Extended Data Fig. 4: WEE1 is acetylated by GCN5 and deacetylated by SIRT1.
From: SIRT1 deacetylates WEE1 and sensitizes cancer cells to WEE1 inhibition
(a) SIRT1 interacts with WEE1. HEK293T cells were transfected with the indicated plasmids for 24 hr. Cell lysates were then prepared and western blot analysis was carried out as indicated. (b) SIRT1 directly interacts with WEE1 in vitro. GST-SIRT1 and MBP-WEE1 were expressed and purified from E. coli and subjected to MBP pull-down assays. (c–e) Association of endogenous SIRT1 with WEE1. Cells were irradiated with 40 J/m2 UV and allowed to recover for the indicated time. Cell lysates were then prepared and western blot analysis was carried out as indicated. (f) GCN5 acetylates WEE1 in vivo. HEK293T cells were transfected with the indicated plasmids for 24 hr. Cell lysates were then prepared and western blot analysis was carried out as indicated. (g) HEK293T cells were transfected with the indicated plasmids for 24 hr. Cell lysates were then prepared and western blot analysis was carried out as indicated. (h) WEE1 forms a complex with GCN5. HEK293T cells were transfected with the indicated plasmids for 24 hr. Cell lysates were then prepared and western blot analysis was carried out as indicated. (i) Association of endogenous WEE1 with GCN5. MDA-MB-231 cells were irradiated with 40 J/m2 UV and allowed to recover for the indicated time. Cell lysates were then prepared and western blot analysis was carried out as indicated. (j) WEE1 directly interacts with GCN5 in vitro. GST-GCN5 and MBP-WEE1 were expressed and purified from E. coli and subjected to MBP pull-down assays. (k) SIRT1 mediates WEE1 deacetylation. HEK293T cells were transfected with the indicated plasmids for 24 hr. Cell lysates were then prepared and western blot analysis was carried out as indicated. Data in A-K were independently replicated at least three times, with similar results.
