The CLIP-170 load relative to EB3 at MT plus-ends is greater inside than outside AChR clusters and is reduced by PI3-K inhibition and by deletion of Clasp2. (a) Comets in AChR clusters in GFP-Clip-170^ki/ki myotubes revealed by immunolabeling of EB3 and of GFP, to visualize CLIP-170. Regions outlined by boxes within and outside AChR cluster are shown enlarged at bottom. Agrin-induced AChR clusters in blue, GFP-CLIP-170 in green, EB3 in red. Note increased load of CLIP-170 at comets in AChR cluster (arrowheads) compared with comets outside cluster lacking increased CLIP-170 (asterisks). Bars: (top) 10 µm; (bottom) 5 µm. (b) Same as panel a but after blockade of PI3K with ZSTK474. Note reduction in CLIP-170 load in PI3-K–blocked compared with nonblocked myotubes. Bars: (top) 10 µm; (bottom) 5 µm. Asterisks same as in panel a. (c) Quantification of CLIP-170 staining on EB3-stained comets inside relative to that of comets outside AChR clusters. The elevated CLIP-170 load at agrin-induced AChR clusters observed in wild-type myotubes is abolished by PI3-K inhibition and by genetic deletion of Clasp2. EB3 staining is similar inside and outside AChR clusters. Percentages are means ± SEM from 84 to 245 comets analyzed in wild-type, ZSTK474-blocked, and Clasp2^−/− myotubes (4 cells each). *, P < 0.05; **, P < 0.01, two-sided t test.