Fig. 5. AChR clustering and stabilization are defective in AChR-ß^3F/3F myotubes. (A) Agrin-induced AChR clustering is attenuated in AChR-ß^3F/3F muscle fibers. Wild-type and AChR-ß^3F/3F myotubes were treated with agrin, and AChRs were labeled with Alexa Fluor 594--BGT. Scale bar: 50 µm. (B) Quantification of AChR cluster size and number in wild-type and AChR-ß^3F/3F myotubes. The size and number of AChR clusters that form independent of agrin are similar in wild-type and AChR-ß^3F/3F myotubes (P>0.05, Mann-Whitney). Agrin induces a 3.5-fold increase in the number of AChR clusters in wild-type myotubes, and a 1.4-fold increase in AChR-ß^3F/3F myotubes (P<0.05, Mann-Whitney). (C) Wild-type and AChR-ß^3F/3F myotubes were treated with or without agrin, and AChRs were labeled with ¹²⁵I--BGT. Myotubes were incubated in medium containing 0.05% Triton X-100, which was collected and replaced every 2 minutes for a total of 6 minutes. The amount of ¹²⁵I--BGT extracted at each time point, and the amount remaining bound to the myotubes at the end of the extraction period, was determined. In wild-type myotubes, agrin treatment leads to a significant decrease in the rate of AChR extraction (P<0.05, ANOVA), which is abolished when the cells are pretreated with 20 nM staurosporine. Detergent extraction of AChRs from AChR-ß^3F/3F muscle fibers is significantly faster than from wild-type fibers (^*P<0.05) and is not altered by either agrin or staurosporine treatment (P>0.05 is considered not significant; n.s.).