Figure 3

AKAP350 interacts with EB1 at the centrosomes. (a) Merged images show AKAP350 (green) and EB1 (red) staining in MDCK cells. Arrows indicate centrosomes in mitotic cells, where significant colocalization is observed. Scale bars, 10 μm. (b) MDCK cells with stable expression of AKAP350(1-1029) domain fused to GFP (AKAP350NTD) were prepared as described in Materials and Methods. AKAP350NTD cell lysates (Input) were incubated with an anti-EB1 antibody, and subjected to immunoprecipitation using protein A-sepharose beads. Non-bound material was removed, beads were washed and immunoprecipitates (IP) eluted with sample buffer. A negative control was run by incubating cell lysates with protein A-sepharose beads in the absence of anti-EB1 antibody (EB1-). Samples were analyzed by Western blot using anti-EB1 (top) and anti-GFP (bottom) antibodies. (c) Analysis of EB1/AKAP350 interaction in situ was performed using immuno-FRET by acceptor photobleaching, as described in Materials and Methods. The first image show the DIC channel of a mitotic cell, which was bleached at the centrosome area in the acceptor channel. The second image illustrates in fire pseudocolor the increase in intensity in the bleached area in the donor (AKAP350) channel, after photobleaching of the acceptor (EB1) channel.