Fig. 1. Generation of mice lacking tyrosine phosphorylation sites in the AChR ß-subunit. (A) The structures of the AChR ß-subunit gene, the targeting construct and the targeted locus. (B) Southern blots of genomic DNA, which were digested with HindIII, were hybridized with a probe (indicated in A) that is 3' to the targeting construct. The labeled band in the targeted allele is 2 kb larger than in wild-type DNA because of the insertion of the PGK-neo cassette. (C) Western blots of AChRs, isolated with biotin-conjugated -BGT, were probed with antibodies to phosphotyrosine. The AChR ß-subunit is tyrosine phosphorylated in wild-type but not in AChR-ß^3F/3F mice. (D) Sections of skeletal muscle from P30 AChR-ß^3F/3F and wild-type mice were stained with Alex Fluor 594--BGT and antibodies against a phosphopeptide specific to AChR-ß^Y390-P. Antibodies to AChR-ß^Y390-P label synaptic sites in wild-type but not in AChR-ß^3F/3F mice. Scale bar: 10 µm.