Fig 2: The effect of Torin2 on translation of reporter mRNAs in HeLa cells, and the effect of 4E-BP1 on translation of reporter mRNAs in vitro.(A) Untreated or Torin2-treated (0.25 μM, 1 h) HeLa cells were transfected by reporter Firefly luciferase mRNAs with indicated 5′ UTRs and Renilla luciferase mRNA (as internal control), cultivated for 2 h, harvested and assayed for Firefly and Renilla luciferase (FLuc and RLuc, respectively). Absolute values of FLuc or RLuc activities are presented. Errors are 2 standard deviations. (B) The same as (A) but the FLuc/RLuc ratio is presented. The FLuc/RLuc ratio for the control (untreated cells) was taken as 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. (C), 0.1 pmol C+A+ reporter Firefly luciferase mRNAs with indicated 5′ UTRs were translated in Krebs extract in the presence of increasing amounts of recombinant 4E-BP1 (0.4, 0.8, and 1.6 pmol) or without it. Reaction mixtures were assayed for Firefly luciferase after 45 min incubation at 30 °C. The FLuc activity without addition of 4E-BP1 was taken to be 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. (D) Translation reactions (Krebs extract) with 1.6 pmol 4E-BP1 or without it were analyzed by Western blotting (left panel) or used for analysis of binding of translation initiation factors. The biotinylated, capped luciferase mRNA with β-globin mRNA 5′ UTR (0.1 pmol) was incubated in 10 μl translation reaction mixture (Krebs extract) with or without 1.6 pmol 4E-BP1 and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blotting (right panel).

Fig 3: The staining properties of both phosphorylated and nonphosphorylated antibodies for each protein were evaluated in the tumor tissues of CCPRCC and CCRCC. We confirmed same expression pattern in CCPRCC and CCRCC. TSC1 (anti‐TSC1 antibody, sc‐377386, Santa Cruz Biotechnology), TSC2 (phosphorylated antibody: anti‐TSC2 antibody, ab32554, Abcam/nonphosphorylated antibody: Tuberin/TSC2 antibody, #3612, Cell Signaling Technology), mTOR [phosphorylated antibody: anti‐phospho‐mTOR (Ser2448), #2976, Cell Signaling Technology/nonphosphorylated antibody: mTOR (7C10) Rabbit mAb, #2983, Cell Signaling Technology], and 4EBP1 [phosphorylated antibody: anti‐phospho‐4EBP1 (Thr37/46), #2855, Cell Signaling Technology/nonphosphorylated antibody: non‐phospho‐4EBP1 (Thr46) (87D12) Rabbit mAb, #4923, Cell Signaling Technology].