Figure 3. His₁₂-I573A shows impaired glycosylation.

(A) Following transient transfection cell lysates (30 μg) were resolved on 10% w/v acrylamide gels and blotted with BXP-21 antibody. Transfection reagent only (PEI) served as negative control, WT His₁₂–ABCG2 and His₁₂–M131A serve as positive controls. His₁₂–N596Q is a glycosylation defective form of ABCG2. His₁₂–I573A is exclusively present at an intermediate molecular mass at 24 h and even at 48 h the majority of His₁₂–I573A isoform is not the mature molecular mass. (B). Cell lysates were obtained 24 and 48 h post transfection, denatured and then incubated in the presence or absence of PNGaseF to remove glycosylation before western blotting. His₁₂–N596Q shows no change in molecular mass upon PNGaseF treatment. By contrast, the lower molecular mass band following transfection with the His₁₂–I573A isoform shows a small (estimated < 5 kDa) change in molecular mass upon PNGaseF treatment indicative of the removal of core glycosylation. WT and a representative other mutant (His₁₂–M131A) are fully glycosylated at both 24 and 48 h post transfection. Data are representative of at least three independent transfections.