Abstract
The antigen recognized with monoclonal antibody (mAb) Rip (Rip‐antigen) has been long used as a marker of oligodendrocytes and myelin sheaths. However, the identity of Rip‐antigen has yet to be elucidated. We herein identified the Rip‐antigen. No signal recognized by mAb‐Rip was detected by immunoblot analyses in the rat brain, cultured rat oligodendrocytes, or the oligodendrocyte cell line CG‐4. As this antibody worked very well on immunocytochemistry and immunohistochemistry, Rip‐antigen was immunopurified with mAb‐Rip from the differentiated CG‐4 cells. Eight strong‐intensity bands thus appeared on 5–20% SDS‐PAGE with SYPRO ruby fluorescence staining. To identify these molecules, each band extracted from the gel was analyzed by MALDI‐QIT/TOF mass spectrometry. We found an interesting molecule in the oligodendrocytes from an approximately 44‐kDa band as 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP). To test whether CNP was recognized by mAb‐Rip, double‐immunofluorescence staining was performed by using Alexa Fluor 488‐conjugated mAb‐Rip and Alexa Fluor 568‐conjugated mAb‐CNP in the rat cerebellum, mouse cerebellum, cultured rat oligodendrocytes, and CG‐4 cells. The Rip‐antigen was colocalized with CNP in these cells and tissues. To provide direct evidence that CNP was recognized by mAb‐Rip, rat Cnp1‐transfected HEK293T cells were used for double‐immunofluorescence staining with mAb‐Rip and mAb‐CNP. The Rip‐antigen was colocalized with CNP in rat Cnp1‐transfected HEK293T cells, but the antigen was not detected by mAb‐Rip and mAb‐CNP in mock‐transfected HEK293T cells. Overall, we have demonstrated that the antigen labeled with mAb‐Rip is CNP in the oligodendrocytes. © 2006 Wiley‐Liss, Inc.