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. 2020 Jan 7;11:24. doi: 10.1038/s41467-019-13808-9

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© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2020

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Fig. 6 — a–c BUB3 colocalizes with PML-NB during interphase. U2OS RepID WT and KO cells were exposed to Triton X-100 to remove excess non-chromatin-bound proteins, and immunofluorescence analysis was performed using anti-PML and anti-BUB3 antibodies. Representative images using confocal microscopy (a) and super-resolution microscopy (b) are shown. c Colocalization between BUB3 and PML-NB was analyzed by intensity profiling as indicated in b. d–f BUB3 is degraded in RepID WT cells in which PML has been knocked down. siRNA-PML was transfected into U2OS RepID WT and KO cells, and immunofluorescence analysis was performed using anti-BUB3 and anti-PML antibodies. Representative images are shown without MG132 (d) or with MG132 (e). f Intensities of BUB3 in cells as in d, e. g BUB3 is ubiquitinated by CRL4^CDT2 in PML-deficient RepID WT cells. HIS-ubiquitin plasmid was transfected along with the indicated siRNAs into HCT116 RepID WT cells, and lysates were isolated on Ni-NTA beads, followed by immunoblot analysis. Error bars in all results represent standard deviation from three independent experiments (***p value < 0.001, n.s.; not significant, Student’s t test).