Figure 2

Lack of T91/93 phosphorylation protects from cell death. (a and b) Confocal analysis of CGCs generated from c-Jun-ΔN mice and infected with GFP-tagged lentivirus particles expressing either HA-tagged c-Jun proteins: c-Jun-wt-HA (a) or c-JunA95-HA (b). After infections, cells were cultured for 6 days and then treated as indicated for 18 h. After fixation, nuclei were stained by DAPI and immunostained with HA antibody. (c) Quantification of results shown in a and b. Columns indicate the average percentage number of condensed nuclei present in 10 different fields, with bars indicating S.D. (d) Western blot analysis of CGCs from c-Jun-ΔN mice infected with lentivirus as described in a and b. After infection, cells were cultured either in 25 mM K or shifted to 5 mM K medium for 4 h, as indicated. Control represents not infected CGCs. c-Jun N-terminal phosphorylation was determined by using the indicated c-Jun phospho-specific antibodies. The levels of c-Jun-HA proteins were determined by using the HA antibody. (e) CGCs from c-Jun-ΔN mice were transfected with Jun2-luc reporter plasmids in combination with plasmids expressing either c-Jun-wt-HA or c-JunA95-HA proteins or empty vector (indicated as control). After infection, cells were cultured either in 25 mM K or shifted to 5 mM K medium for 4 h, as indicated. The luciferase activities were normalized to the internal transfection control. Values of CGCs transfected with empty vectors and cultured in 25 mM K were set as onefold induction. Bar graphs represent the mean ±S.D. of three independent assays