KARs are reduced in the cerebellar PSD of Neto2-null mice.

<p>(A) Immunoblots (representative of three experiments) of proteins from cerebellar homogenates and cerebellar PSD fractions of wild-type (Wt) and Neto2-null (<i>Neto2^−/−</i>) mice. Antibodies used for detection are indicated on the left. (B) Histogram showing normalized levels of different proteins in Neto2-null cerebellar homogenates relative to that of wild-type (white bars), and in Neto2-null cerebellar PSD fractions relative to that of wild-type (black bars); **, p<0.01, paired t-test, n = 3.</p

Neto2 is associated with KARs in the cerebellum.

<p>(A) Confocal micrographs of immunostained cerebellar slices. Antibodies used for immunostaining are indicated in the top left corner of each image. In the cerebellum, the NeuN antibody stains the neuronal nuclei of granule cells but does not recognize Purkinje cells. MCL, molecular cell layer; GCL, granule cell layer; Wt, wild-type sections; <i>Neto2^−/−</i>, Neto2-null sections. Scale bar, 100 μm. (B) High-magnification confocal microscopy of the cerebellar granule cell layer immunostained with Neto2, GluK2, NeuN, or synaptophysin antibodies. Scale bar, 20 μm; scale bar (small panels on the right), 5 μm (C) Immunoblot of immunoprecipitates from the cerebellum. Blot: antibody used for immunoblot analysis; IP: immunoprecipitate. The input represents 2% of the material used in the immunoprecipitation experiment.</p

Neto2 associates with GRIP <i>in vivo</i>.

<p>(A, B) Immunoblots of immunoprecipitates from cerebellar (A) or whole brain (B) membrane fractions. Samples were subjected to immunoprecipitation with an anti-GRIP antibody, or with normal rabbit IgGs (IgG), as the negative control. Blot, antibody used for immunoblot analysis; IP, immunoprecipitate.</p

Neto2 increases the interaction between GRIP and GluK2.

<p>Immunoblot of immunoprecipitates from lysates of transfected COS-7 cells. The cDNAs used for transfection are shown above each lane. Neto2Δ7, Neto2 lacking the last seven C-terminal residues. Blot, antibody used for immunoblot analysis; IP, antibody used for immunoprecipitation.</p

Summary of <i>Vsx2-5.3-PRE-Cre</i> transgenic mouse lines.

<p>*“+” indicates Cre immunolabeling restricted to postmitotic presumptive bipolar cells and/or mature retinal bipolar cells</p><p>**“+” indicates reporter expression in the mature retina in bipolar cells and a subset of photoreceptor cells</p

Cre expression precedes expression of the mature bipolar markers PKCα and Cabp5.

<p>At postnatal day 3 (P3) Cre-positive nuclei do not co-localize to Cabp5 (A–C), PKCα (D–F), or with the early bipolar fate marker Bhlhb5 (M–O). Insets (G–I) are high magnification view of Cre-positive (solid arrowhead) and Cre-negative (open arrowhead) nuclei located apical to the Bhlhb5-positive layer (arrow for example). Scale bar (O)  = 20 µm.</p

<i>Vsx2-5.3-PRE-Cre</i> is expressed in a large subset of bipolar neurons in the adult retina.

<p>Immunolabeling for Cre-recombinase under control by the <i>Vsx2-5.3-PRE</i> transgene is localized in nearly all PKCα expressing rod bipolar cells (A–C); a large subset of CaBp5-expressing Type-III a/b, Type-V ON, or rod bipolar cells (D–F); <i>mGlur6-lacZ</i> expressing ON bipolar cells (G–I); and a large subset of PKARIIβ-expressing Type-IIb and OFF bipolar neurons (J–L). Bhlhb5 expressing Type-II OFF bipolar cells do not express Vsx2-5.3-PRE-Cre (M–O). Arrows indicate co-localized cells; solid arrowheads indicate Cre-only cells; open arrowheads indicate Cre-negative cells. Scale bar (N)  = 10 µm.</p

<i>Vsx2-5.3-PRE-Cre</i> is specifically localized to Vsx2-expressing neurons of the inner nuclear layer in the mature retina.

<p>(A–C) The vast majority of Vsx2-labeled nuclei (A) in the inner nuclear layer (boundaries indicated by broken lines) co-label for Cre (B). Arrowheads in (C) indicate Vsx2-positive/Cre-negative nuclei outlined in (B). (D–F) Sox9-positive Müller glial nuclei (D) do not express Cre. Arrowheads (F) show examples of Cre-negative/Sox9-positive nuclei outlined in (E). (G–I) Horizontal cells labelled with Calbindin-D28k (G) do not label for Cre (H). Sections are from >6 week old mice. Scale bar  = 10 µm.</p

Summary of <i>Vsx2-5.3-PRE-Cre</i> expression in the developing retina.

<p>Although Cre immunolabeling is not evident in proliferating RPCs, genetic fate mapping identifies <i>Vsx2-5.3-PRE-Cre</i> activity in bipolar and photoreceptor cells. (A) Following bipolar cell specification, Cre immunolabeling is strongly up-regulated in all bipolar neuron subtypes, with the exception of blhlb5-positive Type 2 OFF cells. TdTomato-positive photoreceptors could be derived from postmitotic bipolar cell/photoreceptor cell precursors. (B) Alternatively, TdTomato expression may result from transient low-level Cre expression in photoreceptor cells derived from either RPCs, or from uncommitted bipolar cell precursors that switch their fate to that of photoreceptor cells.</p

List of Antibodies.

<p>List of Antibodies.</p