The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone
J. Clin. Invest. Jaemin Lee, et al. 118:2950 doi:10.1172/JCI35164 [Go to this article.]

Figure 1
Free Cys thiols in Tg. 293 cells were transiently transfected with an expression vector encoding wild-type Tg, the rdw Tg mutant (G2300R), or empty vector. At 48 hours after transfection, cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for 4 hours in the absence or presence of BFA (5 μg/ml) where indicated. At this time, chase media (lanes labeled “Tg secreted”) and cell lysates (all other lanes) were immunoprecipitated with anti-Tg. The immunoprecipitates were denatured in 2× SDS gel sample buffer lacking reducing agents and mock incubated or incubated with AMS (5 mM, 30°C for 1 hour). At the end of the incubation, samples were boiled in the presence of 20 mM DTT and analyzed by 4% SDS-PAGE and fluorography. A slowed mobility (shift up) of the Tg band after AMS is indicative of free reactive thiols in the Tg molecule that are not apparent in secreted, wild-type Tg.