Figure 5.

Sucrose polysome gradient analysis: aldosterone and dDAVP increase translational efficiency of α- and γ-ENaC mRNA. (A) Equal amounts of cytosolic extracts of control (0.1% ethanol), aldosterone (300 nM) or dDAVP (10 nM) stimulated mCCD cells (S10) were separated by ultracentrifugation (160 000g, 2 h, Beckman SW 41 rotor) through 17–51% sucrose gradients and divided into 12 fractions. Continuous RNA monitoring (OD₂₅₄) of a representative gradient fractionation from the bottom (51% sucrose) to the top (17% sucrose) is shown. (B) Total RNA was prepared and relative levels of α-, β- and γ-ENaC mRNAs were monitored by RT–PCR. −RT controls showed no signals (data not shown). Polysome bound mRNAs sediment in fractions 2–8, free mRNPs and cytosolic proteins in fractions 9–12. A representative figure of three independent experiments is shown.