Fig. 2
SirT6 induces a modification in Suv39h1. a Western blotting of extracts from 293F cells transfected with Myc-Suv39h1 in the presence or absence of HA-tagged SirT1 or SirT6 (lanes 2 and 3, respectively). A SirT6-induced modification in Suv39h1 is indicated (red asterisk). b Endogenous Suv39h1 is also modified upon SirT6 upregulation. Suv39h1 Western-blot of extracts from 293 cells overexpressed or not with SIRT6-HA. c Western blotting of extracts from 293F cells expressing non-tagged Suv39h1 in the presence or absence of either Ubiquitin-HA15 and/or SirT6-HA. The effect of a titration of SirT6-HA (1, 3 and 6 μg transfected) on Suv39h1 (2 μg transfected) was tested (lanes 3–5). A lower exposition of the Suv39h1 main band is also shown. Red and blue asterisks indicate endogenous or HA-tagged ubiquitination in Suv39h1, respectively. d Quantification of the levels of modified Myc-Suv39h1 in the absence or presence SirT6-HA expression. Relative levels (%) of Suv39h1 modification compared to unmodified Suv39h1 are shown. The results were obtained from n = 3 replicas of experiment shown in lanes 1–2 of Fig. 4c. e Analysis, as in a, of Myc-Suv39h1 cotransfected with different HA-tagged SirT6 mutants. WT: SirT6 wild type; HY: H133Y; GA: G60A. f Schematics of the different Myc-tagged constructs of Suv39h1 used in g. g Western blotting with the indicated different Myc-Suv39h1 constructs − / + SirT6-HA. h Fractionation of 293F cells co-transfected with Myc-Suv39h1 and SirT6-HA. Nuclear extracts (NE) and nuclear insoluble pellet (NP) were generated using the Dignam method. NP was step-washed with increasing concentrations of NaCl (from 100–1000 mM). i Schematic summary of the experiment shown in j. j Fractionation of 293 cells transfected with Myc-Suv39h1 and − / + SirT1 or SirT6. Fractionation with the RIPA method generated a soluble fraction (RIPA, lanes 1–3) and a NP, which was further digested with Benzonase (lanes 4–6)
