Fig. 4

SirT6-induced monoubiquitination of Suv39h1 is induced by NF-κB pathway activation. a Suv39h1 levels from 293F cells transfected with Myc-Suv39h1 − / + SirT6-HA and analyzed at different degree of confluency. Cells were all plated at the same time and harvested at the indicated confluency (indicated in %). A quantification of n = 3 experiments is shown in Supplementary Figure 4a. b Western blotting of Myc-Suv39h1 − / + SirT6 expressed in 293F cells in the indicated conditions. C, control; DTB, double thymidine block; SS, serum starvation; NOC, nocodazole. A quantification of n = 3 experiments is shown in Supplementary Figure 4c. c Similar experiment as in b, with the indicated treatments. C, control; HU, hydroxyurea; CPT, camptothecin. FACS analysis of these treatments are included in Supplementary Figure 4b. A quantification of n = 3 experiments is shown in Supplementary Figure 5b. d SirT6 depletion by shRNA. Western blotting of endogenous SirT6 in 293F cells transfected with either scramble shRNA (Sc) or SIRT6 shRNA (Sh6). e Cells in d treated with the indicated conditions. A quantification of n = 3 experiments is shown in Supplementary Figure 5e. f Nuclear fractionation of Suv39h1 monoubiquitination induced by TNFα in 293F cells. Left panel, either total nuclear fraction or nuclear pellet digested with Benzonase (see online Methods) is shown. Right panel, quantification (n = 3) of the relative abundance of Suv39h1mUb vs total Suv39h1 in total nuclear fraction. (T-test; SEM, *p < 0.05, **p < 0.01). g Induction of Suv39h1mUb by TNFα in 293F cells expressing either scramble shRNA or shSIRT6. h Western blotting of extracts from 293F cells transfected with the indicated combinations of SirT6-HA, Myc-Suv39h1, and FLAG-RelA, and incubated in the presence or absence of TNFα before collecting the cells. i Co-immunoprecipitation experiments using anti-FLAG resin of extracts from 293F cells transfected with the indicated combinations of FLAG-RelA and Myc-Suv39h1 and in presence or absence of TNFα