FIG 2.

Exogenous Crk and CrkL are both enriched within AChR aggregates. In vitro-transcribed mRNAs encoding CrkL or both isoforms of the Crk gene, CrkI and CrkII, were fused to GFP and transfected into differentiated C2C12 myotubes. We analyzed soluble cellular lysates at 12 h posttransfection and confirmed the predicted sizes of our GFP fusion proteins by LiCor Odyssey infrared imaging using antibodies specific for CrkL or Crk. In parallel experiments, myotubes plated on laminin-111 were stained with fluorophore-conjugated bungarotoxin to label AChRs as well as Alexa Fluor 488-conjugated antibodies directed against GFP. Significant enrichment of CrkL-GFP, CrkI-GFP, and CrkII-GFP was observed within the area occupied by clustered AChRs and to a lesser degree the gaps within AChR clusters.