Fig. 10. Cytoplasmic and nuclear complexes containing editing factors. Cytoplasmic and nuclear S100 extracts were sedimented through 10%-50% glycerol gradients, fractionated and assayed for p66/ACF and KSRP by western blotting or for co-sedimenting APOBEC-1 by assaying in vitro editing activity. Glycerol gradients were loaded with 60 and 20 mg of cytoplasmic and nuclear S100 extracts, respectively. Gradient fractions from cytoplasmic (A,C,E) or nuclear (B,D,F) S100 extracts were analyzed by western blotting. Fractions are numbered from the top of each gradient and the gradient positions corresponding to 11S, 27S and 60S complexes are indicated. An equal aliquot of each gradient fractions was resolved by SDS PAGE and blotted. A and B are blots reacted with antibodies specific for KSRP; blots C and D were reacted with antibodies against p66/ACF. Poisoned primer extension and gel analysis of in vitro editing activity in gradient fractions from cytoplasmic (E) and nuclear (F) S100 extracts correspond to those immunoblotted in panels A/C and B/D, respectively. The primer extension products from unedited (CAA) and edited (UAA) RNA are indicated. The percent editing in each fraction was determined as described in Fig. 5 and was 2%, 2%, 16%, 14% and 14% for fractions 5-9, respectively, in E, and 15%, 15%, 17%, 4% and 0.5% in fractions 4-8 in F, respectively.