Fig. 3.

Downregulation of death-suppressing Bcl-2 proteins coincides with the induction of apoptosis. (A) RT-PCR analysis of Bcl-x, Bcl-2 and Bcl-w confirmed the RNA profile of Bcl-x as measured by northern blotting and showed that expression of Bcl-2 and Bcl-w were regulated during development. In several independent experiments, the Bcl-2 RT-PCR product was absent in lactation and expressed at low levels in involution, whereas the Bcl-w RT-PCR product was dramatically lower in the I1 and I2 samples than L9 but was not completely absent. (B) Whole tissue lysates were prepared from the developmental samples, separated by polyacrylamide gel electrophoresis, then analyzed by western immunoblotting. Blots were incubated with antibodies directed against Bcl-x, Bcl-2 and Bcl-w. Confirmation of the Bcl-w expression profile was validated using two additional and independently derived antibodies directed against Bcl-w (data not shown). Lysates from M1 cells were included as controls. (C) Whole tissue lysates were prepared from ICR mice in pregnancy (P12) and involution (I2) and directly compared with tissue lysates from brain, mammary gland and uterus of ROSA41 Bcl-w null mutant animals (–/–) and their wild-type littermates (+/+) (Ross et al., 1998). Western blotting confirmed the identity of the detected protein as Bcl-w.