Fig. 1

Development of a cell based system that recapitulates incomplete splicing of Htt. a Schematic showing the elements of the Htt minigenes. b Three minigene constructs differed only in the length of 5′ intron sequences (short: 917 bp; medium: 1848 bp; long: 3160 bp). One construct contained only the coding sequence for exon 1 HTT and the first 916 bp of intron 1 (ex1 only). c 3′RACE analysis showed that the cryptic polyA site at 677 bp into intron 1 (arrowhead) was only used in the long minigene lines with a threshold of about 40 CAGs. d Proteins that will be expressed from these constructs: Splicing will generate an exon 1-exon 2-FLAG fusion protein (control and expanded CAG). In the case of an expanded CAG, an exon 1 HTT protein will also be generated due to incomplete splicing of exon 1 to exon 2. e Overlay image of spliced (FLAG) and exon 1 HTT (S830) containing fragments. HTT fragments were immunoprecipitated (IP) with 3B5H10 coupled magnetic beads and immunoprobed (IB) with antibodies as indicated (please see Supplementary Fig. 2 for complete protein analysis). FLAG-tag detects the properly spliced exon 1–exon 2 fragment (2) (see also Fig. 1d). The S830 antibody recognized Q50 and Q100 containing spliced (2), as well as incompletely spliced fragments (1: Q50, 3: Q100). Q7 containing fragments were not detected because they are not efficiently immunoprecipitated with the anti-polyQ antibody 3B5H10. PL parent line; M marker