Figure 4.
Disappearance of Bim, Puma, and Bad in Chlamydia-infected cells. (A) Analysis of SDS and Triton X-100 extracts of various cell lines. Cells were infected with C. trachomatis or left uninfected and harvested after 24 h of infection or as indicated. SDS extracts from MCF-7 cells (top) or Triton X-100 extracts (bottom) were subjected to Western blotting with antibodies against Bim (isoform BimEL is detected) and chlamydial HSP60 (as control for chlamydial infection). Detection of tubulin served as a loading control. SDS extracts were used in the initial experiments to exclude the possibility of Bim degradation during extraction. (B) Rifampin treatment prevents degradation of Bim by both chlamydial species. Hep2 cells were infected with C. trachomatis (24 h before analysis) or C. pneumoniae (as indicated) in the presence or absence of 10 μg/ml rifampin. Triton X-100 extracts were analyzed by Western blotting. (C) No disappearance of Bim is seen in Legionella-infected cells. Jurkat and Hep2 cells were infected with L. pneumophila for 8 h. Triton X-100 extracts were analyzed by Western blotting (ns, nonspecific band). Caspase 3–like activity was measured in cell extracts as described above to confirm infection, which is known to cause caspase activation. (D) Kinetics of Bim disappearance. At the indicated time points after infection, Triton X-100 extracts of Hep2 cell samples were taken and analyzed by anti-Bim Western blotting. The figure shows typical results from at least three independent experiments. (E) Disappearance of Puma and Bad in infected cells. Hep2 or HeLa cells were infected with C. trachomatis. 24 h later, the expression of Puma or Bad was assessed by Western blotting.