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. 2022 May 20;13:2835. doi: 10.1038/s41467-022-30264-0

Fig. 5. Identification of Trim21 as a mediator of CDK2 autophagic degradation induced by HHT treatment.

Fig. 5

A Representative co-immunoprecipitation assay in THP-1 cells treated with HHT (40 ng/mL). B Western blot analysis in THP-1 cells treated with 1 μM of 17-AAG. C Western blot analysis in THP-1 cells were pretreated with NH4Cl (20 mM) or MG132 (5 μM) (upper panel) and Chloroquine (50 μM) or lactacystin (10 μM) (lower panel), together with HHT treatment for 9 hours. D Two days after ATG7 or GFP siRNA transfection, THP-1 cells were treated with HHT for 9 hours and subjected to western blot analysis. E HEK293 cells stably expressing 3xFLAG-CDK2 were treated with either HHT (50 ng/mL) or DMSO for 6 hours and proteins were separated by SDS–PAGE. The specific band in the HHT treatment lane (as indicated) was cut for LC/MS analysis. Co-immunoprecipitation assay by CDK2 antibody (F) or Trim21 antibody (G) in cells either treated with HHT (50 ng/mL) or DMSO for 6 hours, followed by western blot analysis. H Trim21 domains and the deletion mutant were constructed as indicated. I HEK293 cells stably expressed 3xFLAG-CDK2 were transfected with either Trim21-HA plasmid or Trim21-HA-ΔC plasmid and treated with HHT (50 ng/mL) for 6 hours, followed by co-immunoprecipitation with FLAG-beads and western blot analysis. J The complex model of Trim21 and CDK2 proteins. The protein-protein interaction residues on Trim21 (green-colored cartoons) C-terminal region to protein IGHG1 (PDB -code: 2IWG) are displayed as blue-colored sticks. The cyclin A protein is also superimposed as gray cartoons (PDB -code: 1FIN) to show its overlap with Trim21. The model of HHT bound to CDK2 is superimposed as pink sticks. K HEK293 cells transfected with Trim21-HA plasmid and 3xFLAG-CDK2 or 3xFLAG-CDK2-5Rs plasmid were treated with HHT (50 ng/mL) for 3 hours, followed by co-immunoprecipitation with HA-beads and western blot analysis. L Representative co-immunoprecipitation assay in mononuclear cells isolated from one primary AML patient were treated with HHT (40 ng/mL) or DMSO for 3 hours. Source data are provided as a Source data file.