A) Biochemical confirmation of TSGA10 constructs expression by using total lysates (60 μg of each) of the yeast cells transformed by TSGA10 constructs (Fulllength (FL), the carboxyl terminus 1 (CT1), carboxyl terminus 2 (CT2) or amino terminus (NT)), as well as, control empty vector (pGBKT7) run in 10% SDS-PAGE and visualized with anti-myc antibody (because pGBKT7 contains a c-Myc epitope tag). B) The table shows the exact number of TSGA10 aminoacids in its various constructs (Full –FL-, CT1, CT2 and NT). It also summarises the results of interaction of TSGA10 amino and carboxyl termini constructs with ODF2 using yeast transformation and mating and its confirmation by X-Gal assay. C) Representative yeast colonies in the selective medium (-Leu/-His and –Leu/-His/-Trp/-Ade) after transformation of TSGA10 and ODF2 constructs as well as schematic figure of the TSGA10 bait constructs subcloned in pGBKT7 (Clontech), sequence domains of both the carboxyl terminus (C) and amino terminus (N) regions of mouse TSGA10 (see marked starting and ending amino acid residues). Colony lift assay shows positive interaction between ODF2 and both full-length (FL) or the carboxyl terminus 1 (CT1) of TSGA10, whereas incubation with the carboxyl terminus 2 (CT2), or the amino terminus (NT) of TSGA10 did not result in any interaction. The interactions in the colony lift assay were examined after a 24-h incubation of filter paper in 5-bromo-4-chloro-3-indolyl- -D-galactopyranoside (X-gal) solution. This suggests that only the full-length TSGA10 and the CT1 terminus (55 KD) of TSGA10, but not the CT2 or N-termini interact with ODF2.

Figure 1: Yeast two-hybrid interaction between ODF2 and various regions of TSGA10. The yeast two-hybrid system was used to test physical association between TSGA10 and ODF2. The full-length TSGA10-bait construct was used for screening of the testis library; its N,C-termini half bait constructs were used to test any possible interaction with the interacting ODF2 prey.