Figure 1.

rTAG-1 expression in the CNS of plp^Tg(rTag-1) mice. A–F, Immunohistochemistry in cryosections of adult cerebellar white matter using specific antibodies against rTAG-1 in combination with an oligodendrocyte marker (Olig2; A–C), or with a paranodal marker (Caspr; D–F). rTAG-1 protein is detected on Olig2⁺ cells (C) and in juxtaparanodes (F) of Tag-1^−/−;plp^Tg(rTag-1) mice. As expected, rTAG-1 is not detected in Tag-1^+/− (A, D) or Tag-1^−/− (B, E) animals. G, Distribution of TAG-1 in axonal and myelin fractions. Two-month-old brain tissues were purified and used for WB analysis (as described in Materials and Methods) for TAG-1 and axonal as well as myelin proteins. The four lanes represent the different animals used. Lane 1, Tag-1^−/−; lane 2, Tag-1^+/−; lane 3, Tag-1^+/−;plp^Tg(rTag-1); lane 4, Tag-1^−/−;plp^Tg(rTag-1). TAG-1 detected in Tag-1^−/−;plp^Tg(rTag-1) specifically originates from the myelin fraction. Ha, Live immunostaining of cultured mixed glial cells from Tag-1^−/−;plp^Tg(rTag-1) P2 brains. TAG-1 (red) is found on the cell surface of Olig2⁺ cells (green). Hb, WB analysis for TAG-1 and Coomassie blue staining of the supernatant medium from glial cells after 3 d in culture. TAG-1 is detected in the medium only in Tag-1^−/−;plp^Tg(rTag-1) mice, and is absent from the homozygous mutants. Lane 1, Tag-1^−/−; lane 2, Tag-1^−/−;plp^Tg(rTag-1). I, Immunohistochemistry in cryosections from cerebellum of adult Tag-1^−/−;plp^Tg(rTag-1) mice for TAG-1 (green) and the neuronal marker NeuN (red). The transgenic protein is clearly not expressed by neurons. J, Immunocytochemistry in P2 dissociated cortices from Tag-1^+/+ and Tag-1^−/−;plp^Tg(rTag-1) mice with anti-TAG-1 (green) and anti-NeuN (red) antibodies. TAG-1 is expressed by neuronal cells in Tag-1^+/+ but not in the transgenic mice. To-Pro3 (blue) is used for the visualization of the nuclei in A–F, I, and J. Scale bars, 10 μm. GCL, Granule cell layer; WM, white matter; NFM, neurofilaments M.