Fig 6.

Charged residues in the Bob1 C terminus regulate protein stability. (A) Protein alignment of the C-terminal 44 amino acids from mouse (top row) and catfish (bottom row). (B) (Top left) Full-length mBob1 (instable) and mcBob1 (stable) migration in an SDS-polyacrylamide gel (expression in HEK293 cells); (top right) fusions of the C termini of mBob1 or cfBob1 (see panel A) to the C terminus of GFP after expression in K46 cells; (bottom) electrophoresis of the same lysates from the top panels in SDS-urea gels. (C) Median GFP/Tomato ratios for C-terminal acidic and basic tags fused to GFP-mBob1 relative to those for GFP only (GFP) and GFP-mBob1 (mBob1). WEHI-231 murine B cells were analyzed at 24 h posttransfection. (D) Point mutations in the C terminus of mBob1 affecting stability. WEHI-231 cells were analyzed at 16 h posttransfection. †, mutations of mBob1 to the corresponding residue in cfBob1; ‡, stabilizing mutations identified by mBob1 random mutagenesis.