Figure 3. In situ hybridization and im- munohistochemistry showing OAP-1 expression in mouse forebrain. (A) Dark field photomicrograph of adult rat forebrain after in situ hybridization using 35S–OAP-1 cRNA shows dense neuronal expression of OAP-1 mRNAs in the CA1 region of a P10 mouse hippocampus. The area under the square is shown in B. (B) A high power bright field photomicrograph showing expression of OAP-1 mRNA in oligodendrocytes of the corpus callosum (arrowheads). (C) Using an immunohistochemistry anti– OSP/claudin-11 antibody (Texas red secondary antibody) showed staining primarily in white matter tracts and not in pyramidal cells and astrocytes (large arrow). Small arrows denote the outer borders of corpus callosum. (D) In contrast to OSP/claudin-11, immuno- histochemistry using anti–OAP-1 anti- body (FITC secondary antibody) in the same section demonstrated widespread staining in white matter tracts and in areas containing pyramidal cells and astrocytes (large arrow). mCD63, mCD82, mTspan-6, and hTspan-3) revealed highly conserved amino acids specific for tetraspanins (Fig. 1 A). There are four sites of potential NH2-linked glycosylation in extracellular domain 2. For OAP-1 and other TM4SF proteins, conserved sequences are primarily within the putative transmembrane domains analyzed by a Kyte-Doolittle plot (Fig. 1 B). In contrast, the extracellu- lar domains are more divergent in terms of length, se- quence, and degree of glycosylation. Sequence similarity between murine OAP-1 and human tetraspanin Tspan-3 suggests that they are homologues. OAP-1 mRNA Is Expressed in Oligodendrocytes OAP-1 is widely expressed in brain and other tissues. Northern blot analysis revealed a 1.9-kb mRNA present in all the tissues tested, including spinal cord, brain, testes, skeletal muscle, heart, kidney, spleen, and lung (Fig. 2 A). A second band of z4 kb was also present in much lower levels. This may represent an alternate transcript or an- other gene product with high homology to OAP-1. North- ern blot analysis on RNA from purified primary mouse oligodendrocyte cultures and from the CIMO cell line confirmed the presence of OAP-1 expression in oligoden- drocytes (Fig. 2 B and see Fig. 7 A). Western blot analysis revealed a single band of z31 kD in O2A oligodendrocyte progenitor cells (Fig. 2 C). Two bands of z31 and 60 kD were present in mature oligodendrocytes and mouse brain homogenates, although under strong reducing conditions (20% b-mercaptoethanol and 50 mM DTT) only the 31- kD band was apparent. The band was eliminated by prein- cubation of the OAP-1 antibody with peptide (Fig. 2 C). These data suggest that the antibody specifically recog- nized OAP-1 protein and that the 60-kD band was a dimer of OAP-1 protein. Using in situ hybridization, we found that OAP-1 mRNA was expressed in all areas of the mouse brain with high levels in hippocampal pyramidal neurons and moder- ate levels in oligodendrocytes of the corpus callosum (Fig. 3, A and B). Unlike OSP/claudin-11 staining in myelin tracts (Fig. 3 C), immunohistochemistry demonstrated that OAP-1 expression was more widespread, consistent with in situ hybridization experiments (Fig. 3 D). OSP/Claudin-11, OAP-1, and b1 Integrin Form a Complex To determine if the association of OSP/claudin-11 with OAP-1 occurs in vivo, we performed immunoprecipita- tion. Homogenates were precipitated under relatively stringent conditions (1% Triton X-100) with either anti– OSP/claudin-11 or anti–OAP-1 antibodies. Western blots of the precipitated proteins confirmed OSP/claudin-11– OAP-1 association in both mouse brain (Fig. 4 A) and pri- mary oligodendrocyte homogenates (Fig. 4 B). Most if not all TM4SFs (for example, CD81, CD9, CD53, CD63, and CD82) are known to associate with a vast range of integrins in various types of cells (for review see Maecker et al., 1997). This is also true for OAP-1, since it was immu- noprecipitated with anti–b1 integrin (Fig. 4, A and B). The Tiwari-Woodruff et al. OSP/Claudin-11, b1 Integrin, and OAP-1 Form a Complex 299