Supplementary Figure 6: KDM4B demethylates Akt1.
From: AKT methylation by SETDB1 promotes AKT kinase activity and oncogenic functions
a, IB analyses of GST pull-down and WCL derived from HEK293 cells transfected with the indicated constructs. b-e, Coomassie staining image to illustrate mammalian purified GST-KDM4 (b) and bacterially purified GST-KDM4B catalytic domains (d), which were used for in vitro demethylation assays with Akt1 methylated peptides (c) or cell purified HA-Akt1 (insulin treated before harvesting) (e) as substrates. f-l, IB analyses of GST pull-down and WCL derived from HEK293 cells transfected with the indicated constructs. m-o, IB analysis of WCL derived from A375 cells (m), OVCAR5 cells (n) or HEK293 cells (o) lentivirally infected with shRNAs against KDM4B. p, IB analysis of WCL derived from AKT1K140/142R-edited HEK293 cells infected with lentivirus against KDM4B. q,r, IB analysis of cell fractionations separated from HEK293 cells infected with lentiviruses against KDM4B. s, IB analysis of cell fractionations separated from HEK293 cells infected with shRNA against KDM4B. t, HEK293 cells were transfected with indicated constructs and serum starved for 12 hrs. Resulting cells were stimulated with insulin (0.1 μM) in different time points before harvesting for GST pull-down and IB analysis. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.
