Fig. 3. Aire mutants with increased PML association are impaired in transcriptional activity and are hyper-SUMOylated.

a Transcriptional activity of mAire and Sp110-CARD swap, where Aire CARD was swapped with Sp110 CARD. Experiments were performed as in Fig. 1g and presented as mean ± s.d., n = 3. Bottom right, WB of mAire and histone H3. b Representative fluorescence microscopy images of FLAG-tagged WT, Sp110-CARD swap and K53A/E54A mAire variants in 4D6 cells. Note that 4D6 cells were used as these cells show more distinct PML bodies and are flatter than 293 T, allowing more robust analysis of Aire foci and their endogenous PML body localization. Cells were immunostained with anti-FLAG (mAire) and anti-PML. Right, quantification of the Aire foci colocalized with endogenous PML bodies from automated image analysis (see Supplementary Fig. 3a, b for the definition of colocalization). n = number of Aire foci examined per sample. Statistical significance comparisons were calculated using a two-tailed Student’s t-test for two population proportions where each population consists of all individual Aire foci examined. ***p = 5.57e-26 and 6.46e-15 for Sp110-CARD swap and K53A/E54A, respectively. See also Supplementary Fig. 3c for FLAG-tagged hAire stably expressed in 4D6 cells. c SUMO modification analysis of WT mAire and K53A/E54A. FLAG-tagged mAire was co-expressed with HA-SUMO1 or -SUMO2 in 293 T cells. Cells were treated with MG132 (10 μM) for 24 h before harvesting. mAire proteins were immunoprecipitated (IPed) using anti-FLAG beads under semi-denaturing condition and analyzed by anti-HA WB. d Schematic of chromatin fractionation analysis of Aire. 293 T cells were transfected with mAire expressing plasmids for 48 h before harvesting. Solubility of Aire and chromatin (as measured by Histone H3) before and after MNase treatment was analyzed by WB. e Chromatin fractionation analysis of mAire WT and K53A/E54A. Experiments were performed as in (d).