1 Introduction AIRE [1–3] [4,5] [6] in vitro [4] [7] [8,9] [10,11] [12] [5,13] [13,14] [15,16] in vitro [5,16] [17,18] [17,19] [17] [20] [21] [22] [20,23] hXOR [24] [22] [18] in vitro in vitro 2 Materials and methods 2.1 Plasmids Nco Kpn Eco Xho [5] Eco Sal Hin 2.2 Expression and purification of GST fusion proteins Section 2.1 [5] Escherichia coli 2 2 2.3 GST pull-down and mass spectrometry [25] 2 2 http://www.matrixscience.com/ 2.4 Transfections and immunoprecipitations in vitro 6 [3] 2 2 [3] 2 2 2 [26] 2 2 2.5 Computer prediction of AIRE phosphorylation http://scansite.mit.edu/cgi-bin/motifscan_seq [27] http://www.cbs.dtu.dk/services/NetPhos [28] 2.6 Phosphorylation assays 32 32 Sections 2.3 and 2.4 Fig. 3 2 3 4 2 3 4 2.7 Mice, thymic stromal cell isolation, cell sorting and RT-PCR [29] 2.8 Luciferase reporter assays 4 3 Results 3.1 DNA-PK complex proteins co-purify with AIRE PHD domain Fig. 1 Fig. 1 Table 1 Table 1 3.2 Full-length AIRE interacts with DNA-PK in vivo Fig. 2 Fig. 2 [26,30] [26,31] Fig. 2 Fig. 2 Fig. 2 Fig. 2 3.3 DNA-PK phosphorylates the AIRE protein [32] 32 Fig. 3 32 [32] in vitro Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 4 Fig. 4 Fig. 4 Fig. 4 in vitro 3.4 T68 and S156 are the phosphorylation sites on the AIRE protein [4,5] in vitro [4] Figs. 4A and 5A Fig. 5 Fig. 5 3.5 DNA-PK is expressed in thymic medullary epithelial cells (mTEC) [17] [1–3] [29] Aire DNA-PK involucrin loricrin involucrin loricrin [33] [34] Fig. 6 involucrin loricrin DNA-PKcs Aire Fig. 6 3.6 T68A and S156A mutations downregulate the transactivation activity of AIRE [13–15,35] loricrin involucrin Fig. 7 Fig. 7 4 Discussion [36] [37] [4] [17,24] [38] [39] [18] [40] [41,42] [43] [44] [10,11] Fig. 2 [17] in vivo, [4] in vitro [4] in vitro [4,5] [15,16] in vitro [4] [45,46] [47] [48,49] [5,13,15,16] in vivo [1–3] [14,16] [50] [51] Fig. 6