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Figure 2 In Vitro Effect of Everolimus on the mTOR Pathway and Protein Synthesis in M and SMC

(A) Western blot analysis of everolimus-binding protein FKBP12 and phosphorylated (Ser2448) and total mTOR. β-Actin served as a loading control. (B) Western blot analysis of phosphorylated and total p70 S6 kinase in cells treated with everolimus (10 µmol/l) for 0 to 12 h showed dephosphorylation of the downstream mTOR target p70 S6 kinase at site Thr389 and to a lesser extent at sites Thr421/Ser424. Everolimus treatment also resulted in an increased expression of the downstream mTOR target 4E-BP1 without changing its phosphorylation at site Thr37/46. This corresponds to a relative reduction of 4E-BP1 phosphorylation. In contrast, phosphorylation of initiation factor eIF2 and elongation factor eEF2 increased. All of these effects were similar between M and SMC. All protein bands correspond to their molecular weight (size markers not shown). (C) Administration of everolimus (10 µmol/l) resulted in a significant reduction of de novo protein synthesis in both M and SMC. Cycloheximide (10 µg/ml; CHX) was used as a positive control. Versus control: **p < 0.01; ***p < 0.001. 4E-BP1 = 4E-binding protein 1; eEF2 = eukaryotic elongation factor 2; eIF2 = eukaryotic initiation factor 2; FKBP12 = FK506-binding protein 12; mTOR = mammalian target of rapamycin; other abbreviations as in Figure 1.