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  • 99
    Thermo Fisher alexa fluor antibodies
    Expression of Ca 2+ channels in human osteoclasts. At the end of the CBM cultures, immunofluorescence was performed on mature osteoclasts, using polyclonal rabbit antibodies directed against Ca 2+ channels (L-, T-, and R-channels and TRPV-5) and goat anti-rabbit secondary antibodies conjugated to <t>Alexa</t> Fluor 546, and counterstaining was performed with <t>DAPI.</t> Images are representative of three independent experiments.
    Alexa Fluor Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher alexa fluor 488 antibody
    Multivalent RNA Binding Stimulates Super-order Assembly of Dynamic MRJP-3 RNPs and Isolation of RJ RNA Partners of MRJP-3 (A) The multivalent interaction of MRJP-3 with RNA is reversible. MRJP-3-bound ssRNA ∗ was introduced to increasing quantities of unlabeled ssRNA. ssRNA ∗ (0.04 μM) and MRJP-3 (31.3 μM) were used in all ssRNA ∗ - and protein-containing treatments. Unlabeled DNA ladder served as labeling control. (B) MRJP-3 RNPs are affected by the protein/ssRNA mole ratio. Images of RNPs formed at various mole ratios of MRJP-3 and <t>Alexa</t> <t>Fluor-488</t> labeled ssRNA (ssRNA ∗ ). Scale bar represents 10 μm. (C) RNA mediates super-order assembly of MRJP-3 oligomers, resulting in RNP formation in soluble RJ fraction. RJ buffer or 4.28 μM Alexa Fluor-633 labeled MRJP-3 (MRJP-3 ∗ ) was introduced to 50% soluble RJ fraction. ssRNA (0.15 μM) or ssRNA ∗ was used in RNA-containing treatments. Scale bar represents 2 μm. (D) MRJP-3 binds both endogenous ( Apis mellifera ) and exogenous (viral) RNA. Only viruses with a mapped fraction of at least 1% are shown. Points are individual biological replicates. Bars represent the mean across replicates. Horizontal lines indicate tests for significant enrichment of viral RNA over bee RNA in the MRJP-3 bound fraction, but not in RJ ( ∗ p
    Alexa Fluor 488 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 403 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher alexa fluor conjugated antibodies
    NPM T199A suppresses centrosome amplification and chromosome instability. (A) Passage 2 p53 − / − MEFs were transiently transfected with plasmids encoding FLAG epitope-tagged NPM and the NPM/B23 mutant (NPM T99A ). As a control, an empty vector was transfected. After neomycin selection, cell lysates were obtained and then probed with anti-FLAG antibodies. (B) The transfectants described in the legend to panel A were fixed, immunostained with <t>anti-γ-tubulin</t> polyclonal antibodies, and detected with <t>Alexa</t> Fluor 488-conjugated secondary antibodies. Cells were counterstained with DAPI. The number of cells with ≥3 centrosomes in a population of at least 200 cells was statistically analyzed by fluorescence microscopy. Each group included three transfected MEFs. P values (relative to the result for the transfected vector control) were 0.006963 for NPM T199A and 0.560677 for wild-type NPM. (C) Proliferating E13.5 mouse embryonic fibroblasts of the indicated genotypes were fixed, and nuclei were visualized with DAPI. The frequencies of micronucleus formation in a population of 500 cells were calculated for the indicated genotypes. P values (relative to the result for the transfected vector control) were 0.011338 for NPM T199A and 0.353737 for wild-type NPM. (D) Frequencies of γ-H2AX foci in cells with the indicated genotypes were calculated. P values (relative to the result for the transfected vector control) were 0.002591 for NPM T199A and 0.476327 for wild-type NPM.
    Alexa Fluor Conjugated Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 917 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher alexa fluor 488
    Endocytosis of NogoΔ20 is required for NogoΔ20-induced growth cone collapse in hippocampal neurons. (A and B) Morphology of noncollapsed (A, arrow) and collapsed growth cones (B, arrowhead) of E19 hippocampal neurons at DIV 4 was visualized by staining of F-actin with <t>phalloidin–Alexa</t> <t>Fluor</t> 488 (green). (C) Quantification of the proportion of collapsed growth cones after incubation with 300 nM NogoΔ20 (shaded bars) or with 300 nM NogoΔ21 (open bars). (D and E) The morphology of hippocampal neurons infected with immunodeficient recombinant adenovirus containing HA-tagged wt Pincher (D, red) or HA-tagged dn PincherG68E (E) upon treatment for 30 min with 300 nM NogoΔ20 was visualized with phalloidin (green). (F) Most growth cones remained uncollapsed when HA-tagged dn PincherG68E was overexpressed (open bars) compared with wt Pincher (shaded bars). (G and H) Growth cone morphology of hippocampal neurons upon treatment with 40 nM semaphorin 3A overexpressing either wt HA-Pincher protein (G) or dn HA-PincherG68E protein (H). (I) Treatment with 40 nM semaphorin 3A for 30 min leads to growth cone collapse in the presence of both wt HA-Pincher (shaded bars) and dn HA-Pincherg68E (open bars). Data represent the mean of three (semaphorin 3A) or four (NogoΔ20) independent experiments ± SEM (90 neurons per group and experiment). Asterisks mark highly significant differences between wt and dn Pincher-infected hippocampal neurons (***, P
    Alexa Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 71255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of Ca 2+ channels in human osteoclasts. At the end of the CBM cultures, immunofluorescence was performed on mature osteoclasts, using polyclonal rabbit antibodies directed against Ca 2+ channels (L-, T-, and R-channels and TRPV-5) and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 546, and counterstaining was performed with DAPI. Images are representative of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: TRPV-5 Mediates a Receptor Activator of NF-?B (RANK) Ligand-induced Increase in Cytosolic Ca2+ in Human Osteoclasts and Down-regulates Bone Resorption *

    doi: 10.1074/jbc.M109.075234

    Figure Lengend Snippet: Expression of Ca 2+ channels in human osteoclasts. At the end of the CBM cultures, immunofluorescence was performed on mature osteoclasts, using polyclonal rabbit antibodies directed against Ca 2+ channels (L-, T-, and R-channels and TRPV-5) and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 546, and counterstaining was performed with DAPI. Images are representative of three independent experiments.

    Article Snippet: Alexa Fluor antibodies and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen.

    Techniques: Expressing, Immunofluorescence

    Localization of TRPV-5 by confocal imaging. A , at the end of the CBM cultures, immunofluorescence was performed on mature osteoclasts cultured on plastic. The expression of TRPV-5 and of the CTR was evaluated by immunofluorescence double labeling using specific antibodies, and visualized by confocal microscopy using anti-mouse/Alexa Fluor 633 (TRPV-5, red ) and anti-goat/Alexa Fluor 488 (CTR, green ) secondary antibodies, and counterstaining was performed with DAPI ( blue ). Cross-sectional ( top ) and reconstructed images along the z axis ( left bottom ) are shown. The scale bar represents 10 μm. Fluorograms of two regions are presented: the basolateral membrane ( 2 ) and the region close to the plastic surface ( 1 ) ( green pixels , y axis; red pixels , x axis on fluorogram). B , CBMs were settled on bovine bone slices, and cells were cultured for 3 weeks. At the end of the culture periods, the expression of TRPV-5 and of V-ATPase was evaluated by immunofluorescence using specific antibodies and visualized with anti-mouse/Alexa Fluor 633 (TRPV-5, red ) and anti-rabbit/Alexa Fluor 488 (V-ATPase, green ) secondary antibodies. Confocal microscopy was performed to determine the localization of TRPV-5 and V-ATPase, and one representative cell is shown. Optical slices were taken every 0.8 μm along the z axis, starting from the top of the cell and ending at the level of the bone slice; three sample slices are shown with each channel (DAPI, V-ATPase, and TRPV-5) followed by a merged reconstitution.

    Journal: The Journal of Biological Chemistry

    Article Title: TRPV-5 Mediates a Receptor Activator of NF-?B (RANK) Ligand-induced Increase in Cytosolic Ca2+ in Human Osteoclasts and Down-regulates Bone Resorption *

    doi: 10.1074/jbc.M109.075234

    Figure Lengend Snippet: Localization of TRPV-5 by confocal imaging. A , at the end of the CBM cultures, immunofluorescence was performed on mature osteoclasts cultured on plastic. The expression of TRPV-5 and of the CTR was evaluated by immunofluorescence double labeling using specific antibodies, and visualized by confocal microscopy using anti-mouse/Alexa Fluor 633 (TRPV-5, red ) and anti-goat/Alexa Fluor 488 (CTR, green ) secondary antibodies, and counterstaining was performed with DAPI ( blue ). Cross-sectional ( top ) and reconstructed images along the z axis ( left bottom ) are shown. The scale bar represents 10 μm. Fluorograms of two regions are presented: the basolateral membrane ( 2 ) and the region close to the plastic surface ( 1 ) ( green pixels , y axis; red pixels , x axis on fluorogram). B , CBMs were settled on bovine bone slices, and cells were cultured for 3 weeks. At the end of the culture periods, the expression of TRPV-5 and of V-ATPase was evaluated by immunofluorescence using specific antibodies and visualized with anti-mouse/Alexa Fluor 633 (TRPV-5, red ) and anti-rabbit/Alexa Fluor 488 (V-ATPase, green ) secondary antibodies. Confocal microscopy was performed to determine the localization of TRPV-5 and V-ATPase, and one representative cell is shown. Optical slices were taken every 0.8 μm along the z axis, starting from the top of the cell and ending at the level of the bone slice; three sample slices are shown with each channel (DAPI, V-ATPase, and TRPV-5) followed by a merged reconstitution.

    Article Snippet: Alexa Fluor antibodies and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen.

    Techniques: Imaging, Immunofluorescence, Cell Culture, Expressing, Labeling, Confocal Microscopy

    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

    Immunofluorescence and quantitative flow cytometry analysis of h10H5 uptake. A , R− MEFs transfected with WT or all the mutant IGF-IRs (or vector alone, which gave no specific signal; not shown) were incubated for 60 min with h10H5 in the presence of lysosomal protease inhibitors to inhibit lysosomal degradation, fixed, and permeabilized, and the total antibody was detected with Cy3-anti-human IgG. Scale bar for overlays with lysosomes. B , R− MEFs transfected with WT or mutant IGF-IR were incubated on ice for 1 h with Alexa Fluor 488-labeled h10H5, washed, and chased for 20 or 60 min in the presence of lysosomal protease inhibitors, and then any remaining surface antibody was quenched with anti-Alexa Fluor 488 antibodies. Following flow cytometry measurements, the percentage of internalized h10H5 at each time point (after correcting for any losses or incomplete quenching) was calculated from three independent duplicate experiments, each normalized to WT as 100%, with mean and Standard Deviation of the Mean shown. *, p = 0.05; **, p = 0.01; ***, p = 0.001 versus WT (by Student's t test).

    Journal: The Journal of Biological Chemistry

    Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

    doi: 10.1074/jbc.M111.288514

    Figure Lengend Snippet: Immunofluorescence and quantitative flow cytometry analysis of h10H5 uptake. A , R− MEFs transfected with WT or all the mutant IGF-IRs (or vector alone, which gave no specific signal; not shown) were incubated for 60 min with h10H5 in the presence of lysosomal protease inhibitors to inhibit lysosomal degradation, fixed, and permeabilized, and the total antibody was detected with Cy3-anti-human IgG. Scale bar for overlays with lysosomes. B , R− MEFs transfected with WT or mutant IGF-IR were incubated on ice for 1 h with Alexa Fluor 488-labeled h10H5, washed, and chased for 20 or 60 min in the presence of lysosomal protease inhibitors, and then any remaining surface antibody was quenched with anti-Alexa Fluor 488 antibodies. Following flow cytometry measurements, the percentage of internalized h10H5 at each time point (after correcting for any losses or incomplete quenching) was calculated from three independent duplicate experiments, each normalized to WT as 100%, with mean and Standard Deviation of the Mean shown. *, p = 0.05; **, p = 0.01; ***, p = 0.001 versus WT (by Student's t test).

    Article Snippet: Cells were then chilled, detached with 5 m m EDTA in PBS, and either fixed immediately in 2% paraformaldehyde (unquenched) or after quenching for 1 h on ice with 25 μg/ml anti-Alexa Fluor 488 (Molecular Probes).

    Techniques: Immunofluorescence, Flow Cytometry, Cytometry, Transfection, Mutagenesis, Plasmid Preparation, Incubation, Labeling, Standard Deviation

    Mitotic signaling regulates cytoplasmic localization of SIRT1. DU145 cells were cultured in the chamber slide with DMEM+10% FBS. The immunofluorescence was performed with anti-SIRT1 antibody (sc 74504, Santa Cruz 1:1000, RT 1hr) and Alexa Fluor 488 anti-mouse (Invitrogen, 1:200, RT 1hr). Nuclear protein was stained with DAPI (1:10,000). A. DU145 cells were cultured with DMEM+10% FBS. B. The DU145 cells that were cultured in the DMEM+10% FBS were shifted to serum-free DMEM for 48 hr. C. After culturing the DU145 cells in serum-free medium for 48hr, the medium was replaced with DMEM+10% FBS for 24hr. D. DU145 cells that were cultured in DMEM+10%FBS were treated with hydroxyurea for 24 hr.

    Journal: International Journal of Biological Sciences

    Article Title: Aberrant Cytoplasm Localization and Protein Stability of SIRT1 is Regulated by PI3K/IGF-1R Signaling in Human Cancer Cells

    doi:

    Figure Lengend Snippet: Mitotic signaling regulates cytoplasmic localization of SIRT1. DU145 cells were cultured in the chamber slide with DMEM+10% FBS. The immunofluorescence was performed with anti-SIRT1 antibody (sc 74504, Santa Cruz 1:1000, RT 1hr) and Alexa Fluor 488 anti-mouse (Invitrogen, 1:200, RT 1hr). Nuclear protein was stained with DAPI (1:10,000). A. DU145 cells were cultured with DMEM+10% FBS. B. The DU145 cells that were cultured in the DMEM+10% FBS were shifted to serum-free DMEM for 48 hr. C. After culturing the DU145 cells in serum-free medium for 48hr, the medium was replaced with DMEM+10% FBS for 24hr. D. DU145 cells that were cultured in DMEM+10%FBS were treated with hydroxyurea for 24 hr.

    Article Snippet: The cells were blocked with 3% BSA, treated with anti-SIRT1 antibodies, then incubated with Alexa Fluor secondary antibodies (Invitrogen) and stained with DAPI.

    Techniques: Cell Culture, Immunofluorescence, Staining

    SIRT1 localization determined by both Immunofluorescence and immunobolt analysis. DU145 cells were cultured in the chamber slide with DMEM+10% FBS for the immunoflurorescence analysis ( left panel ). A. Anti-SIRT1 (C05187, Epitomics, 1:100, RT 1hr) and Alexa Fluor 488 goat anti-rabbit (Invitrogen, 200x, RT 1hr). B. Anti-hSir2 (856, 1:500, RT 1hr) and Alexa Fluor 488 goat anti-rabbit (Invitrogen, 200x, RT 1hr). C. Anti-SIRT1 (SC-19857, Santa Cruz, 1:50. RT 1hr) and Alexa Fluor 546 donkey anti-goat (Invitrogen, 100x, RT 1hr). D. Anti-SIRT1 (07-131, Milipore, 1:50, RT 1hr) and Alexa Fluor 488 goat anti-rabbit (Invitrogen, 200x, RT 1hr). E. Anti-SIRT1 (sc-74504, Santa Cruz, 1:1000, RT 1hr) and Alexa Fluor 488 goat anti-mouse (Invitrogen, 200x, RT 1hr). F. Anti-SIRT1 (05-707, upstate, 1:1000, RT 1hr) and Alexa Fluor 594 goat anti-mouse (Invitrogen, 100x, RT 1hr). Cytoplasmic and nuclear fractions of DU145 cells were isolated and same amount of nuclear and cytoplasmic protein were subjected to SDS gels, and immunoblots were performed ( right panel ). A. Anti-SIRT1 (C05187, Epitomics, 1:500, RT 1hr). B. Anti-hSir2 (856, 1:1000, RT 1hr). C. Anti-SIRT1 (SC-19857, Santa Cruz, 1:200, 4 o C O/N). D. Anti-SIRT1 (07-131, Milipore, 1:500, 4 o C O/N). E. Anti-SIRT1 (sc-74504, Santa Cruz, 1:1000, RT 1hr). F. Anti-SIRT1 (05-707, upstate, 1:1000, RT 1hr).

    Journal: International Journal of Biological Sciences

    Article Title: Aberrant Cytoplasm Localization and Protein Stability of SIRT1 is Regulated by PI3K/IGF-1R Signaling in Human Cancer Cells

    doi:

    Figure Lengend Snippet: SIRT1 localization determined by both Immunofluorescence and immunobolt analysis. DU145 cells were cultured in the chamber slide with DMEM+10% FBS for the immunoflurorescence analysis ( left panel ). A. Anti-SIRT1 (C05187, Epitomics, 1:100, RT 1hr) and Alexa Fluor 488 goat anti-rabbit (Invitrogen, 200x, RT 1hr). B. Anti-hSir2 (856, 1:500, RT 1hr) and Alexa Fluor 488 goat anti-rabbit (Invitrogen, 200x, RT 1hr). C. Anti-SIRT1 (SC-19857, Santa Cruz, 1:50. RT 1hr) and Alexa Fluor 546 donkey anti-goat (Invitrogen, 100x, RT 1hr). D. Anti-SIRT1 (07-131, Milipore, 1:50, RT 1hr) and Alexa Fluor 488 goat anti-rabbit (Invitrogen, 200x, RT 1hr). E. Anti-SIRT1 (sc-74504, Santa Cruz, 1:1000, RT 1hr) and Alexa Fluor 488 goat anti-mouse (Invitrogen, 200x, RT 1hr). F. Anti-SIRT1 (05-707, upstate, 1:1000, RT 1hr) and Alexa Fluor 594 goat anti-mouse (Invitrogen, 100x, RT 1hr). Cytoplasmic and nuclear fractions of DU145 cells were isolated and same amount of nuclear and cytoplasmic protein were subjected to SDS gels, and immunoblots were performed ( right panel ). A. Anti-SIRT1 (C05187, Epitomics, 1:500, RT 1hr). B. Anti-hSir2 (856, 1:1000, RT 1hr). C. Anti-SIRT1 (SC-19857, Santa Cruz, 1:200, 4 o C O/N). D. Anti-SIRT1 (07-131, Milipore, 1:500, 4 o C O/N). E. Anti-SIRT1 (sc-74504, Santa Cruz, 1:1000, RT 1hr). F. Anti-SIRT1 (05-707, upstate, 1:1000, RT 1hr).

    Article Snippet: The cells were blocked with 3% BSA, treated with anti-SIRT1 antibodies, then incubated with Alexa Fluor secondary antibodies (Invitrogen) and stained with DAPI.

    Techniques: Immunofluorescence, Cell Culture, Isolation, Western Blot

    Activity and PKC-dependent phosphorylation of MARCKS at the single cell level. (A) Cells were stimulated as labeled, fixed, stained with a polyclonal MARCKS antibody and counterstained with an Alexa Fluor-488 secondary antibody to detect total MARCKS

    Journal: Molecular Biology of the Cell

    Article Title: Cortical F-Actin, the Exocytic Mode, and Neuropeptide Release in Mouse Chromaffin Cells Is Regulated by Myristoylated Alanine-rich C-Kinase Substrate and Myosin II

    doi: 10.1091/mbc.E09-03-0197

    Figure Lengend Snippet: Activity and PKC-dependent phosphorylation of MARCKS at the single cell level. (A) Cells were stimulated as labeled, fixed, stained with a polyclonal MARCKS antibody and counterstained with an Alexa Fluor-488 secondary antibody to detect total MARCKS

    Article Snippet: The cells were then washed for 5 min in 1× PBS and labeled with an Alexa Fluor-488 secondary antibody (Invitrogen).

    Techniques: Activity Assay, Labeling, Staining

    Analysis of nuclear localization of HNF-1A protein variants in HeLa cells. Cells were transiently transfected for 24 h and Xpress-epitope–tagged HNF-1A protein variants detected by immunofluorescence. A : Subcellular localization in a minimum of 200 cells was assessed for each HNF1A variant. The percentage of cells with nuclear accumulation alone is presented. B : Representative images of cells of the two most impaired nuclear localization variants. One MODY3 variant (p.Q466*) with abnormal subcellular localization was included as a control. HNF-1A was detected using tag-specific antibody and Alexa Fluor 488 (green). DNA staining (DAPI) is shown in blue. In more detail, the cytoplasmic signals of the cells expressing p.R131Q were more uniform, whereas the cells expressing p.H514R revealed a pattern resembling aggregated particles. For the purpose of clarity, the nuclei and cell membrane have been marked with a white line.

    Journal: Diabetes

    Article Title: Functional Investigations of HNF1A Identify Rare Variants as Risk Factors for Type 2 Diabetes in the General Population

    doi: 10.2337/db16-0460

    Figure Lengend Snippet: Analysis of nuclear localization of HNF-1A protein variants in HeLa cells. Cells were transiently transfected for 24 h and Xpress-epitope–tagged HNF-1A protein variants detected by immunofluorescence. A : Subcellular localization in a minimum of 200 cells was assessed for each HNF1A variant. The percentage of cells with nuclear accumulation alone is presented. B : Representative images of cells of the two most impaired nuclear localization variants. One MODY3 variant (p.Q466*) with abnormal subcellular localization was included as a control. HNF-1A was detected using tag-specific antibody and Alexa Fluor 488 (green). DNA staining (DAPI) is shown in blue. In more detail, the cytoplasmic signals of the cells expressing p.R131Q were more uniform, whereas the cells expressing p.H514R revealed a pattern resembling aggregated particles. For the purpose of clarity, the nuclei and cell membrane have been marked with a white line.

    Article Snippet: HNF-1A was detected using primary antibody anti-Xpress and secondary antibody Alexa Fluor 488 (Thermo Fisher).

    Techniques: Transfection, Immunofluorescence, Variant Assay, Staining, Expressing

    In vitro S. pombe microtubule dynamics assay. A , schematic diagram of S. pombe microtubule dynamics assay. GMPCPP stabilized polarity-marked microtubule seed assembled from Alexa Fluor 488- and Alexa Fluor 680-labeled pig brain tubulin. Only the center of the seed is attached to the surface by anti-Alexa Fluor 488 antibody. Dynamic non-fluorescently labeled S. pombe microtubules grown from seeds were observed by dark field illumination. B , merged fluorescence images of GMPCPP stabilized, polarity-marked pig microtubule seed ( pig Alexa-MT ). Green , Alexa Fluor 488; red , Alexa Fluor 680. Polarity is indicated by − or +. The plus end of the seed has a longer Alexa Fluor 680-labeled region ( upper panel ), a dark field image showing pig microtubule seed plus elongated S. pombe microtubules ( middle panel ), and the merged images ( lower panel ). Red broken lines show the ends of the seed, and yellow broken lines show the ends of the elongated S. pombe microtubules. Arrows indicate the dynamic S. pombe microtubule elongated from the stabilized microtubule seed. Scale bar : 10 μm. C , kymographs of microtubule length change over time. The left panel shows a diagram of a typical example. Time is indicated by the vertical axis , and length is indicated by the horizontal axis . Rescue ( r ) and catastrophe ( c ) events are labeled. Regrowth of shrinking microtubules from the seed ( yellow arrow ) were not counted as rescues. Scale bars : vertical , 5 min; horizontal , 20 μm. + and − ends of microtubule are indicated. D , enlargement of catastrophe events from the yellow rectangle in C. Scale bars : vertical , 30 s; horizontal , 5 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Mal3 Masks Catastrophe Events in Schizosaccharomyces pombe Microtubules by Inhibiting Shrinkage and Promoting Rescue *

    doi: 10.1074/jbc.C109.052159

    Figure Lengend Snippet: In vitro S. pombe microtubule dynamics assay. A , schematic diagram of S. pombe microtubule dynamics assay. GMPCPP stabilized polarity-marked microtubule seed assembled from Alexa Fluor 488- and Alexa Fluor 680-labeled pig brain tubulin. Only the center of the seed is attached to the surface by anti-Alexa Fluor 488 antibody. Dynamic non-fluorescently labeled S. pombe microtubules grown from seeds were observed by dark field illumination. B , merged fluorescence images of GMPCPP stabilized, polarity-marked pig microtubule seed ( pig Alexa-MT ). Green , Alexa Fluor 488; red , Alexa Fluor 680. Polarity is indicated by − or +. The plus end of the seed has a longer Alexa Fluor 680-labeled region ( upper panel ), a dark field image showing pig microtubule seed plus elongated S. pombe microtubules ( middle panel ), and the merged images ( lower panel ). Red broken lines show the ends of the seed, and yellow broken lines show the ends of the elongated S. pombe microtubules. Arrows indicate the dynamic S. pombe microtubule elongated from the stabilized microtubule seed. Scale bar : 10 μm. C , kymographs of microtubule length change over time. The left panel shows a diagram of a typical example. Time is indicated by the vertical axis , and length is indicated by the horizontal axis . Rescue ( r ) and catastrophe ( c ) events are labeled. Regrowth of shrinking microtubules from the seed ( yellow arrow ) were not counted as rescues. Scale bars : vertical , 5 min; horizontal , 20 μm. + and − ends of microtubule are indicated. D , enlargement of catastrophe events from the yellow rectangle in C. Scale bars : vertical , 30 s; horizontal , 5 μm.

    Article Snippet: Polarity-marked GMPCPP microtubule seeds were attached by anti-Alexa Fluor 488 antibody ( , Invitrogen) to Sigmacote (Sigma)-coated coverglasses in flow cells.

    Techniques: In Vitro, Labeling, Fluorescence

    Colocalization of EBO-Z and MBG pseudotypes with the caveola protein marker CAV-1 after infection of HeLa cells. (A to I) Images of cells with Ctxn B-FITC (A to C), tfn-FITC (D to F), and the VSV, EBO-Z, and MBG pseudotypes (G, H, and I, respectively); (C) Ctxn B-FITC and CAV-1 colocalization following incubation of cells with Ctxn B-FITC for 30 min at 37°C; (F) absence of colocalization of tfn-FITC and CAV-1 following incubation of cells with tfn-FITC for 30 min at 37°C; (G) absence of colocalization of VSV and CAV-1 following incubation of cells with VSV pseudotypes for 30 min at 37°C; (H and I) colocalization of EBO-Z and MBG with CAV-1 following a 90-min incubation of cells with EBO-Z (H) and MBG (I) pseudotypes at 37°C. Following the incubations, all samples were fixed in paraformaldehyde (2%), permeabilized in Triton X-100 (0.1%), and immunostained for p24 (to detect pseudotypes) and/or CAV-1 (to detect caveolae). p24 was detected with an FITC-conjugated mouse anti-p24 antibody (Coulter Corp.), and the FITC signal was amplified with Alexa sandwich antibodies (Alexa Fluor 488-conjugated goat polyclonal anti-FITC antibody and Alexa Fluor 488-conjugated donkey anti-goat IgG (H+L) conjugate (Molecular Probes, Inc.). CAV-1 was detected with a rabbit polyclonal antibody against CAV-1 (BD Transduction Laboratories) and a Texas Red-conjugated anti-rabbit IgG (H+L) F(ab′) 2 fragment (Jackson ImmunoResearch Laboratories, Inc.). Cell images were taken under oil at a ×60 magnification with a Bio-Rad Radiance2100 laser scanning confocal microscope system operated via LaserSharp2000 software. For intracellular analyses of cells, the z -axis position was computer controlled and the basal and apical membranes were set as the start and stop points, respectively. All images were at least 2 μm from either vantage point. Immunostained cells were excited with krypton or argon lasers at 488 nm (Alexa Fluor 488 and FITC) and 568 nm (Texas Red), and fluorescence signals were collected with barrier filters for Alexa Fluor 488 and FITC and for Texas Red. Regions of colocalization were generated by digital overlay with Photoshop 6 (Adobe Systems) and appear yellow (C and F to I). For comparison purposes, images collected with green and red barrier filters were digitally colorized to green (A and D) and red (B and E), respectively, also with Photoshop 6.

    Journal: Journal of Virology

    Article Title: Association of the Caveola Vesicular System with Cellular Entry by Filoviruses

    doi: 10.1128/JVI.76.10.5266-5270.2002

    Figure Lengend Snippet: Colocalization of EBO-Z and MBG pseudotypes with the caveola protein marker CAV-1 after infection of HeLa cells. (A to I) Images of cells with Ctxn B-FITC (A to C), tfn-FITC (D to F), and the VSV, EBO-Z, and MBG pseudotypes (G, H, and I, respectively); (C) Ctxn B-FITC and CAV-1 colocalization following incubation of cells with Ctxn B-FITC for 30 min at 37°C; (F) absence of colocalization of tfn-FITC and CAV-1 following incubation of cells with tfn-FITC for 30 min at 37°C; (G) absence of colocalization of VSV and CAV-1 following incubation of cells with VSV pseudotypes for 30 min at 37°C; (H and I) colocalization of EBO-Z and MBG with CAV-1 following a 90-min incubation of cells with EBO-Z (H) and MBG (I) pseudotypes at 37°C. Following the incubations, all samples were fixed in paraformaldehyde (2%), permeabilized in Triton X-100 (0.1%), and immunostained for p24 (to detect pseudotypes) and/or CAV-1 (to detect caveolae). p24 was detected with an FITC-conjugated mouse anti-p24 antibody (Coulter Corp.), and the FITC signal was amplified with Alexa sandwich antibodies (Alexa Fluor 488-conjugated goat polyclonal anti-FITC antibody and Alexa Fluor 488-conjugated donkey anti-goat IgG (H+L) conjugate (Molecular Probes, Inc.). CAV-1 was detected with a rabbit polyclonal antibody against CAV-1 (BD Transduction Laboratories) and a Texas Red-conjugated anti-rabbit IgG (H+L) F(ab′) 2 fragment (Jackson ImmunoResearch Laboratories, Inc.). Cell images were taken under oil at a ×60 magnification with a Bio-Rad Radiance2100 laser scanning confocal microscope system operated via LaserSharp2000 software. For intracellular analyses of cells, the z -axis position was computer controlled and the basal and apical membranes were set as the start and stop points, respectively. All images were at least 2 μm from either vantage point. Immunostained cells were excited with krypton or argon lasers at 488 nm (Alexa Fluor 488 and FITC) and 568 nm (Texas Red), and fluorescence signals were collected with barrier filters for Alexa Fluor 488 and FITC and for Texas Red. Regions of colocalization were generated by digital overlay with Photoshop 6 (Adobe Systems) and appear yellow (C and F to I). For comparison purposes, images collected with green and red barrier filters were digitally colorized to green (A and D) and red (B and E), respectively, also with Photoshop 6.

    Article Snippet: The cells were then stained for intracellular p24 with a fluorescein isothiocyanate (FITC)-conjugated mouse anti-p24 antibody (Coulter Corp.), and the FITC signal was amplified with Alexa Fluor 488 sandwich antibodies (Molecular Probes, Inc.).

    Techniques: Marker, Infection, Incubation, Amplification, Transduction, Microscopy, Software, Fluorescence, Generated

    Multivalent RNA Binding Stimulates Super-order Assembly of Dynamic MRJP-3 RNPs and Isolation of RJ RNA Partners of MRJP-3 (A) The multivalent interaction of MRJP-3 with RNA is reversible. MRJP-3-bound ssRNA ∗ was introduced to increasing quantities of unlabeled ssRNA. ssRNA ∗ (0.04 μM) and MRJP-3 (31.3 μM) were used in all ssRNA ∗ - and protein-containing treatments. Unlabeled DNA ladder served as labeling control. (B) MRJP-3 RNPs are affected by the protein/ssRNA mole ratio. Images of RNPs formed at various mole ratios of MRJP-3 and Alexa Fluor-488 labeled ssRNA (ssRNA ∗ ). Scale bar represents 10 μm. (C) RNA mediates super-order assembly of MRJP-3 oligomers, resulting in RNP formation in soluble RJ fraction. RJ buffer or 4.28 μM Alexa Fluor-633 labeled MRJP-3 (MRJP-3 ∗ ) was introduced to 50% soluble RJ fraction. ssRNA (0.15 μM) or ssRNA ∗ was used in RNA-containing treatments. Scale bar represents 2 μm. (D) MRJP-3 binds both endogenous ( Apis mellifera ) and exogenous (viral) RNA. Only viruses with a mapped fraction of at least 1% are shown. Points are individual biological replicates. Bars represent the mean across replicates. Horizontal lines indicate tests for significant enrichment of viral RNA over bee RNA in the MRJP-3 bound fraction, but not in RJ ( ∗ p

    Journal: Molecular Cell

    Article Title: A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly

    doi: 10.1016/j.molcel.2019.03.010

    Figure Lengend Snippet: Multivalent RNA Binding Stimulates Super-order Assembly of Dynamic MRJP-3 RNPs and Isolation of RJ RNA Partners of MRJP-3 (A) The multivalent interaction of MRJP-3 with RNA is reversible. MRJP-3-bound ssRNA ∗ was introduced to increasing quantities of unlabeled ssRNA. ssRNA ∗ (0.04 μM) and MRJP-3 (31.3 μM) were used in all ssRNA ∗ - and protein-containing treatments. Unlabeled DNA ladder served as labeling control. (B) MRJP-3 RNPs are affected by the protein/ssRNA mole ratio. Images of RNPs formed at various mole ratios of MRJP-3 and Alexa Fluor-488 labeled ssRNA (ssRNA ∗ ). Scale bar represents 10 μm. (C) RNA mediates super-order assembly of MRJP-3 oligomers, resulting in RNP formation in soluble RJ fraction. RJ buffer or 4.28 μM Alexa Fluor-633 labeled MRJP-3 (MRJP-3 ∗ ) was introduced to 50% soluble RJ fraction. ssRNA (0.15 μM) or ssRNA ∗ was used in RNA-containing treatments. Scale bar represents 2 μm. (D) MRJP-3 binds both endogenous ( Apis mellifera ) and exogenous (viral) RNA. Only viruses with a mapped fraction of at least 1% are shown. Points are individual biological replicates. Bars represent the mean across replicates. Horizontal lines indicate tests for significant enrichment of viral RNA over bee RNA in the MRJP-3 bound fraction, but not in RJ ( ∗ p

    Article Snippet: Immunostaining was performed by the Dako Autostainer Link 48 with the Envision Flex kit system (Dako) according to the manufacturer’s instructions using 1:250 diluted Alexa Fluor-488 antibody (Thermo Fisher, Cat. A-11094).

    Techniques: RNA Binding Assay, Isolation, Labeling

    The Honeybee Jelly Harbors RNA-Binding Activity (A) Experimental design for RNA detection in RJ. Hives were fed with a 10% sucrose solution with or without the addition of Alexa Fluor-488-labeled dsRNA (dsRNA ∗ ). (B) Immunohistochemistry-based detection of dsRNA ∗ in RJ samples, which were reacted with Alexa Fluor-488 antibody. Scale bar represents 25 μm. (C) RJ proteins bind dsRNA. dsRNA-binding activity was tested using EMSA. Treatments included dsRNA mixed in RJ buffer, 10% RJ mixed with dsRNA, 10% RJ digested by Proteinase K (PK) and then mixed with dsRNA, 10% RJ mixed with dsRNA and then digested by PK, 10% RJ mixed with dsRNA and PK buffer, 27.3 μM purified BSA mixed with dsRNA, 10% RJ only, and 10% RJ only digested by PK. dsRNA (0.05 μM) was applied in all dsRNA-containing treatments. (D) Precipitation dynamics of dsRNA-protein complexes in RJ. Two percent RJ was mixed with increasing dsRNA concentrations. (E) MRJP-3 and its prion-like TRR. Amino acid sequence in bold: secretion signal peptide. Amino acid sequence highlighted in color: tandem repeats. Alignment of the tandem repeats, QN (in gray) and positively charged amino acids (in red). .

    Journal: Molecular Cell

    Article Title: A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly

    doi: 10.1016/j.molcel.2019.03.010

    Figure Lengend Snippet: The Honeybee Jelly Harbors RNA-Binding Activity (A) Experimental design for RNA detection in RJ. Hives were fed with a 10% sucrose solution with or without the addition of Alexa Fluor-488-labeled dsRNA (dsRNA ∗ ). (B) Immunohistochemistry-based detection of dsRNA ∗ in RJ samples, which were reacted with Alexa Fluor-488 antibody. Scale bar represents 25 μm. (C) RJ proteins bind dsRNA. dsRNA-binding activity was tested using EMSA. Treatments included dsRNA mixed in RJ buffer, 10% RJ mixed with dsRNA, 10% RJ digested by Proteinase K (PK) and then mixed with dsRNA, 10% RJ mixed with dsRNA and then digested by PK, 10% RJ mixed with dsRNA and PK buffer, 27.3 μM purified BSA mixed with dsRNA, 10% RJ only, and 10% RJ only digested by PK. dsRNA (0.05 μM) was applied in all dsRNA-containing treatments. (D) Precipitation dynamics of dsRNA-protein complexes in RJ. Two percent RJ was mixed with increasing dsRNA concentrations. (E) MRJP-3 and its prion-like TRR. Amino acid sequence in bold: secretion signal peptide. Amino acid sequence highlighted in color: tandem repeats. Alignment of the tandem repeats, QN (in gray) and positively charged amino acids (in red). .

    Article Snippet: Immunostaining was performed by the Dako Autostainer Link 48 with the Envision Flex kit system (Dako) according to the manufacturer’s instructions using 1:250 diluted Alexa Fluor-488 antibody (Thermo Fisher, Cat. A-11094).

    Techniques: RNA Binding Assay, Activity Assay, RNA Detection, Labeling, Immunohistochemistry, Binding Assay, Purification, Sequencing

    MRJP-3 Is a Multivalent RNA-Binding Oligomer (A) Taxonomy tree analysis suggests that the MRJP-3 tandem-repeats region evolved in the genus Apis and is associated with jelly secretion. (B) Purified MRJP-3 binds dsRNA and ssRNA as demonstrated by EMSA. MRJP-3 was incubated with increasing concentrations of dsRNA or ssRNA. Additional controls: dsRNA and ssRNA only, MRJP-1 mixed with 43.1 nM dsRNA or 0.3 μM ssRNA, and MRJP-3 only. Protein (42.8 μM) was applied in all MRJP-3- or MRJP-1-containing treatments. (C) MRJP-3 efficiently binds ssRNA that is 18 nt and longer. Binding activity was tested using ssRNA substrates with different lengths and analyzed by EMSA. ssRNA (19.4 pmol) and proteins (42.8 μM) were applied in all ssRNA- and/or protein-containing treatments. (D) Binding curves of Alexa Fluor-488-labeled 22 nt ssRNA and dsRNA to MRJP-3 in RJ buffer conditions (left and right curves, respectively). Calculated and estimated equilibrium K d values are shown as dashed lines. (E) The TRR of MRJP-3 is predicted to be intrinsically disordered by the PONDR VSL2 and IUPred algorithms. (F) The TRR of MRJP-3 is required for RNP formation. Proteins (13.6 μM) and ssRNA ∗ (0.2 μM) were applied in all RNA- and/or protein-containing treatments. Scale bar represents 1 μm. .

    Journal: Molecular Cell

    Article Title: A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly

    doi: 10.1016/j.molcel.2019.03.010

    Figure Lengend Snippet: MRJP-3 Is a Multivalent RNA-Binding Oligomer (A) Taxonomy tree analysis suggests that the MRJP-3 tandem-repeats region evolved in the genus Apis and is associated with jelly secretion. (B) Purified MRJP-3 binds dsRNA and ssRNA as demonstrated by EMSA. MRJP-3 was incubated with increasing concentrations of dsRNA or ssRNA. Additional controls: dsRNA and ssRNA only, MRJP-1 mixed with 43.1 nM dsRNA or 0.3 μM ssRNA, and MRJP-3 only. Protein (42.8 μM) was applied in all MRJP-3- or MRJP-1-containing treatments. (C) MRJP-3 efficiently binds ssRNA that is 18 nt and longer. Binding activity was tested using ssRNA substrates with different lengths and analyzed by EMSA. ssRNA (19.4 pmol) and proteins (42.8 μM) were applied in all ssRNA- and/or protein-containing treatments. (D) Binding curves of Alexa Fluor-488-labeled 22 nt ssRNA and dsRNA to MRJP-3 in RJ buffer conditions (left and right curves, respectively). Calculated and estimated equilibrium K d values are shown as dashed lines. (E) The TRR of MRJP-3 is predicted to be intrinsically disordered by the PONDR VSL2 and IUPred algorithms. (F) The TRR of MRJP-3 is required for RNP formation. Proteins (13.6 μM) and ssRNA ∗ (0.2 μM) were applied in all RNA- and/or protein-containing treatments. Scale bar represents 1 μm. .

    Article Snippet: Immunostaining was performed by the Dako Autostainer Link 48 with the Envision Flex kit system (Dako) according to the manufacturer’s instructions using 1:250 diluted Alexa Fluor-488 antibody (Thermo Fisher, Cat. A-11094).

    Techniques: RNA Binding Assay, Purification, Incubation, Binding Assay, Activity Assay, Labeling

    NPM T199A suppresses centrosome amplification and chromosome instability. (A) Passage 2 p53 − / − MEFs were transiently transfected with plasmids encoding FLAG epitope-tagged NPM and the NPM/B23 mutant (NPM T99A ). As a control, an empty vector was transfected. After neomycin selection, cell lysates were obtained and then probed with anti-FLAG antibodies. (B) The transfectants described in the legend to panel A were fixed, immunostained with anti-γ-tubulin polyclonal antibodies, and detected with Alexa Fluor 488-conjugated secondary antibodies. Cells were counterstained with DAPI. The number of cells with ≥3 centrosomes in a population of at least 200 cells was statistically analyzed by fluorescence microscopy. Each group included three transfected MEFs. P values (relative to the result for the transfected vector control) were 0.006963 for NPM T199A and 0.560677 for wild-type NPM. (C) Proliferating E13.5 mouse embryonic fibroblasts of the indicated genotypes were fixed, and nuclei were visualized with DAPI. The frequencies of micronucleus formation in a population of 500 cells were calculated for the indicated genotypes. P values (relative to the result for the transfected vector control) were 0.011338 for NPM T199A and 0.353737 for wild-type NPM. (D) Frequencies of γ-H2AX foci in cells with the indicated genotypes were calculated. P values (relative to the result for the transfected vector control) were 0.002591 for NPM T199A and 0.476327 for wild-type NPM.

    Journal: Molecular and Cellular Biology

    Article Title: Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical Mediators of Centrosome Amplification in p53-Null Cells ▿

    doi: 10.1128/MCB.00253-09

    Figure Lengend Snippet: NPM T199A suppresses centrosome amplification and chromosome instability. (A) Passage 2 p53 − / − MEFs were transiently transfected with plasmids encoding FLAG epitope-tagged NPM and the NPM/B23 mutant (NPM T99A ). As a control, an empty vector was transfected. After neomycin selection, cell lysates were obtained and then probed with anti-FLAG antibodies. (B) The transfectants described in the legend to panel A were fixed, immunostained with anti-γ-tubulin polyclonal antibodies, and detected with Alexa Fluor 488-conjugated secondary antibodies. Cells were counterstained with DAPI. The number of cells with ≥3 centrosomes in a population of at least 200 cells was statistically analyzed by fluorescence microscopy. Each group included three transfected MEFs. P values (relative to the result for the transfected vector control) were 0.006963 for NPM T199A and 0.560677 for wild-type NPM. (C) Proliferating E13.5 mouse embryonic fibroblasts of the indicated genotypes were fixed, and nuclei were visualized with DAPI. The frequencies of micronucleus formation in a population of 500 cells were calculated for the indicated genotypes. P values (relative to the result for the transfected vector control) were 0.011338 for NPM T199A and 0.353737 for wild-type NPM. (D) Frequencies of γ-H2AX foci in cells with the indicated genotypes were calculated. P values (relative to the result for the transfected vector control) were 0.002591 for NPM T199A and 0.476327 for wild-type NPM.

    Article Snippet: Chromosome breaks were detected using phosphorylated histone 2A variant X (γ-H2AX) (07-164; Upstate) with the appropriate Alexa Fluor-conjugated antibodies (Molecular Probes).

    Techniques: Amplification, Transfection, FLAG-tag, Mutagenesis, Plasmid Preparation, Selection, Fluorescence, Microscopy

    Endocytosis of NogoΔ20 is required for NogoΔ20-induced growth cone collapse in hippocampal neurons. (A and B) Morphology of noncollapsed (A, arrow) and collapsed growth cones (B, arrowhead) of E19 hippocampal neurons at DIV 4 was visualized by staining of F-actin with phalloidin–Alexa Fluor 488 (green). (C) Quantification of the proportion of collapsed growth cones after incubation with 300 nM NogoΔ20 (shaded bars) or with 300 nM NogoΔ21 (open bars). (D and E) The morphology of hippocampal neurons infected with immunodeficient recombinant adenovirus containing HA-tagged wt Pincher (D, red) or HA-tagged dn PincherG68E (E) upon treatment for 30 min with 300 nM NogoΔ20 was visualized with phalloidin (green). (F) Most growth cones remained uncollapsed when HA-tagged dn PincherG68E was overexpressed (open bars) compared with wt Pincher (shaded bars). (G and H) Growth cone morphology of hippocampal neurons upon treatment with 40 nM semaphorin 3A overexpressing either wt HA-Pincher protein (G) or dn HA-PincherG68E protein (H). (I) Treatment with 40 nM semaphorin 3A for 30 min leads to growth cone collapse in the presence of both wt HA-Pincher (shaded bars) and dn HA-Pincherg68E (open bars). Data represent the mean of three (semaphorin 3A) or four (NogoΔ20) independent experiments ± SEM (90 neurons per group and experiment). Asterisks mark highly significant differences between wt and dn Pincher-infected hippocampal neurons (***, P

    Journal: The Journal of Cell Biology

    Article Title: Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling

    doi: 10.1083/jcb.200906089

    Figure Lengend Snippet: Endocytosis of NogoΔ20 is required for NogoΔ20-induced growth cone collapse in hippocampal neurons. (A and B) Morphology of noncollapsed (A, arrow) and collapsed growth cones (B, arrowhead) of E19 hippocampal neurons at DIV 4 was visualized by staining of F-actin with phalloidin–Alexa Fluor 488 (green). (C) Quantification of the proportion of collapsed growth cones after incubation with 300 nM NogoΔ20 (shaded bars) or with 300 nM NogoΔ21 (open bars). (D and E) The morphology of hippocampal neurons infected with immunodeficient recombinant adenovirus containing HA-tagged wt Pincher (D, red) or HA-tagged dn PincherG68E (E) upon treatment for 30 min with 300 nM NogoΔ20 was visualized with phalloidin (green). (F) Most growth cones remained uncollapsed when HA-tagged dn PincherG68E was overexpressed (open bars) compared with wt Pincher (shaded bars). (G and H) Growth cone morphology of hippocampal neurons upon treatment with 40 nM semaphorin 3A overexpressing either wt HA-Pincher protein (G) or dn HA-PincherG68E protein (H). (I) Treatment with 40 nM semaphorin 3A for 30 min leads to growth cone collapse in the presence of both wt HA-Pincher (shaded bars) and dn HA-Pincherg68E (open bars). Data represent the mean of three (semaphorin 3A) or four (NogoΔ20) independent experiments ± SEM (90 neurons per group and experiment). Asterisks mark highly significant differences between wt and dn Pincher-infected hippocampal neurons (***, P

    Article Snippet: Secondary antibodies used were Alexa Fluor 488, Alexa Fluor 594, avidin-rhodamin, and avidin-FITC (all from Invitrogen).

    Techniques: Staining, Incubation, Infection, Recombinant