Figure 5. OSP/claudin-11 colocalizes with OAP-1 and b1 integrin in oligodendro- cytes. (A) Confocal images of primary mouse oligodendro- cytes shown in the top row were double labeled for OSP/claudin-11 (Texas red) and OAP-1 (FITC) or b1 in- tegrin (FITC). Fused images are shown at the far right and bottom. Most cells showed striking colocalization of OSP/ claudin-11, OAP-1, and b1 in- tegrin. (B) Confocal images of primary mouse oligoden- drocytes shown were double labeled for OAP-1 (Texas Red) and OSP/claudin-11 (FITC) in live primary oligo- dendrocytes. Fused images are showed to the far right. Colocalization of the two proteins was mostly observed in the cell borders. (C) Live primary oligodendrocytes were cultured on slides and immunostained with anti– OAP-1 (top row) and anti– OSP/claudin-11 (bottom row) antibody in the presence and absence of the respective an- tibody peptides. Background staining is also shown. Bars: (A, top row) 10 mM; (A, bot- tom row) 25 mM. measured. CIMO cells overexpressing OSP/claudin-11 protein or OAP-1 protein were confirmed via Western blot analysis (Fig. 7 B). Under permissive conditions, over- expression of OSP/claudin-11 had no significant effect on [3H]thymidine incorporation in CIMO cells nor did over- expression of PLP, MBP, or OSP antisense (in an attempt to lower endogenous OSP/claudin-11) (Fig. 7 C). When the cells were transferred to nonpermissive conditions, the cells overexpressing OSP/claudin-11 incorporated 81-fold more [3H]thymidine compared with those transfected with vector alone and took on a less differentiated appearance. In fact, overexpression of OSP/claudin-11 under these conditions had a 2.5-fold greater proliferative effect than that of a known mitogen, SV40ts58 antigen. There was an increase in cellularity and necessity to passage cells more frequently, consistent with the concept that increase in [3H]thymidine incorporation reflected an increase in pro- liferation. Thus, OSP/claudin-11 had a powerful prolifera- tive effect on differentiated CIMO cells but not on less differentiated CIMO cells. Interestingly, OAP-1 overexpression of CIMO cells increased proliferation greater than twofold compared with vector alone controls Tiwari-Woodruff et al. OSP/Claudin-11, b1 Integrin, and OAP-1 Form a Complex 301