33052-3

    Figure Lengend Snippet: Pulse labelling of nascent DNA in E. coli cells with EdU. ( a ) Pulse labelling experiment in a fast-growing E. coli batch culture (LB medium, 32 °C). Growth curves of cultures with and without EdU are shown in the upper left panel. Culture aliquots were fixed after 2, 3.5 and 5 min and labelled with Alexa Fluor 647 via click-chemistry. Parallel multi-colour imaging was performed as described in the Methods section. Nucleoids were probed using JF 503 ). Increasing EdU incubation time leads to an increase of nucleoid coverage (ratio of the area populated by pulse-labelled DNA and the entire nucleoid) (upper right panel). The lower panel shows representative cells for the indicated EdU pulse durations, labelled for nascent DNA (red hot), the entire nucleoid (cyan hot) and the membrane (green). Outlines were determined in the membrane PAINT image and are shown by dashed lines in the DNA d STORM and PAINT images. ( b ) Pulse labelling during metabolic arrest. MG1655 cells were grown in LB medium at 32 °C. Growth curves of cultures with and without SHX are shown in the upper left panel. The treated culture was split into 3 cultures and EdU was added for the last 30 min of SHX treatment, finally resulting in cells arrested for 30 min, 60 min and 90 min. Parallel multi-colour imaging was performed as described in the Methods section. The nucleoid coverage of nascent DNA decreases with the time of SHX treatment (upper right panel). The lower panel shows representative cells for the different time points during metabolic arrest, the colour-coding is kept identical to ( a ). Bars represent mean values and error bars the respecting standard deviation (scale bars: 1 µm).

    Article Snippet: The DNA of the EdU treated bacteria was labelled with Alexa Fluor 647 azide (Thermo Fisher Scientific) via click chemistry as described previously .

    Techniques: Imaging, Incubation, Standard Deviation

    Annexin-V/Alexa Fluor™ 647 and Propidium iodide stained U87-MG cells post treatment with free and nano GSK (5 µM). Population of cells that are in early apoptosis, late apoptosis and necrosis at 24, 48 and 72 h are mentioned. The first row indicates cells that received no treatment.

    Journal: Bioengineering

    Article Title: GSK461364A, a Polo-Like Kinase-1 Inhibitor Encapsulated in Polymeric Nanoparticles for the Treatment of Glioblastoma Multiforme (GBM)

    doi: 10.3390/bioengineering5040083

    Figure Lengend Snippet: Annexin-V/Alexa Fluor™ 647 and Propidium iodide stained U87-MG cells post treatment with free and nano GSK (5 µM). Population of cells that are in early apoptosis, late apoptosis and necrosis at 24, 48 and 72 h are mentioned. The first row indicates cells that received no treatment.

    Article Snippet: Annexin-V, Alexa Fluor™ 647 conjugate was purchased from Invitrogen™.

    Techniques: Staining

    Seldegs increase the accumulation of antigen-specific antibodies in human endothelial (HMEC-1) cells expressing FcRn-GFP. ( a ) HMEC-1 cells were incubated with 100 nM Alexa 647-labelled 8-18C5 (MOG-specific) or TZB (HER2-specific) in complex with 400 nM MOG-Seldeg/MOG-WT or HER2-Seldeg/HER2-WT for 30 min and chased for 0 (30' P) or 60 min (30' P, 60' C). Mean fluorescence intensities (MFI) of Alexa 647-labelled 8-18C5 or TZB for triplicate samples were determined by flow cytometry. Error bars indicate s.d. ( b , c ) HMEC-1 cells were plated on coverslips and treated as in a , except that Seldegs or control WT proteins were labelled with Alexa 555 and cells were fixed for microscopy. Images of representative cells from multiple cells analysed are shown with GFP, Alexa 555 and Alexa 647 in overlays pseudocoloured green, red and blue, respectively. Representative endosomes in the insets are cropped and expanded. ( d ) HMEC-1 cells were pre-pulsed with Alexa 555-labelled dextran for 2 h, washed and pulsed with 8-18C5 in complex with MOG-Seldeg, MOG-WT and HER2-Seldeg (concentrations and labels as for a ) for 30 min, followed by an 8 h chase. Cells were washed, fixed and imaged, and images for a representative cell from multiple cells analysed are presented. Representative lysosomes in the insets are cropped and expanded. For the overlay, GFP, Alexa 555 and Alexa 647 are pseudocoloured as in b . For b – d , scale bars=5 μm, and for insets, scale bars=0.25 μm. Data shown are representative of at least two independent experiments.

    Journal: Nature Communications

    Article Title: Engineered clearing agents for the selective depletion of antigen-specific antibodies

    doi: 10.1038/ncomms15314

    Figure Lengend Snippet: Seldegs increase the accumulation of antigen-specific antibodies in human endothelial (HMEC-1) cells expressing FcRn-GFP. ( a ) HMEC-1 cells were incubated with 100 nM Alexa 647-labelled 8-18C5 (MOG-specific) or TZB (HER2-specific) in complex with 400 nM MOG-Seldeg/MOG-WT or HER2-Seldeg/HER2-WT for 30 min and chased for 0 (30' P) or 60 min (30' P, 60' C). Mean fluorescence intensities (MFI) of Alexa 647-labelled 8-18C5 or TZB for triplicate samples were determined by flow cytometry. Error bars indicate s.d. ( b , c ) HMEC-1 cells were plated on coverslips and treated as in a , except that Seldegs or control WT proteins were labelled with Alexa 555 and cells were fixed for microscopy. Images of representative cells from multiple cells analysed are shown with GFP, Alexa 555 and Alexa 647 in overlays pseudocoloured green, red and blue, respectively. Representative endosomes in the insets are cropped and expanded. ( d ) HMEC-1 cells were pre-pulsed with Alexa 555-labelled dextran for 2 h, washed and pulsed with 8-18C5 in complex with MOG-Seldeg, MOG-WT and HER2-Seldeg (concentrations and labels as for a ) for 30 min, followed by an 8 h chase. Cells were washed, fixed and imaged, and images for a representative cell from multiple cells analysed are presented. Representative lysosomes in the insets are cropped and expanded. For the overlay, GFP, Alexa 555 and Alexa 647 are pseudocoloured as in b . For b – d , scale bars=5 μm, and for insets, scale bars=0.25 μm. Data shown are representative of at least two independent experiments.

    Article Snippet: 8-18C5 and TZB were fluorescently labelled with Alexa 647 Fluor (ThermoFisher Scientific) with antibody:dye ratios of 1.6 and 3, respectively.

    Techniques: Expressing, Incubation, Fluorescence, Flow Cytometry, Cytometry, Microscopy

    Fluorescent labeling of a natural RNA sample. A total RNA isolate from E. coli was reacted with 2′-N 3 -2′-dUTP and yeast PAP, and further subjected to CuAAC with Alexa Fluor 647 alkyne. Analysis by 8% denaturing PAGE. SYBR Gold scan (left panel), Alexa Fluor 647 scan (middle panel) and overlay (green: radioactivity; magenta: fluorescence; white: both; right panel) are given. The two main bands represent 23S and 16S ribosomal RNA.

    Journal: Nucleic Acids Research

    Article Title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

    doi: 10.1093/nar/gks062

    Figure Lengend Snippet: Fluorescent labeling of a natural RNA sample. A total RNA isolate from E. coli was reacted with 2′-N 3 -2′-dUTP and yeast PAP, and further subjected to CuAAC with Alexa Fluor 647 alkyne. Analysis by 8% denaturing PAGE. SYBR Gold scan (left panel), Alexa Fluor 647 scan (middle panel) and overlay (green: radioactivity; magenta: fluorescence; white: both; right panel) are given. The two main bands represent 23S and 16S ribosomal RNA.

    Article Snippet: Alexa Fluor 647 alkyne, Alexa Fluor 488 alkyne and biotin-alkyne were purchased from Invitrogen.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Radioactivity, Fluorescence

    Creating an internal fluorescent label. ( A ) General reaction scheme for creation of terminal (upper right corner) or internal modifications (lower right corner). An internal modification can be created by first adding an N 3 -modified nucleotide to the 3′-terminus of the RNA sequence, connecting this RNA to a second RNA sequence via splinted ligation, and subjecting the product, with an internal N 3 -modification to CuAAC. ( B ) Splinted ligation of RNA1 and RNA4, employing different ligases (RNL2 and DNL) under different reaction conditions (time and temperature). Analysis by 15% seqPAGE. ( C ) Addition of 2′-N 3 -guanosine to RNA1, followed by splinted ligation to RNA4 (using RNL2), and CuAAC with Alexa Fluor 488/647 alkyne, with or without the use of a helper DNA that forces the modified position into a 9-nt bulge loop. Analysis by 15% seqPAGE. Radioactivity scan (left panel), and an overlay of Alexa Fluor 488 scan (green, middle two lanes) and Alexa Fluor 647 scan (magenta, right two lanes) are shown. ( D ) Formation of 9 nt bulge loop to assist CuAAC. N.R.: no reaction control.

    Journal: Nucleic Acids Research

    Article Title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

    doi: 10.1093/nar/gks062

    Figure Lengend Snippet: Creating an internal fluorescent label. ( A ) General reaction scheme for creation of terminal (upper right corner) or internal modifications (lower right corner). An internal modification can be created by first adding an N 3 -modified nucleotide to the 3′-terminus of the RNA sequence, connecting this RNA to a second RNA sequence via splinted ligation, and subjecting the product, with an internal N 3 -modification to CuAAC. ( B ) Splinted ligation of RNA1 and RNA4, employing different ligases (RNL2 and DNL) under different reaction conditions (time and temperature). Analysis by 15% seqPAGE. ( C ) Addition of 2′-N 3 -guanosine to RNA1, followed by splinted ligation to RNA4 (using RNL2), and CuAAC with Alexa Fluor 488/647 alkyne, with or without the use of a helper DNA that forces the modified position into a 9-nt bulge loop. Analysis by 15% seqPAGE. Radioactivity scan (left panel), and an overlay of Alexa Fluor 488 scan (green, middle two lanes) and Alexa Fluor 647 scan (magenta, right two lanes) are shown. ( D ) Formation of 9 nt bulge loop to assist CuAAC. N.R.: no reaction control.

    Article Snippet: Alexa Fluor 647 alkyne, Alexa Fluor 488 alkyne and biotin-alkyne were purchased from Invitrogen.

    Techniques: Modification, Sequencing, Ligation, Radioactivity

    Detection limit for RNA1 modified with each of the four 2′-N 3 -2′-dNTPs and conjugated with Alexa Fluor 647 alkyne. Samples (same as in Figure 3 A) were diluted and analyzed by 12% seqPAGE in amounts from 25 amol to 2.5 fmol. Fluorescence scan (Typhoon 9400, pixel size: 100 µm, PMT: 800 V, high sensitivity) is shown.

    Journal: Nucleic Acids Research

    Article Title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

    doi: 10.1093/nar/gks062

    Figure Lengend Snippet: Detection limit for RNA1 modified with each of the four 2′-N 3 -2′-dNTPs and conjugated with Alexa Fluor 647 alkyne. Samples (same as in Figure 3 A) were diluted and analyzed by 12% seqPAGE in amounts from 25 amol to 2.5 fmol. Fluorescence scan (Typhoon 9400, pixel size: 100 µm, PMT: 800 V, high sensitivity) is shown.

    Article Snippet: Alexa Fluor 647 alkyne, Alexa Fluor 488 alkyne and biotin-alkyne were purchased from Invitrogen.

    Techniques: Modification, Fluorescence

    Fluorescent labeling of RNA by CuAAC or SPAAC. RNA1, modified with each of the four 2′-N 3 -2′-dNTPs under optimized conditions and further conjugated with fluorescent dyes. Analysis by 15% seqPAGE. Radioactivity scan (upper panel), fluorescence scan (middle panel) and an overlay of both (green: radioactivity; magenta: fluorescence; white: both; lower panel) are given. ( A ) Conjugation with Alexa Fluor 647 alkyne by CuAAC. ( B ) Conjugation with DIBAC Fluor 488 by SPAAC.

    Journal: Nucleic Acids Research

    Article Title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

    doi: 10.1093/nar/gks062

    Figure Lengend Snippet: Fluorescent labeling of RNA by CuAAC or SPAAC. RNA1, modified with each of the four 2′-N 3 -2′-dNTPs under optimized conditions and further conjugated with fluorescent dyes. Analysis by 15% seqPAGE. Radioactivity scan (upper panel), fluorescence scan (middle panel) and an overlay of both (green: radioactivity; magenta: fluorescence; white: both; lower panel) are given. ( A ) Conjugation with Alexa Fluor 647 alkyne by CuAAC. ( B ) Conjugation with DIBAC Fluor 488 by SPAAC.

    Article Snippet: Alexa Fluor 647 alkyne, Alexa Fluor 488 alkyne and biotin-alkyne were purchased from Invitrogen.

    Techniques: Labeling, Modification, Radioactivity, Fluorescence, Conjugation Assay

    Mouse primary and immortalized osteoblast cell proliferation. Proliferation of primary and immortalized cells was immunostained using a mouse monoclonal anti-BrdU antibody (1:100 dilution) after a 4-h BrdU incorporation (30 μM), followed by a 1:1,000 dilution of the secondary antibody (goat-anti-mouse) with Alexa Fluo® 488 green. For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst. Images were obtained by a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei. BrdU positive staining of the primary cells acts as 100% and asterisk (*) shows significant difference between the primary and immortalized cells ( p

    Journal: Cell and tissue research

    Article Title: Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype

    doi: 10.1007/s00441-010-1120-3

    Figure Lengend Snippet: Mouse primary and immortalized osteoblast cell proliferation. Proliferation of primary and immortalized cells was immunostained using a mouse monoclonal anti-BrdU antibody (1:100 dilution) after a 4-h BrdU incorporation (30 μM), followed by a 1:1,000 dilution of the secondary antibody (goat-anti-mouse) with Alexa Fluo® 488 green. For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst. Images were obtained by a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei. BrdU positive staining of the primary cells acts as 100% and asterisk (*) shows significant difference between the primary and immortalized cells ( p

    Article Snippet: The cells were then incubated with a mouse monoclonal anti-BrdU antibody (1:100; Santa Cruz Biotechnology), followed by a 1:1,000 dilution of the secondary antibodies (goat-anti-mouse) with Alexa Fluo® 488 green (Molecular Probes).

    Techniques: BrdU Incorporation Assay, Staining, Incubation, Inverted Microscopy

    Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for Alexa 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.

    Journal: Nature biotechnology

    Article Title: Engineering a HER2-specific antibody-drug conjugate to increase lysosomal delivery and therapeutic efficacy

    doi: 10.1038/s41587-019-0073-7

    Figure Lengend Snippet: Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for Alexa 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.

    Article Snippet: ADCs were labeled with Alexa 488 Fluor (Thermo Fisher Scientific) or radiolabeled with 125 I-Iodogen [Perkin Elmer or MP Biomedical (Solon, OH, USA)] as described previously , except that a 1:1 molar ratio of Alexa 488:ADC was used during the labeling reaction.

    Techniques: Expressing, Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence, Two Tailed Test

    Neuronal calcium sensor (NCS1) in the Golgi apparatus recognizes Ca 2+ signal for focal release of mannosidase-II vesicles. A, mCherrry-MAN2A-expressing U937 macrophages were incubated with latex beads (30 min) or infected with PKH67-labeled H37Rv (1 h). Cells were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-labeled secondary antibody. For the upper panel, latex beads were given green pseudo-color using Imaris ( scale bar, 4 μm). B, upper panel , in U937 macrophages, incubated with beads for 30 min or 1 h, samples were stained with anti-NCS1 antibody. Presence of NCS1 at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments. For lower panel , mCherry-MAN2A ( red )-expressing U937-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-tagged secondary antibody. The images are representative of the 1-h time point. Percent co-localization of H37Rv with mannosidase-II, NCS1, or both mannosidase-II and NCS1 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.). DIC , differential interference contrast. C, co-localization of mannosidase-II and NCS1 in U937 macrophages. U937 macrophages expressing mCherry-MAN2A were stained with anti-NCS1 antibody followed by secondary antibody (pseudo-colored green ) ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. D, siRNA-mediated knockdown of NCS1 was confirmed by Western blottings on the whole cell lysates from the transfected cells. Knockdown was monitored at 48 h post-transfection. E, THP-1-derived macrophages treated with NCS1 siRNA were incubated with GFP expressing E. coli for 5 and 15 min. Cells were stained with anti-mannosidase-II antibody to assess the recruitment of mannosidase-II at the phagosomes in NCS1-depleted cells. Percent co-localization of E. coli with mannosidase-II was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.E., *, p value

    Journal: The Journal of Biological Chemistry

    Article Title: Ca2+-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages *

    doi: 10.1074/jbc.M116.743047

    Figure Lengend Snippet: Neuronal calcium sensor (NCS1) in the Golgi apparatus recognizes Ca 2+ signal for focal release of mannosidase-II vesicles. A, mCherrry-MAN2A-expressing U937 macrophages were incubated with latex beads (30 min) or infected with PKH67-labeled H37Rv (1 h). Cells were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-labeled secondary antibody. For the upper panel, latex beads were given green pseudo-color using Imaris ( scale bar, 4 μm). B, upper panel , in U937 macrophages, incubated with beads for 30 min or 1 h, samples were stained with anti-NCS1 antibody. Presence of NCS1 at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments. For lower panel , mCherry-MAN2A ( red )-expressing U937-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-tagged secondary antibody. The images are representative of the 1-h time point. Percent co-localization of H37Rv with mannosidase-II, NCS1, or both mannosidase-II and NCS1 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.). DIC , differential interference contrast. C, co-localization of mannosidase-II and NCS1 in U937 macrophages. U937 macrophages expressing mCherry-MAN2A were stained with anti-NCS1 antibody followed by secondary antibody (pseudo-colored green ) ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. D, siRNA-mediated knockdown of NCS1 was confirmed by Western blottings on the whole cell lysates from the transfected cells. Knockdown was monitored at 48 h post-transfection. E, THP-1-derived macrophages treated with NCS1 siRNA were incubated with GFP expressing E. coli for 5 and 15 min. Cells were stained with anti-mannosidase-II antibody to assess the recruitment of mannosidase-II at the phagosomes in NCS1-depleted cells. Percent co-localization of E. coli with mannosidase-II was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.E., *, p value

    Article Snippet: The secondary antibodies used in this study are Alexa Fluor 405 and Alexa Fluor 568 conjugates from Life Technologies, Inc.

    Techniques: Expressing, Incubation, Infection, Labeling, Staining, Software, Derivative Assay, Western Blot, Transfection

    Vesicles derived from Golgi apparatus are distinct from recycling endosome vesicles and are recruited independently. A, U937 cells expressing MAN2A-mCherry were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. DIC , differential interference contrast. B, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were incubated with mouse serum-coated latex beads for 30 min or 1 h. At the respective time points, cells were immunostained with anti-VAMP-3 antibody followed by a secondary antibody tagged with Alexa Fluor 405 ( blue ). Presence of VAMP-3 or mannosidase-II at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments ( scale bar, 5 μm). C, mCherry-MAN2A ( red )-expressing THP-1-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-VAMP-3 antibody followed by Alexa Fluor 405-tagged secondary antibody ( blue ). The images are representative of the 1-h time point. For the plots at right , % co-localization of H37Rv with mannosidase-II, VAMP-3, or both mannosidase-II and VAMP-3 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.; scale bar, 4 μm). D, U937 cells were incubated with serum-coated FITC-labeled latex beads in the presence or absence of BfA for 30 min. Samples were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody. Images were analyzed using 3D module in Imaris 7.2, and VAMP3 intensity distribution in the latex beads was calculated. E, U937 cells were incubated with GFP expressing E. coli in the presence or absence of BfA for 30 min. Samples were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody. Images were analyzed using 3D module in Imaris 7.2, and VAMP3 intensity distribution in the latex beads was calculated.

    Journal: The Journal of Biological Chemistry

    Article Title: Ca2+-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages *

    doi: 10.1074/jbc.M116.743047

    Figure Lengend Snippet: Vesicles derived from Golgi apparatus are distinct from recycling endosome vesicles and are recruited independently. A, U937 cells expressing MAN2A-mCherry were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. DIC , differential interference contrast. B, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were incubated with mouse serum-coated latex beads for 30 min or 1 h. At the respective time points, cells were immunostained with anti-VAMP-3 antibody followed by a secondary antibody tagged with Alexa Fluor 405 ( blue ). Presence of VAMP-3 or mannosidase-II at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments ( scale bar, 5 μm). C, mCherry-MAN2A ( red )-expressing THP-1-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-VAMP-3 antibody followed by Alexa Fluor 405-tagged secondary antibody ( blue ). The images are representative of the 1-h time point. For the plots at right , % co-localization of H37Rv with mannosidase-II, VAMP-3, or both mannosidase-II and VAMP-3 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.; scale bar, 4 μm). D, U937 cells were incubated with serum-coated FITC-labeled latex beads in the presence or absence of BfA for 30 min. Samples were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody. Images were analyzed using 3D module in Imaris 7.2, and VAMP3 intensity distribution in the latex beads was calculated. E, U937 cells were incubated with GFP expressing E. coli in the presence or absence of BfA for 30 min. Samples were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody. Images were analyzed using 3D module in Imaris 7.2, and VAMP3 intensity distribution in the latex beads was calculated.

    Article Snippet: The secondary antibodies used in this study are Alexa Fluor 405 and Alexa Fluor 568 conjugates from Life Technologies, Inc.

    Techniques: Derivative Assay, Expressing, Staining, Labeling, Incubation, Software, Infection

    Early recruitment of mannosidase-II at phagosomes and proteomic characterization. A, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were incubated with mouse serum-coated latex beads for 30 min or 1 h. At the respective time points, cells were immunostained with anti-TfR antibody followed by a secondary antibody tagged with Alexa Fluor 405 ( blue ). Presence of TfR or mannosidase-II at the bead surface was calculated in terms of fluorescence intensity using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments ( scale bar, 5 μm). B, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-transferrin receptor antibody followed by Alexa Fluor 405-tagged secondary antibody ( blue ). The images are representative of the 1-h time point. For the plots at right , % co-localization of H37Rv with mannosidase-II, TfR, or both mannosidase-II and TfR was calculated using Imaris 7.2. The data represent the average of more than 150 bacteria from three different experiments (values ± S.D.; scale bar, 5 μm). C, preparation of latex bead phagosomes from THP-1-derived macrophages on a sucrose density gradient (see “Materials and Methods”). D, THP-1-derived macrophages were incubated with latex beads (1 μm size) for 1 h. Phagosomes were isolated using differential density ultracentrifugation, and samples were probed for indicated markers using Western blottings. Molecular mass markers are the closest markers to the band of interest. WCL , whole cell lysate. E, latex beads phagosomes isolated from RAW264.7 macrophages were lysed and resolved on a 10% SDS-PAGE. The molecules below the 25-kDa molecular mass were cut from the gel and analyzed using mass spectrometry to identify enrichment of low molecular weight proteins (see “Materials and Methods”). The list of genes identified was then searched in the AMIGO2.0 database to establish functional association. Finally the representative network was constructed using Cytoscape 2.6.1. The pink nodes and edges in the network denote association with the Golgi apparatus.

    Journal: The Journal of Biological Chemistry

    Article Title: Ca2+-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages *

    doi: 10.1074/jbc.M116.743047

    Figure Lengend Snippet: Early recruitment of mannosidase-II at phagosomes and proteomic characterization. A, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were incubated with mouse serum-coated latex beads for 30 min or 1 h. At the respective time points, cells were immunostained with anti-TfR antibody followed by a secondary antibody tagged with Alexa Fluor 405 ( blue ). Presence of TfR or mannosidase-II at the bead surface was calculated in terms of fluorescence intensity using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments ( scale bar, 5 μm). B, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-transferrin receptor antibody followed by Alexa Fluor 405-tagged secondary antibody ( blue ). The images are representative of the 1-h time point. For the plots at right , % co-localization of H37Rv with mannosidase-II, TfR, or both mannosidase-II and TfR was calculated using Imaris 7.2. The data represent the average of more than 150 bacteria from three different experiments (values ± S.D.; scale bar, 5 μm). C, preparation of latex bead phagosomes from THP-1-derived macrophages on a sucrose density gradient (see “Materials and Methods”). D, THP-1-derived macrophages were incubated with latex beads (1 μm size) for 1 h. Phagosomes were isolated using differential density ultracentrifugation, and samples were probed for indicated markers using Western blottings. Molecular mass markers are the closest markers to the band of interest. WCL , whole cell lysate. E, latex beads phagosomes isolated from RAW264.7 macrophages were lysed and resolved on a 10% SDS-PAGE. The molecules below the 25-kDa molecular mass were cut from the gel and analyzed using mass spectrometry to identify enrichment of low molecular weight proteins (see “Materials and Methods”). The list of genes identified was then searched in the AMIGO2.0 database to establish functional association. Finally the representative network was constructed using Cytoscape 2.6.1. The pink nodes and edges in the network denote association with the Golgi apparatus.

    Article Snippet: The secondary antibodies used in this study are Alexa Fluor 405 and Alexa Fluor 568 conjugates from Life Technologies, Inc.

    Techniques: Expressing, Incubation, Fluorescence, Software, Infection, Labeling, Staining, Derivative Assay, Isolation, Western Blot, SDS Page, Mass Spectrometry, Molecular Weight, Functional Assay, Construct

    Characterization of VCAM-1 + macrophages in the CHT. a, Transgenic Tg(kdrl:eGFP) embryos, stained with an anti-VCAM-1 antibody (magenta, arrows), show that the VCAM-1 + macrophage first appeared in the CHT at 32 h.p.f. b, Tg(kdrl:eGFP) embryos in the vcam1 cas011 , itga4 cas005 or runx1 w84x mutant background are stained with an anti-VCAM-1 antibody (magenta, white arrows) at 54 h.p.f. Signals in itga4 cas005 and runx1 w84x are similar to that in wild-type siblings, whereas there is almost no detectable signal in vcam1 cas011 mutants. c, Schematic diagrams (left) and confocal imaging (right) of VCAM-1 + macrophages (labelled with Alexa Fluor 647 dye-conjugated anti-VCAM-1 antibody by intravascular injection) that patrol the CHT of wild-type embryos. VCAM-1 + macrophages were mainly located intravascularly ( > 91%) with round or unpolarized cell morphology ( > 84%). Cross indicates the original position of VCAM-1 + . DA, dorsal aorta; VC, venous capillaries. Scale bars, 50 μm ( a, b ) and 20 μm ( c ).

    Journal: Nature

    Article Title: VCAM-1+ macrophages guide the homing of HSPCs to a vascular niche

    doi: 10.1038/s41586-018-0709-7

    Figure Lengend Snippet: Characterization of VCAM-1 + macrophages in the CHT. a, Transgenic Tg(kdrl:eGFP) embryos, stained with an anti-VCAM-1 antibody (magenta, arrows), show that the VCAM-1 + macrophage first appeared in the CHT at 32 h.p.f. b, Tg(kdrl:eGFP) embryos in the vcam1 cas011 , itga4 cas005 or runx1 w84x mutant background are stained with an anti-VCAM-1 antibody (magenta, white arrows) at 54 h.p.f. Signals in itga4 cas005 and runx1 w84x are similar to that in wild-type siblings, whereas there is almost no detectable signal in vcam1 cas011 mutants. c, Schematic diagrams (left) and confocal imaging (right) of VCAM-1 + macrophages (labelled with Alexa Fluor 647 dye-conjugated anti-VCAM-1 antibody by intravascular injection) that patrol the CHT of wild-type embryos. VCAM-1 + macrophages were mainly located intravascularly ( > 91%) with round or unpolarized cell morphology ( > 84%). Cross indicates the original position of VCAM-1 + . DA, dorsal aorta; VC, venous capillaries. Scale bars, 50 μm ( a, b ) and 20 μm ( c ).

    Article Snippet: For live labelling of usher cells, VCAM-1 antibody (Abclonal) was conjugated with Alexa Fluor 647 dye and purified by Microscale Protein Labelling Kit (Invitrogen, A30009).

    Techniques: Transgenic Assay, Staining, Mutagenesis, Imaging, Injection

    Confocal LSM images of DCs after LPK NP uptake, in which KLH was labeled with Alexa Fluor ® 647, and lipids were labeled with NBD. Dendritic cells were treated with (A) LPK − NPs, (B) LPK + NPs, and (C) LPK pH NPs, respectively. The intracellular

    Journal: Polymer

    Article Title: In vitro controlled release of antigen in dendritic cells using pH-sensitive liposome-polymeric hybrid nanoparticles

    doi: 10.1016/j.polymer.2015.10.048

    Figure Lengend Snippet: Confocal LSM images of DCs after LPK NP uptake, in which KLH was labeled with Alexa Fluor ® 647, and lipids were labeled with NBD. Dendritic cells were treated with (A) LPK − NPs, (B) LPK + NPs, and (C) LPK pH NPs, respectively. The intracellular

    Article Snippet: Fetal bovine serum (FBS), GM-CSF recombinant mouse protein, minimum essential medium (MEM) α , trypsin/EDTA, CellMask™ Orange Plasma membrane Stain, Alexa Fluor® 647 Hydrazide, tris(triethylammonium) salt were purchased from Life Technologies Corporation (Grand Island, NY).

    Techniques: Labeling

    Confocal LSM images of LPK pH NPs, in which KLH was labeled with Alexa Fluor ® 647, and lipids were labeled with NBD.

    Journal: Polymer

    Article Title: In vitro controlled release of antigen in dendritic cells using pH-sensitive liposome-polymeric hybrid nanoparticles

    doi: 10.1016/j.polymer.2015.10.048

    Figure Lengend Snippet: Confocal LSM images of LPK pH NPs, in which KLH was labeled with Alexa Fluor ® 647, and lipids were labeled with NBD.

    Article Snippet: Fetal bovine serum (FBS), GM-CSF recombinant mouse protein, minimum essential medium (MEM) α , trypsin/EDTA, CellMask™ Orange Plasma membrane Stain, Alexa Fluor® 647 Hydrazide, tris(triethylammonium) salt were purchased from Life Technologies Corporation (Grand Island, NY).

    Techniques: Labeling