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. 2016 Jun 30;291(34):17953–17963. doi: 10.1074/jbc.M116.724401

FIGURE 7.

FIGURE 7.

FBXO3 increases AIRE transcriptional activity. A, mutations of Thr-68 and Ser-156 influence AIRE transcriptional activity. The relative luciferase activity of INSLuc for WT mHis:AIRE (lane 2) and mutant mHis:AIRET68ES156E (lane 3) or mHis:AIRET68AS156A proteins (lane 4) is presented as -fold activation above levels of the empty plasmid vector. Levels of mHis:AIRE and GAPDH (as internal control) proteins are given below the bar graph. B, knockdown of FBXO3 decreases AIRE transcriptional activity. Presented is the relative luciferase activity of INSLuc, which is induced by AIRE, after FBXO3 knockdown (lane 2) or control (lane 1). Values are presented as -fold activation above levels of control siRNA. Levels of mHis:AIRE, FBXO3, and GAPDH (as internal control) proteins are given below the bar graph. C, FBXO3 knockdown affects the expression of AIRE-dependent genes in 293T cells. RT-quantitative PCR analysis was performed on four candidate genes after FBXO3 knockdown in 293T cells. 293T cells co-expressed mHis:AIRE with control (siScr) or FBXO3 targeting siRNAs (siFBXO3) for 72 h before harvesting. Bottom panels present levels of FBXO3, AIRE, and GAPDH (as the internal control) proteins. D, FBXO3 knockdown affects AIRE-dependent genes in the mTECs. RT-quantitative PCR analysis was performed on five candidate genes after FBXO3 knockdown in 1C6 mTECs. 1C6 cells co-expressed mHis:AIRE with control (siScr) or FBXO3 targeting (siFBXO3) siRNAs for 72 h before harvesting. Values and Western blots are as in panel C. For panels A–D, bars represent S.E., n = 3. Student's t test was preformed to measure the significance of the data (*, p < 0.05; **, p < 0.01; ***, p < 0.001).