Regulation of Puma by IL-3 signaling. (A) RT-PCR analysis of changes in Puma mRNA levels following IL-3 withdrawal in wild-type and Bax^-/-;Bak^-/- cells. Total RNA (1.2 μg) extracted from wild-type and Bax^-/-;Bak^-/- cells cultured in the presence or absence of IL-3 was reverse transcribed and 2.5 μL of the reaction volume (dilution 1) and 1:5 serial dilutions of this (dilutions 2 and 3) were used in a PCR to amplify Puma mRNA. RT-PCR analysis of β-actin mRNA is shown as a control. (B) Lysates from 2 wild-type and 2 Bax^-/-;Bak^-/- cells cultured in the presence of IL-3 (0 hours) or in the absence of IL-3 for the indicated time periods were immunoblotted with an antibody to Puma. Probing with an antibody to β-actin was used as a loading control. The arrows indicate Puma and the asterisks a nonspecific band that was observed only in cell lines other than Bax^-/-;Bak^-/- cells. The lysate from Puma^-/- cells shown aligned to the wild-type 2 and Bax^-/-;Bak^-/- 2 clones was run on the same gel, but intervening irrelevant bands have been removed.