Figure 3.
Expression of RNF126, But Not Its Catalytically Inactive Mutant, Promotes Frataxin Ubiquitination
(A) HEK293 cells were transiently transfected with frataxin1–210, HA-tagged ubiquitin (HA-Ub), and a control empty vector, RNF126, or its catalytically inactive mutant. Protein extracts were collected 40 hr post-transfection. Total cell extracts were analyzed by WB with anti-frataxin antibody or anti-tubulin as a loading control. Slower-migrating bands can be detected above the frataxin precursor, corresponding to mono- and polyubiquitinated frataxin.
(B) Cells were transfected as in (A). Mono- and polyubiquitin-conjugated forms of frataxin can be detected by WB with anti-frataxin antibody on anti-HA immunoprecipitates. Control samples (excluded from these panels) were treated with 10 μM of the proteasome inhibitor MG132 as a positive control for the detection of ubiquitinated frataxin.
∗Antibody heavy chain; ∗∗antibody light chain.