Following IL-3 withdrawal, Bax translocates normally from the cytosolic fraction to the membrane fraction in cells lacking Bad or Bim. (A) Cells of the indicated genotypes were cultured in the absence of IL-3 and then fractionated into cytosolic and membrane fractions using digitonin. Lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies to Bax, Bak, and, as a control to demonstrate purity of the fractions, with an antibody to VDAC. (B) Bcl-2 and Bcl-xL expression is maintained following IL-3 withdrawal. Western blots of lysates from cells cultured in the absence of IL-3 for 0, 24, and 48 hours probed with antibodies against Bcl-2 or Bcl-xL. Probing with an antibody to β-actin was used as a loading control. Lysates from equal numbers of cells were loaded in each lane. Asterisks mark a nonspecific band. These blots are the same as shown in Figure 2C, but have been reprobed with the indicated antibodies. The same β-actin controls are shown in Figure 2C and in this panel.