Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Distribution of the GFP-tagged CB2R knock-in mice. (A) The genetic background of the knock-in mice is shown where the GFP reporter gene was inserted within exon 3 of CB2R and the expression of GFP was driven by internal ribosomal entry (IRE) promoter. Cochleae isolated from these GFP-CB2R co-expressing knock-in mice were fixed in 4% paraformaldehyde followed by decalcification and then sectioned. (B) The representative mid-modiolar section shows endogenous GFP fluorescence (green) in different anatomical regions of the cochlea including organ of Corti (OC), stria vascularis (SV), spiral ligament (SL), and spiral ganglion (SG) neuron. (C) Immunolabeling of GFP-tagged CB2R (green) along with hair cell marker, Myo VIIa (red), show colocalization of CB2R in both, the OHCs and IHCs. (D) Co-labeling of these cochleae with Tuj1 (red), a neuronal marker, show the distribution of GFP-CB2R (green) in spiral ganglion neurons. (E) Whole mount sections of cochleae from GFP-CB2R co-expressing knock-in mice show co-localization of GFP-CB2R (green) and myosin VIIa (red) in the OHCs and IHCs of OC. (F) GFP-CB2R whole mounts sections were also co-stained with neuronal marker, Tuj1 (red), and pre-synaptic ribbon marker, CtBP2 (magenta), showing pre-synaptic localization of CB2R. Cell nuclei (blue) are stained with Hoechst stain in (B,E,F) . Studies reported here were repeated in at least three different animals and similar results were obtained.

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Knock-In, Expressing, Isolation, Fluorescence, Immunolabeling, Marker, Labeling, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Distribution of the CB2R in the rat cochlea. (A) Immunolabeling of mid-modiolar cochlear sections with CB2R antibody shows labeling of cells in the organ of Corti (green). Labeling was observed in the outer hair cells (OHC), inner hair cells (IHC), inner pillar cell (IPC), and outer pillar cell (OPC). (B) Co-labeling of the organ of Corti with antibodies for CB2R (green) and myosin VIIa (red) showed co-localization of these proteins in the OHC and IHC. (C) Staining of CB2R was also observed in the intermediate and basal cells of the SV and type II fibrocytes in SL. CB2R (green) showed some overlap with Na + /K + -ATPase α1 subunit (red) in the SV. (D) CB2R was localized to the plasma membranes of SG. Approximately 60% of cells in this section were stained with both CB2R (green) and Hoechst (blue). (E) Co-labeling of CB2R and myosin VIIa in OHCs and IHCs in whole mount preparations of the organ of Corti shows that distribution of CB2R (green) is more widespread than that of myosin VIIa (red). Hoechst was used as a nuclear stain (blue) in (B–E) . Studies reported here were repeated in at least three different animals and similar results were obtained.

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Immunolabeling, Labeling, Staining, Clinical Proteomics

Journal: Frontiers in Cellular Neuroscience

Article Title: The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss

doi: 10.3389/fncel.2018.00271

Figure Lengend Snippet: Trans -tympanic administration of JWH015 prevents cisplatin-induced hearing loss. (A) Baseline ABR thresholds were recorded in Wistar rats, which were then pre-treated with either trans -tympanic AM630 (2.5 nmoles) for 30 min followed by trans -tympanic JWH015 (2.5 nmoles) or individually with trans -tympanic AM630 or JWH015 for 24 h. Cisplatin (11 mg/kg) was then administered (i.p.) after day 1 and post-ABR was performed after 72 h. Pre-treatment with JWH015 diminished cisplatin-induce ABR threshold shift in all the three frequency regions. The protective action of JWH015 was completely blocked by AM630. Trans -tympanic administration of AM630 alone significantly elevated by ABR thresholds. Data (threshold shift) is plotted as mean ± SEM ( n = 12). (B) The animals were sacrificed at the end point and cochleae were collected, fixed by 4% PFA in PBS followed by decalcification for 3 weeks in 100 mM EDTA. The base of the cochlea were dissected out and stained with hair cell marker, myosin VIIa, to determine the hair cell loss. Representative images are shown. Scale bar is 20 μm. (C) The bar graph represents the average number of hair cells per 100 μm area shown in (B) ( n ≥ 3). Pre-treatment with JWH015 reduced cisplatin-induced outer hair cell loss. Administration of AM630 produced no hair cell loss. ∗ p < 0.05 vs. vehicle; ∗∗ p < 0.05 vs. cisplatin; # p < 0.05 vs. JWH015 + cisplatin; and ∗∗∗ p < 0.05 vs. JWH015 (one-way ANOVA).

Article Snippet: Two different myosin VIIa antibodies were used for different purposes: Mouse IgG1 anti-myosin-VIIa antibody was obtained from Developmental Studies Hybridoma Bank (#138-1), while rabbit anti-myosin-VIIa was purchased from Proteus Biosciences (#25-6790).

Techniques: Staining, Marker, Produced

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A role for mutations in AK9 and other genes affecting ependymal cells in idiopathic normal pressure hydrocephalus.

doi: 10.1073/pnas.2300681120

Figure Lengend Snippet: Fig. 2. Expression of iNPH-associated proteins in the ventricular region of the mouse brain. Immunofluorescence micrographs showing immunoreactivity for proteins expressed from iNPH-associated genes in the ependymal layer and subventricular zone of mice. Immunohistochemistry for Cwh43, PRKD1, Rxfp2, Ak9, and Myo7a was performed using cryostat sections from 7-wk-old C57BL6 mice. Cilia were visualized using an antibody directed against acetylated α-tubulin (Ac-tubulin). Nuclei were stained with DAPI. (Scale is 25 µm.)

Article Snippet: Sagittal or coronal cryostat sections of mouse brains were prepared and stained for flu‐ orescence immunohistochemistry using antibodies directed against Cwh43 (1:250, HPA048140, Sigma- Aldrich), Ak9(1:250, HPA030804, Sigma- Aldrich), HAVCR1(1:500, 14791, Cell signaling), Rxfp2 (1:500, sc- 374293, Santa Cruz Bioecology), otogelin (1:200, ARP65594, Aviva Systems Biology), notch 1 receptor (1:500, MA5- 11961, ThermoFisher Scientific), protein kinase D1(1:200, sc- 638, Santa Cruz Bioecology), myosin heavy chain 13 (1:500, PA5- 89942, ThermoFisher Scientific), myosin VIIA (Myo7a) (1:200, HPA028918- , Sigma- Aldrich), spatacsin (SAB3500003, Sigma- Aldrich), or acetylated α- tubulin (1:1,000, sc- 23950, Santa Cruz Bioecology).

Techniques: Expressing, Immunofluorescence, Immunohistochemistry, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A role for mutations in AK9 and other genes affecting ependymal cells in idiopathic normal pressure hydrocephalus.

doi: 10.1073/pnas.2300681120

Figure Lengend Snippet: Fig. 3. Relationship between proteins from iNPH-associated genes and ependymal cilia. (A) High resolution immunohistochemistry showing localization of SPG11/spatacsin, Ak9, HAVCR1/Kim1, Notch1, and otogelin immunoreactivity in ependymal cells. Cilia were visualized using an acetylated α-tubulin antibody (green). (Scale is 5 µm.) (B) Immunocytochemistry for Rxfp2 or Myo7a (red) in cultured mouse ependymal cells. Cilia were visualized using an acetylated α-tubulin antibody (green). Arrows show areas where iNPH-associated protein immunoreactivity colocalizes with cilia. (Scale is 5 µm.)

Article Snippet: Sagittal or coronal cryostat sections of mouse brains were prepared and stained for flu‐ orescence immunohistochemistry using antibodies directed against Cwh43 (1:250, HPA048140, Sigma- Aldrich), Ak9(1:250, HPA030804, Sigma- Aldrich), HAVCR1(1:500, 14791, Cell signaling), Rxfp2 (1:500, sc- 374293, Santa Cruz Bioecology), otogelin (1:200, ARP65594, Aviva Systems Biology), notch 1 receptor (1:500, MA5- 11961, ThermoFisher Scientific), protein kinase D1(1:200, sc- 638, Santa Cruz Bioecology), myosin heavy chain 13 (1:500, PA5- 89942, ThermoFisher Scientific), myosin VIIA (Myo7a) (1:200, HPA028918- , Sigma- Aldrich), spatacsin (SAB3500003, Sigma- Aldrich), or acetylated α- tubulin (1:1,000, sc- 23950, Santa Cruz Bioecology).

Techniques: Immunohistochemistry, Immunocytochemistry, Cell Culture

Journal: Redox Biology

Article Title: Targeting nitrative stress for attenuating cisplatin-induced downregulation of cochlear LIM domain only 4 and ototoxicity

doi: 10.1016/j.redox.2016.10.016

Figure Lengend Snippet: Cisplatin-induced increase in nitrotyrosine is attenuated by SRI110. A) Increased levels of nitrotyrosine as well as condensed nuclei (white arrows) were detected in UBOC1 cells treated with 10 µm cisplatin. Co-treatment with 50 µm SRI110 attenuated the cisplatin-induced increase in nitrotyrosine. Red staining indicates immunoreactivity to anti-nitrotyrosine, blue indicates nuclear staining with DAPI, while green indicates actin staining with phalloidin. The images are representative of four biological replicates. Scale bar, 20 µm. (B) The mean pixel intensities for nitrotyrosine immunostaining was significantly higher in cisplatin treated cells (****P<0.0001), which was attenuated by SRI110 co-treatment (***P<0.001). The results are expressed as mean±standard deviation, n=4. (C) Immunostaining (violet) with anti-myosin VIIa indicated the differentiated state of UBOC1 cells. The image is representative of three biological replicates. Scale bar, 20 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Then the cells were incubated with anti-nitrotyrosine, anti-myosin VIIa (catalog no. sc-32757, sc-74516, Santa Cruz Biotechnology Inc., Santa Cruz, CA) or anti-LMO4 (catalog no. ab39383, Abcam, Cambridge, MA) followed by incubation with Alexa Fluor 568 donkey anti-mouse or Alexa Fluor 647 goat anti-rabbit secondary antibody (catalog no. A10037 or A21244, Life Technologies, Carlsbad, CA) and fluorescein phalloidin (catalog no. F432, Life Technologies).

Techniques: Staining, Immunostaining, Standard Deviation

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Expression of MYO7A from single AAV2 and AAV5 vectors in cultured cells. ( a ) Diagram of the viral vector encoding human MYO7A cDNA. ( b ) Western blot of WT eyecup (lane 1), primary RPE cultures derived from Myo7a -null mice and infected with AAV2- MYO7A (lane 2) or AAV5- MYO7A (lane 3), or not infected (lane 4), and primary RPE cultures derived from Myo7a +/− mice (lane 5). All lanes were immunolabeled with antibodies against actin (as a loading indicator of relative protein loading) and MYO7A. ( c-f ) Immunofluorescence images of primary RPE cell cultures. Cells derived from Myo7a -null mice that were not infected (c), from Myo7a +/− mice (d), or from Myo7a -null mice infected with 1x AAV2-MYO7A (e) or 1x AAV5-MYO7A (f). Scale = 10 μm.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Expressing, Cell Culture, Plasmid Preparation, Western Blot, Derivative Assay, Infection, Immunolabeling, Immunofluorescence

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Expression of MYO7A from single AAV2 and AAV5 vectors in vivo . ( a-e ) EM images of MYO7A immunogold labelling of the connecting cilium and pericilium from rod photoreceptors in a Myo7a -null retina. ( a ) Longitudinal section from an untreated Myo7a -null retina (background label only). ( b, c ) Longitudinal sections from Myo7a -null retinas treated with 1x AAV2- MYO7A ( b ) or AAV5- MYO7A ( c ). Scale = 200 nm. ( d, e ) Transverse sections of connecting cilia from rod photoreceptors in Myo7a -null retinas treated with 1x AAV2- MYO7A ( d ) or AAV5- MYO7A ( e ). Scale = 100 nm. ( f-g ) EM images of RPE cells from Myo7a -null retinas treated with 1x AAV2- MYO7A ( f ) or AAV5- MYO7A ( g ). Scale = 2 μm. BM = Bruch’s Membrane, AP = Apical Processes. Areas indicated by rectangles are enlarged in f’ and g’, in order to show MYO7A immunogold labeling (indicated by circles). Scale = 500 nm. ( h, i ) EM image of a longitudinal section of the connecting cilium and pericilium from a rod (h) and a cone (i) photoreceptor in a Myo7a -null retina, treated with 1x AAV2- MYO7A . The section was double-labeled with MYO7A (12 nm gold) and rod opsin (15 nm gold) antibodies. Rod outer segments were labeled with the opsin antibody, while cones were identified by lack of rod opsin labeling in their outer segments. The sections show just the base of the outer segments. Nearly all the label in the connecting cilium is MYO7A, even in the rod. Scale = 200 nm. ( i-l ) Bar graphs indicating MYO7A immunogold particle density in the rod photoreceptor cilium and pericilium ( j, k ) and in the RPE ( l, m ), following treatment with AAV2- MYO7A ( j, l ) or AAV5- MYO7A ( k, m ) of different concentrations. n=3 animals per condition. Bars indicate SEM.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Expressing, In Vivo, Membrane, Labeling

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Correction of mutant phenotypes, following subretinal injections with AAV2- MYO7A or AAV5- MYO7A . ( a-e ) Correction of melanosome localization. Light micrographs showing the presence of melanosomes in the apical processes of the RPE in a WT retina ( a ) and retinas injected with 1x AAV2- MYO7A ( b ) or AAV5- MYO7A ( c ). Further away from the injection site ( d ), melanosomes are present in the apical processes of some RPE cells, but not in others (arrows indicate apical melanosomes; white lines indicate regions where melanosomes are absent from the apical processes). ( e ) Region distant from injection site, where all RPE cells lack melanosomes in their apical processes. Brackets on left side indicate RPE apical processes. Scale = 8 μm. ( f ) Diagram of a dorso-ventral section through an eyecup, indicating the relative locations of the images shown in a-e. Arrow indicates the site of injection, ONH indicates the optic nerve head. ( g ) Correction of abnormal levels of opsin in the connecting cilium and pericilium of rod photoreceptors. Bar graph showing opsin immunogold gold particle density, along the length of the connecting cilium. Ultrathin sections of retinas from Myo7a -null and WT mice were stained with rod opsin antibody. The Myo7a -null retinas had been either not treated or treated with 1x or 1:100 AAV2- MYO7A or AAV5- MYO7A . N = 3 animals per condition. Bars indicate SEM.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Mutagenesis, Injection, Staining

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Expression of MYO7A from the overlapping AAV2- MYO7A dual vectors. ( a ) Diagram of the the overlapping AAV2- MYO7A dual vectors. The overlapping region contains 1365 bases. ( b ) Western blot of proteins from primary RPE cultures derived from Myo7a -null mice and not infected (lane 1), or infected with AAV2- MYO7A (dual) (lane 2); and primary RPE cultures derived from Myo7a +/− mice (lane 3). All lanes were immunolabeled with anti-MYO7A and anti-actin. ( c ) Western blot of primary cultures derived from Myo7a -null mice and not infected (lane 1), or infected with AAV2- MYO7A (dual (lane 2) or with AAV5- MYO7A single vector (1x) (lane 3). Lanes were immunolabeled with anti-MYO7A and anti-actin. Densitometry of the actin labeling showed that lane 2 was loaded with 3-fold more protein than lane 3; the MYO7A to actin ratio is 7-fold greater in lane 3 compared with lane 2 in this blot. ( d-g ) Immunofluorescence of cultured RPE cells transduced with AAV2-MYO7A(dual). ( d-f ) Primary RPE cultures derived from Myo7a -null mice and ( g ) ARPE19 cells. Scale = 10 μm. ( h ) Bar graph indicating the distribution of MYO7A immunogold particle density among RPE cells from retinas of Myo7a -null mice, injected with AAV2-MYO7A(dual). N = 3 animals.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Expressing, Western Blot, Derivative Assay, Infection, Immunolabeling, Plasmid Preparation, Labeling, Immunofluorescence, Cell Culture, Transduction, Injection

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Correction of mutant phenotypes, following subretinal injections with AAV2- MYO7A (dual). ( a ) Light microscopy of a semi-thin section from a treated Myo7a -null mouse retina. Region shown is near the injection site. Arrows indicate melanosomes in the apical processes. White lines indicate cells that still show the Myo7a- null phenotype, with an absence of melanosomes in the apical processes. Scale = 10 μm. ( b ) Low magnification of an immunoEM image of the RPE from a retina treated with AAV2- MYO7A (dual). As in a , the white line indicates a region that still shows the Myo7a- null phenotype. Rectangle, c, includes melanosomes in the apical region, indicating a corrected RPE cell. Scale = 10 μm. ( c-e ) Higher magnification of regions outlined by rectangles in b . MYO7A immunogold particles are indicated by circles. Scale = 1 μm. ( f ) Bar graph illustrating MYO7A immunogold particle density measured in RPE cells from Myo7a -null retinas, WT retinas, or from Myo7a -null retinas treated with AAV2- MYO7A (dual) and determined to be corrected or not corrected by the location of their apical melanosomes. N = 3 animals per condition. Bars indicate SEM. ( g ) ImmunoEM image of a rod photoreceptor cilium double-labeled with antibodies against MYO7A (small gold particles) and against rod opsin (large gold particles). MYO7A labeling is associated with the connecting cilium and periciliary membrane, indicating expression and correct localization of MYO7A, while this region is devoid of opsin labeling, which is restricted to the disk membranes, consistent with the WT phenotype, thus indicating correction of the mutant phenotype. Scale = 300 nm.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Mutagenesis, Light Microscopy, Injection, Labeling, Membrane, Expressing

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: A . Schematic diagram showing the genetic structure of the integrated EIAV vectors used in this study . EIAV-CMV-MYO7A (UshStat) is based on a non-replicating non-human recombinant lentiviral vector based on the non-pathogenic wild type equine infectious anaemia virus (EIAV). The wild-type EIAV virus has 6 distinct genetic units, however, the majority of these EIAV sequences have been removed to produce a minimal vector system that contains less than 10% of the original viral genome and does not contain any viral promoters or enhancers and there are no coding regions for accessory proteins in either the EIAV genome or in the packaging system. SIN LTR: Self inactivating long term repeat. Neo: Neomycin open reading frame (ORF). CMV: Cytomegalovirus promoter (constitutive). RK: Rhodopsin kinase promoter (photoreceptor specific). eGFP: enhanced green fluorescent protein ORF. MYO7A: Myosin VIIa ORF. WPRE: Woodchuk hepatitis virus post-transcriptional regulatory element. B . Expression analysis of myosin VIIa in 4 weeks mouse eye and HeLa cells transfected with the EIAV-CMV-Null (Null) or UshStat constructs. β-actin was used as loading control. RPE: retinal pigment epithelium. NR: neuroretina. IP/Null: immunoprecipitates of HeLa cells transfected with the null vector. IP/UshStat: immunoprecipitates of HeLa cells transfected with the myosin VIIa vector. IB MYO7A: immunoblot with the mouse anti-myosin VIIa. Molecular weight markers are denoted to the left. C–D : Immunocytochemistry studies of HeLa cells transduced with the null ( C ) or the UshStat vector ( D ) and immunostained for myosin VIIa (red). DAPI was used to counter stain the nucleus. Scale bar: 15 μm.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Recombinant, Plasmid Preparation, Virus, Expressing, Transfection, Construct, Control, Western Blot, Molecular Weight, Immunocytochemistry, Transduction, Staining

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: Shaker1 mouse retinas were co-transduced by a subretinal injection of UshStat and EIAV-CMV-GFP or EIAV-CMV-Null and EIAV-RK-GFP (the GFP vector was used to identify the transduced region of the retina). After 4 weeks the animals were dark-adapted overnight, then light-adapted for 10 minutes under 200 lux illumination. Retinas were double immunostained with antibodies against GFP (green) and α-transducin (red). Panel I : Low magnification image of a retina transduce with UshStat and GFP, showing that the gradient in α-transducin translocation (left to right) parallels GFP expression (indicative of wild type myosin VIIa). Scale bar: 40 μm. Panel II : A–C : Representative examples of an EIAV transduced region of the retina (determine by the presence of GFP), presenting dual immunostaining for GFP and α-transducin ( C ). In the presence of wild type myosin VIIa, α-transducin is translocated to the inner segment (IS). D–F : An untransduced region of the same retina as in A–C does not show α-transducin translocation to the IS upon illumination. G–I : Retinas co-transduced with EIAV-Null-vector and EIAV-RK-GFP (photoreceptor cell-specific promoter) as a control for the effects of subretinal lentiviral transduction on α-transducin translocation. The region of the retina shown was transduced, evidenced by GFP expression in the photoreceptors ( H ) however there was no translocation of α-transducin to the IS ( G and I ). The qualitative results represented in each of the panels are representative images for at least three replicate experiments. RPE = Retinal Pigment Epithelium; OS = Outer Segments; IS = Inner Segments; ONL = Outer Nuclear Layer; OPL = Outer Plexiform Layer. Scale bar: 25 μm. Arrowheads in D and G indicate translocation of α-transducin in individual photoreceptors. Asterisks in I denote reactivity of the secondary anti-mouse antibody with circulation mouse IgGs present in the blood vessels.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Injection, Plasmid Preparation, Translocation Assay, Expressing, Immunostaining, Transduction, Control

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: A–B : Show myosin VIIa immunostaining of shaker1 retinas transduced with the EIAV-CMV-Null vector ( A ) or UshStat ( B ). Myosin VIIa expression can be detected in the RPE, OS and IS regions. C–D : monkey retinas transduced with UshStat ( D ) showed high levels of human myosin VIIa in the RPE and moderate expression in outer and inner segments of the photoreceptor cells. Asterisk in C denotes weak immunostaining of the endogenous myosin VIIa in the RPE layer. Note that the anti-myosin VIIa antibody was titrated so as to only detect the exogenous overexpressed virus-derived wild type myosin VIIa ( B, D ) and not the endogenous protein ( A, C ). Scale bars: 25 μm.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Immunostaining, Transduction, Plasmid Preparation, Expressing, Virus, Derivative Assay

Journal: PLoS ONE

Article Title: EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

doi: 10.1371/journal.pone.0094272

Figure Lengend Snippet: Retinas were co-transduced with EIAV-CMV-Null vector and EIAV-RK-GFP ( A–C ) or UshStat and EIAV-CMV-GFP ( D–F, J ). After 4 weeks, mice were exposed to 6 days continuous light at 2000 lux illumination. Retinas were harvested and dual immunostained for myosin VIIa (red) and GFP (green) ( A–F, J ). Immunostaining conditions were chosen as to only detect the exogenous overexpressed myosin VIIa. G–H : Eosin and hematoxylin histochemical staining of shaker1 mouse retinas transduced with either the EIAV-CMV-Null vector ( G ) or UshStat ( H ). I : Eosin and hematoxylin staining of wild type (untransduced) retina for comparison of relative light dependent degeneration of the ONL that is typically observed (double headed arrow). J : Low magnification image of an UshStat+GFP co-transduced retina, showing the area used for the studies (bracket) relative to the area of injection (syringe). This area corresponds to the juxtaposed ∼0.2 mm from the site of injection. K : Schematic representation of the area of injection represented by red boxes, where right box area corresponds to the injection in the right eye and left box area injection in the left eye. The inferior retina was always used for the injections and ONL counting. Scale bars A–F : 25 μm, G–I : 50 μm. J : 90 μm. Labels are as in . Asterisks in C , F and J denote reactivity of the secondary anti-mouse antibody with circulation mouse IgGs present in the blood vessels.

Article Snippet: For immunoprecipitation studies, 40 μl of protein-A sepharose beads (50% slurry, Sigma, MI) were incubated with 4 μg of rabbit anti-myosin VIIa (Novus Biologicals, CO) overnight at 4°C.

Techniques: Transduction, Plasmid Preparation, Immunostaining, Staining, Comparison, Injection

Journal: Aging Cell

Article Title: Morphological phenotyping of the aging cochlea in inbred C57BL/6N and outbred CD1 mouse strains

doi: 10.1111/acel.14362

Figure Lengend Snippet: List of the primary and secondary antibodies used in the study.

Article Snippet: Myosin VIIa , Rabbit , GTX23481 , GeneTex , 1:100.

Techniques: