Analysis of Smad5 mRNA levels in antisense-treated CD34⁺ cells. RNA from approximately 10,000 CD34⁺ BM cells preincubated with antisense oligonucleotides to the Smad5 gene were analyzed by RT-PCR for the presence of Smad5, Smad2, Smad4, TGF-β receptor subunit 1 (TGF-βR1), TGF-β receptor subunit 2 (TGF-βR2) and TFR (as a nonspecific control) mRNA. Amplifications were done under identical conditions and RNA concentrations and represent time points within the exponential phase of the assay. Samples treated with antisense oligonucleotides to Smad5 (AS1 and AS2) contain barely detectable or undetectable levels of Smad5 mRNA, whereas samples treated with sense oligonucleotides (S1 and S2) or mock-treated samples (control) contain similar amounts of Smad5 message. The levels of all other mRNA species were unaffected for all treatments.