Figure 5
Regulation of Bcl-2 family proteins by hypoxia in Min6 cells and MBECs. (a) Hypoxia changes protein levels of several Bcl-2 family proteins. Min6 cells and MBECs were cultured in normoxia or exposed to hypoxia (1% O2) for the indicated time points. Cells were treated during 8 h with 1 μM thapsigargin (Tg), or 1 μM staurosporine (St). Whole-cell extracts were prepared, and proteins were separated by SDS-PAGE. Levels of HIF-1α, PUMA, Bim, Bad, phosphorylated-Bad (p-Bad S112), Bax, Bcl-xL, Bcl-2 and β-actin were assessed by immunoblotting using specific antibodies recognizing these proteins. PUMA, phosphorylated-Bad and Bax are not recognized in MBECs by these antibodies. (b) Impact of hypoxia on mRNA levels of Bcl-2 family of proteins. Min6 cells were cultured at normoxia or hypoxia (1% O2) at indicated time points. Expression levels of PUMA, Bim, Bad, Bax, Bcl-xL and Bcl-2 mRNA were measured by qRT-PCR and normalized to β-actin (n=4; values are mean±S.D.). (c) Caspase-9 is activated by hypoxia in Min6 cells. Whole-cells extract proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-HIF-1α, anti-procaspase-9, anti-cleaved caspase-9 and anti-β-actin antibodies
