Fig. 5

ITPKB interaction with Trim25 dependent on K48 ubiquitination at sites K793 and K818. a Depletion of Trim25 induces ITPKB protein levels. T98G-R and U118-R cells were transfected with control (Ctrl) or Trim25 siRNAs, and cell lysates were immunoblotted with the indicated antibodies. b T98G-R and U118-R cells were transiently transfected with Trim25 plasmid, and the cell lysates were immunoblotted with the indicated antibodies. c T98G-R cells transfected with Ctrl or Trim25 plasmid were treated with vehicle or MG132 (50 μM) for 3 h. Cell lysates were then immunoblotted with the indicated antibodies. d T98G-R cells transfected with Ctrl or Trim25 siRNAs were treated with CHX (0.1 mg/mL) and harvested at the indicated times. Cells were lysed, and cell lysates were then immunoblotted with the indicated antibodies. e Quantification of the ITPKB protein levels relative to Actin. f T98G-R cells transfected with Trim25 plasmid were treated with CHX (0.1 mg/mL) and harvested at the indicated times. g Quantification of the ITPKB protein levels relative to Actin. h Cells transfected with Ctrl or Trim25 siRNAs were treated with MG132 for 3 h before harvest. Immunoprecipitation with control IgG and ITPKB was performed, followed by immunoblotting with the indicated antibodies. i, j HEK293T cells expressing Flag-ITPKB were transiently transfected with V5-tagged Trim25 and HA-tagged ubiquitin/K48 ubiquitin. After 48 h, cells were treated with MG132 (50 μM) for 3 h. Cell lysates were immunoprecipitated with Anti-FLAG® M2 Magnetic Beads, and then immunoblotted with the indicated antibodies. k HEK293T cells expressing Flag-ITPKB were transiently transfected with indicated HA-K48 lysine-specific mutant constructs. After 48 h, cells were treated with MG132 for 3 h before collection. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibody. l, m Identification of the ubiquitination sites of ITPKB for its K48-specific polyubiquitination. ITPKB stably knockdown HEK293T cells were transiently transfected with indicated constructs. After 48 h, cells were treated with MG132 for 3 h before collection. Cell lysates were immunoprecipitated and then immunoblotted with the indicated antibodies. n–q Trim25 plasmids were co-transfected with a control vector, ITPKB wildtype, or 2KR mutant in T98G-R cells. ROS levels were measured using the DCFDA assay. Cell apoptosis was assessed by the Annexin V-FITC apoptosis kit. Relative cell survival was determined by CCK8 assay. Statistical significance is shown as: *p < 0.01, **p < 0.001