Fig. 7: The deubiquitinase USP9X binds to the JmjN domain of KDM4C to prevent its degradation.

a TAP-MS analysis shows KDM4C interacting proteins in HEK293T cells. The name and number of peptides for each protein identified are listed. b SPC-A1 and H460 cells were transfected with SFB-KDM4C plasmids. 24 h later, cells were harvested and then incubated with S-protein agarose prior to analysis by Western blotting (n = 3). c Endogenous interaction between KDM4C and USP9X in SPC-A1 cells (n = 3). d Schematic description of the domains of KDM4C and the generated deletion mutants. e The JmjN domain of KDM4C is required for its binding to USP9X. S protein beads were used for immunoprecipitation assay and cell lysates were subjected to immunoblotting (n = 3). WCL: whole cell lysate. f SPC-A1 and H460 cells were transfected with a plasmid encoding KDM4C for 24 h and then incubated with MG132 (10 μM) for 4 h prior to harvesting (n = 3). g SPC-A1 cells were transfected with the indicated constructs for 24 h, and MG132 (10 μM) was added for another 4 h. Cell lysates were then subjected to immunoprecipitation using S-protein beads and subsequent analysis by Western blotting (n = 3). h USP9X knockdown results in reduced levels of endogenous KDM4C (n = 3). i SPC-A1 cells were transfected with the indicated siRNAs for 48 h, incubated with DMSO or 10 μM MG132 for another 4 h. Cells were collected and probed for the indicated proteins by Western blotting (n = 3). j Cells were transfected with the indicated siRNAs and plasmids prior to treatment with MG132 (10 μM) for 4 h before collection. The lysates were incubated with S beads overnight and then subjected to immunoblotting (n = 3). k Left panel: SPC-A1 cells transfected with the indicated siRNAs for 48 h were treated with 100 μg/mL of cycloheximide (CHX) and collected at the indicated time points. Immunoblotting was performed to examine the protein level of KDM4C. Right panel: quantification of the KDM4C band intensity (n = 3).