Characterization of STAT5 morpholinos. (A) Two distinct morpholinos (MO1 and MO2) were designed to target sequences around the ATG initiation site of xSTAT5. The ability of each to inhibit STAT5 translation was tested first in vitro. STAT5 protein (top panel) or as a control luciferase protein (bottom panel) was transcribed and translated in vitro using rabbit reticulocyte lysates either alone or in the presence of MO1, MO2, or both. Lanes are 1) DNA template alone, 2) 100 ng MO1, 3) 100 ng MO2, 4) 100 ng MO1 + 100 ng MO2, 5) 200 ng nonspecific control morpholino, and 6) 250 ng MO1 + 250 ng MO2. Translation lysates were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. (B) The activity of the morpholinos was next tested in vivo. Shown is a representative Western blot for xSTAT5, following injection of RNA encoding xSTAT5 alone (control, C) or co-injected with either MO1 or MO2 alone (1 or 2, 10 ng) or both MO1 and MO2 together (1 + 2, 10 ng each). Under these conditions the morpholinos reproducibly result in a substantial inhibition of STAT5 expression.