K48-linked ubiquitiniation of Hax-1 enhanced proteasomal degradation of Hax-1 during apoptosis. A. H1299 cells were transfected with EGFP or EGFP-Hax-1. Forty-eight hours later, cells were treated with or without MG132 (1 μM) for 12 hours. Cell lysates were then subjected to immunoprecipitation with anti-GFP antibodies and the immunoprecipitants were detected with total ubiquitin antibody. B. Similar experiments as A were performed using specific anti-K48 or -K63 ubiquitin antibodies. C. H1299 cells were transfected with EGFP or EGFP-Hax-1 or EGFP-ΔPEST Hax-1 expression constructs. Forty-eight hours later, cells were treated with MG132 (1 μM) for 12 hours. Cell lysates were then immunoprecipitated with anti-GFP antibodies and detected with anti-K48 ubiquitin antibodies. D. EGFP-Hax-1 transfected H1299 cells were treated with DMSO or increased levels of STS (0.2-1 μmol) in the absence or presence of MG132 for 3 hours. Cells were then harvested and subjected to immunoblot analysis with anti-GFP antibody. E. Quantitative analyses of Hax-1 degradation upon STS treatment through three independent experiments were shown.