Figure 4.

Activation of mitochondrial events in Dex-induced apoptosis in EBV-transformed B cells. Cells were treated with 100 μM Dex for the indicated times. Total RNA and proteins were extracted from cell lysates and (A) RT-PCR for XIAP, XAF1, Bax, Puma, Noxa, Bcl-2 and β-actin mRNA and (B) western blot analysis for XAF1, Bax, Puma, Noxa, Bcl-2 and Bcl-xL protein were performed. (C and D) Cells were harvested and then the amount of Bax, XAF1 and Puma in cytosol and mitochondria fractions was determined. The mitochondria marker, COX-IV, and cytosol marker, β-tubulin were used to verify the purity of each fraction performed as described in Materials and methods. To block activation of caspase-9, cells were pretreated with z-LEHD-fmk (20 μM) for 2 h. (E) In binding assay, XAF1 or Puma was immunoprecipitated using specific Ab, followed by immunodetection of Bax in immunoprecipitate as detailed in Materials and methods. Results are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblot analysis.