RT-PCR analysis of <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice.

<p>A. Schematic representation of the location of PCR primers used. Primers RR44, 45, and 46 were used for RT-PCR reactions; primers RR50, 51, 52, and 53 were used for genomic PCR reactions. B. Agarose gel depicting RT-PCR reaction products for mRNA isolated from <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice. Regardless of the primer pair used, only a single band is detected using mRNA from <i>Cacna1f^wt</i> mice. Using mRNA from <i>Cacna1f^nob2</i> mice, however, two bands are visible (see arrows). The relative intensities of the fluorescence signals indicate that the larger-M_r band accounts for ∼90%, and the smaller-M_r band for ∼10%, of the total mRNA.</p

Primer sequences.

<p>Primer sequences.</p

Biophysical properties of Ca_v1.4^wt and Ca_v1.4^nob2 channels, coexpressed with β_2a and α₂–δ₁ subunits in tSA-201 cells.

<p>A. Representative current waveforms for Ca_v1.4^wt (left) and Ca_v1.4^nob2 (right) recorded with 20 mM Ba²⁺ external saline. Horizontal scale bars denote 25 ms, and vertical scale bars 25 pA. B. Average activation (filled symbols) and inactivation (hollow) symbols for Ca_v1.4^wt (squares) and Ca_v1.4^nob2 (circles) recorded with 20 mM Ba²⁺ external saline. Average activation parameters from 11 Ca_v1.4^wt cells and 13 Ca_v1.4^nob2 cells are: V_{act, wt} = −3±4 mV, V_{act, nob2} = −1±4 mV (n = 13); G_{max, wt} = 4±3 nS and G_{max, nob2} = 3±1 nS; S_wt = 9±1 mV and S_nob2 = 8.2±0.8 mV. These values are stastically identical, and are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone-0002538-t003" target="_blank">Table 3</a>. Average inactivation parameters from these cells are V_{inact, wt} = −18±11 mV and V_{inact, nob2} = −22±10 mV, with a large fraction of non-inactivating current for both channels. These values statistically identical and are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone-0002538-t003" target="_blank">Table 3</a>. C. Average half-inactivation potentials for channels recorded with 2 mM Ca²⁺ as charge carrier. Currents were substantially smaller than with 20 mM Ba²⁺, but were distinguishable from background noise, and were obtained using a ramp protocol identical to that previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone.0002538-Doering2" target="_blank">[31]</a>, obtained by ramping voltage from −100 mV to +100 mV over 500 ms. Values were V_{act, wt} = −17±8 mV (average peak current size −9±4 pA) and V_{act, nob2} = −17±6 mV (average peak current size −9±3 pA). The shift observed with switching from 20 mM Ba²⁺ to 2 mM Ca²⁺ as external charge carrier is similar to that we have previously reported for the human Ca_v1.4 channels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone.0002538-McRory1" target="_blank">[16]</a>.</p

Optokinetic Response Data for <i>Cacna1f^nob2</i> and <i>Cacna1f^wt</i> mice.

<p>Optokinetic Response Data for <i>Cacna1f^nob2</i> and <i>Cacna1f^wt</i> mice.</p

Sequencing results of <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice.

<p>A. Sequencing results from the larger-M_r, more intense band. Sequences are given for the DNA (small letters) and corresponding protein (capital letters); the sequence corresponding to wild type Ca_v1.4 protein is highlighted in grey. The ETn transposable element is inserted after the ninth Ca_v1.4 amino acid, and encodes an in-frame stop codon (highlighted in red) which is predicted to result in truncation of the Ca_v1.4 protein after only 25 amino acids. The underlined ETn sequence highlights a repetitive sequence (compare to the beginning of the ETn sequence, beginning at amino acid number 4). B. Same as A., but for the smaller-M_r, less intense band. Note that the inserted ETn element is shorter, and the in-frame stop codon is missing. C. Alignment of the predicted N-terminal amino acid sequences of wild type Ca_v1.4 protein and Ca_v1.4 protein encoded in the smaller-M_r band. A region of approximately 22 amino acids differs in the N-termini of the two clones.</p

A. Representative Coomassie Brilliant Blue-stained 10% PAGE gel from three separate experiments.

<p>N-termini from Ca_v1.4^wt or Ca_v1.4^nob2 were fused to glutathione S-transferase and purified on glutathione beads. Lysates from eye (lanes D–F) or spleen (G–I) were then incubated with the beads, and subsequently washed. Unpurified eye or spleen lysates (5 µL) or purified beads+lysates (40 µL) were loaded onto the gel. Lanes are as follows: A: unpurified eye, B: unpurified spleen, C: protein ladder, D: glutathione-sepharose beads+eye lysates, E: glutathione-sepharose beads+eye lysates+GST-Ca_v1.4 N-terminus, F: glutathione-sepharose beads+eye lysates+GST-Ca_v1.4^nob2 N-terminus, G: glutathione-sepharose beads+spleen lysates, H: glutathione-sepharose beads+spleen lysates+GST-Ca_v1.4 N-terminus, I: glutathione-sepharose beads+spleen lysates+GST-Ca_v1.4^nob2 N-terminus. A prominent band of M_r slightly larger than 37 kDa is observed in lanes E and H, but is absent from lanes F and I. This band was interpreted as a filamin A protein fragment. B. GST-fused N-termini from Ca_v1.4^wt or Ca_v1.4^nob2 were incubated with HA-tagged C-termini of filamin A or filamin B. Only GST-Cav1.4^wt N-terminus was capable of interacting with either filamin A or filamin B. Lanes 1-3 correspond to filamin A (with GST-Cav1.4^wt, GST-Cav1.4^nob2, and GST, respectively); lanes 4–6 correspond to filamin B (with GST-Cav1.4^wt, GST-Cav1.4^nob2, and GST, respectively); lanes 7 and 8 to filamin A and B, respectively (no GST construct); lanes 9–11 GST-Cav1.4^wt, GST-Cav1.4^nob2, and GST, respectively (with no filamin constructs); lane 12 is unpurified GST-Cav1.4^wt lysate; lane 13 is unpurified GST-Cav1.4^nob2 lysate.</p

Western blot of spleen samples from <i>Cacna1f^wt</i>, <i>Cacna1f^nob2</i>, and <i>Cacna1f^G305X</i> mice probed with a Ca_v1.4-specific antibody directed against the C-terminus of the channel.

<p>Full-length protein is visible in all lanes except for <i>Cacna1f^G305X</i>.</p

Summary of biophysical properties of mouse Ca_v1.4^wtand Ca_v1.4^nob2 constructs recorded with 20 mM Ba²⁺ as charge carrier.

<p>s.s. denotes serum starvation for 2 hours; no statistical difference is observed between mouse Ca_v1.4^wt and Ca_v1.4^nob2, or between the constructs when currents were recorded in the absence or presence of 20 µM PD98059 following two hours of serum starvation (p>0.05, one-way ANOVA).</p

Abnormal patterning of the hypertrophic IPL in <i>Pten</i> cKOs.

<p>(A–D) P21 wild-type and <i>Pten</i> cKO retinae labelled in wholemount for TH (A,B) and melanopsin (C,D). Arrowheads in B,D mark increased fasciculation of TH⁺ amacrine cell processes and melanopsin⁺ RGC dendrites in <i>Pten</i> cKO retinae, respectively. (E–H) P7 wild-type and <i>Pten</i> cKO retinae labelled with GFP (green; from <i>Pax6::cre</i> transgene) and Pax6 (red; E,F) or syntaxin (G,H). Bracket in F marks area where <i>Pax6::cre</i> transgene is not expressed. (I–T) P21 wild-type and <i>Pten</i> cKO retinae labelled with syntaxin (I,J), melanopsin (K,L), TH (M,N), calretinin (O,P), ChAT (Q,R) and PKCα (S,T). gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; le, lens; onl, outer nuclear layer; opl, outer plexiform layer; re, retina. Scale bars = 50 µm (A,B,G,H,K–T), 100 µm (C,D,I,J), 600 µm (E,F).</p

PTEN retinal expression and generation of retinal-specific <i>Pten</i> cKO.

<p>(A–E) Co-labeling of P7 retina with PTEN (red) and Brn3b (green, A), calbindin (green, B), Pax6 (green, C), Chx10 (green, D) and rhodopsin (green, E). Blue is DAPI counterstain. Insets to the right of each panel are high magnification images of PTEN⁺ cells, showing co-expression in Brn3b⁺ RGCs (A), calbindin⁺ horizontal cells (B) and Pax6⁺ amacrine cells (C). Insets in D show low levels of PTEN co-expression in Chx10⁺ bipolar cells, while PTEN protein was not detected in rhodopsin⁺ rod photoreceptors (E). (F–I) Schematic illustration of crosses between transgenic animals carrying a Z/AP dual reporter and <i>Pax6</i> α-cre/P0 promoter::<i>Cre-IRES-GFP</i> transgene (hereafter designated <i>Pax6::Cre</i>) (F). Analysis of AP histochemical stain (i.e., recombined cells) in <i>Pax6::Cre</i>;Z/AP double transgenics at E12.5 (G) and in a P7 retinal flatmount (H). β-galactosidase histochemical stain (i.e., non-recombined cells) in a P7 retinal flatmount (I). Inset in G is a high magnification image of the eye, with the asterisk designating a lack of recombination in the central retina. (J) Schematic illustration of crosses between mice carrying a floxed <i>PTEN</i> allele (<i>PTEN^fl</i>) and <i>Pax6::Cre</i> transgene. (K–M) Expression of PTEN in P7 <i>Pten</i>^+/+ (K) and <i>Pten</i> cKO (L) retinal sections. Bracket in L shows central retina where <i>Pten</i> is not deleted and expression is maintained. Insets in K and L show PTEN immunolabeling of retinal flatmounts, confirming that PTEN expression is retained in the central retina in <i>Pten</i> CKOs. PTEN Western blot analysis and densitometry on P21 wild-type and <i>Pten</i> heterozygous and homozygous cKO retinae (M). (N,O) Expression of pAkt^Ser473 in P7 wild-type (N) and <i>Pten</i> cKO (O) retinae. Asterisks in O mark aberrant aggregations of amacrine dendrites in the INL. (P,Q) Western blot analysis and densitometry of pAkt^Ser473/Akt (P) and pS6^Ser235/236/S6 (Q) in P21 wild-type and <i>Pten</i> cKO retinal lysates. p values are denoted as follows: <0.05 *, <0.01 **, <0.005 ***. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; le, lens; on, optic nerve; onbl, outer neuroblast layer; onl, outer nuclear layer; opl, outer plexiform layer; re, retina. Scale bars = 50 µm (A–E,N,O), 2 mm (G), 1 mm (H,I), 600 µm (K,L).</p