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.
Short et al. BMC Cell Biology 2002 3:1 doi:10.1186/1471-2121-3-1