July 1999
Volume 40, Issue 8
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Biochemistry and Molecular Biology  |   July 1999
β-Arrestin–Related Proteins in Ocular Tissues
Author Affiliations
  • Corine Nicolas–Léveque
    From the Institut National de la Santé et de la Recherche Médicale, Paris, France.
  • Ibtissem Ghedira
    From the Institut National de la Santé et de la Recherche Médicale, Paris, France.
  • Jean Pierre Faure
    From the Institut National de la Santé et de la Recherche Médicale, Paris, France.
  • Massoud Mirshahi
    From the Institut National de la Santé et de la Recherche Médicale, Paris, France.
Investigative Ophthalmology & Visual Science July 1999, Vol.40, 1812-1818. doi:
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      Corine Nicolas–Léveque, Ibtissem Ghedira, Jean Pierre Faure, Massoud Mirshahi; β-Arrestin–Related Proteins in Ocular Tissues. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1812-1818.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Proteins of the arrestin family contribute to the regulation of G-protein–mediated transduction. In this study, the presence ofβ -arrestins in ocular tissues was investigated.

methods. Mouse monoclonal and rabbit polyclonal antibodies were raised against the peptide Val-Asp-Thr-Asn-Ile-Leu-Glu-Leu-Asp-Thr-Asn-Asp-Asp-Asp-Ile, a sequence present in β-arrestins 1 and 2 but absent from visual arrestin. These antibodies were used for the immunohistologic detection of β-arrestins in parafin sections of rodent eyes fixed in Bouin’s solution. Reverse transcription–polymerase chain reaction (RT-PCR) analysis of RNA from bovine retina, retinal pigmented epithelial (RPE) cells, lens epithelial cells, and human corneal fibroblasts was performed using β-1 arrestin primers.

results. In the eye, β-arrestin staining predominated in RPE, inner segments of photoreceptors, synaptic spherules of rods, inner plexiform layer and ganglion cell fibers, epithelial cells from ciliary body, and vessels. RT- PCR amplified a 480 bp product, corresponding to the predicted length. The sequence of PCR products from bovine retina and RPE cells was identical with the bovine β-arrestin mRNA.

conclusions. β-arrestins were detected in several ocular tissues. In photoreceptor cells, their specific localization in the synaptic terminals and plexiform layer suggests a role of β-arrestin in synaptic transmission. In other ocular tissues, the presence of β-arrestin may be related either to adrenergic signal transduction or to signal transduction mediated by other G-protein–coupled receptors.

Proteins of the arrestin family are implicated in the desensitization of the seven transmembrane domain receptors, a large family of membrane proteins that are at the origin of several transduction cascades mediated by G proteins. The most extensively studied processes implicating these molecules include the visual transduction cascade in the retina 1 2 3 and theβ -adrenergic signal transduction cascade. 4 Visual arrestin (or S-antigen) binds to phosphorylated rhodopsin leading to rhodopsin desensitization. In contrast, β-arrestin desensitizesβ -adrenergic receptors. 5 β-arrestin also mediates the internalization of the agonist-bound G-protein–coupled receptor 6 and is believed to act as a clathrin adaptor in the endocytosis of the β-2 adrenergic receptor. 7 Norepinephrine, an important neuroregulator, exerts many physiological functions in the eye that are mediated by a family of adrenergic receptors. 8 Knowledge of the distribution of theβ -arrestin family in the eye would therefore be useful for understanding the mechanism of adrenergic signal transduction in ocular tissues. 
Consequently, we investigated the presence of β-arrestin in ocular tissues by RT-PCR analysis and by immunohistochemistry. The results are compatible with the idea that β-arrestins are widely distributed in a number of ocular tissues. 
Methods
Preparation of Monoclonal Antibodies
The peptide Val-Asp-Thr-Asn-Ile-Leu-Glu-Leu-Asp-Thr-Asn-Asp-Asp-Asp-Ile which represents an amino acid sequence present in β-arrestins 1 and 2 but absent from visual arrestin was coupled to keyhole limpets hemocyanin (KLH) according to the method described by the manufacturer (Pierce, Rockford, IL). Mouse monoclonal antibodies (mAbs) were raised against this peptide as previously described by de St. Groth and Scheidegger. 9 Several cell lines were selected based on the anti-β-arrestin peptide, and anti-retinal arrestin activity in their supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA). The positive cells were expanded and cloned by the limiting-dilution technique. Aliquots of antibody-secreting clones were kept frozen at −80°C. 10  
Polyclonal Rabbit Antiserum to the β-Arrestin Peptide
Animals used in these experiments were managed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Two hundred micrograms of the peptide coupled to KLH, was reconstituted in Freund’s complete adjuvant and injected intradermally at four foci into adult fawn Burgundy rabbits. A booster was administered 3 weeks later, and the serum was tested for anti-β-arrestin activity by ELISA. To assess antibody specificity, increasing dilutions of the supernatants (β10D3 and β1D4), and of the anti-β-arrestin serum, were incubated (48 hours, 4°C) with either 40 μg free peptide or visual arrestin (antigen-S). After ultracentrifugation, the supernatants were tested for their anti-β-arrestin activity by ELISA. β-10D3 mAb was finally selected for immunoanalysis of β-arrestin in ocular tissues. The antibody absorbed by the peptide (800 μg peptide/ml β10D3 supernatants) was used as a control. 
Cell Culture
Bovine enucleated eyes, obtained from the local abattoir, were stored for 24 hours at 4°C to reduce adhesion between the neural retina and the pigmented epithelium and were bisected posterior to the ora serrata. After removal of the vitreous body and separation of the retina, the eyecup was rinsed with RPMI-1640 (Gibco, Paisley, Scotland, UK), and 1 ml trypsin (0.25%, Gibco) was introduced into the eyecup, followed by incubation for 1 hour at 37°C. The retinal pigmented epithelium (RPE) cells were pipetted out of the Bruch’s membrane, washed with RPMI by centrifugation (10 minutes, 1500g), and finally resuspended in RPMI 1640 supplemented with 5 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 20% fetal bovine serum. The cells were seeded at 5 × 104 cells/cm2 in tissue culture flasks and incubated at 37°C in 5% CO2 atmosphere. The RPE cells were used after the fourth passage. 
Epithelial cells of the bovine lens were cultured according to Plouet et al. 11 and were kindly provided by Yve Courtois, INSERM, Paris. Normal human corneal fibroblasts were isolated from peripheral cornea, as previously described, 12 and cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 5 mM glutamine, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a 5% CO2 humidified incubator. 
Western Blot Analysis
Bovine retina or cultured RPE cells were homogenized in 10 mM phosphate buffer (pH 7.7) containing a mixture of antiproteases, as previously described. 10 Cell-free extract was obtained by centrifugation at 105,000g (1 hour, 4°C). Soluble extracts of bovine retina or RPE were electrophoresed in 10% sodium dodecyl sulfate (SDS) acrylamide gel, then electrotransferred to polyvinylidene difluoride membranes. The membrane were saturated in 15% nonfat dry milk diluted in phosphate-buffered saline (PBS), washed, and incubated either with the mAb (culture supernatant diluted 1:5) or the rabbit antiserum (diluted 1:500) directed against β-arrestin, for 18 hours at 4°C. Antigen–antibody complexes were detected by successive incubations with sheep anti-mouse or anti-rabbit Ig-biotinylated antibody (diluted 1:500) for 2 hours followed by streptavidin-biotinylated horseradish peroxidase complex (diluted 1:500) for 1 hour. Finally, the immunoperoxidase was developed with 4-chloro-1-naphthol (0.05%) and H2O2 (0.01%). The blots were washed three times with PBS between each of the successive steps. 
Animal Experimentation
Prague virol glaxo rats (X5) were exposed to 500 lux and killed after 40 minutes One eye from each animal was immediately immersed in Bouin’s fixative and embedded in paraffin wax for immunohistochemistry. The retina was dissected from the other eye and kept frozen at −80°C for PCR analysis. 
Immunohistochemistry
Thin paraffin sections were deparaffinized with toluene and incubated successively with the mAbs (undiluted supernatants),1:50 diluted biotinylated anti-mouse Ig sheep antibody (Amersham, Amersham, UK), and streptavidin-fluorescein isothiocyanate complex (1:100, Amersham). All reagents were diluted in PBS containing 1% bovine serum albumin. Each incubation step (60 minutes at room temperature) was followed by extensive washing with PBS. The slides were then mounted (Fluoprep; Biomérieux, Marcyl’Etoile, France), and sections were photographed (400 ASA film; Fuji, Tokyo, Japan) under a fluorescence microscope (Nikon, Tokyo, Japan). 
RNA Preparation
Cell RNA (from bovine retina, RPE cells, lens epithelial cells, and human corneal fibroblasts) was prepared using reagent according to the protocol provided by the manufacturer (Trizol; Gibco). It consisted of an improvement over the single-step RNA isolation method developed by Chomczinski and Sacchi. 13 One microgram total RNA was used for reverse transcription into cDNA. RNA was denatured for 3 minutes at 65°C and incubated for 60 minutes at 43°C in a final volume of 20 μl in the presence of 0.5 mM dNTP (Pharmacia, Uppsala, Sweden), 0.01 M dithiothreitol, 0.5 μg oligo(dT)15 (Promega, Madison, WI), 200 U reverse transcriptase (Superscript RNase H− Gibco) and 1 U RNAsin (Promega) in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl2. This mixture was then heated at 95°C for 5 minutes. 
Polymerase Chain Reaction
Sense and antisense oligonucleotide primer pairs were synthesized to match the sequences of β–arrestin 1 (forward 5′-TCATGTCGGACAAGCCCTTGC-3′, reverse 5′-CACTTTGGGCTTGGGGTGCAT-3′) andβ actin (forward, 5′-CTGGAGAAGAGCTATGAGCTG-3′, reverse 5′-AATCTCCTTCTGCATCCTGTC-3′). PCR was performed using 6 μl cDNA. The assay mixture contained 2.5 U Taq DNA polymerase (Gibco), 200 μM dNTP, 2 μM respective oligonucleotide primers, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2 in a volume of 50 μl. The mixture was overlaid with mineral oil (Sigma) and amplified in a thermal cycler (Crocodile III; Appligene, Illkirch, France). Denaturation was carried out at 93°C for 1 minute (3 minutes in the first cycle) followed by an annealing step at 55°C or 61°C (depending on the primers) for 1 minute and an extension step at 72°C for 1 minute (10 minutes in the last cycle) to a total of 25 cycles for β actin and 35 cycles for β arrestin. The PCR products (10 μl) were electrophoresed on 1.2% agarose (Gibco) gels containing ethidium bromide in 45 mM Tris-borate and 1 mM EDTA (TBE buffer). 14 A negative H2O control was amplified under the same conditions. 15 PCR products were sequenced by a method that combines limited chemical degradation of 3′fluorescent-labeled DNA with densitometric analysis (model 377 automatic sequencer; Applied Biosystems, Foster City, CA). 16  
Results
The peptide VDTNLIELDTNDDDI represents a sequence specific toβ -arrestin and absent from the visual arrestin (Fig. 1) . Monoclonal antibodies directed against this peptide linked to KLH, were selected by the cell lines β10D3 and β1D4, whereas polyclonal antibodies were raised in the rabbit (Fig. 2) . The specificity of the antibody was confirmed by incubating various dilutions of antiserum or mAbs with 40 μg peptide. Incubation with this peptide, but not with S-antigen, neutralized the antibody progressively, and total elimination was observed with a 1:128 dilution of mAb β10D3 (similar results were obtained with mAb β1D4). A strong decrease of immunoreactivity was also observed with the 1:1280 dilution of the antiserum. Thus, the antibody appeared to be specifically directed against the β-arrestin peptide antigen (Fig. 2) . In concurrent experiments, these antibodies did not react with retinal arrestin in the ELISA (results not shown). 
In western blot analysis, bovine retinal or RPE extracts were resolved as a unique band of 48 to 50 kDa, both with the rabbit antiserum (Fig. 3A) and with mAbs β10D3 and β1D4 (Fig. 3 B). This molecular weight is in good agreement with β-arrestin reported previously. 10  
Immunohistochemistry, with the mAb reveled thatβ -ar-restin was present in both segments of the photoreceptor (rod and cone cell layer) the inner segment, the synaptic spherules of rods, and in the outer segments (arrow). Similarly,β -arrestin was present in fibers and granules of outer and inner plexiform layers, and in the ganglion cell layer (Fig. 4C) . Intense staining was also observed in the ciliary body, iris, pars plana (Fig. 5 A), and pars plicata (Fig. 5B) , where both the unpigmented (arrow) and the pigmented (double arrow) epithelia were immunopositive. In the iris (Fig. 5C) , only the vessel wall was stained by these antibodies, whereas no immunoreactivity was observed in the epithelium of iris (Fig. 5C , asterisk). Finally, β-arrestin was also detected in the cytoplasm of RPE cells (Fig. 5D , double arrows) and in the endothelial cells of vessels in the choroid (Fig. 5D , arrow). Similar results were observed with the polyclonal rabbit antiserum (results not shown). 
To determine the levels of mRNAs for β-arrestin 1 and β-actin, PCR was performed with RNA prepared from different ocular tissues. Data in Figure 6 show that the PCR products corresponded to the predicted lengths, according to the primers used for amplification. The mRNA forβ -arrestin was present in bovine retina, bovine RPE cells, bovine lens epithelial cells, and human corneal fibroblasts. The DNA sequences for β-arrestin from retina and RPE were determined by the use of four different chain terminators in a single electrophoretic run. Significant homology was found among amino acid sequences ofβ -arrestin from bovine retina and RPE, bovine retinal arrestin, and bovine β-arrestins 1 and 2 (results not shown). These provide clear evidence for β-arrestin-like proteins in ocular tissues. 
Discussion
The sequence of the peptide used for the preparation of the mAbs to β-arrestins is common to β-arrestins 1 and 2 but absent from other known mammalian arrestins. Therefore these mAbs can specifically detect β-arrestins and possibly other still unknown closely related arrestins. They do not detect the S-antigen (the arrestin involved in phototransduction) or the cone (or X-) arrestin 17 18 that are expressed in the retinal photoreceptor cells. The mRNAs forβ -arrestins 1 and 2 have previously been detected in the retina and in many other nonocular tissues by PCR. 19 To our knowledge, immunodetection of these proteins in the eye has not been previously reported. Attramadal et al. 20 showed the presence of β-arrestins 1 and 2 in sections of rat brain in synaptic junctions and in some neurons. 
We show here that, in the retina, β-arrestins were specifically located in the synaptic spherules of photoreceptor cells. They were also present in the inner segments, the probable site of their synthesis. Furthermore, β-arrestins were also detected in other synaptic bodies and nerve fibers of the inner retinal layers. This localization suggests that these molecules have a role in synaptic transmission in the retina. Until now, adrenergic receptorsβ , 21 β-2, 22 23 and β 1, 2 24 were reported in the retina, but the absence of these receptors 25 also has been reported. Although the cellular localization of these receptors is unknown, the α-adrenergic receptor also was described in the retina. 8 The β-adrenergic receptor kinases 1 and 2, which phosphorylate the β-adrenergic receptor and thus promote the binding of β-arrestin to the receptor, are widely distributed in the brain synapses. 26 These kinases also are able to phosphorylate other receptors. This suggests that β-arrestins could similalrly desensitize other receptors involved in neurotransmission. However, the specificity of each arrestin for the recognition of individual G-protein–coupled receptors remains to be explored. 
β-Arrestin–related proteins may be implicated in synaptic receptor desensitization, but they also could be an adaptor for the adrenergic-related receptors involved in endocytosis of corresponding receptors. 7 Exocytosis, another process in the synaptic function, also is dependent on guanosine triphosphate–binding proteins and arrestin-related proteins may be implicated in this manner. 
The presence of β-arrestins in the epithelial cells in the eye is in agreement with the localization of adrenergic receptors described in oculars tissues. 8 Consequently, these proteins are likely to play an important role in the function of adrenergic receptors. 
 
Figure 1.
 
Sequence comparison of arrestin and β-arrestin: The peptide VDTNLIELDTNDDDI represents an amino acid sequence present inβ -arrestins 1 and 2 but absent from visual arrestins.
Figure 1.
 
Sequence comparison of arrestin and β-arrestin: The peptide VDTNLIELDTNDDDI represents an amino acid sequence present inβ -arrestins 1 and 2 but absent from visual arrestins.
Figure 2.
 
Quantitation and specificity of the anti-β-arrestin activity in the rabbit serum and in the mouse mAb. Serial dilutions of the rabbit serum and β10D3 supernatant were analyzed for β-arrestin activity by ELISA. The antibody could be absorbed with 40 μg peptide (pep.) but not with 40 μg S-antigen (AgS) as described in the Methods section. Each value is the average of three determinations.
Figure 2.
 
Quantitation and specificity of the anti-β-arrestin activity in the rabbit serum and in the mouse mAb. Serial dilutions of the rabbit serum and β10D3 supernatant were analyzed for β-arrestin activity by ELISA. The antibody could be absorbed with 40 μg peptide (pep.) but not with 40 μg S-antigen (AgS) as described in the Methods section. Each value is the average of three determinations.
Figure 3.
 
Western blot analysis of bovine retina (A) and RPE (B) soluble extracts. Immunoblot was developed with the rabbit polyclonal antiserum to β-arrestin peptide (lane A) and the mAb to β-arrestin peptide, β10D3 (lane B) and β1D4 (lane C). All the antibodies recognized a band of 46 to 48 kDa. Preimmune serum and the anti-fibrinogen mAb were used as a control (lane D).
Figure 3.
 
Western blot analysis of bovine retina (A) and RPE (B) soluble extracts. Immunoblot was developed with the rabbit polyclonal antiserum to β-arrestin peptide (lane A) and the mAb to β-arrestin peptide, β10D3 (lane B) and β1D4 (lane C). All the antibodies recognized a band of 46 to 48 kDa. Preimmune serum and the anti-fibrinogen mAb were used as a control (lane D).
Figure 4.
 
Demonstration of β-arrestin by immunofluorescence: Rat eye sections were treated with the absorbed mAb β10D3 to establish a control baseline. The retina (A), ciliary body, and iris (B) were negative in all the cellular layers.β -arrestin was immunolocalized in the photoreceptor layer, the rod and cone layer (RCL), the external plexus (EP), the internal plexus (IP), and the ganglion cell layer (GCL) from light-adapted rats. (C) Original magnification, ×200.
Figure 4.
 
Demonstration of β-arrestin by immunofluorescence: Rat eye sections were treated with the absorbed mAb β10D3 to establish a control baseline. The retina (A), ciliary body, and iris (B) were negative in all the cellular layers.β -arrestin was immunolocalized in the photoreceptor layer, the rod and cone layer (RCL), the external plexus (EP), the internal plexus (IP), and the ganglion cell layer (GCL) from light-adapted rats. (C) Original magnification, ×200.
Figure 5.
 
Immunofluorescent staining of the pars plana and pars plicata of the ciliary body, the iris, and the RPE. The nonpigmented (arrow) and pigmented (double arrow) epithelia exhibited β-arrestin–specific immunofluorescence in the pars plana (A) and pars plicata (B) of the ciliary body. In the iris (C) and choroid (D) only the vessel walls were stained; no immunoreactivity was observed in the epithelium of the iris (C, asterisk). Strong immunoreactivity was observed in the RPE (D, arrow). The β-arrestin–specific immunoreactivity was intense in the cytoplasm of the cells. Original magnification, ×200, section from PVG rat eye. Immunofluorescence reaction with mAbβ -10D3.
Figure 5.
 
Immunofluorescent staining of the pars plana and pars plicata of the ciliary body, the iris, and the RPE. The nonpigmented (arrow) and pigmented (double arrow) epithelia exhibited β-arrestin–specific immunofluorescence in the pars plana (A) and pars plicata (B) of the ciliary body. In the iris (C) and choroid (D) only the vessel walls were stained; no immunoreactivity was observed in the epithelium of the iris (C, asterisk). Strong immunoreactivity was observed in the RPE (D, arrow). The β-arrestin–specific immunoreactivity was intense in the cytoplasm of the cells. Original magnification, ×200, section from PVG rat eye. Immunofluorescence reaction with mAbβ -10D3.
Figure 6.
 
Agarose gel analysis of the PCR products for β-actin and β-arrestin 1. The predicted products were seen in the bovine retina (RB), retinal pigmented epithelial (RPE), lens epithelium (LE), and corneal fibroblasts (CF). The size of the PCR products corresponded to the predicted lengths of 460 and 480 bp for β-arrestin and β-actin, respectively. The size of DNA standards is indicated on the left, in base pairs.
Figure 6.
 
Agarose gel analysis of the PCR products for β-actin and β-arrestin 1. The predicted products were seen in the bovine retina (RB), retinal pigmented epithelial (RPE), lens epithelium (LE), and corneal fibroblasts (CF). The size of the PCR products corresponded to the predicted lengths of 460 and 480 bp for β-arrestin and β-actin, respectively. The size of DNA standards is indicated on the left, in base pairs.
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Figure 1.
 
Sequence comparison of arrestin and β-arrestin: The peptide VDTNLIELDTNDDDI represents an amino acid sequence present inβ -arrestins 1 and 2 but absent from visual arrestins.
Figure 1.
 
Sequence comparison of arrestin and β-arrestin: The peptide VDTNLIELDTNDDDI represents an amino acid sequence present inβ -arrestins 1 and 2 but absent from visual arrestins.
Figure 2.
 
Quantitation and specificity of the anti-β-arrestin activity in the rabbit serum and in the mouse mAb. Serial dilutions of the rabbit serum and β10D3 supernatant were analyzed for β-arrestin activity by ELISA. The antibody could be absorbed with 40 μg peptide (pep.) but not with 40 μg S-antigen (AgS) as described in the Methods section. Each value is the average of three determinations.
Figure 2.
 
Quantitation and specificity of the anti-β-arrestin activity in the rabbit serum and in the mouse mAb. Serial dilutions of the rabbit serum and β10D3 supernatant were analyzed for β-arrestin activity by ELISA. The antibody could be absorbed with 40 μg peptide (pep.) but not with 40 μg S-antigen (AgS) as described in the Methods section. Each value is the average of three determinations.
Figure 3.
 
Western blot analysis of bovine retina (A) and RPE (B) soluble extracts. Immunoblot was developed with the rabbit polyclonal antiserum to β-arrestin peptide (lane A) and the mAb to β-arrestin peptide, β10D3 (lane B) and β1D4 (lane C). All the antibodies recognized a band of 46 to 48 kDa. Preimmune serum and the anti-fibrinogen mAb were used as a control (lane D).
Figure 3.
 
Western blot analysis of bovine retina (A) and RPE (B) soluble extracts. Immunoblot was developed with the rabbit polyclonal antiserum to β-arrestin peptide (lane A) and the mAb to β-arrestin peptide, β10D3 (lane B) and β1D4 (lane C). All the antibodies recognized a band of 46 to 48 kDa. Preimmune serum and the anti-fibrinogen mAb were used as a control (lane D).
Figure 4.
 
Demonstration of β-arrestin by immunofluorescence: Rat eye sections were treated with the absorbed mAb β10D3 to establish a control baseline. The retina (A), ciliary body, and iris (B) were negative in all the cellular layers.β -arrestin was immunolocalized in the photoreceptor layer, the rod and cone layer (RCL), the external plexus (EP), the internal plexus (IP), and the ganglion cell layer (GCL) from light-adapted rats. (C) Original magnification, ×200.
Figure 4.
 
Demonstration of β-arrestin by immunofluorescence: Rat eye sections were treated with the absorbed mAb β10D3 to establish a control baseline. The retina (A), ciliary body, and iris (B) were negative in all the cellular layers.β -arrestin was immunolocalized in the photoreceptor layer, the rod and cone layer (RCL), the external plexus (EP), the internal plexus (IP), and the ganglion cell layer (GCL) from light-adapted rats. (C) Original magnification, ×200.
Figure 5.
 
Immunofluorescent staining of the pars plana and pars plicata of the ciliary body, the iris, and the RPE. The nonpigmented (arrow) and pigmented (double arrow) epithelia exhibited β-arrestin–specific immunofluorescence in the pars plana (A) and pars plicata (B) of the ciliary body. In the iris (C) and choroid (D) only the vessel walls were stained; no immunoreactivity was observed in the epithelium of the iris (C, asterisk). Strong immunoreactivity was observed in the RPE (D, arrow). The β-arrestin–specific immunoreactivity was intense in the cytoplasm of the cells. Original magnification, ×200, section from PVG rat eye. Immunofluorescence reaction with mAbβ -10D3.
Figure 5.
 
Immunofluorescent staining of the pars plana and pars plicata of the ciliary body, the iris, and the RPE. The nonpigmented (arrow) and pigmented (double arrow) epithelia exhibited β-arrestin–specific immunofluorescence in the pars plana (A) and pars plicata (B) of the ciliary body. In the iris (C) and choroid (D) only the vessel walls were stained; no immunoreactivity was observed in the epithelium of the iris (C, asterisk). Strong immunoreactivity was observed in the RPE (D, arrow). The β-arrestin–specific immunoreactivity was intense in the cytoplasm of the cells. Original magnification, ×200, section from PVG rat eye. Immunofluorescence reaction with mAbβ -10D3.
Figure 6.
 
Agarose gel analysis of the PCR products for β-actin and β-arrestin 1. The predicted products were seen in the bovine retina (RB), retinal pigmented epithelial (RPE), lens epithelium (LE), and corneal fibroblasts (CF). The size of the PCR products corresponded to the predicted lengths of 460 and 480 bp for β-arrestin and β-actin, respectively. The size of DNA standards is indicated on the left, in base pairs.
Figure 6.
 
Agarose gel analysis of the PCR products for β-actin and β-arrestin 1. The predicted products were seen in the bovine retina (RB), retinal pigmented epithelial (RPE), lens epithelium (LE), and corneal fibroblasts (CF). The size of the PCR products corresponded to the predicted lengths of 460 and 480 bp for β-arrestin and β-actin, respectively. The size of DNA standards is indicated on the left, in base pairs.
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