Loss of MCT1, MCT3, and MCT4 Expression in the Retinal Pigment Epithelium and Neural Retina of the 5A11/Basigin-Null Mouse

Abstract

purpose. The neural retina expresses multiple monocarboxylate transporters (MCTs) that are likely to play a key role in the metabolism of the outer retina. Recently, it was reported that targeting of MCT1 and -4 to the plasma membrane requires association with 5A11/basigin (CD147). In the present study, the hypothesis that reduced amplitudes in the electroretinograms in the 5A11/basigin null mouse (Bsg ^−/−) may be linked to altered expression of MCTs was studied.

methods. The expression and subcellular distribution of MCTs in Bsg ^−/− mice was analyzed by immunofluorescence microscopy with isoform-specific antibodies. Protein expression was analyzed by Western blot analysis, and mRNA expression was examined with RT-PCR.

results. Immunofluorescence labeling of tissue sections from the Bsg ^−/− mice revealed a dramatic reduction in labeling with MCT antibodies. There was a loss of MCT1 labeling in the apical membrane of the RPE and in the neural retina. MCT3, which is expressed in the basolateral membrane of the RPE wild-type mouse, was expressed at very low levels in both the apical and basolateral membranes of the Bsg ^−/− mouse. There was no change in expression or distribution of the glucose transporter (GLUT)-1 in the RPE and retina of the Bsg ^−/− mouse. Western blot analysis of detergent-soluble lysates prepared from wild-type and Bsg ^−/− eyes confirmed that the levels of MCT1, MCT3, and MCT4 protein were severely reduced in Bsg ^−/− mice. RT-PCR analyses of mRNA levels from wild-type and Bsg ^−/− mice demonstrated that the MCT1 transcript was expressed at normal levels in Bsg ^−/− mice.

conclusions. In Bsg ^−/− mice, there is a severe reduction in accumulation of the MCT1 and -3 proteins in the RPE and a concomitant reduction in MCT1 and -4 in the neural retina supporting a role for 5A11/basigin in the targeting of these transporters to the plasma membrane. Decreased expression of MCT1 and -4 on the surfaces of Müller and photoreceptor cells may compromise energy metabolism in the outer retina, leading to abnormal photoreceptor cell function and degeneration.

In the retina, glucose is metabolized through aerobic glycolysis producing substantial quantities of lactate. Whereas lactate was once viewed merely as a end product of glucose metabolism, it is now known that lactate can be used by photoreceptor cells to fuel oxidative phosphorylation. ¹ Excess lactate is transported out of the retina by the RPE. The transport of lactate across the plasma membrane is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). ² Nine members of this family have been identified based on sequence homologies but only MCT types 1 through 4 have been functionally characterized. ^{3 4 5 6} These isoforms are distinguished by their kinetic properties and tissue distribution. 

Multiple MCT isoforms have been detected in the RPE and retina. ^{7 8 9} The RPE expresses MCT1 in the apical membrane and MCT3 in the basolateral membrane. ¹⁰ In the retina, MCT1 is highly expressed in Müller cells, photoreceptor inner segments, and microvessels forming the inner blood–retinal barrier. ¹¹ MCT2 is detected in Müller cell end feet and in glial processes surrounding retinal microvessels, ⁹ and MCT4 is expressed in the inner retina. ⁸ The expression of multiple MCTs in the retina and RPE is consistent with the complex role of lactate in normal retinal metabolism. 

5A11/basigin (5A11/Bsg) is a widely expressed transmembrane glycoprotein belonging to the immunoglobulin superfamily. This protein has been identified independently by a number of different laboratories and is described in the literature as 5A11, ^{12 13} gp42, ¹⁴ basigin, ¹⁵ neurothelin, ^{16 17} CE9, ¹⁸ EMMPRIN, ¹⁹ and CD147 ²⁰ . Although the expression and distribution of 5A11/Bsg has been extensively characterized, the function(s) of this protein in different tissues has not been clearly determined. 5A11/Bsg has been identified as a Müller cell membrane protein that plays a role in glial cell maturation. ¹¹ RPE cells and endothelial cells, which form the inner and blood–retinal barriers express high levels of 5A11/Bsg, although a role for 5A11/Bsg in these barriers has never been established. 5A11/Bsg (EMMPRIN) expressed by tumor cells has been shown to stimulate the production of matrix metalloproteinases (MMPs) by stromal fibroblasts. ^{19 21} 5A11/Bsg was found in coprecipitation studies to associate specifically with β1-integrin ^{22 23} and with the monocarboxylate transporters MCT1 and -4. ^{24 25} Coexpression of MCTs with 5A11/Bsg in transiently transfected cells facilitates the targeting of the proteins to the plasma membrane. ²⁴  

Mice with a targeted deletion of the 5A11/Bsg gene (Bsg ^−/−) are sterile, have deficits in learning and memory, and are blind. ^{26 27 28} In the first two months of age, the retinal morphology appears relatively normal; however, there is a reduction in the amplitude of the electroretinograms (ERGs) at eye opening when compared with those in control littermates. ^{27 29} A decrease in the amplitude of the ERG can result from an alteration in energy metabolism in the retina. ^{30 31} Because MCTs are involved in metabolic coupling in the retina, we tested the hypothesis that MCT expression was altered in the Bsg ^−/− mouse. Based on our findings, we propose that the loss of expression of MCT in the RPE and retina of the Bsg ^−/− mouse contributes to the abnormal ERG. 

Materials and Methods

Animals

The 5A11/Bsg-knockout mouse strain was generated using a neomycin gene-insertion strategy, as described previously. ²⁰ Heterozygote breeding was performed at the Whitney Laboratory (University of Florida, St. Augustine, FL). The genotype of offspring mice was determined with PCR of genomic DNA isolated from tail snips performed with a commercial system (Easy-DNA; Invitrogen, Carlsbad, CA). The PCR primers and reaction conditions followed published protocols. ³² Care and handling of these animals was in accordance with the guidelines established by the University of Florida Institutional Animal Care and Use Committee (IACUC). All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Antibodies

Isoform-specific antibodies were produced against the 18-mer synthetic oligopeptide corresponding to the carboxyl terminal amino acids of MCT3 and -4. The antibodies were purified with immunoaffinity columns prepared with peptide antigen linked to agarose. The specificity of the antibodies was confirmed by performing Western blot and immunocytochemical analyses in the presence or absence of the peptide antigen. ¹⁰ MCT1 and glucose transporter (GLUT)-1 antibodies were gifts from Ian Simpson (Pennsylvania State University, Hershey, PA). The 5A11/Bsg antibody was produced in rabbits against an extracellular domain of basigin. ²⁹  

Immunocytochemical Analysis

Primary fixation of mouse retinas was by perfusion with 4% paraformaldehyde in 0.1 M cacodylate (pH 7.4) followed by enucleation and immersion in the same solution for 1 hour at 4°C. The tissue was transferred through two 30-minute changes of Carnoy fluid (60% reagent alcohol, 30% chloroform, 10% acetic acid) and brought to room temperature. Tissues were transferred to 100% ethanol, cleared with aniline-methylsalicylate (1:1), and embedded in paraffin. Tissues were cut in 10-μm-thick sections, mounted on gelatin-coated slides, deparaffinized, and rehydrated in a graded series of ethanol. 

Sections were preincubated in phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) and 0.1% Tween 20 for 1 hour at room temperature. Primary antibodies were diluted in PBS containing 1% BSA and 0.1% Tween 20 and incubated with the sections for 1 hour at room temperature, as previously described. ¹⁰ The primary antibodies were detected using carboxymethylindocyanine (Cy3)-coupled secondary antibody (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in the same buffer as the primary antibodies. Sections were examined by microscope (Microphot FX; Nikon, Tokyo, Japan) equipped with a digital camera (Optronics, Goleta, CA) and imaging software (Bioquant, Nashville, TN). 

Western Blot Analyses

Detergent soluble lysates of retina and RPE were prepared as previously described. ¹⁰ Briefly, the anterior segment of the eye was removed with a sharp razor blade. The posterior eyecup was placed in PBS, and the retina was removed and immediately frozen. The RPE-choroid-sclera was frozen in a separate tube. Two eyes were collected from both Bsg ^−/− and Bsg ^+/+ mice for each time point and extracted separately with ice-cold lysis buffer (1% Triton X-100, 10 mM imidazole, 100 mM KCl, 1 mM EDTA, 5 mM MgCl₂, and protease inhibitors; Complete Mini, Roche Molecular Biochemicals, Indianapolis, IN) for 30 minutes on ice. Lysates were cleared by centrifugation at 12,000 rpm for 15 minutes at 4°C, and supernatants were removed and diluted with an equal volume of 2× SDS sample buffer. Protein concentrations of the supernatants were determined with the bicinchoninic acid (BCA) reagent (Pierce, Rockford, IL). Detergent lysates from various mouse tissues were prepared by the extraction methods detailed earlier. Intestinal epithelial cells were isolated by incubating segments of the small intestine in PBS containing 15 mM EDTA, as previously described for isolation of RPE from embryonic chick eyes. ³³ Cell lysates containing equal amounts of proteins were separated on 4% to 12% SDS-polyacrylamide gels and then electrophoretically transferred to membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in primary and secondary antibodies with a Western blot immunodetection system (Western Breeze Chemiluminescent; Invitrogen). 

RT-PCR Analyses

5A11/Bsg-null mice and wild-type littermates were killed according to accepted protocols, and the eyes were immediately removed. Neural retinas were isolated and homogenized (TRI-Reagent; MRC, Cincinnati, OH) and the total RNA was extracted according to the manufacturer’s protocol. RT-PCR to determine expression of MCT1 and -4 was performed with 1 μg total retinal RNA and Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ), according to the manufacturer’s protocol. The MCT1 primer set was as follows: MCT1 forward, 5′-GTGCAGCA-GCCAAGGAGCCC; MCT1 reverse, 5′-CCATGGCCAGTCCGTTGGCC. The PCR cycling protocol used was as follows: 96°C for 1 minute, 55°C for 5 minutes, and polymerization at 72°C for 5 minutes, and repeated for 30 cycles, which was determined to be in the linear range. 

For real-time RT-PCR analyses, relative amounts of MCT1 mRNA present within normal and 5A11/Bsg-null mouse retinas were determined with green fluorescent dye (SYBR Green; Applied Biosystems, Foster City, CA) on a sequence detection system (ABI PRISM 7000; Applied Biosystems). Primers specific for mouse MCT1 and 18S ribosomal RNA were designed on computer (Primer Express software; Applied Biosystems). All tests were performed in triplicate. The default one-step RT-PCR protocol (40 cycles) was used. Expression of MCT1 was normalized with 18S ribosomal RNA. Expression within the null retina, compared with that found in the normal retina was evaluated with the mathematical equations pertinent to sequence detection (SYBR Green; Applied Biosystems), as directed by the manufacturer. The amount of MCT1 mRNA in the wild-type mouse retina was assigned a value of 1. 

Results

Immunocytochemical Analysis of MCT1 and -3 in the Retina and RPE of Bsg ^−/− Mice

In the present study, immunofluorescence analysis was used to examine the expression and targeting of MCT1 and -3 in the retinas of wild-type and Bsg ^−/− mice. Paraffin sections of posterior eyecups prepared from 8-week-old wild-type and Bsg ^−/− mice were labeled with antibodies to 5A11/Bsg (Figs. 1A 1B) , MCT1 (Figs. 1C 1D) , and MCT3 (Figs. 1E 1F) . Images were collected from wild-type and Bsg ^−/− mice using the same integrated exposure time. In the wild-type mouse, 5A11/Bsg antibody labeled apical and basolateral membranes of the RPE, Müller glial cells and endothelial cells of the inner retinal capillaries (Fig. 1A) . A similar labeling pattern was seen in the neural retina of the wild-type mouse with antibody to MCT1 (compare Figs. 1A 1C ). MCT1 was expressed in the apical membrane of the RPE (Fig. 1C) , whereas MCT3 expression was restricted to the basolateral membrane (Fig. 1E) . 

In the Bsg ^−/− mouse, no labeling was observed with the 5A11/Bsg antibody (Fig. 1B) . MCT1 was not detected in the plasma membrane of Müller cells or endothelial cells in the Bsg ^−/− mouse (Fig. 1D) . The apical portions of the Müller cells had what appeared to be intracellular inclusions that constituted the primary site of residual MCT1 labeling within the neural retina proper (Fig. 1D , asterisk). MCT1 was not expressed at detectable levels in the apical membrane of the RPE of the Bsg ^−/− mouse (Fig. 1D , arrowhead). There was only faint MCT3 labeling in apical and basolateral membranes of the RPE in the Bsg ^−/− mouse (Fig. 1F) . 

To determine whether the loss of MCTs in the Bsg ^−/− mouse was specific for this family of solute transporters, expression of GLUT1 was examined in wild-type and Bsg ^−/− mice. Immunofluorescence analysis revealed that GLUT1 was expressed at high levels in the RPE and retinas of wild-type and Bsg ^−/− mice (Fig. 2) . 

Immunoblot Analysis of MCT Isoform Expression in the 5A11/Bsg-Null Mouse

The results of the immunofluorescence analysis indicated a loss of expression or accumulation of MCTs in Bsg ^−/− mice. To ensure that the reduced immunolabeling was not a result of epitope masking, immunoblot analyses were performed by using detergent lysates prepared from RPE-choroid-sclera and retinas of Bsg ^−/− mice and their wild-type littermates. Two ages were examined; postnatal day (P)20, when retinas of the Bsg ^−/− appear relatively normal and photoreceptor cells are still present, and 1 year of age, when there is nearly a total loss of photoreceptor cells. ^{27 29}  

Western blot analysis of the RPE-choroid-sclera lysates is presented in Figure 3 . In samples prepared from both young (P20) and old (1 year) mice, there was a substantial reduction in the levels of both MCT1 and -3 proteins in the Bsg ^−/− mice compared with levels in age matched wild-type mice (Fig. 3) . GLUT1 expression was relatively unchanged in the RPE-choroid-sclera of Bsg ^−/− mice compared with that in the age-matched control. 

The expression of MCT1 and -4 in retinas of Bsg ^−/− and wild-type mice was examined by Western blot analysis (Fig. 4A) . In detergent lysates of neural retinas prepared from 20-day Bsg ^−/− mice, there was a decrease in MCT1 and -4 proteins compared with levels in age-matched control. At 1 year of age, only very low levels of MCT1 and -4 were detected in retinal lysates prepared from Bsg ^−/− mice. In contrast, MCT2 was expressed at similar levels in retinas of control and Bsg ^−/− mice. This is consistent with the report that MCT2 does not associate with CD147. ²⁴ GLUT1 was abundantly expressed in lysates prepared from both Bsg ^−/− and Bsg ^+/+ mice. In the 1-year-old animals, after photoreceptor cell degeneration has become protracted, there was a decrease in the level of GLUT1 protein (Fig. 4A) . 

RT-PCR Analysis of MCT1 mRNA in the Mouse Retina

To determine whether the loss of expression of MCT protein in the Bsg ^−/− mouse is due to a loss of transcription, the expression of MCT1 mRNA was examined by standard and real-time RT-PCR. Retinas from age-matched (P20 and 1 year) control and Bsg ^−/− mice were evaluated. As shown in Figure 4B , the transcript for MCT1 was present in Bsg ^−/− mice at both ages tested. However, a standard RT-PCR did not provide information about the amounts of transcript present within a given sample. To determine the amount of MCT1 transcript within the Bsg ^−/− mouse retina, compared with that found within a normal retina, real-time analyses using the green fluorescence (SYBR Green; Applied Biosystems) technology were performed (Fig. 4C) . MCT1 transcript was present within the Bsg ^−/− retina at a similar or slightly elevated level, compared with that in the normal mouse retina. 

Colocalization of 5A11/Bsg with MCT1 and -3 in the Apical and Basolateral Membranes of RPE

In the present study, we demonstrate by immunofluorescence and immunoblot analyses that there was a dramatic reduction in MCT1 and -3 protein in the RPE of 5A11/Bsg null mice. This observation suggests 5A11/Bsg plays a role in the trafficking of both MCTs to the RPE plasma membrane. Previous studies in rat have suggested that 5A11/Bsg (EMMPRIN) polarizes to the apical membrane of the RPE of adult animals. ³⁴ Another report stated that in chicken, 5A11/Bsg is localized basally in the RPE. ¹¹ To clarify the issue of 5A11/Bsg localization in the vertebrate RPE, we reexamined the expression of 5A11/Bsg in chicken, rat, and mouse RPE, where polarized expression of MCT3 in the basolateral membrane has also been demonstrated. ^{2 3 13} As shown in Figure 5 , 5A11/Bsg was abundantly expressed in both the apical and the basolateral plasma membranes in chick, rat, and mouse RPE. The colocalization of 5A11/Bsg with both MCT1 and -3 is consistent with the loss of expression of both of these transporters in the Bsg ^−/− mouse. 

Expression of 5A11/Bsg and MCT1 in Mouse Tissues

Western blot analysis was used to compare the relative abundance of 5A11/Bsg in different mouse tissues (Fig. 6) . The differences in the mobility of 5A11/Bsg in samples results from differences in glycosylation. ^{12 18} As shown in Figure 6 , 5A11/Bsg was expressed at higher levels in the RPE than in intestine or liver and was more abundant in retina than in the brain. Duplicate blots were probed with MCT1 antibody. The pattern of MCT1 expression mirrored 5A11/Bsg expression; MCT1 was expressed at higher levels in retina and RPE than in other tissues. 

Discussion

Mice with a targeted deletion of the 5A11/Bsg gene have abnormal ERGs beginning at an early age, when histology of the retina appears normal, suggesting an alteration in ion transport. ²⁷ In the present work, there was a specific loss of MCT proteins in the RPE and neural retina of Bsg ^−/− mice. The results of these studies demonstrate that 5A11/Bsg is necessary for proper targeting of MCTs to the plasma membrane and suggest that MCTs are essential for maintaining normal electrical activity in the retina. ^{27 29}  

In both the neural retina and the RPE of Bsg ^−/− mice, there was a reduction in accumulation of MCT1, even at early time points when the morphology of the retina appeared relatively normal. MCT1 is the primary lactate carrier in the outer retina and is found in Müller cells and inner segments of the photoreceptor cells. ^{7 8 9} Expression of MCT1 on Müller cells and photoreceptor cell membranes provides a mechanism for metabolic coupling between glial and neuronal cells. ¹ Müller cells depend primarily on glycolysis for energy production and export lactate to the photoreceptor cells, where it is used as a substrate for oxidative phosphorylation. ¹ In the absence of MCT1, lactate from Müller cell metabolism is no longer available for energy metabolism in the outer retina, which could lead to a decrease in the amplitude of the ERG. The observation supports the hypothesis that glial glycolysis is essential for normal electrical activity in the retina. ³⁰  

In the adult mouse, MCT4 is expressed in the inner layers of the neural retina but not in the RPE (Philp NJ, unpublished observation, 2002). Western blot analysis revealed that there was a reduction in expression of MCT4 in the Bsg ^−/− mouse. Our results are consistent with the findings that antibodies against 5A11/Bsg (CD147) coimmunoprecipitate MCT4 ²⁴ and demonstrate in vivo that 5A11/Bsg is necessary for accumulation of normal levels of MCT4. 

5A11/Bsg is expressed at higher levels in the RPE than in other epithelia and parallels the high level of expression of MCT1 and -3 in these cells. 5A11/Bsg colocalizes with MCT1 and -3 in the apical and basolateral membranes of the RPE, respectively (Fig. 1) . In the Bsg ^−/− mouse RPE, both MCT3 and MCT1 achieve only barely detectable levels of the protein. Therefore, on the basis of this observation, it seems likely that 5A11/Bsg plays a role in protein translation or trafficking of MCT1 and -3 to the RPE plasma membrane. The observation that MCT1 and -3 reside on opposite sides of the RPE indicates that polarization of these proteins is controlled by some mechanism independent of the role of 5A11/Bsg—that is, 5A11/Bsg is necessary but not sufficient for polarized targeting of MCT1 and -3 in the RPE. 

The loss of MCT1, -3, and -4 accumulation in the RPE and retina of the 5A11/Bsg-null mouse appears to be a direct result of the loss of expression of 5A11/Bsg and not a consequence of photoreceptor cell degeneration. Support for this hypothesis comes from the observation that the expression and distribution of GLUT1 was similar in both the Bsg ^−/− and wild-type mice. In addition, the expression of the Müller cell–specific proteins glutamine synthetase and carbonic anhydrase was not affected in Bsg ^−/− mice. ²⁷ A loss of MCT expression was not observed in other mouse models of retinal degeneration, such as the Rpe65-null mouse and the rd mouse (Philp NJ, unpublished observations, 2002). MCT1 mRNA was expressed in retinas of the Bsg ^−/− mouse, suggesting that CD147 regulates the targeting of MCT proteins to the plasma membrane but does not regulate gene expression. When MCT1 ²⁵ and -3 ³⁵ cDNA constructs are overexpressed in COS cells, protein accumulates in the endoplasmic reticulum and is not targeted to the plasma membrane. In contrast, when COS cells are cotransfected with MCT1 and 5A11/Bsg constructs, both proteins are detected in the plasma membrane. ²⁵ These studies support the hypothesis that CD147 is essential for targeting of MCTs to the plasma membrane. 

The failure of certain MCTs to achieve normal levels in the plasma membranes of neural retina and RPE cells may well provide an explanation for the absence of normal visual activity in the Bsg ^−/− mouse. Additional work is needed to validate this conclusion, but it certainly is likely that a decrease in lactate transport could have a dramatic and negative impact on the complex physiology of the visual apparatus. 

 Supported by Grants EY12042 (NJP) and F32EY13918 (JDO) from the National Eye Institute and IBN-0113697 (PJL) from the National Science Foundation.

 Submitted for publication June 6, 2002; revised August 16 and September 27, 2002; accepted October 18, 2002.

 Disclosure: N.J. Philp, None; J.D. Ochrietor, None; C. Rudoy, None; T. Muramatsu, None; P.J. Linser, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Immunocytochemical localization of 5A11/Bsg, MCT1 and -3 in the RPE and retina of Bsg ^+/+ and Bsg ^−/− mouse. Immunofluorescence microscopy of paraffin sections of eyes from 20-week-old Bsg ^+/+ (A, C, E) and Bsg ^−/− mice (B, D, F) labeled with antibodies to 5A11/Bsg (A, B), MCT1 (C, D), and MCT3 (E, F). Arrows: basolateral membrane of the RPE; arrowheads: the apical membrane of the RPE. IN, inner nuclear layer; IP, inner plexiform layer; IS, inner segments; ON, outer nuclear layer; OP, outer plexiform layer; OS, outer segments; RPE, retinal pigment epithelium. (D, ⋆) position of abnormal intracellular MCT1 in retina. Small arrows: capillaries.

Figure 2.

 

Immunohistochemical localization of GLUT1 in the RPE and retina of Bsg ^+/+ and Bsg ^−/− mice. Immunofluorescence microscopy of paraffin sections of eyes from 20-week-old Bsg ^+/+ (A) and Bsg ^−/− mice (B) labeled with antibodies to GLUT1. Arrows: basolateral membrane of the RPE; arrowheads: apical membrane of the RPE.

Figure 3.

 

Western blot analysis of transporter proteins in the RPE of Bsg ^+/+ and Bsg ^−/− mice at P20 and 1 year of age. Detergent lysates were prepared from microdissected RPE-choroid-sclera (RPE). Identical blots were probed with MCT3 and -1 and GLUT1 antibodies.

Figure 4.

 

Western blot and RT-PCR analyses of MCTs and GLUT1 in neural retinas of Bsg ^+/+ and Bsg ^−/− mice. ( A) Detergent lysates were prepared from retinas, and duplicate blots were probed with MCT1, -2, and -4, and GLUT1 antibodies. (B) Standard and real-time RT-PCR analyses of MCT1 mRNA expression in retinas of P20 and 1-year-old Bsg ^+/+ and Bsg ^−/− mice. For standard RT-PCR, 1 μg total retina RNA was used. For real-time RT-PCR, all tests were performed in triplicate, and expression of MCT1 was normalized to 18S ribosomal RNA.

Figure 5.

 

Immunohistochemical localization of 5A11/Bsg RPE of chicken, rat, and mouse. Immunofluorescence microscopy of frozen sections of eyes from 1-day-old chicken (A), adult rat (B), and adult mouse (C). Arrows: basolateral membrane of the RPE; arrowheads: apical membrane of the RPE.

Figure 6.

 

Western blot analysis of 5A11/Bsg expression in mouse tissues. Detergent extracts of mouse tissues were prepared, and protein (10 μg/lane) was separated on 4% to 12% gradient gels and transferred to polyvinylidene difluoride membranes. Duplicate blots were probed with 5A11/Bsg and MCT1 antibodies.

The authors thank Dian Wang and Tatiana P. Moroz for technical support.