Fig. 5. Recovery of p66/ACF during rat liver nuclear and cytoplasmic fractionation. (A) Normal rat liver nuclear and cytoplasmic S100 extracts were prepared and subjected to an in vitro editing reaction and poisoned primer extension quantification of editing. RNA editing was quantified by PhosphorImager densitometry as the number of counts in edited RNA (UAA) divided by the sum of the counts in UAA and the unedited RNA (CAA) times 100. The reactions were performed in triplicate and shown with the s.e.m. (B) 25 µg of protein from nuclear (N) and cytoplasmic (C) S100 extracts were resolved by SDS PAGE and stained with Coomassie blue (left pair of lanes) or western blotted with anti-ACF as described in Materials and Methods (right pair of lanes). The migration of molecular mass marker proteins is shown to the left (Mr). (C) Chemiluminescence from western blots in B were quantified, setting the density in the cytoplasmic lane to an arbitrary unit of 1 (first column). The total amount of protein in each fraction was calculated (second column) and this value was multiplied by the relevant number in column 1 to give the estimated amount of total p66/ACF in each fraction (column three). The relative percent p66/ACF in each fractions (column four) was calculated assuming that the sum of the nuclear and cytoplasmic p66/ACF equalled the total tissue p66/ACF. On average, 35 ml of cytoplasmic S100 extract at 39 mg protein/ml and 2 ml of nuclear S100 extract at 21 mg protein/ml were obtained.

Return to article