Functionality of EGFP-Rheb, EGFP-mTOR and DsRed-raptor determined by S6-kinase activity. EGFP-Rheb, EGFP-mTOR and DsRed-raptor were expressed in HEK cells and gels were run and blotted with the respective antibodies to Rheb, mTOR, raptor and S6-kinase (thr389). The expressed level of EGFP-Rheb is shown in Row A, lanes 2, 5 and 7 but the antibody was unable to pick up endogenous levels. The endogenous levels of mTOR can be seen in Row B, lanes 1, 2, 4 and 7 (with increased density when EGFP-mTOR is expressed (lanes 3, 5 and 6, high MW too close for large migration difference). The endogenous levels of raptor can be seen in Row C, lanes 1, 2, 3, and 5, with lanes 4, 6 and 7 showing DsRed-raptor expression.
Yadav et al. BMC Cell Biology 2013 14:3 doi:10.1186/1471-2121-14-3