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Thiol reductase activity of intracellular and secreted GILT mutants. Intracellular and secreted GILT were immunoisolated from the cell extract and supernatant of COS-7 cells expressing wild-type, C91S, C98S, C200S, C211S, and C222S GILT. Eluates from the cell extract (A) and supernatant (B) were immunoblotted for GILT expression. (C and D) Eluates (70 and 30 μl) were assayed for reduction of F(ab′)2. (E and F) Subsequent quantitation. [Ctrl(−): buffer at pH 5; purified GILT: 1 μg.]
