Fig. 4: OTUD3 deubiquitylates MYL12A and activates Akt signaling.

A Cell-free MYL12A ubiquitination experiment: GST-MYL12A and His-OTUD3 proteins, expressed and purified from bacteria, were incubated with commercial E1, UbE2D3 (E2), CHIP (E3), Hsp70, and ubiquitin at 37 °C for 2 h. The mixture underwent GST pulldown, and Western blot analysis was conducted using anti-His antibodies. B Farage cells were transfected with the specified constructs, followed by treatment with MG132 for 8 h before collection. Whole-cell lysates underwent Ni2+ beads pulldown, and Western blot analysis was performed using anti-Myc and anti-His antibodies to detect ubiquitinated MYL12A. C OTUD3 knock-down Stable Farage cells were co-transfected with Flag-OTUD3 WT or C76A mutant and HA-Ub. Cells were treated with MG132 (20 µM, 8 h) before collection. IP of cell lysates was performed using anti-MYL12A antibodies, followed by detection of MYL12A ubiquitination using anti-HA antibodies. D Farage cells were co-transfected with His-MYL12A, Flag-OTUD3, and plasmids encoding HA-Ub Lys0, Lys48-only, or Lys63-only. Subsequently, the ubiquitination of MYL12A was analyzed. E Cell-free ubiquitination experiments of MYL12A were performed using OTUD3 WT or C76A in conjunction with Lys48-only plasmids. F HEK293T cells were transfected with empty plasmid or Flag-OTUD3, HA-Ub, and either wild-type His-MYL12A or K-to-R mutant. Cell lysates underwent IP with anti-Flag beads, followed by immunoblot analysis using anti-HA or anti-Flag antibodies. G Plasmids encoding MYL12A or OTUD3 shRNA-resistant were transfected into MYL12A-knockout Farage cells with stable knockdown of OTUD3 using shRNA. Subsequently, analysis of Akt phosphorylation was conducted. H, I Protein expression in various components of the Akt pathway under normal conditions or after rupatadine treatment in all groups.