Figure 4.

Enforced expression of constitutively active Akt1 delays Puma upregulation in FDM cells response to IL-3 deprivation. (a) WT cells expressing iAkt1 or ieGFP were cultured in the presence or absence of 4-OHT for 24 h and then deprived of IL-3 for the indicated times. Cell lysates were resolved on SDS-PAGE and western blots probed with the indicated antibodies. Anti-HA antibody was used to detect the presence of induced Akt. (b) WT cells expressing iAkt1 or ieGFP were cultured in the presence or absence of 4-OHT for 24 h before IL-3 was withdrawn for the indicated times. RNA was extracted and reverse transcribed and Q-PCR was performed to measure Puma or Mcl-1 mRNA levels. mRNA levels are expressed relative to the mRNA levels of two reference genes, Hmbs and Polr2a, using the ΔΔCT method. Data represent means and standard errors of three independent clones assayed in three independent experiments. (c) Bax^−/−;Bak^−/− FDM cells expressing iAkt1 or ieGFP were treated as described in (a). Proteins in cell lysates were resolved on SDS-PAGE and western blots probed with the indicated antibodies