Figure 4. Expression of Bcl2 family members.. (A) RT-PCR of bcl2 family members. cDNA was prepared from total RNA from p65 null (vector) and reconstituted cells (p65). Specific oligonucleotides for each gene (and three pairs for Noxa) were used to determine expression. GAPDH expression was used as a control. Bax expression was detected by immunoblot. (B) Northern Blot for Noxa after genotoxic treatments. Total RNA was extracted from p65 null and reconstituted cells after treatment with 10 μM etoposide or 5 mJ UV-irradiation for the times indicated. Expression of Noxa, and GAPDH as control, was revealed by blotting with specific radio-labeled probes. (C) Expression of Noxa sensitizes p65 null MEFs to genotoxic agents. Cloned murine Noxa was introduced into p65 null cells by retroviral transfer and sensitivity to etoposide and UV-irradiation compared. Noxa cloned in the anti-sense orientation was used as a control. After selection cells were treated with 10 μM etoposide or 5 mJ UV-irradiation for 24 hr and apoptosis assessed by flow cytometry as described in Figure 1. Results are representative of three different viral clones for both control and Noxa. Northern blotting confirmed Noxa expression.