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Fig. 3. The pro-apoptotic activity of ATAP requires an amphipathic property. (A) Schematic representation of the GFP-ATAP constructs in which point mutations were introduced into the hydrophobic rich (HR) region of the ATAP. (B) Cell survival was measured by PI exclusion in the HEK293 cells transfected with 1 µg GFP-ATAP mutant constructs 24 hours after transfection. (C) Flag-mHR3 peptide showed reduced pro-apoptotic activity. HEK293 cells were co-transfected with 0.1 µg pCMV-
-gal reporter plasmid and 1 µg of the Flag-ATAP or Flag-mHR3 expression plasmids. 24 hours after co-transfection, cell viability was measured by -galactosidase activity (left). The relative expression levels of the Flag-tagged peptides were determined by western blotting with an antibody against the Flag tag epitope (right). (D) Involvement of amphipathic nature of ATAP in the apoptotic function of HCCS1. HEK293 cells were co-transfected with 0.1 µg pCMV--gal reporter plasmid and 1.0 µg HCCS1-GFP or HCCS1-GFP (E46Q/E53Q) containing mutations corresponding to mHR3 of the Bfl1 ATAP. 24 hours after co-transfection, cell viability was measured by -galactosidase activity. Data are expressed as the mean ± s.e.
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