(A.) Alignments of the five members of the human NSE4/EID family: EID1, EID2, EID2b, EID3/NSE4b and NSE4a. The red box indicates Nse3/MAGE-binding domain; hatched and crosshatched boxes indicate kleisin and kleisin-like motifs, respectively (based on Fig. 1B and C; [10]); grey boxes indicate regions of homology between EID1 and 2, respectively. (B. and C.) GST-His-S-tagged fragments of each of the NSE4/EID family members were pre-bound to S-protein agarose beads and incubated with in vitro translated MAGEA1 (aa 1-309; panel B) and necdin (aa 1-321; panel C). The indicated NSE4/EID proteins correspond to the MAGE-interacting domain of EID1(aa146-177), EID2(aa197-225), EID2b(aa135-161), NSE4b/EID3(aa106-135) and NSE4a(aa150-179). In lanes 8-12, immunoblotting of bound fragments are shown in the upper panel and bound radioactive MAGE proteins (MAGEA1 and necdin) in the lower panel. Control, GST-His-S protein alone.