Figure 2.

Tagging cassettes for BAC recombineering and characterization of tagged dysbindin in the brains of BAC transgenic mouse lines. (A) The tag was inserted immediately after the ATG start codon and consists of a 3×FLAG tag for immunopurification and detection, a 6×His spacer for TEV protease cleavage, a tobacco etch virus (TEV) protease cleavage site, and a Strep tag for the second affinity purification and detection. The reporter cassette was inserted immediately after the stop codon and consists of an internal ribosome entry site (IRES) and a bacterial promoter (GB2) in front of the gene encoding Venus fluorescent protein. (B) Immunoblot analysis of total extracts from wild-type, sandy, and BAC transgenic mouse brains using anti-dysbindin and anti-FLAG antibodies. (C) SG analysis of dysbindin-containing complex(es) in the P2 synaptosome fraction of the BAC transgenic mouse brains with and without DSP cross-linking. Equal aliquots from individual fractions were resolved by SDS-PAGE and analyzed by immunoblotting using the anti-dysbindin antibody. (D) The blots in panel C were reprobed with the anti-FLAG antibody. * indicates nonspecific bands.