Figure 2.

Increased Bax/Bcl-2 ratio and cytochrome c release in RanBP9-transfected cells. HT22 cells were transfected with vector or RanBP9 for 24 h, and cells were incubated in medium containing 10 or 2% FBS for another 24 h. (a) Equal protein amounts of cell lysates were subjected to immunoblotting for Flag, Bax, Bcl-2. Note the relative decrease in Bcl-2 and increase in Bax levels in RanBP9-transfected cells. (b) Representative images of EGFP, EGFP-RanBP9, and Bax immunofluorescence. (c) Quantitation of Bax immunofluorescence intensity from EGFP or EGFP-RanBP9-transfected cells (n=4 each). Error bars represent S.E.M. (d) Quantitative RT-PCR analysis of Bax mRNA levels normalized to GAPDH (n=4 each). Error bars represent S.E.M. (e) HT22 cells were transfected with vector or RanBP9 for 48 h, and equal protein amounts of cell lysates were subjected to immunoblotting for Bax. Note the increase in SDS-resistant 46-kD dimeric bax induced by RanBP9 transfection. (f) Thirty-six hours after transfection of EGFP or EGFP-RanBP9, HT22 cells cultured in 10% FBS were subjected to immunofluorescence for cytochrome c. A representative image shows widespread diffuse cytochrome c staining in EGFP-RanBP9-transfected cells, suggestive of release to cytosol. (g) Thirty-six hours after transfection of EGFP or EGFP-RanBP9, HT22 cells cultured in 10% FBS were subjected to biochemical isolation of mitochondria and cytosol. Representative experiment shows the localization of RanBP9 in both cytosol and mitochondria as well as release of cytochrome c from mitochondria to cytosol in RanBP9-transfected cells. Timm50 was used as a marker of mitochondria