Supplementary Figure 5: PIP3 enhances the interaction of SETDB1 with Akt1 PH motif.

a-c, A cartoon illustration of the workflow describing the method for in vitro binding assays (a). Briefly, 293T cells transfected with different Akt1 encoding constructs were harvested for GST pull-down assay after treatment with the PI3K inhibitor, LY294002. The pull-down Akt1 was eluted from the beads (b) and incubated with flag-bead immunoprecipitated SETDB1 (c) in the presence/absence of PIP₃ (20 μM) for 8 hrs in 4 degrees. Then the flag-beads were washed 3 times and subjected for IB analysis. d,e, His-Akt1 was purified from insect cells and eluted (d), then subjected to in vitro binding assay in the presence or absence of PIP₃, and detected with IB analysis (e). f, HCT116-PTEN^-/- and counterpart cells were starved and stimulated with IGF (100 ng/ml), then subjected to IB analysis. g,h, IB analysis of GST pull-down and WCL derived from HEK293 cells transfected with the indicated constructs. i-k, IB analysis of cell fractionations separated from SETDB1 knockdown A375 cells (i), AKT1^K140/142R-edited and parental HEK293 cells (j) and Setdb1-deleted MEFs (k). Where indicated, MEFs were treated with 4-OHT (500 nM) or vehicle for 48 hrs, and resulting cells were serum-starved for 12 hrs and insulin-stimulated in different time points before harvest. l,m, IB analysis of WCL derived from HeLa cells transfected with C-terminal GFP-fusion Akt1. Cells were serum-starved 12 hrs and stimulated with or without insulin for 30 minutes, then subjected to DAPI staining and detected with fluorescence microscope. The nuclear DNA was stained with DAPI. Scale bar, 20 μm. The experiment in m was performed twice, independently, with similar results (m). n, A schematic illustration of the proposed model in which full Akt activation is achieved by a coordinated action of the SETDB1-mediated Akt methylation events and the canonical PI3K-PDK1 dependent kinase cascades leading to Akt phosphorylation. o-r, IB analysis of GST pull-down (o,q), his-tagged ubiquitination (p,r) and WCL derived from HEK293 cells transfected with indicated encoding constructs. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.