Journal: bioRxiv

Article Title: Dyrk1a gene dosage controls bipolar cell development and retinal connectivity

doi: 10.64898/2026.03.15.710015

Figure Lengend Snippet: (A) Schematic representation of electroretinogram (ERG) components. An initial negative deflection (a-wave) is observed upon photoreceptor stimulation (arrow) that is followed by a subsequent positive deflection (b-wave) that corresponds to bipolar cell activity. ERG waveform quantification involves measuring the amplitude (μV) and latency (ms) of the a- and b-waves to increasing stimulus intensities (cd.s/m 2 ) and thus provides an evaluation of retinal function. (B) Representative dark-adapted scotopic ERG waveforms recorded from Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animals (6 – 8 weeks) in response to a single flash stimulus (log 1.48 cd.s/m 2 ). Comparable a-wave responses are observed for both groups while b-wave amplitudes are reduced in heterozygous animals. Thick lines represent genotype-mean responses while thin lines represent individual mice. (C – D) Quantifcation of photoreceptor amplitude (C) and latency (D) responses in Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animals (6 – 8 weeks) in repsonse to a series of increasing scotopic flash stimuli (log cd.s/m 2 ). No significant differences are observed between the groups. (E – F) Quantification of bipolar cell amplitude (E) and latency (F) responses in Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animal (6 – 8 weeks) towards increasing scotopic flash stimuli (log cd.s/m 2 ). Heterozygous animals have a significant reduction in bipolar cell amplitude response at higher stimulus intensities (E) albeit with similar latency (F). (G) Representative light-adapted photopic ERG waveforms recorded from Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animals (6 – 8 weeks) in response to a single UV flash stimulus (log 1.00 cd.s/m 2 ). Comparable a-wave responses are observed for both groups while b-wave amplitudes are reduced in heterozygous animals. Thick lines represent genotype-mean responses while thin lines refer to individual mice. (H) Quantification of bipolar cell amplitude response in Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animal (6 – 8 weeks) in response to an increasing photopic UV stimulus intensity series (log cd.s/m 2 ). Heterozygous animals have a significant reduction in bipolar cell amplitude response at higher stimulus intensities. (I) Representative light-adapted photopic ERG waveforms recorded from Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animals (6 – 8 weeks) in response to a single green flash stimulus (log 1.48 cd.s/m 2 ). Comparable a-wave responses are observed for both groups while b-wave amplitudes are reduced in heterozygous animals. Thick lines represent genotype-mean responses while thin lines refer to individual mice. (J) Quantification of bipolar cell amplitude response in Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animal (6 – 8 weeks) in response to a photopic green stimulus intensity series (log cd.s/m 2 ). Heterozygous animals have a significant reduction in bipolar cell amplitude response at higher stimulus intensities. (K – R) Representative immunohistochemical staining of coronal eye sections taken from Lhx2-Cre and Lhx2-Cre:Dyrk1a +/f adult mice (6 – 8 weeks) across four designated dorsoventral quadrants (D1, D2, V3 and V4) spanning the entire retina relative to the optic nerve head. OPL ribbon synapse assemblies appear comparable in both groups (K – R, arrowheads) and were composed of dense photoreceptor terminals (vGlut1 + ) that overlapped with well organised bipolar cell dendritic processes (PKCα + ). Additionally, the linear alignment of all ribbon junctions (Pikachurin + ) indicated intact photoreceptor and bipolar cell synapses. All data represents the mean ± SEM. Statistical differences were calculated using a two-way ANOVA followed by a Sidak’s multiple comparison test. p-values are denoted as follows: **p≤ 0.01, ***p≤ 0.001 and ****p≤ 0.0001. Scale bar: (K – R) 5 µm. Abbreviations: D, dorsal; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; V, ventral.

Article Snippet: The following primary antibodies and dilutions were used in this study: Calbindin (1:300, Sigma-Aldrich, #C9848); Calretinin (1:1000, Swant, #CG1); cleaved Caspase3 (Asp175) (1:500, Cell Signalling, #9661S); chAT (1:100, Millipore, #AB144P); Chx10 (1:50, SCBT, #SC365519); Dyrk1a (1:100, Novus, #NBP1-84032); Isl1/2 (recognises both Isl1 and Isl2 proteins) (1:500; DSHB, #39.4D5); M-opsin (1:500, Proteintech, #30975-1-AP); Pax6 (1:100, DSHB, #Pax6); Pikachurin (1:200, Proteintech, #14578-1-AP); Rhodopsin (1:500, Millipore, #MABN15); S-opsin (1:500, Proteintech, #24660-1-AP); TH (1:300, Millipore, #AB152); vGlut1 (1:2000, Millipore, #AB5905).

Techniques: Activity Assay, Immunohistochemical staining, Staining, Comparison

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A) Scheme of the pre-synaptic compartment at the first visual synapse. (B) In vitro co-immunoprecipitation of GPR179 with Pikachurin in HEK293 cells transfected with the indicated myc- or HA-tagged constructs. Immunoprecipitated proteins were detected by western blotting using specific antibodies. (C) Live staining of Pikachurin (red) in transfected HEK293 cells shows a predominant localization within the extracellular matrix. Incubation with conditionedmedia of HEK293 cells expressing the ecto-GPR179-Fc (green) shows co-localization with the anti-HA antibody staining (red). Negative controls are cells transfected with empty vector (pcDNA3.1) or incubated with Fc fragment. DAPI in blue. (D) Heparan sulfate modification of Pikachurin is not required for the interaction with GPR179. GPR179-myc was immunoprecipitated from cell lysate oftransfected HEK293 cells. GPR179-conjugated beads were mixed with conditioned media from cells transfected with Pikachurin-mCherry and treated with heparinase III or buffer. Cells transfected with empty vector were used as immunoprecipitation (IP) specificity control. (E) Multiple amino acid sequence alignment of pikachurin from 197 species. Identical amino acids are highlighted in orange and aligned with domain topology(below). (F) Schematics of Pikachurin structural domains and deletion constructs generated to study binding determinants to GPR179. Each construct is fused with mCherry at its C terminus. Amino acid numbers are shown at the bottom of each construct. (G) Pull-down of Pikachurin-derived mutants from HEK293 cell lysates by GPR179 ectodomain fused to an Fc fragment. Fc and mCherry are used as negativecontrols.

Article Snippet: 16 ng of each sample were used in each real-time PCR (TaqMan Gene Expression Assay ID probes: Rgs7 : Mm01317058_m1; Rgs11 : Mm01309856_m1; Gpr179 : Mm00615352_m1; Egflam : Mm01298063_m1; Applied Biosystems).

Techniques: In Vitro, Immunoprecipitation, Transfection, Construct, Western Blot, Staining, Incubation, Expressing, Plasmid Preparation, Modification, Control, Sequencing, Generated, Binding Assay, Derivative Assay

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A) Scheme of the experimental paradigm (left). Pull-down assay of endogenous Pikachurin from retina lysates using affinity-purified ecto-GPR179-Fc expressedin transfected HEK293 cells. Fc fragment was used as negative control. (B) Confocal images of retina sections show co-localization of Pikachurin (green) and GPR179 (red) in the retina OPL. DAPI in blue. (C and D) Detection of the complex GPR179/Pikachurin (red) using proximity ligation assay (PLA) in retina cross-sections from wild-type mice. Primary antibodies were co-incubated with conditioned media of HEK293 cells expressing either the Fc fragment (C) or the ecto-GPR179-Fc as negative control (D). Dashed-line boxes indicate the region of the merged image reported with a higher magnification. PKCα (green) was used as a marker of ON-BC, and nuclei were labeled by DAPI (blue). (E) Quantification of PLA particles in the OPL or inner nuclear layer (INL) (negative control) of retina cross-sections in each condition. Data are mean ± SEM (n = 5–7, ***p < 0.001, Student’s t test). (F and G) Representative western blots (F) and quantification (G) of Pikachurin and GPR179 protein levels in retina lysates at different developmental stages. Equal amounts of total protein were loaded on a single gel, and specific antibodies were used to detect indicated proteins (n = 3). (H) Immunohistochemistry (IHC) of WT retinas showing expression pattern of Pikachurin (green) and GPR179 (red) at different developmental stages. Immunostaining of adult Gpr179 nob5 retina (bottom) shows similar Pikachurin accumulation at synaptic sites. (I) Quantification of GPR179- and Pikachurin-positive puncta at the OPL during retina development in WT mice. Data are mean ± SEM (n = 3, *p < 0.05, Student’s t test).

Article Snippet: 16 ng of each sample were used in each real-time PCR (TaqMan Gene Expression Assay ID probes: Rgs7 : Mm01317058_m1; Rgs11 : Mm01309856_m1; Gpr179 : Mm00615352_m1; Egflam : Mm01298063_m1; Applied Biosystems).

Techniques: Pull Down Assay, Affinity Purification, Transfection, Negative Control, Proximity Ligation Assay, Incubation, Expressing, Marker, Labeling, Western Blot, Immunohistochemistry, Immunostaining

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A and B) Western blot analysis of the indicated signaling molecules in retina samples from WT and Pika −/− mice (A) and quantification normalized to GAPDH expression and reported as a percentage of WT (n = 8 WT, n = 5 Pika −/− ) (B). (C) qRT-PCR of the indicated genes in WT and Pika −/− retinas (n = 5 mice/genotype). (D and E) Representative immunohistochemistry of retina sections from WT and Pika −/− mice using antibodies against mGluR6 (red), GPR179, RGS7, and RGS11 (green) (D) and related quantification (E) (n = 3). (F) In vivo retina electroporation experiments. Pika −/− mice were electroporated at P0 with the outlined photoreceptor-specific construct for Pikachurin overexpression, and the retinas were harvested after 3 weeks and prepared for IHC. (G) Representative confocal image of an individual electroporated retina immunostained with anti-HA (red) and anti-EYFP (green) antibodies. EYFP-negativeregions (left) were used as control for quantification, while EYFP-positive regions (right) represent a successful electroporation. (H and I) Representative confocal images (H) and quantification (I) of synaptic accumulation of overexpressed Pikachurin-HA in control versus electroporated regions. Five to ten different regions of retina from three different mice were used. (J and K) Representative IHC in control and electroporated retina regions in Pika −/− (J) and quantification of synaptic accumulation (K) of GPR179 (left) and RGS11 (right). DAPI in blue. Five to ten different regions of retina from three different mice were used. Data are mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, Student’s t test).

Article Snippet: 16 ng of each sample were used in each real-time PCR (TaqMan Gene Expression Assay ID probes: Rgs7 : Mm01317058_m1; Rgs11 : Mm01309856_m1; Gpr179 : Mm00615352_m1; Egflam : Mm01298063_m1; Applied Biosystems).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Immunohistochemistry, In Vivo, Electroporation, Construct, Over Expression, Control

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A) Schematics of the construct used for in vivo retina electroporation in WT mice. (B) IHC of WT retinas overexpressing Pikachurin-HA in photoreceptors using antibodies against HA (red) or EYFP (green). DAPI in blue. (C and D) IHC of control and electroporated regions in WT mice (C) and quantification of synaptic accumulation (D) of GPR179 (left) and RGS11 (right). DAPI in blue. Five to ten different regions of retina from three different mice were used. (E) Schematics of the dominant-negative construct expressing ectoGPR179myc under control of ON-BC-specific mGluR6 promoter used for in vivo electroporation of WT retinas. (F) IHC using antibodies against EYFP (green) and myc (red). (G and H) Representative IHC in control and electroporated retina regions in WT mice (G) and quantification of synaptic accumulation (H) of GPR179 (left) and RGS11 (right). DAPI in blue. Five to ten different regions of retina from three different mice were used. (I) IHC of mGluR6 (green) and ELFN1 (red) in retina cross-sections of WT and Pika -/− . (J) IHC of mGluR6 and ELFN1 at a higher magnification. (K) CoIP of ELFN1 and mGluR6 in retina samples from WT and Pika −/− mice. Retinas from mGluR6 −/− mice were used as a control for antibody specificity. Data are mean ± SEM (*p < 0.05 and ***p < 0.001, Student’s t test).

Article Snippet: 16 ng of each sample were used in each real-time PCR (TaqMan Gene Expression Assay ID probes: Rgs7 : Mm01317058_m1; Rgs11 : Mm01309856_m1; Gpr179 : Mm00615352_m1; Egflam : Mm01298063_m1; Applied Biosystems).

Techniques: Construct, In Vivo, Electroporation, Control, Dominant Negative Mutation, Expressing

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A) Representative western blots and quantification of the indicated proteins in retina samples from WT and Dmdmdx-4Cv mice. Data are mean ± SEM (n = 5 mice/genotype, *p < 0.05 and ***p < 0.001, Student’s t test). (B) Confocal images of retina sections from WT and Dmdmdx-4Cv mice stained with mGluR6 (red) and TRPM1 (green). (C) Representative confocal images of WT and Dmdmdx-4Cv mice stained with antibodies against mGluR6, GPR179, RGS7, RGS11, and Pikachurin. (D) Quantification of mGluR6, GPR179, RGS7, RGS11, and Pikachurin accumulation at the dendritic tips of ON-BCs. Nuclei were stained with DAPI (blue). Data are mean ± SEM (n = 3 mice/genotype, *p < 0.05, Student’s t test). (E) Representative IHC of retina sections from WT and DG cKO mice using antibodies against mGluR6 (red) and GPR179 or RGS7 or RGS11 (green). (F) Quantification of synaptic accumulation of the indicated proteins in WT and DG cKO mice. Data are mean ± SEM (n = 3 mice/genotype, *p < 0.05 and **p < 0.01, Student’s t test). (G–I) Representative traces of ERG responses of dark-adapted WT (black) and (G) Dmdmdx-4Cv (blue; n = 6 WT and n = 7 Dmdmdx-4Cv), (H) Pika−/− (red; n = 3 WT and n = 3 Pika−/−), and (I) DG cKO (orange; n = 3 WT and n = 3 DG cKO) mice at 1 cd s/m2. (J) Correlation between level of RGS protein accumulation (average of RGS7 and RGS11 content quantified from experiments presented in D and F) at the dendritic tips of ON-BCs and delay in b-wave implicit time (quantified and averaged from experiments presented in G–I). (K) Schematic model of the transsynaptic macromolecular complex of DGC-Pikachurin and GPR179-RGS proteins.

Article Snippet: 16 ng of each sample were used in each real-time PCR (TaqMan Gene Expression Assay ID probes: Rgs7 : Mm01317058_m1; Rgs11 : Mm01309856_m1; Gpr179 : Mm00615352_m1; Egflam : Mm01298063_m1; Applied Biosystems).

Techniques: Western Blot, Staining

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet:

Article Snippet: 16 ng of each sample were used in each real-time PCR (TaqMan Gene Expression Assay ID probes: Rgs7 : Mm01317058_m1; Rgs11 : Mm01309856_m1; Gpr179 : Mm00615352_m1; Egflam : Mm01298063_m1; Applied Biosystems).

Techniques: Recombinant, Cloning, Expressing, Software, Gene Expression

Journal: Journal of Neurophysiology

Article Title: A missense mutation in Grm6 reduces but does not eliminate mGluR6 expression or rod depolarizing bipolar cell function

doi: 10.1152/jn.00888.2016

Figure Lengend Snippet: Retinal anatomy. A: by light microscopy, no difference was observed between control and Grm6nob8 retinas. B: by immunohistochemistry, no difference was noted in the cellular localization of calbindin, PKCα, or PSD95 between control, Grm6nob8, and Grm6−/− retinas. Ribeye and pikachurin appeared correctly localized but at reduced levels in the Grm6nob8 retina. PNA was also correctly localized but had an uneven distribution in Grm6nob8 that is similar to that in the Grm6−/− retinas. Scale bars: 100 µm (A) and 5 µm (B). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer.

Article Snippet: Other primary antibodies and dilutions used were anti-calbindin (1:1,000; Swant, Marly, Switzerland); anti-PKCα (1:1,000; Sigma-Aldrich, St. Louis, MO); anti-PSD95 (1:1,000; Chemicon, Temecula, CA); anti-Ribeye (1:1,000; BD Biosciences, San Jose, CA), anti-pikachurin (1:1,000; Wako Chemicals, Richmond, VA), anti-GPR179 (1:4,000; Peachey et al. 2012a ), anti-TRPM1 (1:2,000; Cao et al. 2012 ), anti-RGS11 (1:2,000; Cao et al. 2012 ), and anti-RGS7 (1:2,000; Cao et al. 2008).

Techniques: Light Microscopy, Immunohistochemistry

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A) Scheme of the pre-synaptic compartment at the first visual synapse. (B) In vitro co-immunoprecipitation of GPR179 with Pikachurin in HEK293 cells transfected with the indicated myc- or HA-tagged constructs. Immunoprecipitated proteins were detected by western blotting using specific antibodies. (C) Live staining of Pikachurin (red) in transfected HEK293 cells shows a predominant localization within the extracellular matrix. Incubation with conditionedmedia of HEK293 cells expressing the ecto-GPR179-Fc (green) shows co-localization with the anti-HA antibody staining (red). Negative controls are cells transfected with empty vector (pcDNA3.1) or incubated with Fc fragment. DAPI in blue. (D) Heparan sulfate modification of Pikachurin is not required for the interaction with GPR179. GPR179-myc was immunoprecipitated from cell lysate oftransfected HEK293 cells. GPR179-conjugated beads were mixed with conditioned media from cells transfected with Pikachurin-mCherry and treated with heparinase III or buffer. Cells transfected with empty vector were used as immunoprecipitation (IP) specificity control. (E) Multiple amino acid sequence alignment of pikachurin from 197 species. Identical amino acids are highlighted in orange and aligned with domain topology(below). (F) Schematics of Pikachurin structural domains and deletion constructs generated to study binding determinants to GPR179. Each construct is fused with mCherry at its C terminus. Amino acid numbers are shown at the bottom of each construct. (G) Pull-down of Pikachurin-derived mutants from HEK293 cell lysates by GPR179 ectodomain fused to an Fc fragment. Fc and mCherry are used as negativecontrols.

Article Snippet: The following commercial antibodies were used: rat anti-HA (Roche), rabbit anti-myc (GenScript), rabbit anti-Pikachurin (Wako), mouse anti-b-DG (Novocastra), mouse anti-PKCα (Abcam), mouse anti-GAPDH (Millipore) mouse anti-human-Fc (ThemoFisher Scientific), chicken anti-GFP (Genscript), rabbit anti-RFP (Rockland) and chicken anti-RFP (Rockland).

Techniques: In Vitro, Immunoprecipitation, Transfection, Construct, Western Blot, Staining, Incubation, Expressing, Plasmid Preparation, Modification, Sequencing, Generated, Binding Assay, Derivative Assay

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A) Scheme of the experimental paradigm (left). Pull-down assay of endogenous Pikachurin from retina lysates using affinity-purified ecto-GPR179-Fc expressedin transfected HEK293 cells. Fc fragment was used as negative control. (B) Confocal images of retina sections show co-localization of Pikachurin (green) and GPR179 (red) in the retina OPL. DAPI in blue. (C and D) Detection of the complex GPR179/Pikachurin (red) using proximity ligation assay (PLA) in retina cross-sections from wild-type mice. Primary antibodies were co-incubated with conditioned media of HEK293 cells expressing either the Fc fragment (C) or the ecto-GPR179-Fc as negative control (D). Dashed-line boxes indicate the region of the merged image reported with a higher magnification. PKCα (green) was used as a marker of ON-BC, and nuclei were labeled by DAPI (blue). (E) Quantification of PLA particles in the OPL or inner nuclear layer (INL) (negative control) of retina cross-sections in each condition. Data are mean ± SEM (n = 5–7, ***p < 0.001, Student’s t test). (F and G) Representative western blots (F) and quantification (G) of Pikachurin and GPR179 protein levels in retina lysates at different developmental stages. Equal amounts of total protein were loaded on a single gel, and specific antibodies were used to detect indicated proteins (n = 3). (H) Immunohistochemistry (IHC) of WT retinas showing expression pattern of Pikachurin (green) and GPR179 (red) at different developmental stages. Immunostaining of adult Gpr179 nob5 retina (bottom) shows similar Pikachurin accumulation at synaptic sites. (I) Quantification of GPR179- and Pikachurin-positive puncta at the OPL during retina development in WT mice. Data are mean ± SEM (n = 3, *p < 0.05, Student’s t test).

Article Snippet: The following commercial antibodies were used: rat anti-HA (Roche), rabbit anti-myc (GenScript), rabbit anti-Pikachurin (Wako), mouse anti-b-DG (Novocastra), mouse anti-PKCα (Abcam), mouse anti-GAPDH (Millipore) mouse anti-human-Fc (ThemoFisher Scientific), chicken anti-GFP (Genscript), rabbit anti-RFP (Rockland) and chicken anti-RFP (Rockland).

Techniques: Pull Down Assay, Affinity Purification, Transfection, Negative Control, Proximity Ligation Assay, Incubation, Expressing, Marker, Labeling, Western Blot, Immunohistochemistry, Immunostaining

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A and B) Western blot analysis of the indicated signaling molecules in retina samples from WT and Pika −/− mice (A) and quantification normalized to GAPDH expression and reported as a percentage of WT (n = 8 WT, n = 5 Pika −/− ) (B). (C) qRT-PCR of the indicated genes in WT and Pika −/− retinas (n = 5 mice/genotype). (D and E) Representative immunohistochemistry of retina sections from WT and Pika −/− mice using antibodies against mGluR6 (red), GPR179, RGS7, and RGS11 (green) (D) and related quantification (E) (n = 3). (F) In vivo retina electroporation experiments. Pika −/− mice were electroporated at P0 with the outlined photoreceptor-specific construct for Pikachurin overexpression, and the retinas were harvested after 3 weeks and prepared for IHC. (G) Representative confocal image of an individual electroporated retina immunostained with anti-HA (red) and anti-EYFP (green) antibodies. EYFP-negativeregions (left) were used as control for quantification, while EYFP-positive regions (right) represent a successful electroporation. (H and I) Representative confocal images (H) and quantification (I) of synaptic accumulation of overexpressed Pikachurin-HA in control versus electroporated regions. Five to ten different regions of retina from three different mice were used. (J and K) Representative IHC in control and electroporated retina regions in Pika −/− (J) and quantification of synaptic accumulation (K) of GPR179 (left) and RGS11 (right). DAPI in blue. Five to ten different regions of retina from three different mice were used. Data are mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, Student’s t test).

Article Snippet: The following commercial antibodies were used: rat anti-HA (Roche), rabbit anti-myc (GenScript), rabbit anti-Pikachurin (Wako), mouse anti-b-DG (Novocastra), mouse anti-PKCα (Abcam), mouse anti-GAPDH (Millipore) mouse anti-human-Fc (ThemoFisher Scientific), chicken anti-GFP (Genscript), rabbit anti-RFP (Rockland) and chicken anti-RFP (Rockland).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Immunohistochemistry, In Vivo, Electroporation, Construct, Over Expression

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A) Schematics of the construct used for in vivo retina electroporation in WT mice. (B) IHC of WT retinas overexpressing Pikachurin-HA in photoreceptors using antibodies against HA (red) or EYFP (green). DAPI in blue. (C and D) IHC of control and electroporated regions in WT mice (C) and quantification of synaptic accumulation (D) of GPR179 (left) and RGS11 (right). DAPI in blue. Five to ten different regions of retina from three different mice were used. (E) Schematics of the dominant-negative construct expressing ectoGPR179myc under control of ON-BC-specific mGluR6 promoter used for in vivo electroporation of WT retinas. (F) IHC using antibodies against EYFP (green) and myc (red). (G and H) Representative IHC in control and electroporated retina regions in WT mice (G) and quantification of synaptic accumulation (H) of GPR179 (left) and RGS11 (right). DAPI in blue. Five to ten different regions of retina from three different mice were used. (I) IHC of mGluR6 (green) and ELFN1 (red) in retina cross-sections of WT and Pika -/− . (J) IHC of mGluR6 and ELFN1 at a higher magnification. (K) CoIP of ELFN1 and mGluR6 in retina samples from WT and Pika −/− mice. Retinas from mGluR6 −/− mice were used as a control for antibody specificity. Data are mean ± SEM (*p < 0.05 and ***p < 0.001, Student’s t test).

Article Snippet: The following commercial antibodies were used: rat anti-HA (Roche), rabbit anti-myc (GenScript), rabbit anti-Pikachurin (Wako), mouse anti-b-DG (Novocastra), mouse anti-PKCα (Abcam), mouse anti-GAPDH (Millipore) mouse anti-human-Fc (ThemoFisher Scientific), chicken anti-GFP (Genscript), rabbit anti-RFP (Rockland) and chicken anti-RFP (Rockland).

Techniques: Construct, In Vivo, Electroporation, Dominant Negative Mutation, Expressing

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet: (A) Representative western blots and quantification of the indicated proteins in retina samples from WT and Dmdmdx-4Cv mice. Data are mean ± SEM (n = 5 mice/genotype, *p < 0.05 and ***p < 0.001, Student’s t test). (B) Confocal images of retina sections from WT and Dmdmdx-4Cv mice stained with mGluR6 (red) and TRPM1 (green). (C) Representative confocal images of WT and Dmdmdx-4Cv mice stained with antibodies against mGluR6, GPR179, RGS7, RGS11, and Pikachurin. (D) Quantification of mGluR6, GPR179, RGS7, RGS11, and Pikachurin accumulation at the dendritic tips of ON-BCs. Nuclei were stained with DAPI (blue). Data are mean ± SEM (n = 3 mice/genotype, *p < 0.05, Student’s t test). (E) Representative IHC of retina sections from WT and DG cKO mice using antibodies against mGluR6 (red) and GPR179 or RGS7 or RGS11 (green). (F) Quantification of synaptic accumulation of the indicated proteins in WT and DG cKO mice. Data are mean ± SEM (n = 3 mice/genotype, *p < 0.05 and **p < 0.01, Student’s t test). (G–I) Representative traces of ERG responses of dark-adapted WT (black) and (G) Dmdmdx-4Cv (blue; n = 6 WT and n = 7 Dmdmdx-4Cv), (H) Pika−/− (red; n = 3 WT and n = 3 Pika−/−), and (I) DG cKO (orange; n = 3 WT and n = 3 DG cKO) mice at 1 cd s/m2. (J) Correlation between level of RGS protein accumulation (average of RGS7 and RGS11 content quantified from experiments presented in D and F) at the dendritic tips of ON-BCs and delay in b-wave implicit time (quantified and averaged from experiments presented in G–I). (K) Schematic model of the transsynaptic macromolecular complex of DGC-Pikachurin and GPR179-RGS proteins.

Article Snippet: The following commercial antibodies were used: rat anti-HA (Roche), rabbit anti-myc (GenScript), rabbit anti-Pikachurin (Wako), mouse anti-b-DG (Novocastra), mouse anti-PKCα (Abcam), mouse anti-GAPDH (Millipore) mouse anti-human-Fc (ThemoFisher Scientific), chicken anti-GFP (Genscript), rabbit anti-RFP (Rockland) and chicken anti-RFP (Rockland).

Techniques: Western Blot, Staining

Journal: Cell reports

Article Title: Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors

doi: 10.1016/j.celrep.2018.08.068

Figure Lengend Snippet:

Article Snippet: The following commercial antibodies were used: rat anti-HA (Roche), rabbit anti-myc (GenScript), rabbit anti-Pikachurin (Wako), mouse anti-b-DG (Novocastra), mouse anti-PKCα (Abcam), mouse anti-GAPDH (Millipore) mouse anti-human-Fc (ThemoFisher Scientific), chicken anti-GFP (Genscript), rabbit anti-RFP (Rockland) and chicken anti-RFP (Rockland).

Techniques: Recombinant, Clone Assay, Expressing, Software

Journal: eNeuro

Article Title: Cone Synaptic Function is Modulated by the Leucine-Rich Repeat Adhesion Molecule LRFN2

doi: 10.1523/ENEURO.0120-23.2024

Figure Lengend Snippet: Immunohistochemical reagents used in experiments

Article Snippet: Pikachurin , 1:2,000 , Wako Chemicals , 011-22631;.

Techniques: Immunohistochemical staining

Journal: eNeuro

Article Title: Cone Synaptic Function is Modulated by the Leucine-Rich Repeat Adhesion Molecule LRFN2

doi: 10.1523/ENEURO.0120-23.2024

Figure Lengend Snippet: Synaptic proteins pikachurin, ribeye and ELFN2 are localized normally in absence of LRFN2. A , Representative confocal images after immunohistochemistry for Pikachurin (top row), Ribeye (middle row), and ELFN2 (bottom row) in transverse retinal sections from control and Lrfn2 −/− mouse retina. Scale bar = 5 µm. OPL, outer plexiform layer. Bi , Representative western blots of retinal lysates for DBC signalplex proteins, Bii , Quantification of signalplex protein levels from western blots of retinal lysates from 3 mice of each genotype (mean ± 95% CI). Data were normalized to the control sample. β-actin was used as a loading control.

Article Snippet: Pikachurin , 1:2,000 , Wako Chemicals , 011-22631;.

Techniques: Immunohistochemistry, Western Blot