Figure 2.
Silencing of RNF126 Promotes Accumulation of Frataxin Precursor and Mature Forms by Preventing Its Degradation
(A) 293 Flp-In cell line stably expressing frataxin1–210 was transfected with the indicated siRNA pools. Cell extracts were collected 48 or 72 hr post-transfection and analyzed by WB with anti-frataxin, anti-RNF126, and anti-tubulin antibody. Pre, precursor; int, intermediate; mat, mature frataxin.
(B and C) Graphs representing relative frataxin precursor (B) or mature frataxin (C) abundance as quantitated by densitometric analysis of the blots in (A) and normalized with tubulin levels.
(D) 293 Flp-In cell line stably expressing frataxin1–210 was transfected with the indicated siRNA pools. Then, 48 hr after transfection, cells were treated with 100 nM actinomycin D for the indicated times. Cell extracts were analyzed by western blot with anti-frataxin, anti-RNF126, and anti-tubulin antibody.
(E) Graph representing relative frataxin precursor abundance as quantitated by densitometric analysis of the blot in (D) and normalized with tubulin levels.
Data represent the mean ± 1 SEM from four independent experiments. The p values were calculated with Student’s t test and were statistically significant (∗∗p < 0.01) compared to non-targeting siRNA-transfected cells.