Determination and confirmation of a precise region within the SID as the TA10 binding domain. A. PCR fragments of different size and location within the smallest prey clone from the initial screen were generated and labeled from 1 to 17. B. The two-hybrid yeast strain containing the TA10 bait plasmid was transformed with the PCR products and a digested prey plasmid. The interactions of the prey fragment were tested by spotting in media lacking histidine (left image). A β-galactosidase qualitative observation was also done to confirm the interaction (right image). C. Alignment of the 17 PCR products with the human giantin amino acid sequence narrowed the putative TA10 binding domain to a 79-amino-acid long region (blue, labeled as "a") between amino acids 1462 to 1540 of the full human sequence (corresponding to amino acids 1399 to 1478 in the rat homologue). A fragment of 63 amino acids upstream of this region (labeled as "b") was used as negative control (see D and E). D. Confirmation using immunofluorescence. HeLa cells were transfected with a plasmid containing GFP fused to either the fragment found using the gap repair approach (called giantin-TA10BDshort, a) or the negative control (called giantin-79aa control, b). Only overexpression of the former was recognized by hTA10. E. Confirmation by Western blotting. The same two HeLa cell populations described in D were harvested and prepared for Western blotting. Only giantin-TA10BDshort (a) but not giantin-79aa control (b) was recognized by hTA10 while both were detected with anti-GFP.