The B-boxes of MID1 are sufficient to bind Alpha 4. (A) Yeast two-hybrid analysis shows that the MID1 B-boxes (pPC86-M1BB) alone can interact with Alpha 4 (pDBLeu-Alpha 4) (31). The wild-type MID1-Alpha 4 interaction (30) and a control with the pPC86-M1BB and no interaction partner (29) were included for comparison. (B) Immunofluorescence assay highlights the importance of the B-boxes for Alpha 4 binding. Subcellular distribution of the GFP-fused MID1 coiled coil domain, GFP-M1CC (a) and myc-Alpha 4 (b) in the same cell shows that the two proteins do not co-localise, as seen in the merged image (c). However, the MID1 fragment, GFP-M1BBCC (d) and the myc-Alpha 4 (e) do co-localise in cytoplasmic speckles as seen in the merged image (f). Both merged images, (c) and (f), also show DAPI stain (blue), which indicates the position of the nucleus.