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. 2009 Aug 15;20(16):3628–3637. doi: 10.1091/mbc.E09-03-0211

Figure 3.

Figure 3.

Function of Trx1 on Cys22,30 and Cys250 of ASK1. (A) Schematic representation of the different cysteine substitution mutants used. Represented are the N-terminal (N), kinase (K), and C-terminal (C) domain of ASK1. Residue numbers at domain boundaries are indicated. Numbers in bold or in white within each domain indicate which cysteine residue(s) have been replaced by alanine residue(s). (B through D) 293T cells were transfected with pRK-Flag-Trx1, pRK-Flag-Trx1C35S, pRK-Flag-Trx1C32S, pcDNA3-ASK1-HA, or pcDNA3-NΔCys-HA alone, or pRK-Flag-Trx1, pRK-Flag-Trx1C35S, or pRK-Flag-Trx1C32S together with either pcDNA3-ASK1-HA, pcDNA3-NΔCys-HA, pcDNA3-C250CΔCys-HA, or pcDNA3-C22,30CΔCys-HA. Twenty-four hours after transfection, the cells were left untreated (−) or exposed (+) to 100 μM (B), 1 mM (C), or 1,5 mM (D) H2O2 for 1 min (D) or the time indicated (B and C). Total cell (B and INPUT in C and D) or anti-HA immunoprecipitated (IP:HA in C and D) extracts were analyzed by Western blot with either anti-HA (HA) or anti-Flag (Flag). All samples were migrated on nonreducing SDS gel except for the INPUT samples in C and D and the anti-HA immunoprecipitated extracts analyzed with anti-HA labeled “reducing” in D. Band 1 indicates the expected positions of monomeric ASK1 (150 kDa). In B, bands 3 and 4 point the positions of ASK1 DBM as in C and D, together with bands 2A and 2B, they indicate the positions of Trx1 or Trx1C35S covalent associations with either or NΔCys, C250CΔCys or C22,30CΔCys.