Journal: BioMed Research International

Article Title: Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

doi: 10.1155/2015/747864

Figure Lengend Snippet: Detection of Receptor Tyrosine Kinases on HL-LECs (P4). (a) and (b) (immunofluorescence): green fluorescence corresponds to the surface expression of vascular endothelial growth factor receptor- (VEGFR-) 2 and VEGFR-3, respectively, in cultured HL-LECs. (c): representative image of a negative control in which the primary antibody was omitted from the reaction. Nuclei are shown by the blue fluorescence of DAPI. (d)–(j) (immunoperoxidase): the brownish precipitate on the surface of HL-LECs on each panel corresponds respectively to the expression of Platelet-Derived Growth Factor Receptor- (PDGFR-) beta, Epidermal Growth Factor Receptor- (EGFR-) 1, Fibroblast Growth Factor Receptor- (FGFR-) 1, type 1 Insulin-like Growth Factor Receptor (IGF-1R), the Hepatocyte Growth Factor receptor c-MET, Tropomyosin-related kinases A (TrkA), and neurotrophin p75 receptor (p75 NTR ). (k): representative image of a negative control in which the primary antibody was omitted from the reaction. Nuclei are counterstained with Haematoxylin. Scale bars: (a), (d), (e), (f), (g), (h), (i), (j), and (k) = 50 μ m; (b) and (c) = 250 μ m.

Article Snippet: Briefly, after fixation, chamber slides of HL-LECs were immersed in 3% hydrogen peroxide solution for 10 minutes and then incubated with mouse anti-human D2-40 (1 : 50; o/n 4°C; Biocare Medical, Concord, CA, USA), rabbit anti-human LYVE-1 (1 : 50; o/n 4°C; Abcam, Cambridge, UK), rabbit anti-human FGFR-1 (1 : 40; o/n 4°C; Cell Signaling, Beverly, MA, USA), mouse anti-human EGFR (1 : 40; o/n 4°C; Zymed-Invitrogen, Grand Island, NY, USA), rabbit anti-human c-MET (according to manufacturer's recommendations; Ventana-Roche), rabbit anti-human IGF-1R (1 : 30; o/n 4°C; Cell Signaling), rabbit anti-human PDGFR-beta (1 : 25; o/n 4°C; Abcam), rabbit anti-human TrkA (1 : 40; o/n 4°C; Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit anti-human p75 NTR (1 : 70; o/n 4°C; Millipore, Darmstadt, Germany), respectively.

Techniques: Immunofluorescence, Fluorescence, Expressing, Cell Culture, Negative Control, Derivative Assay

Journal: Frontiers in Endocrinology

Article Title: Bifidobacterium adolescentis Alleviates Liver Steatosis and Steatohepatitis by Increasing Fibroblast Growth Factor 21 Sensitivity

doi: 10.3389/fendo.2021.773340

Figure Lengend Snippet: Primers for RT-qPCR.

Article Snippet: The primary antibodies used for western blotting were against Phospho-NF-κBp65 (Ser536) (3033, Cell Signaling Technology), NF-κBp65(8242, Cell Signaling Technology), KLB (AF2619, R&D), FGFR1 (12511, Cell Signaling Technology), extracellular signal−regulated protein kinase 1/2 (Erk1/2, 9102, Cell Signaling Technology), phosphorylated (p)- Erk1/2 (Thr202/Tyr204) (9101, Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH,5174, Cell Signaling Technology).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Bifidobacterium adolescentis Alleviates Liver Steatosis and Steatohepatitis by Increasing Fibroblast Growth Factor 21 Sensitivity

doi: 10.3389/fendo.2021.773340

Figure Lengend Snippet: B. adolescentis supplementation alleviated FGF21 resistance. Mice were grouped and treated as in Figure 1 . (A) The serum level of FGF21 (n = 6 in each group). (B) FGF21 levels in liver measured by ELISA (n = 6 in each group). (C) The transcription of FGFR1 in liver (n = 6 in each group). (D) The transcription of KLB in liver (n = 6 in each group). (E) The expression of FGFR1 and β-Klotho in liver detected by western blot analysis. (F) Quantification of the FGFR1 expression normalized to GAPDH (n = 3). (G) Quantification of the KLB expression normalized to GAPDH (n = 3). (H) Experiment schematics for FGF21 response test. The experiment schematic was created using BioRender.com . (I) Phosphorylation of Erk1/2 (Thr202/Tyr204) before and after rmFGF21 administration (2mg/kg) detected by western blot analysis. (J) Quantification analysis of the fold change of Erk1/2 phosphorylation (n = 4-6). FGF21, fibroblast growth factor; FGFR1, Fibroblast growth factor receptor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KLB, β-klotho; rmFGF21, recombinant mouse fibroblast growth factor 21. (K) The transcription of FGFR1 in hep1-6 after the treatment with LPS. (L) The transcription of KLB in hep1-6 after the treatment with LPS. Data are presented as mean ± SEM. Significance was determined by one-way ANOVA with Fisher’s LSD multiple-comparison analysis. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The primary antibodies used for western blotting were against Phospho-NF-κBp65 (Ser536) (3033, Cell Signaling Technology), NF-κBp65(8242, Cell Signaling Technology), KLB (AF2619, R&D), FGFR1 (12511, Cell Signaling Technology), extracellular signal−regulated protein kinase 1/2 (Erk1/2, 9102, Cell Signaling Technology), phosphorylated (p)- Erk1/2 (Thr202/Tyr204) (9101, Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH,5174, Cell Signaling Technology).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Recombinant

Journal: The EMBO Journal

Article Title: Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

doi: 10.15252/embj.2020107182

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal FGFR1 antibody D8E4 , Cell Signaling Technology , 9740.

Techniques: Recombinant, Transfection, Molecular Weight, Marker, Protease Inhibitor, Cell Culture, Spectroscopy, Imaging, Plasmid Preparation, Negative Control, Mutagenesis, Clone Assay, Generated, Software, Microscopy

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: U3DT cells at different stages of mitosis and cytokinesis were fixed but not permeabilized, stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies, and counterstained with DAPI stain (blue). Yellow represents the degree of colocalization. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A‒D) Images of staining with Alexa Fluor 488-conjugated WGA (green) and DAPI (blue) at interphase (A), metaphase (B), and anaphase and telophase (C, D). (D) Maximum projection of 15 Z-staged-images stained with Alexa Fluor 488-WGA and DAPI. (E) U3DT cell at telophase, stained with anti-GPC5 antibody (red), Alexa Fluor 488-WGA (green), and DAPI (blue). (F) Blebs of U3DT cell at telophase stained with anti-FGFR1 (brown), Alexa Fluor 488-WGA (green), and DAPI (blue). (G) Blebs of U3DT cells at telophase stained with anti-Rab11 rabbit antibodies (magenta), Alexa Fluor 488-WGA (green), and DAPI (blue). Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Electron microscopic image of negative-stained EVs. The inset shows a magnification of the boxed EV. (B) Size distribution of vesicles in EV preparations measured by image software (n = 110). (C) (F) (I) (L) GPC5-immunostained EVs (red) stained for Rab11 (green) (C), FGFR1 (green) (F), CD63 (green) (I), and ARF6 (green) (L). In (I) and (L), an Alexa Fluor 647- conjugated anti-GPC5 antibody was used to detect of GPC5-ositive particles. (D) (G) (J) (M) Line scan determination of the red bars in (C), (F), (I), and (L): GPC5 (red), others (green). (E) (H) (K) (N) Distribution of GPC5-positive particles identified by scan determinations in (D), (G), (J), and (M). n = 1,516, (E), 630 (H), 573 (K), and 835 (N). Scale bars: (A), 500 nm; (C), (F), (I), (L), 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining, Software

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A, D) Images of control cells. (B, E) Images of EV-treated cells. (D, E) Merged images of UE6E7T-3 cells (D) and EV-treated cells (E) were stained for GPC5 (red) and FGFR1 (green). (C) Quantitation of GPC5 (pixel sum per cell) in control UE6E7T-3 cells (blue) or cells cultured with EVs for 1 day (red). n = 67 (blue) and 78 (red). (F, G) FACS pattern of GPC5 in UE6E7T-3 cells (F) and in cells cultured with EVs for 1 day (G). ⇔ indicates GPC5-positive cells. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining, Quantitation Assay, Cell Culture

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Control cells fixed immediately after scratching. The wide of the scratched area was ca. 500μm. (B‒D) Mock (B), non-targeting siRNA (NT)-treated (C), and GPC5-siRNA (KD)-treated (D) cells were cultured in appropriate medium containing 25 nM FGF2 for 23 h after scratching. Cells were fixed and immunofluorescence images were acquired using a Leica SP-8 microscope equipped with a 20x objective. (E) The number of cells that moved into the scratched area or the removed insert area was counted after incubation of control (without siRNA) cells (yellow), NT cells (red), and KD cells (blue) in appropriate medium containing (red and blue columns) or lacking (brown and sky blue columns) FGF2 at 37°C for 23 h. (F‒H) Immunofluorescence images of cells treated with mock (F), non-targeting siRNA (G), or GPC5-targeting siRNA (H) for 72 h after removing an insert from a μ -Dish. Cells were stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies. (I) Quantification of the percentage of cells with blebs at telophase. Control (mock) cells (yellow), NT cells (red) and KD cells (blue) were incubated in appropriate medium containing FGF2 at 37°C for 23 h. (J‒L) Immunofluorescence images of mock (J), non-targeting siRNA-treated (K), and GPC5-siRNA-treated (L) cells after 72 h. Cells were stained with WGA (green). White arrows indicate blebs in a telophase cell (J, K). The numbers in parentheses in (E, I) are the number of images observed. Scale bar: (F‒H), 100 μm; (J‒L), 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Cell Culture, Immunofluorescence, Microscopy, Incubation, Staining

Journal: eLife

Article Title: Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

doi: 10.7554/eLife.52027

Figure Lengend Snippet: ( A ) Schematic diagram of optogenetically-driven Nlgn1 tyrosine phosphorylation using optoFGFR1. Phosphorylated Nlgn1 is expected to recruit PSD-95 that serves as a platform for trapping AMPARs. ( B ) Scheme representing the 470 LED array that is placed in the incubator and used to illuminate COS-7 cells or organotypic slices contained in a six-well plate. ( C ) pTyr and Nlgn1 immunoblots of proteins extracted from COS-7 cells and immunoprecipitated with anti-Nlgn1 antibodies. Cells expressed either no Nlgn1, Nlgn1 alone, Nlgn1 + constitutively active (CA) FGFR1, Nlgn1 + optoFGFR1, and Nlgn1-Y782F + optoFGFR1. In the first lane, the starting material (SM) from non-transfected cells reveals numerous tyrosine phosphorylated proteins, whereas a single band is present in the Nlgn1 IP samples (black arrowhead). Cells were either kept in the dark (- light), or exposed to alternative 470 nm light and pulses (1 s light pulse every 1 s) for 15 min (+ light). ( D ) Corresponding starting material immunoblotted with HA and FGFR1 antibodies, respectively. The arrowheads represent HA-tagged Nlgn1, HA-tagged optoFGFR1, or constitutively active FGFR1.

Article Snippet: After blocking with 5% non-fat dried milk in Tris-buffered saline Tween-20 (TBST; 28 mM Tris, 137 mM NaCl, 0.05% Tween-20, pH 7.4) for 45 min at room temperature, membranes were probed for 1 hr at room temperature or overnight at 4°C with mouse anti-phosphotyrosine (1:1000, Cell Signaling Technology 9411S), rabbit anti-Nlgn1 (1:1000, Synaptic systems 129013), rabbit anti-FGFR1 (1:1000, Cell Signaling Technology D8E4), or rat anti-HA (1:1000, Roche 3F10).

Techniques: Western Blot, Immunoprecipitation, Transfection

Journal: eLife

Article Title: Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

doi: 10.7554/eLife.52027

Figure Lengend Snippet: CA1 Neurons were co-electroporated with plasmids encoding tdTomato, shRNA against endogenous Nlgn1 containing a GFP reporter, shRNA resistant AP-tagged Nlgn1 -WT or -Y782F, biotin ligase (BirA ER ), and HA-tagged opto Fgfr1 . ( A, B ) Confocal images showing tdTomato (red) and GFP (green). Biotinylated Nlgn1 and optoFGFR1 were stained in different slices using streptavidin-Atto647 and anti-HA antibody, respectively (magenta). ( C, D ) Scatter plots of AMPAR- and NMDAR-mediated EPSCs, respectively, for electroporated versus paired non-electroporated neurons (control cell) in the indicated conditions. Representative traces (black, blue or violet) normalized to control (grey) are shown as insets. ( E, F ) Average of AMPAR-and NMDAR-mediated EPSCs, respectively, normalized to the control (100%) in the different conditions. Data were compared to the control condition by the Wilcoxon matched-pairs signed rank test and between themselves using one-way ANOVA followed by Tukey's multiple comparison (**p<0.01, ns: not significant).

Article Snippet: After blocking with 5% non-fat dried milk in Tris-buffered saline Tween-20 (TBST; 28 mM Tris, 137 mM NaCl, 0.05% Tween-20, pH 7.4) for 45 min at room temperature, membranes were probed for 1 hr at room temperature or overnight at 4°C with mouse anti-phosphotyrosine (1:1000, Cell Signaling Technology 9411S), rabbit anti-Nlgn1 (1:1000, Synaptic systems 129013), rabbit anti-FGFR1 (1:1000, Cell Signaling Technology D8E4), or rat anti-HA (1:1000, Roche 3F10).

Techniques: shRNA, Staining

Journal: eLife

Article Title: Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

doi: 10.7554/eLife.52027

Figure Lengend Snippet:

Article Snippet: After blocking with 5% non-fat dried milk in Tris-buffered saline Tween-20 (TBST; 28 mM Tris, 137 mM NaCl, 0.05% Tween-20, pH 7.4) for 45 min at room temperature, membranes were probed for 1 hr at room temperature or overnight at 4°C with mouse anti-phosphotyrosine (1:1000, Cell Signaling Technology 9411S), rabbit anti-Nlgn1 (1:1000, Synaptic systems 129013), rabbit anti-FGFR1 (1:1000, Cell Signaling Technology D8E4), or rat anti-HA (1:1000, Roche 3F10).

Techniques: Transfection, Construct, Clone Assay, Plasmid Preparation, Electroporation, shRNA, Recombinant, Sequencing, Immunoprecipitation, Software

Journal: bioRxiv

Article Title: Subcellular localization of glypican-5 associates with dynamic cell motility and cell communication of the human mesenchymal stem cell line U3DT

doi: 10.1101/863720

Figure Lengend Snippet: (A) U3DT cells at different stages of mitosis and cytokinesis were fixed but not permeabilized, stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies, and counterstained with DAPI stain (blue). Yellow represents the degree of colocalization. Scale bar, 5 μm. (B) Cells expressing the same marker as in (A) but with permeabilized cells. Scale bar, 5 μm.

Article Snippet: Antibodies and fluorescence-conjugated reagent used in this study are as follows; Anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594-conjucated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647-conjucated anti-GPC5 antibody (R&D Systems, Inc.), Anti-FGFR1 Xp Rabbit mAb (D8E4, Cell Signaling Tec.), Anti-Rab 11A antibody (A-6: sc-166912, Santa Cruz Biotech), Anti-acetyl-alpha-tubulin antibody (D20G3, #5335, Cell Signaling Tec.) rabbit mAb, Alexa Fluor 488-conjucated Wheat germ agglutinin (W1126: Invitrogen), Anti-CD63 (MX-49.129.5: sc-5275, Santa Cruz Biotech), Anti-CD63 (cl 3-13: Fuji Film Co.), Anti-ARF6 (3A-1: sc-7971, Santa Cruz Biotech,), Anti-FGFR-1 (D8E4) XP Rabbit mAb (Cell Signaling).

Techniques: Staining, Expressing, Marker

Journal: bioRxiv

Article Title: Subcellular localization of glypican-5 associates with dynamic cell motility and cell communication of the human mesenchymal stem cell line U3DT

doi: 10.1101/863720

Figure Lengend Snippet: (A–D) Images of staining with Alexa Fluor 488-conjugated WGA and DAPI at interphase (A), metaphase (B), and anaphase and telophase (C–D). (D) Maximum projection of 15 Z-staged-images stained with Alexa Fluor 488-WGA and DAPI. (E) U3DT cell at telophase, stained with anti-GPC5 antibody (red) and Alexa Fluor 488-WGA (green). (F) Blebs of U3DT cell at telophase stained with anti-FGFR1 (brown), Alexa Fluor 488-WGA (green), and DAPI (blue). (G) Blebs of U3DT cells at telophase stained with anti-Rab11A antibodies (magenta), Alexa Fluor 488-WGA (green), and DAPI (blue). Scale bar, 5 μm.

Article Snippet: Antibodies and fluorescence-conjugated reagent used in this study are as follows; Anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594-conjucated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647-conjucated anti-GPC5 antibody (R&D Systems, Inc.), Anti-FGFR1 Xp Rabbit mAb (D8E4, Cell Signaling Tec.), Anti-Rab 11A antibody (A-6: sc-166912, Santa Cruz Biotech), Anti-acetyl-alpha-tubulin antibody (D20G3, #5335, Cell Signaling Tec.) rabbit mAb, Alexa Fluor 488-conjucated Wheat germ agglutinin (W1126: Invitrogen), Anti-CD63 (MX-49.129.5: sc-5275, Santa Cruz Biotech), Anti-CD63 (cl 3-13: Fuji Film Co.), Anti-ARF6 (3A-1: sc-7971, Santa Cruz Biotech,), Anti-FGFR-1 (D8E4) XP Rabbit mAb (Cell Signaling).

Techniques: Staining

Journal: bioRxiv

Article Title: Subcellular localization of glypican-5 associates with dynamic cell motility and cell communication of the human mesenchymal stem cell line U3DT

doi: 10.1101/863720

Figure Lengend Snippet: (A) Electron microscopic image of negative-stained EVs. The inset shows a magnification of the boxed EV. (B) Size distribution of vesicles in EV preparations measured by image software (n = 110). (C) (F) (I) (L) GPC5-immunostained EVs (red) stained for Rab11 (green) (C), FGFR1 (green) (F), CD63 (green) (I), and ARF6 (green) (L). (D) (G) (J) (M) Line scan determination of the red bars in (C), (F), (I), and (L): GPC5 (red), others (green). (E) (H) (K) (N) Quantification of scan determinations in (D), (G), (J), and (M). n = 1,516, (E), 630 (H), 704 (K), and 406 (N). Scale bars: (A), 500 nm; (C), (F), (I), (L), 2 μm.

Article Snippet: Antibodies and fluorescence-conjugated reagent used in this study are as follows; Anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594-conjucated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647-conjucated anti-GPC5 antibody (R&D Systems, Inc.), Anti-FGFR1 Xp Rabbit mAb (D8E4, Cell Signaling Tec.), Anti-Rab 11A antibody (A-6: sc-166912, Santa Cruz Biotech), Anti-acetyl-alpha-tubulin antibody (D20G3, #5335, Cell Signaling Tec.) rabbit mAb, Alexa Fluor 488-conjucated Wheat germ agglutinin (W1126: Invitrogen), Anti-CD63 (MX-49.129.5: sc-5275, Santa Cruz Biotech), Anti-CD63 (cl 3-13: Fuji Film Co.), Anti-ARF6 (3A-1: sc-7971, Santa Cruz Biotech,), Anti-FGFR-1 (D8E4) XP Rabbit mAb (Cell Signaling).

Techniques: Staining, Software

Journal: bioRxiv

Article Title: Subcellular localization of glypican-5 associates with dynamic cell motility and cell communication of the human mesenchymal stem cell line U3DT

doi: 10.1101/863720

Figure Lengend Snippet: (A–B) Images of control cells. (C–D) Images of EV-treated cells. (B) (D) Merged images of UE6E7T-3 cells (B) and EV-treated cells (D) stained for GPC5 (red) and FGFR1 (green). (E) Histogram of GPC5 (pixel sum per cell) in control UE6E7T-3 cells (blue) or of cells cultured with EVs for 1 day (red). n = 29 (blue) and 46 (red). (F–G) FACS pattern of GPC5 in UE6E7T-3 cells (F) and in cells cultured with EVs for 1 day (G). Scale bar, 5 μm.

Article Snippet: Antibodies and fluorescence-conjugated reagent used in this study are as follows; Anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594-conjucated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647-conjucated anti-GPC5 antibody (R&D Systems, Inc.), Anti-FGFR1 Xp Rabbit mAb (D8E4, Cell Signaling Tec.), Anti-Rab 11A antibody (A-6: sc-166912, Santa Cruz Biotech), Anti-acetyl-alpha-tubulin antibody (D20G3, #5335, Cell Signaling Tec.) rabbit mAb, Alexa Fluor 488-conjucated Wheat germ agglutinin (W1126: Invitrogen), Anti-CD63 (MX-49.129.5: sc-5275, Santa Cruz Biotech), Anti-CD63 (cl 3-13: Fuji Film Co.), Anti-ARF6 (3A-1: sc-7971, Santa Cruz Biotech,), Anti-FGFR-1 (D8E4) XP Rabbit mAb (Cell Signaling).

Techniques: Staining, Cell Culture