FIG. 3.
The intrinsic apoptotic pathway is affected by decreased levels of Prep1. (A) Total extracts from E14.5 Prep1+/+ (+/+) and Prep1i/i (i/i) MEFs, untreated (U) or 4, 8, or 12 h after UV irradiation (UV-C; 254 nm; 1,000 J/m2), were fractionated in 12% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with active caspase 9 and 3 monoclonal and polyclonal antibodies, respectively. β-Actin was used for normalization. Representative results from one of three independent experiments are shown. (B) Densitometric analysis of the β-actin-normalized immunoblots in panel A. A.U., arbitrary units. (C) Twenty-four hours after treatment, total extracts were prepared from untreated (U), 200 μM etoposide-treated, or UV-C light (254 nm; 60 J/m2)-irradiated MEFs from WT and Prep1i/i littermate embryos; fractionated in SDS-PAGE; transferred to PVDF membranes; and immunoblotted with active caspase 9 and β-actin antibodies. Representative results from one of three independent experiments are shown. (D) β-Actin-normalized densitometric analysis of the immunoblots shown in panel C. (E) MEFs from WT and hypomorphic embryos were treated (or not) with actinomycin D (1 μg/ml) for 30 min and then with 0.1, 1, and 10 ng/ml of TNF-α. Total extracts were prepared 6 h after treatment, fractionated, and transferred, and active caspase 8 protein levels were probed with specific polyclonal antibodies. β-Actin was used for normalization. Representative results from one of three independent experiments are shown. U, untreated. (F) β-Actin-normalized densitometric analysis of the immunoblot shown in panel E. (G) MEFs from WT, Prep+/i (+/i), and Prep1i/i littermates were either treated (or not) with 200 μM etoposide for 24 h, irradiated with UV-C light (254 nm; 60 J/m2), or treated with actinomycin D and 1 ng/ml of TNF-α for 6 h (see above). Immunoblotting was performed with anti-active caspase 3 and anti-α-tubulin polyclonal and monoclonal antibodies, respectively. Representative results from one of three independent experiments are shown. U, untreated. (H) α-Tubulin-normalized densitometric analysis of the immunoblot shown in panel G.