FIGURE 5.

FBXO3 destabilizes AIRE. A, knockdown of FBXO3 increases amounts of steady state AIRE protein. 293T cells co-expressed mHis:AIRE, control (scrambled) siRNA (siScr) or FBXO3 siRNA (siFBXO3). Whole cell lysates were probed with anti-Myc or anti-FBXO3 antibodies by Western blotting. GAPDH served as a loading control. B, knockdown of FBXO3 stabilizes AIRE. Cells were transfected with mHis:AIRE, control siRNA (siScr), or siRNA targeting FBXO3 (siFBXO3) for 48 h. 100 μg/ml cycloheximide (CHX) was added to cells, and the disappearance of AIRE was followed by Western blotting with specific antibodies (anti-Myc, anti-FBXO3, and anti-GAPDH) for the indicated times (0, 1, 4, and 8 h). C, densitometric analysis of mHis:AIRE Western blot from panel B. Protein bands were quantified using ImageJ software, and AIRE protein half-life was calculated. Bars represent the S.E. of three independent experiments (S.E., n = 3).