Figure 2.

The three AnkG groups display different anti-apoptotic activities. (a) Schematic representation of the AnkG variants used in this study. Amino acids are shown above each of the schematic diagrams. Striped area represents altered amino acids as compared to the reference AnkG_NM. (b) CHO-FcR cells were transiently transfected with GFP, GFP-AnkG_NM, GFP-AnkG_Soyta or GFP-AnkG_F3. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and probed with an antibody against GFP. Depicted is one representative immunoblot of three independently performed experiments with similar results. (c) CHO-FcR cells expressing GFP, GFP-AnkG_NM, GFP-AnkG_Soyta or GFP-AnkG_F3 were treated with staurosporine for 4 h. The cells were fixed, permeabilized and the nuclei were stained with DAPI. The nuclear morphology of at least 100 GFP-expressing cells was scored in three independent experiments. Error bars indicate ± SD. **p < 0.01; ***p < 0.001, n.s. not significant. (d) HEK293 cells were co-transfected with plasmids encoding HA-tagged p32, GFP-tagged AnkG_NM, AnkG_Soyta and AnkG_F3. The proteins were precipitated from the cell lysates using a GFP-trap. Immunoblot analysis was used to detect p32 (anti-HA) and AnkG-protein variants (anti-GFP) in the lysates (pre-IP) and in the precipitates (IP). Shown is a representative experiment out of three independent experiments with similar results. (e) Cell lysates from HEK293 cells transiently expressing HA-tagged AnkG_NM, AnkG_Soyta and AnkG_F3 were incubated with Flag-tagged importin-α1 bound to an anti-Flag M2 affinity gel. Cell lysates and pull-down fractions were subjected to immunoblot analysis using anti-HA and anti-Flag antibodies. Shown is a representative experiment from three independent experiments with similar results. *Unspecific band. (f) Representative immunofluorescence micrographs show CHO-FcR cells transiently expressing GFP-AnkG_NM, GFP-AnkG_Soyta or GFP-AnkG_F3 (all green) stained with DAPI to visualize the nuclei (blue). Scale bars, 10 µm.