Fig. 5

DAPK and DDX20 form a protein complex via TRIM25 in cells. A, B The Coomassie blue staining gel of purified protein. Red asterisks indicate the HIS-sumo DAPK 364 which is a HIS-sumo fused DAPK domain containing 1–364 amino acids (A), 0: bacteriophage lysis solution, 1: supernatant after sonication lysis and centrifugation, 2: molecular weight marker, 3–4: protein solution collected after 20 mM imidazole elution, 5–7: protein solution collected after 50 mM imidazole elution, 8: protein solution collected after 500 mM imidazole elution. Yellow asterisks indicate the HIS-sumo DDX20 244 which is a HIS-sumo fused DDX20 domain containing 1–244 amino acids. B. 0: molecular weight marker, 1: bacteriophage lysis solution, 2: supernatant after high-speed centrifugation of bacteriophage ultrasonically lysed, 3–4: protein solution collected after 20 mM imidazole elution, 5–8: protein solution collected after 50 mM imidazole elution, 9: protein solution collected after 500 mM imidazole elution. C In vitro HIS pulldown assay analysis of the interaction of purified protein DAPK and DDX20. D Co-immunoprecipitation assay of DDX20-FLAG and HA-DAPK in whole cell lysates of 293T cells transfected with an shRNA targeting TRIM25. E Co-immunoprecipitated exogenous HA-DAPK and MYC-TRIM25 proteins were detected by Western blotting with anti-HA and anti-Myc antibodies, respectively. F Co-IP assay detecting the association of HA-DAPK, MYC-TRIM25 and DDX20-FLAG in 293T cells expressing the indicated plasmids. Whole cell lysates were immunoprecipitated with anti-FLAG antibody. G Effect of overexpression of HA-DAPK or HA-DAPK(K42A), a kinase dead mutant of DAPK, on the ubiquitination of DDX20-FLAG in 293T cells. HIS ubiquitylation assays were performed as described in Materials and Methods. H Western blotting of TRIM25 and GAPDH proteins in 293T cells transfected with HA-DAPK or HA-DAPK(K42A). Whole cell lysates were treated with phosphorylase inhibitor and N-Ethylmaleimide. INPUT: Whole lysate, IP: immunoprecipitation, PD: pull down, IB: immunoblot. *p < 0.05, between the indicated groups. Data are shown as the mean ± SD of three independent experiments