Fig. 2.

Ablation of PrxII impedes formation of disulfide HMGB1. (A, B) Immortalized WT or PrxII KO MEFs were incubated for 30 min in the absence or presence of PMA (50 or 250 nM) or LPS (0.25 or 1 μg/mL), after which WCLs were subjected to nonreducing SDS-PAGE and immunoblotting. #: nonspecific bands, M, monomer; D, dimer. (C, D) Immortalized PrxII KO MEFs that had been transfected with expression vectors for PrxII (C172S) or PrxII (WT) were incubated for 30 min in the absence or presence of GOX (0.5 or 5 mU/mL) or H₂O₂ (50 or 250 μM), after which WCLs were subjected to nonreducing SDS-PAGE. Means ± SEM (n = 5 for A and B, n = 3 for C and D). *P < 0.05, **P < 0.01 versus PrxII KO (A and B) or empty vector (Ev, C and D), t-test. (E) Determination of disulfide formation of HMGB1 Cys²³–Cys⁴⁵ and free thiol of Cys¹⁰⁶ by LC-MS/MS analysis. WCLs of GOX exposed Myc-HMGB1 in HEK293T cells were incubated with NEM and then treated with DTT and NEM for disulfide HMGB1 analysis.