Determination of the binding domain of ALKBH5 with DDX3. (a) Schematic diagram of full-length ALKBH5 and the corresponding partial deletion constructs; summary of the results of interaction with DDX3 was shown. (b) Deletion of the N-terminal domain, D-domain, or C-terminal domain of ALKBH5 showed interaction with full-length DDX3, examined with IP and co-IP. (c) Deletion of the DSBH domain of ALKBH5 abolished its interaction with DDX3 when examined with IP and co-IP. (d) DSBH domain of ALKBH5 alone interacted with full-length DDX3, examined with IP and co-IP. (e) ATP domain of DDX3 interacted with DSBH domain of ALKBH5, examined with IP and co-IP. (f) DSBH domain of ALKBH5 interacted with ATP domain of DDX3, examined with IP and co-IP. (b–f) All IP and co-IP were performed in triplicates, and representative results are shown. (g) The predicted interaction between ATP domain of DDX3 (DDX3211–402) and DSBH domain of ALKBH5 (ALKBH5190–293). DDX3211–402 is labeled in green, and ALKBH5190–293 is labeled in red. The circle indicates the interacting region of these two domains, and a magnified view displays residues involved in formation of intermolecular hydrogen bonds. The hydrogen bonds are presented in blue dash lines.