Copyright information:Taken from "Characterization of the SECIS binding protein 2 complex required for the co-translational insertion of selenocysteine in mammals"Nucleic Acids Research 2005;33(16):5172-5180.Published online 9 Sep 2005PMCID:PMC1214547.© The Author 2005. Published by Oxford University Press. All rights reserved () Reactions containing 6 pmol XH-CTSBP2, 15 pmol purified salt-washed rat ribosomes (+ribs) and 25 pmol P-UTP-labeled wild-type (wt) or mutant SECIS element (ΔAUGA, ΔAAAAC) as indicated were layered onto 200 µl of 20% sucrose cushions followed by ultracentrifugation. Each supernatant (S) or pellet (P) (5%) was analyzed by SDS–PAGE and western analysis using an anti-Xpress tag antibody (top panel). The percent of XH-CTSBP2 in the pellet fractions is indicated below the panel as determined by densitometry. RNA was extracted from the remainder of the supernatant and pellet fractions and subjected to 5% denaturing PAGE (bottom panel). Lanes 1–3 correspond to the input SECIS RNAs, lanes 4–7 are derived from reactions lacking SECIS RNA, lanes 8–13 are derived from reactions containing all components, and lanes 14–19 correspond to reactions lacking SBP2. () Wild-type or mutant (ΔAUGA) full-length GPX-4 mRNA (200 or 1000 ng as indicated) was added to an translation reaction containing pre-translated, S-labeled full-length SBP2 (FLSBP2). After 30 min of continued translation, EDTA was added to a final concentration of 5 mM in order to release mRNA from polysomes. The reactions were then layered onto sucrose cushions and analyzed for ribosome binding as described in (A). Radiolabeled SBP2 was detected by PhosphorImager analysis.