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. 2014 Mar 20;289(19):13132–13141. doi: 10.1074/jbc.M113.520734

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© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 1. — Nicotinamide specifically inhibits S6K1 phosphorylation. COS-7 cells were treated with 100 nm Torin 1, 5 mm NAM, 5 nm TSA, or NT for the indicated times. Total cell lysates were prepared and assessed by immunoblotting (WB) with the indicated antibodies. A, NAM, TSA, and NT induce cellular protein acetylation. Samples treated for 3 h from C–E were analyzed by immunoblotting with anti-acetyl-lysine and actin antibodies. B, Torin 1, a specific mTOR kinase inhibitor, suppresses both Thr-389 phosphorylation of S6K1 (pT389-S6K1) and Thr-37/46 and Ser-65 phosphorylation of 4EBP1 (pT37/46–4EBP1 and pS65–4EBP1) in a similar dose-dependent manner. C, NAM specifically attenuates S6K phosphorylation. pS235/236-S6, Ser-235/236 phosphorylation of S6. D, inhibition of Class I/II HDACs by TSA does not affect S6K1 phosphorylation. E, inhibitory effect of nicotinamide on S6K1 phosphorylation is augmented by TSA co-treatment.