Figure 3.

A 47 kDa. EB-1/AIDA-1 protein interacts with coilin. (A) Cytoplasmic and nuclear extracts from HeLa cells were subjected to Western blotting with anti-AIDA-1 antibodies. (B) Myc-coilin interacts in vivo with EB-1/AIDA-1 nuclear proteins. HeLa cells were transfected with myc-coilin and nuclear extract was subjected to immunoprecipitation (IP) with anti-AIDA-1 antibodies followed by SDS-PAGE and Western blotting with anti-myc antibodies. Normal rabbit serum (NRS) was used as a negative IP control. The input lane accounts for 1/20 the amount of lysate used in the IP reactions. (C) HeLa and melanoma cell lysates were incubated with a GST-tagged coilin fragment (C214) or GST alone, followed by Western blotting with anti-AIDA-1 antibodies (upper panels). Pulldowns using HeLa lysate are shown in lanes 3 and 4. Melanoma cell lysate was used in the reactions shown in lanes 5 and 6. Only the 47 kDa. EB-1/AIDA-1 protein binds to the coilin fragment. The blot was subsequently probed with anti-GST antibody to verify the loading of the GST and GST-C214 beads (lower panel). The input lanes account for 1/20 the amount of lysate used in the pulldown reactions. (D) Purified T7-tagged AIDA-1c protein was run as a control to mark the position of the 47 kDa. protein from nuclear extract (NE). The blot was probed with anti-AIDA-1 antibody.