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. 2002 Dec 27;100(1):289–294. doi: 10.1073/pnas.0136913100

Figure 2.

Figure 2

DISC-1 protein expression in tissues and cells. Major forms of DISC-1 (100 and 75 kDa) were detected by antibodies against both the N-terminal side (amino acids 347–600 and 212–537) and C-terminal side (amino acids 601–854) of the breakpoint. The specificities were also confirmed by preabsorption experiments. (A) High expression of DISC-1 protein in brain, heart, liver, and kidney, and weak expression in thymus. DISC-1 of 100 kDa is predominant in brain and heart. In adult brains, DISC-1 is enriched in the forebrain. DISC-1 expression is developmentally regulated with an enhanced expression of the 100-kDa band at E20.5. (B) DISC-1 protein expression in cell lines. DISC-1 is expressed in both neuronal and glial cells, as well as some peripheral cells. In PC12 cells, differentiation is associated with augmented expression. (C) DISC-1 distribution is analyzed by subcellular fractionation and immunofluorescent staining. Endogenous DISC-1 protein is enriched in P1 and P2 fractions of differentiated PC12 cells and in P2 of HeLa cells. Exogenously expressed full-length (FL) DISC-1 tagged with HA also occurs in P1 and P2 fractions, whereas mutant DISC-1 with the C-terminal deletion (Mut-HA, ΔC) distributes in P1, P2, and P3 fractions. Another mutant of DISC-1 lacking 290 amino acids of the N-terminal region (ΔN) distributes in all fractions, including the supernatant. Immunofluorescent staining of DISC-1 was analyzed under a confocal microscope. Endogenous and exogenous DISC-1 was stained in red; the nucleus was stained in blue with Hoechst 33258. Dotted DISC-1 staining is observed in the cytoplasm, especially in the perinuclear regions of undifferentiated PC12 cells and HeLa cells. In differentiated PC12 cells, DISC-1 occurs not only in cytoplasm, but also in neurites with higher density at the terminal regions (white arrow indicates DISC-1 in the terminus). Exogenously expressed DISC-1 tagged with HA in COS cells was stained with an anti-HA Ab (red). The dotted staining in perinuclear regions similar to centrosomes reflects full-length DISC-1 (DISC-1 FL). Mutant DISC-1 with the C-terminal deletion occurs in the cytoplasm with smaller dotted structures than DISC-1 FL. The N-terminal truncated form of DISC-1 (DISC-1 ΔN) distributes diffusely in the cytoplasm of COS cells.