Interaction of DRBD18 with the editing machinery. (A) Interaction with RESC factors. (Left) DRBD18 was immunoprecipitated from mitochondrially enriched cell extracts that were treated with either RNase inhibitor (−RNase) or an RNase cocktail (+RNase) using α-DRBD18 antibody crosslinked protein A beads. Bound proteins were analyzed by western blot with native antibodies against RESC factors. (Right) RESC proteins (RESC13-MHT, RESC14-MHT, and RESC2-PTP) were precipitated from mitochondrially enriched cell extracts treated with either RNase inhibitor (−RNase) or an RNase cocktail (+RNase) using IgG beads. Target proteins were analyzed using native antibodies or α-Myc antibodies; associated DRBD18 was detected with α-DRBD18 antibodies. (B) As in A, left, but blotting for RNA editing holoenzyme components using native antibodies. (C) As in A, left, but blotting for RNA editing auxiliary factors using native antibodies. In A (left), B, and C, all pull-down lanes were loaded at 1000× input. A (right) was loaded as follows: For RESC13, DRBD18 was 1000× input and RESC13 was 200× input; for RESC14, both DRBD18 and RESC14 were 1000× input; for RESC2, DRBD18 was 1600× input and RESC2 was 100× input. The blots shown are representative of three biological replicates.
