Extended Data Fig. 4: ARBS assays to monitor the effect of FXN and FDX2 on persulfide generation, transfer and reduction.
From: Cross-regulation of [2Fe–2S] cluster synthesis by ferredoxin-2 and frataxin
a–f, ARBS assays monitoring: (a) persulfide generation on NFS1 in the absence (0 eq. FDX2) or presence of 10 eq. FDX2, and persulfide generation on NFS1 in the NFS1-apo-ISCU2 complex in the absence of FDX2 and FXN (0 eq. FDX2/FXN) or presence of 10 eq. FDX2 or 10 eq. FXN. (b) persulfide transfer from NFS1 to ISCU2 in the absence (0 eq. FXN) or presence of 1, 5 or 10 eq. FXN, (c) persulfide reduction on ISCU2 by the addition of a preincubated FDX2-FDXR-NADPH complex in the absence (0 eq. FXN) or presence of 1 or 10 eq. FXN. NP and P labelling corresponds to non-persulfidated (before the addition of L-cysteine) and persulfidated (after the addition of L-cysteine), respectively, (d) persulfide transfer from pre-persulfidated NFS1 to ISCU2 in the absence (0 eq. FDX2) or presence of 3, 5, or 10 eq. FDX2, (e) persulfide transfer from NFS1 to ISCU2 in either the absence (0 eq. FDX2) or presence of 1,3, 5, 7, or 10 eq. FDX2, (f) persulfide transfer from NFS1 to ISCU2 in the presence of 1eq FXN and either the absence (0 eq. FDX2) or presence of 1 or 10 eq. FDX2. Persulfidated protein bands are annotated as NFS1–SSH and ISCU2–SSH, and non-persulfidated protein bands are annotated as NFS1-SH and ISCU2-SH. ISCU2 is loaded with iron unless otherwise stated. For gel source data, see Supplementary Figs. 2 and 3. (a–f, representative ARBS assays from n = 3 independent experiments with similar results).
