Fig. 3

DAPK suppresses the ubiquitination on the 1–244 domain of DDX20. A Effect of HA-DAPK overexpression on DDX20-FLAG ubiquitination in 293T cells. Precipitates were pulled down by nickel beads, and ubiquitylated target protein was detected using an anti-FLAG antibody. The proteins of HA-DAPK, DDX20-FLAG and GAPDH were detected by Western blotting with respective antibodies. Right: Quantitative analysis of relative ubiquitinated DDX20-FLAG in cells expressing HA-DAPK or not. B Effect of HA-DAPK overexpression on the ubiquitination of endogenous DDX20 in 293T cells. Whole cell lysates (containing HA-DAPK) were immunoprecipitated using an anti-DDX20 antibody and blotted with an anti-Ubiquitin antibody. The working concentration of MG132 was 20 μM. C The co-localization of GFP-DAPK (green) and DDX20-mcherry (red) were detected. Scale bar denotes 10 μm. D, E HA-DAPK and DDX20-FLAG plasmids were transfected into 293T cells. Protein complexes were co-immunoprecipitated with anti-HA (D)or Flag antibodies (E), and DDX20 and DAPK were detected, respectively. F Co-immunoprecipitation assay of HA-DAPK and different DDX20 domains in the whole cell lysates of 293T cells expressing indicated proteins. G Effect of HA-DAPK overexpression on the different domains of DDX20 in 293T cells. H Effect of HA-DAPK overexpression on the ubiquitination of truncated domains DDX20 in 293T cells. INPUT: Whole lysate, IP: immunoprecipitation, PD: pull down, IB: immunoblot. ***p < 0.001, between the indicated groups