Restoration of AHSP binding stabilizes α globin K99E. (A) GST pull-down assay. Radiolabeled WT or K99E α globins were incubated with Sepharose beads linked to GST-WT AHSP or GST-AHSP altered specificity mutants, and AHSP-bound α globin was visualized as described in Figure 2. (B) Bicistronic vectors encoding α globin K99E and the indicated forms of AHSP were introduced into E coli, and Western blotting was used to examine protein expression, as described in Figure 5. (C) Human α globin K99E and various forms of AHSP were stably expressed in MEL cells. (Left panel) Agarose gel electrophoresis of reverse transcribed PCR products generated using specific primers for human α globin and mouse Gapdh. Note that human α globin cDNA expression is roughly equivalent in all MEL lines, except for the negative control in lane 2. (Right panel) Western blot analysis of cytosolic extracts from MEL cells expressing α globin K99E and various AHSP mutants. Note that expression of K99E α globin protein is specifically enhanced by coexpression of altered specificity AHSP mutants Q25K and D29R.