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. Author manuscript; available in PMC: 2017 Nov 1.
Published in final edited form as: Clin Cancer Res. 2016 Jun 7;22(21):5383–5393. doi: 10.1158/1078-0432.CCR-16-0609

Figure 2.

Figure 2

FL-associated RRAGC mutations are intrinsically mTOR activating. Upper: HEK293T cells stably retrovirally infected expressing HA-tagged RRAGC wild type or various RRAGC mutants were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS; +) or alternatively for the last 1 h of culture in RPMI1640 medium that was free of the amino acid leucine and supplemented with dialyzed 10% FBS (−). Cells were harvested and prepared for immunoblotting with various antibodies as indicated. Short and long exposures for RPS6KB/S6K (S6K) and p-Thr389-RPS6KB/S6K (p-S6K) are shown. CHO-IR + INS: Insulin receptor transfected CHO cells stimulated with insulin (immunoblot controls). Measurements of HA-RRAGC expression levels were aided by the slower migration of the HA-tagged RRAGC WT and mutant proteins in SDS-PAGE resulting in a doublet band. Lower: Displayed are combined quantitation results (p-S6K/total S6K) from 3 independent experiments using ImageJ densitometry results indexed to the measurements for RRAGC wt.