Extended Data Fig. 3: Disulfide reduction is impaired in the context of the nucleosome. | Nature Structural & Molecular Biology

Extended Data Fig. 3: Disulfide reduction is impaired in the context of the nucleosome.

From: Histone dynamics play a critical role in SNF2h-mediated nucleosome sliding

Extended Data Fig. 3

(a) Native gel remodeling assay with saturating SNF2h (1 µM), saturating ATP, and 15 nM cy3-nucleosomes as in Fig. 1. Nucleosomes containing the oxidized sCX2 bonds were generated by oxidizing the H3C110A sCX2 octamer using CuPhe, and then assembling nucleosomes. Treatment of these nucleosomes with excess DTT as in Yan et al. fails to reverse the remodeling defect. (b) Scheme for the samples run in C. Nucleosomes treated with DTT were either directly added to non-reducing SDS-PAGE loading buffer or quenched with 500 mM N-Ethyl Maleimide freshly dissolved in DMSO (final [DMSO] ≈ 10%(v/v)). Additionally, a condition where N-Ethyl Maleimide and DTT were added simultaneously is included to evaluate the efficacy of the quench. (c) SDS-PAGE of samples treated as in B. Samples with reducing agent quenched prior to running on the gel are near-completely oxidized. The experiments shown here were performed once.

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