Figure 1

(a) Epitope-tagged DISC1 constructs. Schematic representation of the epitope-tagged constructs coexpressed in HEK293 cells. The continuous line is indicative of the genomic sequence of DISC1 with the numbered exons shown as rectangles. An N-terminal Myc and/or FLAG epitope was added to all DISC1 constructs. Known DISC1 (L, Lv and S) and novel isoforms termed ‘DISC1 LΔ78T10’ (deletion of exons 7 and 8; termination at exon 10), DISC1 LΔ78T9 (deletion of exons 7 and 8; termination at exon 9), DISC1 LΔ3 (deletion of exon 3) and Esv1 (insertion in exon 3) were used. (b) DISC1 self-association. Short isoforms bind to full-length DISC1. All FLAG-tagged DISC1 isoforms (Lv, LΔ3, LΔ78T10, LΔ78T9, S and Esv1) co-immunoprecipitate with myc-tagged, full-length DISC1 (L). Precipitated proteins were resolved on SDS-PAGE and immunoblotted with an antibody against FLAG DISC1. N=4 separate experiments. (c and e) Inputs used in co-immunoprecipitation. Lysates used in experiment were subjected to SDS-PAGE in parallel with the directly immunoprecipitated proteins. Anti-FLAG and myc immunoblots of inputs show comparable protein expression present during immunoprecipitation and bands at expected molecular weights. (d) Differential interactions of truncated DISC1 proteins. Essential proteins known to interact with full-length DISC1 (L) were examined for interaction with short isoforms. After coexpression of Myc-tagged DISC1 isoforms with FLAG-tagged DISC1-interactors, lysates were subjected to immunoprecipitation with anti-Myc DISC1 and immunoblotted with an anti-FLAG antibody. First panel: all DISC1 isoforms bind FEZ1. Second panel: DISC1 isoforms co-immunoprecipitate with GSK3B. Third panel: DISC1L and Lv interact with NDEL1. Fourth panel: DISC1LΔ3 and LΔ78 show reduced binding with PDE4B. N=3–4 separate experiments. (f) Quantification of DISC1 self-associations. Densitometry readings of immunoprecipitated bands were normalized to full-length DISC1L. Numbers indicate the coexpressed DISC1 isoforms (1: DISC1 Lv, 2: DISC1 LΔ3, 3: DISC1 LΔ78T10, 4: DISC1 LΔ78T9, 5: DISC1 S and DISC1 Esv1). (g) Quantification of DISC1 interactions with FEZ1, GSK3β, PDE4B and NDEL1. Densitometry readings of immunoprecipitated bands were normalized to input bands as follows 1; DISC1L, 2: DISC1 Lv, 3: DISC1 LΔ3, 4: DISC1 LΔ78T10, 5: DISC1 LΔ78T9, 6: DISC1 S and 7: DISC1 Esv1. Error bars show s.d. and asterisks are given at P<0.05.