Figure 1.

The mTOR kinase is a Trx1 substrate. A, schematic of how the Trx1 substrate-trapping mutant (Trx1C35S) works. B, Trx1C35S traps mTOR in the heart. After 3 h of ischemia, FLAG-Trx1C35S was immunoprecipitated (IP) with anti-FLAG antibody. C, Trx1C35S traps mTOR in cultured cardiomyocytes. FLAG-Trx1C35S-HA was expressed via an adenovirus vector. Cardiomyocytes were incubated with 100 μm H₂O₂ for 30 min, and then FLAG-Trx1C35S-HA was immunoprecipitated with anti-FLAG antibody with or without 1 mm DTT. D, stable interaction between endogenous mTOR and Trx1 is not observed in cultured cardiomyocytes. Cardiomyocytes were treated with 100 μm H₂O₂ for 30 min, and endogenous mTOR was immunoprecipitated. E, mTOR intermolecular disulfide formation in primary cultured cardiomyocytes. Cardiomyocytes were incubated with 100 μm H₂O₂ for 30 min (left panel) or 500 μm H₂O₂ for 10 min (right panel). F, mTOR forms disulfide bonds with either mTOR itself or other proteins. FLAG-mTOR and Myc-mTOR were expressed in HEK293 cells. After H₂O₂ treatment (3 mm, 30 min), FLAG-mTOR was immunoprecipitated with anti-FLAG antibody. G, data interpretation of the results shown in F. E and F, Western blot analyses were performed after SDS-PAGE under non-reducing (without 2ME) conditions. Ox, oxidized form; Red, reduced form. Western blot results were verified by two (B–D and F) and three (E) additional independent experiments.