MID1/Alpha 4 and MID2/Alpha 4 interactions are maintained in all MID domain-specific deletions except for those involving the B-boxes. (A) Yeast two-hybrid analysis shows that the MID B-boxes are required for interaction with Alpha 4. The interaction of Alpha 4 with MID1ΔBB (21), or MID2ΔBB (23), is compared to its interaction with MID1ΔCC (22), or MID2ΔCC (24). (B) Subcellular localisation of myc tagged-Alpha 4 when co-expressed in Cos-1 cells with MID1 domain-specific deletions as GFP fusion proteins. Fluorescence detection of GFP-MID1ΔRF (a), GFP-MID1ΔBB (d), GFP-MID1ΔCC (g), GFP-MID1ΔFNIII (j), and GFP-MID1ΔCTD (m). Anti-myc antibody detection of myc-Alpha 4 in the same cells as expressing the various MID1 domain-specific deletions (b,e,h,k,n). Overlay of the GFP and anti-myc images of the same cells merged with DAPI stain of nuclei (c,f,i,l,o). All merged images, with the exception of (f) show co-localisation of myc-Alpha 4 with the various MID1 domain deletions. In (f), myc-Alpha 4 fails to co-localise with GFP-MID1ΔBB in small cytoplasmic aggregates.