Ubiquitination-resistant Bfl-1 point mutants KKK/RRR and ST/DD coexpressed with p53DD induce lymphomagenesis with kinetics and efficiencies similar to that of Bfl-1ΔC. (A) Similar to wild-type Bfl-1 and Bfl-1ΔC, Bfl-1 point mutants KKK/ RRR and ST/DD selectively interact with endogenous Bak but not Bax, although Bfl-1ΔC and ST/DD did so with reduced efficiency. Coimmunoprecipitation of endogenous Bak or Bax with transiently transfected 2xMyc-tagged Bfl-1, Bfl-1ΔC, ST/DD, or KKK/RRR in HeLa cells. IN indicates 1/10 input; IP, immunoprecipitation with anti-Myc; IgG, immunoprecipitation with control mouse immunoglobulin. (B) Bfl-1, KKK/RRR, and Bcl-xL localize to mitochondria as seen by colocalization with DS-Red2-Mito tracker, whereas Bfl-1ΔC and ST/DD show diffuse localization by GFP fluorescence. (C) Helical wheel diagram showing that the ST/DD substitution adds significant negative charge on one side of the helix predicted in the C-terminus of Bfl-1. Basic (red), acidic (blue), nonpolar (orange), polar (green), and aromatic (brown) amino acids are indicated. (D) Kaplan-Meier curves showing similar kinetics of tumorigenesis in NCR nude mice injected intravenously with FL5.12 cells coexpressing Bfl-1 mutants GFP-Bfl-1ΔC (red, n = 4), KKK/RRR (blue, n = 4), or ST/DD (purple, n = 4) along with p53DD (GFP-Bfl-1ΔC/p53DD vs GFP-Bfl-1 KKK/RRR/p53DD, P = .401; GFP-Bfl-1ΔC/p53DD vs GFP-Bfl-1 ST/DD/p53DD, P = .469; GFP-Bfl-1KKK/RRR/p53DD vs GFP-Bfl-1 ST/DD/p53DD, P = .240).