Journal: Acta Neuropathologica Communications

Article Title: Anti-tau conformational scFv MC1 antibody efficiently reduces pathological tau species in adult JNPL3 mice

doi: 10.1186/s40478-018-0585-2

Figure Lengend Snippet: AAV5-scFv-MC1 transduction in vivo . a Representative images of JNPL3 mice brain sections, 4 weeks post injection ( n = 6): the AAV5-scFv-MC1 injected brain showed clear expression of the recombinant antibody in the hippocampus using an anti-Myc antibody (red). Nuclei were stained with DAPI (blue). No signal was detected in the not injected brains (Zeiss Axio Imager Apotome, bar: 500 μm). b western blotting on hippocampus lysates of injected (Inj.) or not-injected (Not Inj.) JNPL3 mice, using anti-Myc antibody. Neuronal N-cadherin was used as a marker of neuronal integrity, and actin was used as a housekeeper

Article Snippet: Mouse anti-N-Cadherin A60 (BD, Franklin Lakes, NJ), mouse anti-actin Ab5 (BD) were diluted 1:5000; anti-Myc-tag 9B11 (Cell Signaling, Danvers, MA) was diluted 1:1000.

Techniques: Transduction, In Vivo, Injection, Expressing, Recombinant, Staining, Western Blot, Marker

Journal: Acta Neuropathologica Communications

Article Title: Anti-tau conformational scFv MC1 antibody efficiently reduces pathological tau species in adult JNPL3 mice

doi: 10.1186/s40478-018-0585-2

Figure Lengend Snippet: Brain parenchyma expression and diffusibility. ScFv-MC1 is expressed and released in the extracellular milieu: anti Myc-tag staining performed 4 months after the one time intracranial injection with AAV5-eGFP ( a-g ) ( n = 15), AAV5-CAG-scFvMC1 ( h-n ) (n = 15) and AAV5-GFAP-scFvMC1 ( o-u ) (n = 15). Representative images from the whole hippocampus (Olympus BH-2 bright field microscope, bar: 500 μm), CA1, subiculum, dentate gyrus (DG), entorhinal cortex (EC), frontal cortex and HB (bar: 100 μm). The scFv is visualized as a brown signal and shows extensive expression in hippocampi from treated mice, with spreading in areas distant from the site of injection. ( v ) Immunoblot analysis of lysates from the hippocampus, cortex and hindbrain: AAV5-eGFP injected (1), AAV5-CAG-scFMC1 injected (2), AAV5-GFAP-scFvMC1 injected (3). Three representative lysates from each group of treatment were pulled together and loaded on SDS-PAGE (40μg proteins of hippocampus; 300μg proteins of cortex; 150μg proteins of HB plus HRP substrate enhancer when developing). Anti Myc-tag was used to track the scFv in the brain parenchyma; actin was used as a housekeeper and N-cadherin as a marker of neuronal integrity. Of note, immunoblots images shown represent optimal exposure samples to visualize differences within the brain areas

Article Snippet: Mouse anti-N-Cadherin A60 (BD, Franklin Lakes, NJ), mouse anti-actin Ab5 (BD) were diluted 1:5000; anti-Myc-tag 9B11 (Cell Signaling, Danvers, MA) was diluted 1:1000.

Techniques: Expressing, Staining, Injection, Microscopy, Western Blot, SDS Page, Marker

Journal: International Journal of Molecular Sciences

Article Title: Inactivation of PTEN and ZFHX3 in Mammary Epithelial Cells Alters Patterns of Collective Cell Migration

doi: 10.3390/ijms24010313

Figure Lengend Snippet: Effect of PTEN and ZFHX3 knockouts on morphology, cell–cell adherences and proliferation activity of MCF10A cells. ( A ) Phase contrast view and E-cadherin AJs (immunofluorescence) of cells cultivated in depleted medium (1% of horse serum) and no EGF (epidermal growth factor). Red arrows point to gaps between cells. Scale bars 100 μm for phase contrast pictures and 20 μm for immunofluorescence. ( B ) Quantification of E-cadherin linear and non-linear AJs within islets of parental and KOs cells. ( C ) Quantification of gaps between cells in cell islets. The data ( B , C ) represent 3 independent experiments, Sample size (the number of calculated AJs) for each experiment is: Parental 155, 114, 199; PTEN KO 86, 87, 159; ZFHX3 KO 98, 91, 161; DKO 136, 116, 189 junctions for ( B , C ). ( D ) Analysis of proliferation of obtained cells. The percentage of cells in S-phase was determined as a ratio of EdU—positive nuclei/DAPI-stained nuclei. The data represent 3 independent experiments. Measurements were performed from 6 different fields of view ( N = 600−1000). Statistical analysis was performed by a contingency Chi square test in ( B) ; unpaired t -test in panel ( C) , Kruskal–Wallis test in panel ( D ); * p < 0.05, ** p < 0.01, **** p < 0.0001 and ns: non-significant.

Article Snippet: For Western blot analysis and immunostaining, the following antibodies were used: mouse monoclonal anti-E-cadherin clone 36, mouse monoclonal anti-N-cadherin clone 32, mouse monoclonal anti-P-cadherin (BD Transduction Laboratories, USA); mouse monoclonal anti-vimentin V9 antibodies; mouse monoclonal anti-β-actin, clone C4; rabbit polyclonal anti-ZFHX3, clone ab119909, (Abcam); rabbit polyclonal anti-PTEN, clone 9552 (Cell Signaling); mouse monoclonal anti-GAPDH, clone, AM4300 (Invitrogen, Waltham, MA, USA); mouse monoclonal Anti-pan-Cytokeratin (Sigma-Aldrich, USA).

Techniques: Activity Assay, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Inactivation of PTEN and ZFHX3 in Mammary Epithelial Cells Alters Patterns of Collective Cell Migration

doi: 10.3390/ijms24010313

Figure Lengend Snippet: EMT features in gene-edited MCF10A cell lines. ( A ) Alteration of expression of E-, N- and P-cadherins; vimentin; and cytokeratins (5, 6, 8), and actin was used for normalization of protein loading (Western blots). ( B ) Quantification of Western blots by densitometry. Mean ± SEM from 3 independent experiments. ( C ) Immunofluorescence staining of islets for vimentin (green), E-cadherin (red) and nuclei (blue). Scale bars: 50 μm for islet images. ( D ) Quantification of vimentin-positive and -negative cells within islets or at their periphery. ( E ) Top images: immunofluorescence staining of wound edges for vimentin (green), E-cadherin (red) and nuclei (blue). Scale bars: 100 μm. Middle images: confocal microscopy of E-cadherin immunofluorescence in wound healing experiments. Nuclei are in cyan. Note the radial AJ in ZFHX3 KO. Arrows indicate the magnified junctions displayed below. Scale bars 18 μm for main immunofluorescence images and 5 μm for magnified junctions. Three independent experiments. Sample size (the number of calculated Vim + cells) for each experiment is: parental 193, 236, 438; PTEN KO 190, 235, 481; ZFHX3 KO 150, 264, 424; DKO 250, 289, 356 cells for ( D ). Statistical analysis: unpaired t -test in panels ( B ); contingency Chi square test in ( D ); * p < 0.05, ** p < 0.01, **** p < 0.0001 and ns: non-significant.

Article Snippet: For Western blot analysis and immunostaining, the following antibodies were used: mouse monoclonal anti-E-cadherin clone 36, mouse monoclonal anti-N-cadherin clone 32, mouse monoclonal anti-P-cadherin (BD Transduction Laboratories, USA); mouse monoclonal anti-vimentin V9 antibodies; mouse monoclonal anti-β-actin, clone C4; rabbit polyclonal anti-ZFHX3, clone ab119909, (Abcam); rabbit polyclonal anti-PTEN, clone 9552 (Cell Signaling); mouse monoclonal anti-GAPDH, clone, AM4300 (Invitrogen, Waltham, MA, USA); mouse monoclonal Anti-pan-Cytokeratin (Sigma-Aldrich, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Confocal Microscopy

Journal: Neural Plasticity

Article Title: Tumor Necrosis Factor Alpha Mediates GABA A Receptor Trafficking to the Plasma Membrane of Spinal Cord Neurons In Vivo

doi: 10.1155/2012/261345

Figure Lengend Snippet: Quantitative western blotting reveals a nonsignificant trend towards an increase in plasma membrane GABA A R. (a) Representative examples of Western Blots of membrane-enriched homogenate fractions from an albumin subject and a TNF α subject that were run on the same gel. GABA A R (red) and plasma membrane protein N-Cadherin (NCad; green) bands were visualized and quantified using the Odyssey IR Imaging System (Li-Cor). (b) Linear intensity quantification of Western Blots of the P2 fraction yields a trend suggesting an increase in GABA A R : NCad ratio following TNF α injection ( P = 0.315, n = 10 albumin subjects, n = 8 TNF α subjects). Bars represent group intensity means averaged across 2 Western Blots. Error bars indicate SEM.

Article Snippet: The membrane was then reblocked and reincubated in a primary antibody solution containing 1 : 800 mouse anti-N-Cadherin primary antibody (BD Biosciences, 610920).

Techniques: Western Blot, Imaging, Injection