Figure 1

TRAIL signaling proteins are altered when BNIP3 is upregulated in the nucleus or knocked down. (a) Total cell lysates from U251 parental, U251 overexpressing BNIP3 targeted to the nucleus (BNIP3-NLS) and BNIP3 knocked down (BNIP3-shRNA) were western blotted for BNIP3, DR5, BID, TRAIL, DR4, caspase 3, caspase 8, FLIP, Bcl-xL and actin as a loading control. These blots represent three independent experiments. (b) U87 cells were transfected with BNIP3-NLS expression vector, vector alone, shRNA against BNIP3 or scrambled vector. Lysate was western blotted for DR5 and actin as loading control. (c) Protein was isolated from mature astrocytes as described above and then western blotted for DR5, TRAIL, AIF and 8 and FAS. Blots were stripped and reprobed for actin as a loading control. (d) Immunohistochemistry was performed on 5 μM sections from the brains of 30-week BNIP3 wild type and knockout mice using the DAKO EnVision system and DR5 antibody (Atlas). Images were taken in matched regions of the hippocampus in both animals. Scale bar indicates 100 μM. (e) Total cell lysates were generated from cryopreserved brain tissue extracted from wild type, heterozygous and BNIP3-null adult mice. Lysates were analyzed for BNIP3 expression by western blot, with actin as a loading control; each lane represents a different mouse. Mice were killed by cervical dislocation to minimize hypoxia at the time of death, and brain tissue was removed within 5 min