Fig. 2.

Increase in the AChR ε-mRNA by transient stimulation with neuregulin. A, Stimulation protocol. Stimulation of myotubes with NRG began at time 0; after an incubation for the indicated time, the cells were washed and incubated in neuregulin-free medium. At 24 hr, cells were lysed to isolate total RNA, which was then blotted for AChR mRNA expression. B, Activation of ERK kinase by neuregulin. Top, C2C12 myotubes stimulated continuously without (control) or with 1 nm neuregulin for the indicated times. Bottom, C2C12 myotubes stimulated with 1 nm neuregulin for 1 min and then incubated in neuregulin-free medium for the indicated times. Control C2C12 myotubes were treated with an equal volume of DMEM (vehicle) without neuregulin for 5 min. ERK kinase was immunoprecipitated and assayed using MBP as a substrate in vitro with [γ-³²P]ATP. The phosphorylated MBP was resolved on a 15% SDS gel and exposed to x-ray film. C, Northern blot radiograms. C2C12 myotubes were stimulated as described in A. Control C2C12 myotubes were treated with the same volume of DMEM (without neuregulin) for 24 hr. Twenty micrograms of total RNA were probed with [³²P]-labeled ε-subunit probe. D,Histograms showing mean ± SD of three different samples. The mRNA levels in the absence of neuregulin were considered to be 100%.CONST, Continuous stimulation;ST, stimulation.