Figure 5.

In Vitro Ubiquitination Assay

(A) In vitro ubiquitination assay was carried out with purified recombinant E1, UbcH5b as the E2 ubiquitin-conjugating enzyme, and different doses of GST-RNF126 as the E3 ubiquitin ligase, together with Ub, ATP, and recombinant frataxin precursor as a substrate. The reaction mixture was incubated for 60 min at 30°C. Reaction was stopped by the addition of 4× sample buffer. Proteins were separated on SDS-PAGE and analyzed by a WB with anti-frataxin antibody.

(B) Ubiquitination assay was carried out with the indicated E2 ubiquitin-conjugating enzymes and 200 nM RNF126. Proteins were analyzed as in (A).

(C) Ubiquitination assay was carried out as in (B), in the presence of the indicated concentration of 1,10-phenanthroline. Proteins were analyzed as in (A).

Frataxin precursor, monoubiquitinated frataxin, and polyubiquitinated frataxin are indicated by arrows. Fxn precursor s.e. indicates a short exposure of the blot to appreciate precursor levels. ^∗Non-specific bands.