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.
